Later Stages Of Fermentation Research Articles

Correlation analysis of key enzyme activities and aroma compounds during fermentation of simulated juice system with Saccharomyces cerevisiae

Key enzymes play a variety of vital roles in the metabolic pathways of aroma compounds during fermentation with yeasts. In this study, the activities of key enzymes and the contents of selected aroma compounds were determined during fermentation of a simulated juice system with Saccharomyces cerevisiae, and then correlation analyses on key enzyme activities and aroma compounds were performed. The results showed that pyruvic acid accumulated in large quantities with the rapid decrease in glucose during the early stage of fermentation and then decreased significantly. The key aroma compounds increased gradually during fermentation and then decreased slightly and fluctuated within a certain range at the later stage of fermentation. Moreover, the activities of hexokinase (HK), 6-phosphofructokinase (PFK-1) and pyruvate kinase (PK) in glycolysis significantly increased with the consumption of glucose during fermentation, and there were significant positive correlations between the key enzymes of glycolysis, while the glycolytic enzymes demonstrated no obvious correlations with pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH). PK and PDC were positively correlated with the synthesis of pyruvic acid. PK also had significant positive correlations with the formation of esters and alcohols, but not fatty acids. ADH was significantly negatively correlated with the formation of ethyl caprylate and nerolidol.

Proteomic profiling and integrated analysis with transcriptomic data bring new insights in the stress responses of Kluyveromyces marxianus after an arrest during high-temperature ethanol fermentation

BackgroundThe thermotolerant yeast Kluyveromyces marxianus is a potential candidate for high-temperature fermentation. When K. marxianus was used for high-temperature ethanol fermentation, a fermentation arrest was observed during the late fermentation stage and the stress responses have been investigated based on the integration of RNA-Seq and metabolite data. In order to bring new insights into the cellular responses of K. marxianus after the fermentation arrest during high-temperature ethanol fermentation, quantitative proteomic profiling and integrated analysis with transcriptomic data were performed in this study.ResultsSamples collected at 14, 16, 18, 20 and 22 h during high-temperature fermentation were subjected to isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic profiling and integrated analysis with transcriptomic data. The correlations between transcripts and proteins for the comparative group 16 h vs 14 h accounted for only 4.20% quantified proteins and 3.23% differentially expressed proteins (DEPs), respectively, much higher percentages of correlations (30.56%–59.11%) were found for other comparative groups (i.e., 18 h vs 14 h, 20 h vs 14 h, and 22 h vs 14 h). According to Spearman correlation tests between transcriptome and proteome (the absolute value of a correlation coefficient between 0.5 and 1 indicates a strong correlation), poor correlations were found for all quantified proteins (R = − 0.0355 to 0.0138), DEPs (R = − 0.0079 to 0.0233) and the DEPs with opposite expression trends to corresponding differentially expressed genes (DEGs) (R = − 0.0478 to 0.0636), whereas stronger correlations were observed in terms of the DEPs with the same expression trends as the correlated DEGs (R = 0.5593 to 0.7080). The results of multiple reaction monitoring (MRM) verification indicate that the iTRAQ results were reliable. After the fermentation arrest, a number of proteins involved in transcription, translation, oxidative phosphorylation and fatty acid metabolism were down-regulated, some molecular chaperones and proteasome proteins were up-regulated, the ATPase activity significantly decreased, and the total fatty acids gradually accumulated. In addition, the contents of palmitic acid, oleic acid, C16, C18, C22 and C24 fatty acids increased by 16.77%, 28.49%, 14.14%, 26.88%, 628.57% and 125.29%, respectively.ConclusionsThis study confirmed some biochemical and enzymatic alterations provoked by the stress conditions in the specific case of K. marxianus: such as decreases in transcription, translation and oxidative phosphorylation, alterations in cellular fatty acid composition, and increases in the abundance of molecular chaperones and proteasome proteins. These findings provide potential targets for further metabolic engineering towards improvement of the stress tolerance in K. marxianus.

