Improving the performance of industrial ethanol‐producing yeast by expressing the aspartyl protease on the cell surface

The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell‐surface anchored expressed in industrial ethanol‐producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild‐type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98–99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild‐type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.     

Using RFLP-mtDNA for the rapid monitoring of the dominant inoculated yeast strain in industrial wine fermentations

The analysis of restriction fragment length polymorphism of mitochondrial DNA (mtDNA-RFLP) has been applied as a test to monitor the abundance of the starter yeast strain during industrial wine fermentations without previous isolation of yeast colonies. For white wine fermentations, we performed a rapid assay consisting in taking a sample of fermenting must, purifying the DNA from harvested cells, and obtaining the restriction patterns by digestion with the endonuclease HinfI. The same protocol, but adding an overnight cultivation step before DNA purification, was also applied to red wine fermentations. The results were compared with those obtained from the subsequent characterisation of strains, for the same samples, by analysis of the electrophoretic karyotype of isolated yeast colonies. In all cases, when the inoculated strain was dominant within the yeast population, the rapid assay anticipated the result by showing the coincidence between the restriction profiles obtained from both total cells and the inoculated strain. The results were obtained at 11 or 23 h after sampling for white- or red-wine fermentations respectively. This method allows a rapid intervention of the wine-producer if the presence of the inoculated yeasts has suffered a sudden decrease in any phase of the fermentation process.     

Influence of pre‐cure freezing on the profile of volatile compounds during the processing of Iberian hams

BACKGROUND: This work was designed to study the effect of pre‐cure freezing of raw thighs from Iberian pigs on the profile of volatile compounds during the processing of hams. RESULTS: Generation of volatile compounds during Iberian ham processing was similar in both pre‐cure frozen and refrigerated hams, the main differences being at the final stage. The levels of 2‐methylbutanal, 2‐methyl‐1‐butanol, 2,3‐butanediol and 2‐heptanol were significantly higher in dry‐cured hams that were pre‐cure frozen than in refrigerated ones, whereas the content of most detected esters was statistically lower in pre‐cure frozen than in refrigerated hams. CONCLUSION: The effect of pre‐cure freezing of Iberian ham on the profile of volatile compounds during ripening was not remarkable. Few differences were found in the final product, which would not greatly modify the aroma and flavour features of the dry‐cured hams. Copyright © 2010 Society of Chemical Industry     

Effect of cold pre‐treatment duration before freezing on frozen bread dough quality

The effect of cold pre‐treatment (CT) duration prior to freezing on the quality of a standard bread dough was investigated. Doughs held at 0 °C or 10 °C for 1 h or 3 h before air‐blast freezing were compared with standard dough frozen after 0.5 h at 0 °C (0 °C/0.5 h) and fresh (unfrozen) dough. Cumulative gas production measured in a risograph was used to quantify the dough quality after storage at ?18 ± 0.1 °C for 1, 7 or 17 days. Relative to fresh dough, gas production significantly reduced after freezing for all treatments. The doughs with CT at 0 °C for 1 or 3 h or 10 °C for 1 h had significantly higher gas production after freezing and less rapid decline in gas production during frozen storage than the doughs with the 0 °C/0.5 h CT. The 10 °C/3 h CT gave no gas production benefit after freezing and had the most rapid decline in gas production during frozen storage.     

Low‐temperature wine‐making using yeast immobilized on pear pieces

A biocatalyst was prepared by the immobilization of Saccharomyces cerevisiae AXAZ‐1 yeast cells on pear pieces and tested for grape must fermentation in both batch and continuous conditions. The immobilized yeast cells were stable and active even at low temperatures (<10 °C). Wine production under batch fermentation at 8 °C was completed within 15 days while at 3 °C it took 36 days. In continuous fermentation, the bioreactor was operated for 33 days, then stored for 12 days at 10 °C, and re‐run for another 15 days without any diminution of the ethanol productivity. Total acidity of the produced wines remained within the ranges usually observed in dry wines, while volatile acidity was found in rather increased levels. The concentrations of higher alcohols (1‐propanol, isobutyl alcohol and amyl alcohols) were relatively low, while ethyl acetate was detected at up to 118 mg l^?1, contributing to the fruity aroma of the wines produced. Preliminary sensory evaluations carried out in the laboratory indicated the fine quality of the produced wines. Copyright © 2004 Society of Chemical Industry     

