Characterization of Kloeckera apiculata strains from the Friuli regionin Northern Italy

Forty-nine strains of Kloeckera apiculata, isolated from the Friuli region in Italy, were differentiated on the basis of fermentation behaviour and production of secondary compounds in two different grape musts at 18 °C. The isolates exhibited a controlled production of acetic acid, only in a few cases more that 1 g/l. In Moscato grape must the strains exhibited a more uniform behaviour for the production of higher alcohols, ethyl acetate and acetoin than in red grapes. In general, higher levels of ethanol, glycerol and acetic acid were produced in red grape must fermentation. Apiculate strains behaved differently in the two musts, with different metabolic phenotypes dominating the fermentation process. The existence of different metabolic phenotypes correlated with the must composition underlines the need to perform a selection of indigenous apiculate yeasts to obtain the desired consistent products.     

Acetaldehyde production in Saccharomyces cerevisiae wine yeasts

Abstract Eighty-six strains of Saccharomyces cerevisiae were investigated for their ability to produce acetaldehyde in synthetic medium and in grape must. Acetaldehyde production did not differ significantly between the two media, ranging from a few mg/l to about 60 mg/l, and was found to be a strain characteristic. The fermentation temperature of 30°C considerably increased the acetaldehyde produced. This study allowed us to assign the strains to different phenotypes: low, medium and high acetaldehyde producers. The low and high phenotypes differed considerably also in the production of acetic acid, acetoin and higher alcohols and can be useful for studying acetaldehyde production in S. cerevisiae , both from the technological and genetic point of view.     

Biotechnological suitability of Saccharomycodes ludwigii for fermented beverages

Nineteen Saccharomycodes ludwigii strains were tested for the production of secondary products in grape must fermentation. A predominant metabolic pattern characterized by high production of isobutyl alcohol, acetoin and ethyl acetate was obtained. The occurrence of some strains producing enhanced amounts of these compounds suggests a potential utilization of this species for industrial applications. Feijoa juice was inoculated with a selected S'codes ludwigii strain in comparison to a control strain of S. cerevisiae and evaluation of the fermented products was carried out by 30 consumers with respect to the odour, flavour and taste. The sample fermented by S'codes ludwigii was characterized by a fresh odour with a fruity flavour, identified as flavour of apple and kiwi-fruit. This product was compared to apple juice, with a more acid taste. Despite the high concentrations of acetic acid, this beverage might be considered a potential summer refreshing drink, addressed to a target of consumers who prefer fruit drinks that leave a slightly acid and little sugary taste in the mouth.     

Comparative metabolic profiling to investigate the contribution of <Emphasis Type="Italic">O. oeni</Emphasis> MLF starter cultures to red wine composition

In this research work we investigated changes in volatile aroma composition associated with four commercial Oenococcus oeni malolactic fermentation (MLF) starter cultures in South African Shiraz and Pinotage red wines. A control wine in which MLF was suppressed was included. The MLF progress was monitored by use of infrared spectroscopy. Gas chromatographic analysis and capillary electrophoresis were used to evaluate the volatile aroma composition and organic acid profiles, respectively. Significant strain-specific variations were observed in the degradation of citric acid and production of lactic acid during MLF. Subsequently, compounds directly and indirectly resulting from citric acid metabolism, namely diacetyl, acetic acid, acetoin, and ethyl lactate, were also affected depending on the bacterial strain used for MLF. Bacterial metabolic activity increased concentrations of the higher alcohols, fatty acids, and total esters, with a larger increase in ethyl esters than in acetate esters. Ethyl lactate, diethyl succinate, ethyl octanoate, ethyl 2-methylpropanoate, and ethyl propionate concentrations were increased by MLF. In contrast, levels of hexyl acetate, isoamyl acetate, 2-phenylethyl acetate, and ethyl acetate were reduced or remained unchanged, depending on the strain and cultivar evaluated. Formation of ethyl butyrate, ethyl propionate, ethyl 2-methylbutryate, and ethyl isovalerate was related to specific bacterial strains used, indicating possible differences in esterase activity. A strain-specific tendency to reduce total aldehyde concentrations was found at the completion of MLF, although further investigation is needed in this regard. This study provided insight into metabolism in O. oeni starter cultures during MLF in red wine.     