Effects of cpxR on the growth characteristics and antibiotic production of Xenorhabdus nematophila

SummaryCpxR is a global response regulator that negatively influences the antimicrobial activities of Xenorhabdus nematophila. Herein, the wildtype and ΔcpxR mutant of X. nematophila were cultured in a 5‐l and 70‐l bioreactor. The kinetic analysis showed that ΔcpxR significantly increased the cell biomass and antibiotic activity. The maximum dry cell weight (DCW) and antibiotic activity of ΔcpxR were 20.77 ± 1.56 g L−1 and 492.0 ± 31.2 U ml−1 and increased by 17.28 and 97.33% compared to the wildtype respectively. Xenocoumacin 1 (Xcn1), a major antimicrobial compound, was increased 3.07‐fold, but nematophin was decreased by 48.7%. In 70‐l bioreactor, DCW was increased by 18.97%, while antibiotic activity and Xcn1 were decreased by 27.71% and 11.0% compared to that in 5‐l bioreactor respectively. Notably, pH had remarkable effects on the cell biomass and antibiotic activity of ΔcpxR, where ΔcpxR was sensitive to alkaline pH conditions. The optimal cell growth and antibiotic activity of ΔcpxR occurred at pH 7.0, while Xcn1 was increased 5.45‐ and 3.87‐fold relative to that at pH 5.5 and 8.5 respectively. These findings confirmed that ΔcpxR considerably increased the biomass of X. nematophila at a late stage of fermentation. In addition, ΔcpxR significantly promoted the biosynthesis of Xcns but decreased the production of nematophin.

Differences in protein content and foaming properties of cloudy beers based on wheat malt content

To investigate differences in protein content, all barley malt beer, wheat/barley malt beer and all wheat malt beer were brewed, and the protein during mashing, wort, fermentation and beer determined. It was shown that protein was mainly extracted during mashing and the protein rest phase, decreased in the early stages of fermentation and remained almost steady during wort boiling and cooling, in the middle and late stages of fermentation. By separating beer foam from beer, similar protein bands of 51.7, 40.0, 27.3, 14.8, 6.5 and < 6.5 kDa appeared in the three beers, defoamed beers and beer foams using the sodium dodecyl sulphate polyacrylamide gel electrophoresis. Quantitatively, protein bands of 6.5–14.8 and <6.5 kDa had the highest contents in the three beers. Unique bands at 34, 29.2, 23.0, 19.7 and 17.7 kDa were found in beer, defoamed beer and beer foam from wheat beer and all-wheat malt beer, respectively. Wheat beer foam showed the best foam stability and the protein in all barley malt beer showed the best migration to the foam. The beer foam properties were influenced by not only protein content but also protein characteristics and/or origin. It is suggested that the barley malt contributed the beer foam ‘skeleton protein’ while protein components from wheat malt kept the foam stable. © 2018 The Institute of Brewing & Distilling

Evolution of Flavanol Glycosides during Red Grape Fermentation.

Monomeric and dimeric flavanol glycosides were quantified by UHPLC-MRM in Syrah (SYR) and Grenache (GRE) grapes and in their corresponding wines for the first time. Quantities were extremely variable depending on grape tissue (seeds or skins) and during fermentation. Overall, 22 monomeric and dimeric mono- and diglycosides were determined with concentrations ranging from 0.7 nanograms to 0.700 micrograms per gram of grape tissue, and 0 to 60 micrograms per liter for wines. The evolution of the glycosides’ composition during winemaking suggests that almost all these compounds originate in the grapes themselves and display different extraction kinetics during winemaking. One isomer of the monomeric (epi) flavanol monoglycosides seemed to be biosynthesized by yeasts during wine fermentation. The sharp decrease in concentration of some isomers at the late stages of fermentation or after pressing suggests that some grape glycosidase activities convert these compounds into non-glycosylated flavanols.