Disruption of the ABC transporter genes PDR5, YOR1, and SNQ2, and their participation in improved fermentative activity of a sake yeast mutant showing pleiotropic drug resistance

Clotrimazole-resistant mutants from sake yeasts show improved fermentative activity in sake mash and pleiotropic drug resistance (PDR). The PDR mechanism is interpreted by overexpression of ATP-binding cassette (ABC) transporters, which extrude various kinds of drugs out of a cell. In a clotrimazole-resistant mutant, CTZ21, isolated from the haploid sake yeast HL69, the levels of mRNA for three major ABC transporter genes, PDR5, SNQ2, and YOR1, markedly increased. These three genes of CTZ21 were disrupted to investigate which participated in the improved fermentative activity of CTZ21. The fermentative activities of Δpdr5 and Δsnq2 strains of CTZ21 were reduced to that of HL69 in the initial and middle stages of fermentation. In the last stage, however, the sake meter [(1/gravity-1) × 1443] of the Δpdr5 and Δsnq2 strains rose faster than that of HL69. On the other hand, a Δyor1 strain of CTZ21 fermented sake mash in a manner nearly identical to that of CTZ21 until the last stage of fermentation. But in the last stage, fermentation of the Δyor1 slowed down compared with that of CTZ21. A Δyor1 strain of HL69 also exhibited much reduced fermentative activity in the middle and last fermentation stages. The YOR1 gene seems necessary for sake fermentation to be completed efficiently. The ATP content in sake mash brewed with CTZ21 was drastically decreased throughout the whole fermentation period. This low ATP level was restored to a medium level in the cases of both the Δpdr5 and Δsnq2 strains of CTZ21. In contrast, the Δyor1 of CTZ21 exhibited a low ATP level in sake mash in the same manner as CTZ21. These results suggest that the low ATP level of CTZ21 contributes to a certain extent its improved fermentative activity in the initial and middle stages of sake fermentation.     

Reduction of acrylamide formation in French fries by microwave pre‐cooking of potato strips

In this study, the effect of microwave pre‐cooking of potato strips on the resultant acrylamide levels in French fries was investigated. Control and microwaved (10, 20, and 30 s at 850 W) samples were fried at 150, 170 and 190 °C for predetermined times. Surface and core temperatures of potato strips were acquired during frying, and acrylamide content in the surface and the core regions were determined separately. The results showed that microwave application prior to frying resulted in a marked reduction of acrylamide level in the surface region, whereas a slight increase was noted for the core region. When the potato strips were subjected to frying after a microwave pre‐cooking step, acrylamide content in the whole potato strip was reduced by 36%, 41%, and 60% for frying at 150, 170, and 190 °C, respectively, in comparison to the control. Copyright © 2006 Society of Chemical Industry     

Changes in aromatic profile of fresh and dried fig – the role of pre‐treatments in drying process

The aromatic profile of volatiles in fresh figs (FF), fresh figs frozen in liquid nitrogen (NF) and dried figs (DF) (dark variety Petrova?a Crna) was characterised by HS–SPME followed by GC–MS. Figs were dried in a pilot plant cabinet dryer using different pre‐treatments to preserve the dried fruit: sulphuring and immersion in a solution of citric acid and ascorbic acid (separately). The adaptability of thin‐layer drying models to whole figs was investigated. Fresh figs and fresh figs frozen in liquid nitrogen differed mainly in the amount of aldehydes. The highest abundance of volatile compounds in dried figs was found in figs pre‐treated with sulphur dioxide and the control, compared to samples immersed in the acid solutions. Preservation was the most successful for the group of terpenes and terpenic compounds, quite good for some esters and ketones, whereas aldehydes were not affected by the used pre‐treatment.     