Characterization of kinetic parameters and the formation of volatile compounds during the tequila fermentation by wild yeasts isolated from agave juice

The production of aroma compounds during tequila fermentation using four native yeast strains isolated from agave juice was quantified at controlled (35 degrees C) and uncontrolled temperatures (room temperature) by gas chromatography (FID). Three of the four strains were identified as Saccharomyces cerevisiae (MTLI 1, MALI 1 and MGLI 1) and one as Kloeckera apiculata (MALI 2). Among the aroma compounds produced, acetaldehyde has the highest accumulation at the controlled temperature and before 50% of sugar was consumed. The S. cerevisiae strains produced ethyl acetate in almost the same quantity at a concentration of 5 mg/L and the K. apiculata produced six-times more (30 mg/L) than the S. cerevisiae strains, independent of the fermentation temperature. The rate and amount of 1-propanol, amyl alcohols and isobutanol production were affected by the type of yeast used. The K. apiculate strain produced 50% less of the higher alcohols than the Saccharomyces strains. The results obtained showed that indigenous isolated yeasts play an important role in the tequila flavor and suggest that mixtures of these yeasts may be used to produce tequila with a unique and desirable aroma.     

Glycerol and other fermentation products of apiculate wine yeasts

Ninety-six strains of apiculate wine yeasts were studied for their ability to produce glycerol, acetaldehyde, ethyl acetate, sulphur dioxide and hydrogen sulphide in synthetic medium. Hanseniaspora guilliermondii produced smaller quantities of glycerol, acetaldehyde and hydrogen sulphide than Kloeckera apiculata , whereas the production of ethyl acetate and sulphur dioxide was found to be similar. Strains characterized by different capacities and properties were found for both species. The existence of apiculate strains differing in secondary compound production is of technological interest, as these yeasts constitute potential flavour producers. Selected strains of apiculate yeasts might favour an enhanced flavour formation and yield desirable characteristics to the final product.     

Secondary products formation as a tool for discriminating non-Saccharomyces wine strains

A total of 78 strains of non-Saccharomyces yeasts were isolated: 30 strains of Kloeckera apiculata, 20 of Candida stellata, 8 of Candida valida and 20 of Zygosaccharomyces fermentati. The diversity of yeast species and strains was monitored by determining the formation of secondary products of fermentation, such as acetaldehyde, ethyl acetate and higher alcohols. Within each species, the strains were distinguishable in phenotypes through the production of different amounts of by-products. In particular, a great variability was found in C. stellata, where six different phenotypes were identified by means of the production of acetaldehyde, ethyl acetate, isobutanol and isoamyl alcohol. At different stages of the spontaneous fermentation different phenotypes of the non-Saccharomyces yeasts were represented, characterized by consistent differences in some by-products involved in the wine bouquet, such as acetaldehyde.     

Higher alcohol and acetic acid production by apiculate wine yeasts

P. ROMANO, G. SUZZI, G. COMI AND R. ZIRONI. 1992. Ninety-six strains of apiculate wine yeasts were investigated for their ability to produce higher alcohols and acetic acid in synthetic medium. Less isoamyl alcohol and more n -propanol and isobutanol were formed by Hanseniaspora guilliermondii than by Kloeckera apiculata. The latter produced twice as much acetic acid as H. guilliermondii. The production of higher alcohols and acetic acid was found to be a characteristic of individual strains and was statistically significant. In a multivariate analysis of higher alcohol production two main groupings were formed at 86%S, corresponding to the taxa H. guilliermondii and K. apiculata. Strains that produced low amounts (50 mg/1) of acetic acid, comparable with that of Saccharomyces cerevisiae, were found in both species of apiculate yeasts.     

Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice

Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juice supplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classified as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classified as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxic compounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenyl ethyl acetate and ethyl acetate. The methanol concentration was significantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic profile than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.     