Enhancing the production of cephalosporin C through modulating the autophagic process of Acremonium chrysogenum

BackgroundAutophagy is used for degradation of cellular components and nutrient recycling. Atg8 is one of the core proteins in autophagy and used as a marker for autophagic detection. However, the autophagy of filamentous fungi is poorly understood compared with that of Saccharomyces cerevisiae. Our previous study revealed that disruption of the autophagy related gene Acatg1 significantly enhanced cephalosporin C yield through reducing degradation of cephalosporin biosynthetic proteins in Acremonium chrysogenum, suggesting that modulation of autophagic process is one promising way to increase antibiotic production in A. chrysogenum.ResultsIn this study, a S. cerevisiae ATG8 homologue gene Acatg8 was identified from A. chrysogenum. Acatg8 could complement the ATG8 mutation in S. cerevisiae, indicating that Acatg8 is a functional homologue of ATG8. Microscope observation demonstrated the fluorescently labeled AcAtg8 was localized in the cytoplasm and autophagosome of A. chrysogenum, and the expression of Acatg8 was induced by nutrient starvation. Gene disruption and genetic complementation revealed that Acatg8 is essential for autophagosome formation. Disruption of Acatg8 significantly reduced fungal conidiation and delayed conidial germination. Localization of GFP-AcAtg8 implied that autophagy is involved in the early phase of conidial germination. Similar to Acatg1, disruption of Acatg8 remarkably enhanced cephalosporin C yield. The cephalosporin C biosynthetic enzymes (isopenicillin N synthase PcbC and isopenicillin N epimerase CefD2) and peroxisomes were accumulated in the Acatg8 disruption mutant (∆Acatg8), which might be the main reasons for the enhancement of cephalosporin C production. However, the biomass of ΔAcatg8 decreased drastically at the late stage of fermentation, suggesting that autophagy is critical for A. chrysogenum cell survival under nutrition deprived condition. Disruption of Acatg8 also resulted in accumulation of mitochondria, which might produce more reactive oxygen species (ROS) which promotes fungal death. However, the premature death is unfavorable for cephalosporin C production. To solve this problem, a plasmid containing Acatg8 under control of the xylose/xylan-inducible promoter was introduced into ∆Acatg8. Conidiation and growth of the recombinant strain restored to the wild-type level in the medium supplemented with xylose, while the cephalosporin C production maintained at a high level even prolonged fermentation.ConclusionsOur results demonstrated inducible expression of Acatg8 and disruption of Acatg8 remarkably increased cephalosporin C production. This study provides a promising approach for yield improvement of cephalosporin C in A. chrysogenum.

Impacts of dietary silver nanoparticles and probiotic administration on the microbiota of an in-vitro gut model

Ingestion of silver nanoparticles (AgNPs) is inevitable linked to their widespread use in food, medicines and other consumer products. However, their effects on human microbiota at non-lethal concentrations remain poorly understood.In this study, the interactions among 1 μg mL−1 AgNPs, the intestinal microbiota, and the probiotic Bacillus subtilis (BS) were tested using in-vitro batch fermentation models inoculated with human fecal matter.Results from metagenomic investigations revealed that the core bacterial community was not affected by the exposure of AgNPs and BS at the later stage of fermentation, while the proportions of rare species changed drastically with the treatments. Furthermore, shifts in the Firmicutes/Bacteriodetes (F/B) ratios were observed after 24 h with an increase in the relative abundance of Firmicutes species and a decrease in Bacteroidetes in all fermentation cultures. The co-exposure to AgNPs and BS led to the lowest F/B ratio.Fluorescent in-situ hybridization analyses indicated that non-lethal concentration of AgNPs negatively affected the relative percentage of Faecalibacterium prausnitzii and Clostridium coccoides/Eubacterium rectales taxa in the fermentation cultures after 24 h. However, exposure to single and combined treatments of AgNPs and BS did not change the overall diversity of the fecal microflora.Functional differences in cell motility, translation, transport, and xenobiotics degradation occurred in AgNPs-treated fermentation cultures but not in AgNPs+BS-treated samples.Compared to the control samples, treated fecal cultures showed no significant statistical differences in terms of short-chain fatty acids profiles, cytotoxic and genotoxic effects on Caco-2 cell monolayers.Overall, AgNPs did not affect the composition and diversity of the core fecal microflora and its metabolic and toxic profiles. This work indicated a chemopreventive role of probiotic on fecal microflora against AgNPs, which were shown by the decrease of F/B ratio and the unaltered state of some key metabolic pathways.