Nitrogen starvation induces expression of Lg‐FLO1 and flocculation in bottom‐fermenting yeast

When exponentially growing cells of bottom‐fermenting yeast were starved for nitrogen or were grown on proline (a non‐preferred nitrogen source), flocculation was induced. This flocculation was not induced by starvation for either carbon or amino acids. Expression of Lg‐FLO1, which is required for flocculation of bottom‐fermenting yeast, was also found to be induced by starvation for nitrogen. This suggests that the flocculation of bottom‐fermenting yeast is under the control of a nitrogen catabolite repression (NCR)‐like mechanism. Copyright © 2012 John Wiley & Sons, Ltd.     

Occurrence of 20S RNA and 23S RNA replicons in industrial yeast strains and their variation under nutritional stress conditions

We have characterized industrial yeast strains used in the brewing, baking, and winemaking industries for the presence or absence of cytoplasmic single-stranded 20S and 23S RNAs. Furthermore, the variation of intracellular concentrations of these replicons in brewing and laboratory strains under nutritional stress conditions was determined. Our results show a correlation between the relative abundance of these replicons and exposure of yeast to nutritionally stressful conditions, indicating that these RNAs could be employed as molecular probes to evaluate the exposure of 20S(+) and/or 23S(+) yeast strains to stress situations during industrial manipulation. During this study, several 20S(-)23S(+) Saccharomyces cerevisiae strains were isolated and identified. This is the first time that a yeast strain containing only 23S RNA has been reported, demonstrating that 20S RNA is not required for 23S RNA replication.     

Construction of self‐cloning industrial brewer's yeast with SOD1 gene insertion into PEP4 prosequence locus by homologous recombination

Superoxide dismutase (SOD, encoded by SOD1), which can scavenge active oxygen free radicals, is an ideal endogenous antioxidase in beer. In this study, the SOD1 expression cassette was constructed, and this cassette contained the PGK1 promoter, the PGK1 terminator and the SOD1 gene fused to the signal sequence of the yeast mating pheromone α‐factor (MFα1_s). One of the prosequences of the PEP4 gene (encoding proteinase A, PrA) in Saccharomyces cerevisiae strain S‐6 was replaced by the SOD1 expression cassette via homologous recombination and the self‐cloning strain S54PS, which could improve the antioxidant capability and foam stability of beer, was successfully obtained. Fermentation results showed that the SOD activity of the final beer brewed with S54PS was increased by 21.06%. Accordingly, the DPPH‐radical scavenging activity of S54PS increased by 30.6% compared with that yielded by the parental strain S‐6. Furthermore, the PrA activity of S54PS was always lower than that of the parental strain at all stages of beer fermentation. The head retention of the beer (255 ± 4 s) was better than that of the parental strain (224 ± 1 s). Hence, this research implies that S54PS exhibits good brewing performance and can be applied to improve the industrial brewing process. Copyright © 2016 The Institute of Brewing & Distilling     

酵母多糖产生菌的筛选和发酵条件的研究

16种酵母菌通过初筛后，复筛时在培养基中加入磷酸二氢钾、碳源和氮源，筛选出高产多糖的酵母菌种——产脂酵母。确定此产脂酵母最佳发酵条件为：磷酸二氢钾0．6％，蔗糖6％，硝酸铵0．30％，玉米浆0．7％。     

The influence of vitamin E supplementation on yeast fermentation

Ethanol is a well‐known toxic metabolite of yeast, affecting cell growth, and it is a major factor responsible for reduced ethanol production. Therefore, yeast strains that can withstand high ethanol concentrations are highly desirable. The effects of vitamin E supplementation and enhanced cell membrane fluidity in ethanol fermentations were studied. The results showed that ethanol tolerance was significantly improved by vitamin E supplementation, which correlated with the increased level of polyunsaturated fatty acids. The data showed that, after 0.1 g L^?1 vitamin E was added to the highest ethanol concentration, one could attain 118.7 g L^?1, in contrast to 106.4 g L^?1. The experimental results indicated that the increase in vitamin E suppressed unsaturated fatty acid oxidation, resulting in enhanced ethanol tolerance and increased production yield. Copyright © 2016 The Institute of Brewing & Distilling