Disruption of the acetate kinase (<Emphasis Type="Italic">ack</Emphasis>) gene of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> results in delayed acetate production

In microorganisms, the enzyme acetate kinase (AK) catalyses the formation of ATP from ADP by de-phosphorylation of acetyl phosphate into acetic acid. A mutant strain of Clostridium acetobutylicum lacking acetate kinase activity is expected to have reduced acetate and acetone production compared to the wild type. In this work, a C. acetobutylicum mutant strain with a selectively disrupted ack gene, encoding AK, was constructed and genetically and physiologically characterized. The ack (-) strain showed a reduction in acetate kinase activity of more than 97% compared to the wild type. The fermentation profiles of the ack (-) and wild-type strain were compared using two different fermentation media, CGM and CM1. The latter contains acetate and has a higher iron and magnesium content than CGM. In general, fermentations by the mutant strain showed a clear shift in the timing of peak acetate production relative to butyrate and had increased acid uptake after the onset of solvent formation. Specifically, in acetate containing CM1 medium, acetate production was reduced by more than 80% compared to the wild type under the same conditions, but both strains produced similar final amounts of solvents. Fermentations in CGM showed similar peak acetate and butyrate levels, but increased acetoin (60%), ethanol (63%) and butanol (16%) production and reduced lactate (-50%) formation by the mutant compared to the wild type. These findings are in agreement with the proposed regulatory function of butyryl phosphate as opposed to acetyl phosphate in the metabolic switch of solventogenic clostridia.     

Oenological properties of Hanseniaspora osmophila and Kloeckera corticis from wines produced by spontaneous fermentations of normal and dried grapes

Strains of Hanseniaspora osmophila and Kloeckera corticis, isolated from wines produced by spontaneous fermentations of normal and dried grapes, were characterized for their fermentation behavior with and without SO(2) at 25 degrees C. All isolates behaved as glucophilic yeasts and yielded ethanol at concentrations of about 9% (v/v); acetic acid, acetaldehyde, ethyl acetate and acetoin were always produced to high concentrations. SO(2) addition had no significant effect on growth yield and fermentation rate. These metabolic features were maintained in the presence of 400 g l(-1) of sugars and at 15 degrees C, and were quite similar to those shown by Saccharomycodes ludwigii. Therefore, H. osmophila and K. corticis should be considered detrimental yeast species, particularly in fermentations of musts from dried grapes.     

Reduction of Ethanol Yield and Improvement of Glycerol Formation by Adaptive Evolution of the Wine Yeast Saccharomyces cerevisiae under Hyperosmotic Conditions

There is a strong demand from the wine industry for methodologies to reduce the alcohol content of wine without compromising wine''s sensory characteristics. We assessed the potential of adaptive laboratory evolution strategies under hyperosmotic stress for generation of Saccharomyces cerevisiae wine yeast strains with enhanced glycerol and reduced ethanol yields. Experimental evolution on KCl resulted, after 200 generations, in strains that had higher glycerol and lower ethanol production than the ancestral strain. This major metabolic shift was accompanied by reduced fermentative capacities, suggesting a trade-off between high glycerol production and fermentation rate. Several evolved strains retaining good fermentation performance were selected. These strains produced more succinate and 2,3-butanediol than the ancestral strain and did not accumulate undesirable organoleptic compounds, such as acetate, acetaldehyde, or acetoin. They survived better under osmotic stress and glucose starvation conditions than the ancestral strain, suggesting that the forces that drove the redirection of carbon fluxes involved a combination of osmotic and salt stresses and carbon limitation. To further decrease the ethanol yield, a breeding strategy was used, generating intrastrain hybrids that produced more glycerol than the evolved strain. Pilot-scale fermentation on Syrah using evolved and hybrid strains produced wine with 0.6% (vol/vol) and 1.3% (vol/vol) less ethanol, more glycerol and 2,3-butanediol, and less acetate than the ancestral strain. This work demonstrates that the combination of adaptive evolution and breeding is a valuable alternative to rational design for remodeling the yeast metabolic network.     