Coproduction of menaquinone-7 and nattokinase by Bacillus subtilis using soybean curd residue as a renewable substrate combined with a dissolved oxygen control strategy

Numerous physiological functions of menaquinone-7 (MK-7) act to reduce vascular calcification, suggesting that MK-7 may be a potential therapy for Alzheimer’s and Parkinson’s disease, and in this study, we attempted to increase the concentration of MK-7 synthesized by Bacillus subtilis natto, a standard nattokinase (NK) producing strain. Different Bacillus subtilis isolates demonstrated positive correlations between MK-7 and NK concentrations. Response surface methodology (RSM) was employed to optimize a culture medium for the simultaneous production of these molecules; the optimized medium contained the following components (%, w/v): soybean curd residue, 12.2; soya peptone, 5.7; lactose, 2.6; and K2HPO4, 0.6. The fermentation process was subsequently optimized based on online feedback control of fermentation process parameters. The dissolved oxygen (DO) concentration played an important role in the production of MK-7 and NK. With increased DO concentrations, the cell growth rate and NK activity increased. In contrast, at low DO concentrations, the concentration of MK-7 rapidly increased during the late fermentation stage. Thus, in this study, the production of MK-7 and NK by Bacillus subtilis was accomplished using soybean curd residue through medium optimization and DO control. This novel coproduction strategy was developed by controlling the aeration rate during the fermentation process. The concentrations of MK-7 and NK achieved in this study reached 91.25 mg/L and 2675.73 U/mL, respectively.

RNA-Seq-based transcriptomic analysis of Saccharomyces cerevisiae during solid-state fermentation of crushed sweet sorghum stalks

Abstract Bioethanol production based on solid-state fermentation (SSF) of sweet sorghum stalks has been demonstrated to have great potential due to the its low pollution and low cost. A novel S. cerevisiae strain TSH3 exhibited better SSF performance compared with BY4743 during SSF of sweet sorghum stalks. High-quality total RNA of S. cerevisiae was extracted from SSF mixture and the global gene expression profiles during SSF were studied using RNA-Seq. Compared with BY4743, TSH3’s genes related to ribosome biogenesis, amino acid and coenzyme metabolism during early fermentation stage, secondary metabolite biosynthesis, metabolism in diverse environment during middle fermentation stage, and lipid metabolism during late fermentation stage were up-regulated; while the genes involved in fatty acid metabolism and peroxisome during early fermentation stage, ribosome biogenesis during middle fermentation stage, and mitotic cell cycle during late fermentation stage were down-regulated. Further dynamic analysis of TSH3’s transcriptome reveals its three different metabolic stages: 1) ribosome biogenesis and respiration-fermentation transition; 2) biosynthesis of secondary metabolites and stress resistance; 3) plasma membrane related metabolism for stress resistance. These findings provided insight into the S. cerevisiae transcriptome during SSF of sweet sorghum stalks and suggest that TSH3 would be an ideal candidate for SSF-based bioethanol production.

Microbial diversity and component variation in Xiaguan Tuo Tea during pile fermentation.

Xiaguan Tuo Tea is largely consumed by the Chinese, but there is little research into the microbial diversity and component changes during the fermentation of this tea. In this study, we first used fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) and chemical analysis methods to determine the microbial abundance and diversity and the chemical composition during fermentation. The FISH results showed that the total number of microorganisms ranges from 2.3×102 to 4.0×108 cells per gram of sample during fermentation and is mainly dominated by fungi. In the early fermentation stages, molds are dominant (0.6×102~2.8×106 cells/g, 0~35 d). However, in the late stages of fermentation, yeasts are dominant (3.6×104~9.6×106 cells/g, 35~56 d). The bacteria have little effect during the fermentation of tea (102~103 cells/g, <1% of fungus values). Of these fungi, A. niger (Aspergillus niger) and B. adeninivorans (Blastobotrys adeninivorans) are identified as the two most common strains, based on Next-generation Sequencing (NGS) analysis. Peak diversity in tea was observed at day 35 of fermentation (Shannon–Weaver index: 1.195857), and lower diversity was observed on days 6 and 56 of fermentation (Shannon–Weaver index 0.860589 and 1.119106, respectively). During the microbial fermentation, compared to the unfermented tea, the tea polyphenol content decreased by 54%, and the caffeine content increased by 59%. Theanine and free amino acid contents were reduced during fermentation by 81.1 and 92.85%, respectively.

Industrial Applications of Selected JFS Articles

Industrial Applications of Selected <i>JFS</i> Articles

红条茶加工中香气物质的动态变化

以桃源大叶种茶鲜叶为原料，按照经典红条茶加工工艺将其加工成红条茶，采用同时蒸馏萃取法(SDE)制备精油及GC-MS分析方法，研究其加工过程中香气物质的动态变化，以期为高香红条茶的加工提供参考。结果表明，加工成的红条茶共有110种香气组分，相对含量较高有22种，其中醇类9种，脂肪酸类3种，醛类5种，脂类3种，酮类2种；萎凋期间，醇类、脂类香气组分的相对含量逐渐增加，醛类、酮类、烃类、酸类香气组分的相对含量则逐渐下降；揉捻期间，醇类、酯类、醛类、酮类香气组分的相对含量递增，酸类、烃类香气组分的相对含量下降；发酵期间，醇类、醛类、酮类香气组分的相对含量持续增加，而酯类香气组分的相对含量先增后降，烃类和酸类香气组分的相对含量小幅下降；干燥期间，各类香气物质相对含量均下降。总体而言，从萎凋开始到发酵中后期，红条茶特征香气组分的相对含量呈逐渐增加趋势，而从发酵后期至干燥结束则呈逐渐降低趋势，在生产实际中，可通过适当延长萎凋时间、缩短发酵后期时间及干燥时间，有利于加工高香红条茶。 The black tea processed with traditional technology by Taoyuan leaves was used as the research object, and the aroma components were analyzed by simultaneous distillation and extraction (SDE) combining GC-MS technique, which provided reference for the processing of high-aroma black tea. The results show that the differences of aroma components of black tea were studied by chemical analysis. 110 species of aroma components were detected by GC-MS in totally, and 22 of them were high in content, including 9 types of alcohols, 3 types of fatty acids, 5 types of aldehydes, 3 types of lipids and 2 types of ketones. During the withering period, the relative content of alcohols and lipids increased gradually. The relative content of aldehydes, ketones, hydrocarbons and acids gradually decreased. The relative content of alcohols, lipids, aldehydes, and ketones showed an increasing trend during the period of rolling, and the relative content of acids and hydrocarbons decreased. During the fermentation period, the relative content of alcohols, aldehydes and ketones continued to increase, and the relative content of lipids was first increased and then decreased by the end of fermentation, and the relative content of hydrocarbons and acids decreased slightly. During the drying period, the relative content of all kinds of substances is decreased. As whole, from the beginning of the withering to the middle and late of fermentation, the relative content of characteristic aroma of black tea gradually increased, and then decreased from the later stage of fermentation to the end of drying. In the production, it is conducive to the processing of high-aroma black tea by moderately prolonging the withering time, shortening the time of late fermentation and drying.

Performance and mechanism analysis of succinate production under different transporters in Escherichia coli

Succinic acid is a platform chemical with potential for bio-based synthesis. However, the production of bio-based succinate is limited because of insufficient succinate efflux capacity in the late stage of fermentation. In the present study, three different transporters, which have been reported to be responsible for C4-dicarboxylates transport, were employed for investigation of the transport capacity of succinate in Escherichia coli. After engineered strains were constructed, the fermentative production of succinic acid was studied in serum bottles and 3 L of fermentor. The results demonstrated that engineered strain showed better efflux capacity than control strain under high concentration of succinate. The highest production of succinate was 68.66 g/L, while the NCgl2130 transporter may be the best candidate for succinate export in E. coli. Further research showed that the expression levels and relative enzyme activities involved in the metabolic pathway all increased markedly, and the maximum activities of PPC, PCK, PYK, and MDH increased by 1.50, 1.38, 1.28, and 1.27-fold in recombinant E. coli AFP111/pTrc99a-NCgl2130, respectively. Moreover, the maximum level of intracellular ATP increased by 23.79% in E. coli AFP111/pTrc99a-NCgl2130. Taken together, these findings indicated that engineered transporters can improve succinate production by increasing key enzyme activities and intracellular ATP levels. To the best of thew authors’ knowledge, this is the first report on a mechanism to improve succinate production by engineered transporters. This strategy set up a foundation for improving the biosynthesis of other C4-dicarboxylates, such as fumaric acid and malic acid.