Production of Acetic Acid and Other Volatile Compounds by Leuconostoc citrovorum and Leuconostoc dextranicum

Single-strain milk cultures of Leuconostoc dextranicum are capable of reducing added acetaldehyde, propionaldehyde, and butanone to the corresponding alcohols at 30 C. L. dextranicum and L. citrovorum reduced propionaldehyde to n-propanol quantitatively in 30 hr, and the reduction of this compound paralleled culture growth. Under unagitated conditions, these organisms produced large amounts of acetic acid and ethyl alcohol. The yield of acetic acid increased when cultures were agitated during growth. This increase in acetic acid production was accompanied by a 20- to 70-fold decrease in ethyl alcohol. The addition of acetaldehyde to the fermentation caused a reduction in the final concentration of acetic acid.     

Production of higher alcohols,ethyl acetate,acetaldehyde and other compounds by 14Saccharomyces cerevisiae wine strains isolated from the same region (Salnés,N.W. Spain)

Fourteen strains of the yeastSaccharomyces cerevisiae were isolated from three wineries in the Salnés wine region (N.W. Spain) at the three different periods of the natural fermentation. Each wild yeast was screened for production of acetaldehyde, ethyl acetate, isobutanol,n-propanol, amylic alcohol and other important enological compounds during laboratory scale fermentations of grape juice. After 25 days at 20°C, the analytical results evidenced variations in the production of acetaldehyde (from 13.1 to 24.3 mg/l), isobutanol (from 27.7 to 51.1 mg/l), amyl alcohols (from 111 to 183 mg/l) and ethyl acetate (from 19.3 to 43.7 mg/l). Although isolated from the same wine region, differences in the wine composition were observed depending on the particular yeast strain used for the vinification experiments.     

Enhanced Glycerol Content in Wines Made with Immobilized Candida stellata Cells

Screening tests carried out for 10 strains of Candida stellata confirmed high levels of glycerol production, although a low fermentation rate and reduced ethanol content were observed. To overcome the poor competition with Saccharomyces cerevisiae, fermentation tests with immobilized C. stellata cells, alone or in combination with S. cerevisiae, have been carried out. The immobilization of C. stellata cells consistently reduced the fermentation length when compared with that obtained with free cells, immobilized cells exhibiting about a 30-and a 2-fold improvement in fermentation rate compared with rates for C. stellata and S. cerevisiae free cells, respectively. Moreover, immobilized C. stellata cells produced a twofold increase in ethanol content and a strong reduction in acetaldehyde and acetoin production in comparison with levels for free cells. The evaluation of different combinations of C. stellata immobilized cells and S. cerevisiae showed interesting results with regard to analytical profiles for practical application in wine making. In fact, analytical profiles of combinations showed, apart from a high glycerol content, a reduction in the amounts of acetic acid and higher alcohols and a consistent increase in succinic acid content in comparison with values for the S. cerevisiae control strain. Sequential fermentation first with immobilized C. stellata cells and then after 3 days with an added inoculum of S. cerevisiae free cells was the best combination, producing 15.10 g of glycerol per liter, i.e., 136% more than the S. cerevisiae control strain produced. Fermentation with immobilized C. stellata cells could be an interesting process by which to enhance glycerol content in wine.     