Inducing hyperosmotic stress resistance in succinate-producing Escherichia coli by using the response regulator DR1558 from Deinococcus radiodurans

Abstract Bio-based succinate synthesis process suffers from low production efficiency, especially in the late stage of fermentation because of high osmotic stress. In this study, DR1558 (a response regulator in tow-component signal transduction systems) from Deinococcus radiodurans was overexpressed in Escherichia coli Suc260. High concentration glucose and NaCl impact experiment results showed that the recombinant strain (E. coli Suc270) displayed higher tolerance to osmotic stress. Fermentation in a 3-L fermenter showed that the dry cell weight, glucose consumption, and succinic acid titer of Suc270 were 2.06, 90.2, and 55.4 g/L, which is an increase of 1.50-, 1.38-, and 1.26-fold, respectively, compared to those of Suc260. Moreover, the lag phase was shortened by 10 h compared to that of the control strain Suc260. Real-time PCR results showed that DR1558 could enhance the expression level of the genes involved in glucose utilization, ATP synthesis, and osmoprotectant (glycine betaine, trehalose) synthesis pathways. Intracellular ATP and osmoprotectant content analysis further showed that Suc270 accumulated much more intracellular ATP, glycine betaine, and trehalose than Suc260. In conclusion, DR1558 could affect ATP metabolism and the synthesis of osmoprotectants and thus enhance the cell’s tolerance against osmotic stress.

Relationship Between Fatty Acid Concentrations in Wine Yeasts and Sugar Fermentation at Different Temperatures

The effects of temperature and fermentation stage on the cellular concentrations of unsaturated and saturated fatty acids on the fermentation abilities of four differentSaccharomyces cerevisiae wine yeasts were studied at three temperatures. The total unsaturated and saturated fatty acid concentrations were determined at six fermentation stages. Unsaturated fatty acid concentration decreased and the concentration of saturated fatty acids increased as fermentation proceeded. Saturated fatty acid concentration of the yeast cells correlated positively with high specific fermentation rates during the later stages of fermentation in contrast to the belief that fluid membranes with high unsaturated fatty acid concentrations enhance ethanol tolerance and, therefore, fermentation performance.

고구마를 이용한 된장의 품질 특성

The effect of sweet potato on the quality of Doenjang was investigated during fermentation. Viable cells of yeast decreased gradually after 4 weeks of fermentation, but those of aerobic bacteria increased in the late stage. Amylase activity of Doenjang was higher in the late stage of fermentation, while neutral protease maintained high activity during fermentation. Hunter L and b values of Doenjang decreased gradually during fermentation, while a value was increased. The pH of Doenjang decreased gradually until 10 weeks of fermentation, and the titratable acidity was low in the sweet potato added groups. The acid value was low in the Shinyulmi sweet potato added Doenjang. Water activity and oxidation-reduction potential of Doenjang decreased during fermentation. Reducing sugar of Doenjang decreased in the middle stage of fermentation, and it was low in sweet potato added groups. The alcohol content of Doenjang decreased after 2 weeks of fermentation. Amino and ammonia-type nitrogen of Doenjang increased during fermentation and reached the maximum after 10 and 12 weeks of fermentation, respectively. After 12 weeks fermentation, 8% of Shinyulmi sweet potato added Doenjang was more favorable taste, flavor and overall acceptability (p&lt;0.05) than the control or the Yeonwhangmi sweet potato added groups.

Dynamics of microbial community during the extremely long-term fermentation process of a traditional soy sauce.