清香型白酒中乳酸菌和酵母菌的相互作用

【目的】探究清香型白酒中不同乳酸菌和酵母菌的相互作用,了解不同菌株的发酵性能,为更深入地认识白酒发酵机理、实现发酵过程优化提供理论基础。【方法】利用程序控温和固态发酵模拟清香型白酒酿造环境,测定纯培养和共培养中菌株的理化指标、活菌数以及主要代谢产物的变化。【结果】Saccharomyces cerevisiae YJ1糖消耗快产乙醇和酯类物质多,Lactobacillus plantarum JMRS4糖消耗快产酸较多。共培养中乳酸菌对Saccharomyces cerevisiae YJ1的生长和产乙醇抑制较大,对Candida aaseri MJ7产乙醇几乎无影响。乳酸菌对Pichia kudriavzevii MJ14的生物量和乙醇代谢抑制作用较小,还对其产己酸乙酯、乙酸乙酯和异戊醇等代谢产物有促进作用;而反过来Pichia kudriavzevii MJ14对3株乳酸菌产乳酸均有抑制作用,对产乙酸则有促进作用。【结论】建立了一种固态培养方法,结合清香型白酒发酵温度变化规律,有效模拟了实际发酵环境。Pichia kudriavzevii MJ14在与乳酸菌共培养中受到的抑制较小并能有效抑制乳酸菌产乳酸,Saccharomyces cerevisiae YJ1能代谢产生多种风味物质,对清香型白酒酿造有重要意义。     

Influence of Williopsis saturnus yeasts in combination with Saccharomyces cerevisiae on wine fermentation

Aim: To examine the growth and survival of Williopsis saturnus strains along with wine yeast Saccharomyces cerevisiae in grape must. Methods and Results: For this study, fermentations were performed in sterilized grape must at 18°C. Inoculum level was 5 × 10⁶ cells per ml for each yeast. The results showed that W. saturnus yeasts exhibited slight growth and survival depending on the strain, but they died off by day 5. Saccharomyces cerevisiae, however, dominated the fermentation, reaching the population of about 8 log CFU ml^?1. It was observed that ethanol formation was not affected. The concentrations of acetic acid, ethyl acetate and isoamyl acetate were found higher in mixed culture experiments compared to control fermentation. The results also revealed that higher alcohols production was unaffected in general. Conclusion: Fermentations did not form undesirable concentrations of flavour compounds, but production of higher levels of acetic acid in mixed culture fermentations may unfavour the usage of W. saturnus in wine making. Significance and Impact of the Study: This study provides information on the behaviour of W. saturnus together with S. cerevisiae during the alcoholic fermentation.     

Furfural, 5-hydroxymethyl furfural, and acetoin act as external electron acceptors during anaerobic fermentation of xylose in recombinant Saccharomyces cerevisiae

The electron acceptors acetoin, acetaldehyde, furfural, and 5-hydroxymethylfurfural (HMF) were added to anaerobic batch fermentation of xylose by recombinant, xylose utilising Saccharomyces cerevisiae TMB 3001. The intracellular fluxes during xylose fermentation before and after acetoin addition were calculated with metabolic flux analysis. Acetoin halted xylitol excretion and decreased the flux through the oxidative pentose phosphate pathway. The yield of ethanol increased from 0.62 mol ethanol/mol xylose to 1.35 mol ethanol/mol xylose, and the cell more than doubled its specific ATP production after acetoin addition compared to fermentation of xylose only. This did, however, not result in biomass growth. The xylitol excretion was also decreased by furfural and acetaldehyde but was unchanged by HMF. Thus, furfural present in lignocellulosic hydrolysate can be beneficial for ethanolic fermentation of xylose. Enzymatic analyses showed that the reduction of acetoin and furfural required NADH, whereas the reduction of HMF required NADPH. The enzymatic activity responsible for furfural reduction was considerably higher than for HMF reduction and also in situ furfural conversion was higher than HMF conversion.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.     

Isolation and characterization of mutants resistant to amino acid analogues obtained from Saccharomyces cerevisiae strains

Mutants resistant to the amino acid analogues dl-thiaisoleucine, dl-4-azaleucine, 5,5,5-trifluoro-dl-leucine and l-O-methylthreonine, were isolated from Saccharomyces cerevisiae wine yeast strains. The fermentative production of secondary metabolites by the mutants was tested in grape must. Higher alcohols, acetaldehyde and acetic acid concentration varied depending on strain and analogue. Most of the mutants produced increased amounts of amyl alcohol. A remarkable variability in the level of n-propanol, isobutanol, acetaldehyde and acetic acid was observed. In practical application, the use of mutants resistant to amino acid analogues can improve the quality of wines by reducing or increasing the presence of some secondary compounds.