Soy sauce produced by long-term natural fermentation is a traditional specialty in Asia, with a reputation for superior quality and rich flavour. In this study, both culture-dependent and culture-independent approaches were used to investigate the microbial diversity and community dynamics during an extremely long-term (up to 4 years) natural fermentation of Xianshi Soy Sauce, a national intangible cultural heritage. Genera of Bacillus, Aspergillus and Cladosporium were detected by both methods above. The relative abundance of the genera Bacillus and Weissella was significantly higher in the late stage than in the early one, while the genera Klebsiella and Shimwellia were opposite (P < 0.05). For microbial community structure, subsequent analyses showed that obvious changes occurred with fermentation time, while there was a fair homogeneousness among samples of the same year, especially during the late fermentation stage. The clustering analysis tended to separate the fermented mashes of the 4th year from the earlier stages, suggesting the necessity of the long fermentation period for developing distinctive microbiota and characteristic quality-related compounds. This is the first report to explore the temporal changes in microbial dynamics over a period of 4 years in traditional fermentation of soy sauce, and this work illustrated the importance of isolation of appropriate strains to be used as starter cultures in brewing processes. © 2016 Society of Chemical Industry.

Medium optimization based on yeast's elemental composition for glutathione production in Saccharomyces cerevisiae

The production of glutathione (GSH) or GSH enriched yeast is still in the focus of research driven by a high industrial interest. In this study, an optimal growth rate for GSH production via Saccharomyces cerevisiae Sa-07346 was investigated. To further improve the fermentation process in a way that it is independent of lots, the influence of different WMIX medium compositions on biomass and GSH production was studied. Thereby, the fermentation medium was adjusted based on yeast's elemental composition. The resulting chemically defined fermentation medium led to high cell densities in fed-batches. Therefore, it has the potential to be applied successfully for other high cell density yeast fermentation processes. As cysteine is the key component for GSH production, different cysteine addition strategies were studied and finally, a continuous cysteine feeding was applied in the late stage of fermentation. Thereby, a GSH concentration of 1459±57mg/l was reached by continuously feeding cysteine, which meant an increase to 253% compared to the control without cysteine addition (577mg/l GSH).

Promoter engineering of the Saccharomyces cerevisiae RIM15 gene for improvement of alcoholic fermentation rates under stress conditions

A loss-of-function mutation in the RIM15 gene, which encodes a Greatwall-like protein kinase, is one of the major causes of the high alcoholic fermentation rates in Saccharomyces cerevisiae sake strains closely related to Kyokai no. 7 (K7). However, impairment of Rim15p may not be beneficial under more severe fermentation conditions, such as in the late fermentation stage, as it negatively affects stress responses. To balance stress tolerance and fermentation performance, we inserted the promoter of a gluconeogenic gene, PCK1, into the 5'-untranslated region (5'-UTR) of the RIM15 gene in a laboratory strain to achieve repression of RIM15 gene expression in the glucose-rich early stage with its induction in the stressful late stage of alcoholic fermentation. The promoter-engineered strain exhibited a fermentation rate comparable to that of the RIM15-deleted strain with no decrease in cell viability. The engineered strain achieved better alcoholic fermentation performance than the RIM15-deleted strain under repetitive and high-glucose fermentation conditions. These data demonstrated the validity of promoter engineering of the RIM15 gene that governs inhibitory control of alcoholic fermentation.

Establishing the relative importance of damaged starch and fructan as sources of fermentable sugars in wheat flour and whole meal bread dough fermentations

It is generally believed that maltose drives yeast-mediated bread dough fermentation. The relative importance of fructose and glucose, released from wheat fructan and sucrose by invertase, compared to maltose is, however, not documented. This is surprising given the preference of yeast for glucose and fructose over maltose. This study revealed that, after 2h fermentation of wheat flour dough, about 44% of the sugars consumed were generated by invertase-mediated degradation of fructan, raffinose and sucrose. The other 56% were generated by amylases. In whole meal dough, 70% of the sugars consumed were released by invertase activity. Invertase-mediated sugar release seems to be crucial during the first hour of fermentation, while amylase-mediated sugar release was predominant in the later stages of fermentation, which explains why higher amylolytic activity prolonged the productive fermentation time only. These results illustrate the importance of wheat fructan and sucrose content and their degradation for dough fermentations.