Evolutionary engineering of Wickerhamomyces subpelliculosus and Kazachstania gamospora for baking

The conventional baker’s yeast, Saccharomyces cerevisiae , is an indispensable baking workhorse of all times. Its monopoly coupled to its major drawbacks such as streamlined carbon substrate utilisation base and a poor ability to withstand a number of baking associated stresses prompt the need to search for alternative yeasts to leaven bread in the era of increasingly complex consumer lifestyles. Our previous work identified the inefficient baking attributes of Wickerhamomyces subpelliculosus and Kazachstania gamospora as well as preliminarily observations of improving fermentative capacity of potential alternative baker’s yeasts using evolutionary engineering. Here we report the characterisation and improvement in baking traits in five out of six independently evolved lines incubated for longer time and passaged for at least 60 cycles relative to their parental strains as well as the conventional baker’s yeast. In addition, evolved clones produced bread with a higher loaf volume when compared to bread baked with either ancestral strain or the control conventional baker’s yeast. Remarkably, our approach improved the yeasts’ ability to withstand baking associated stresses, a key baking trait exhibited poorly in both the conventional baker’s yeast and their ancestral strains. W. subpelliculosus evolved the best characteristics attractive for alternative baker’s yeasts as compared to the evolved K. gamospora strains. These results demonstrate the robustness of evolutionary engineering in development of alternative baker’s yeasts.

Dietary fibers in the prevention of type 2 diabetes mellitus

This chapter reviews how dietary fibers can be used to prevent Type 2 diabetes mellitus. Dietary fibers are a heterogenous group of food compounds. The physicochemical properties of dietary fibers determine their effects on gastrointestinal and metabolic health, including effect on gastro-intestinal transit, glycemic response, microbial composition and fermentative capacity. Most fiber rich foods contain insoluble, prebiotic and viscous fibers in varying ratios. The chapter begins by discussing underlying mechanisms of action of insoluble fibers. It then goes on to discuss the various dietary fiber intervention studies, specifically focusing on prebiotic fibers and soluble viscous fibers. The chapter concludes by highlighting the importance of consuming high levels dietary fiber and whole grains and their positive impact on metabolic health.

DOP27 The fibre fermentative capacity of the gut microbiota is diminished in children with Crohn’s Disease and it is independent of disease activity or treatment with exclusive enteral nutrition

Background Induction of clinical remission with exclusive enteral nutrition (EEN), has been associated with accompanying changes in the concentration of short chain fatty acids (SCFA) (a biomarker of fibre fermentation) in faeces of children with Crohn’s disease (CD) 1. Here, we assessed the fibre fermentative capacity of the gut microbiota of children with active CD in vitro, before, during and after EEN and compared with healthy children. Methods 44 faecal samples from 14 children (female, n=7, age, median [Q1, Q3]: 14.1 [11.1, 15.1] years) with active CD were collected before, during (4 weeks) and at the end of EEN (8 weeks) and after food reintroduction (median [Q1, Q3]: 21 [16, 31] days post-EEN). All children had achieved clinical remission at the end of EEN (weighted Paediatric Crohn’s Disease Activity Index <12.5). A single faecal sample was collected from 11 healthy children (female, n=4, age, median [Q1, Q3]: 12.4 [9.6, 13.0] years). 24-hour in vitro batch fermentations were performed using 4 fibre substrates (pectin, high-resistant maize starch, wheat bran and a mixture of the three). Net production of SCFA was measured with gas chromatography. Results Compared to healthy children, the total production of SCFA was significantly lower in children with CD, for all 4 fibre substrates, and regardless of the study timepoint (Figure 1). Net production of SCFA remained unchanged during EEN and at food reintroduction, and for all fibre substrates (Figure 1). No significant association with levels of faecal calprotectin was observed at any of the timepoints. Acetate production was significantly lower in children with CD compared to healthy children for all fibre substrates except for resistant maize starch (Figure 1). Likewise, except for pectin, production of butyrate was significantly lower in children with CD than healthy controls (Figure 1). Production of propionate did not significantly differ between any of the groups. Legend: Production of SCFA after 24-hour in vitro fermentation of faecal samples from CD and healthy children. Asterisks indicate significant differences. A: before EEN, B: 4-week EEN, C: End of EEN, D: Food reintroduction, HC: Healthy children Conclusion Fibre fermentative capacity is independent of disease activity in patients with CD and remains lower compared to healthy controls. It might be unlikely that the mechanism of action of EEN is mediated by modulation of fibre fermenting bacteria. Reference 1. Gerasimidis K, Bertz M, Hanske L, Junick J, Biskou O, Aguilera M, et al. Decline in presumptively protective gut bacterial species and metabolites are paradoxically associated with disease improvement in pediatric Crohn’s disease during enteral nutrition. Inflamm Bowel Dis. 2014;20(5):861–71.

Profile of Volatile Compounds of On-Farm Fermented and Dried Cocoa Beans Inoculated with Saccharomyces cerevisiae KY794742 and Pichia kudriavzevii KY794725

This study aimed to identify the volatile compounds in the fermented and dried cocoa beans conducted with three distinct inoculants of yeast species due to their high fermentative capacity: Saccharomyces cerevisiae, Pichia kudriavzevii, the mixture in equal proportions 1:1 of both species, and a control fermentation (with no inoculum application). Three starter cultures of yeasts, previously isolated and identified in cocoa fermentation in the municipality of Tomé-Açu, Pará state, Brazil. The seeds with pulp were removed manually and placed in wooden boxes for the fermentation process that lasted from 6 to 7 days. On the last day of fermentation, the almonds were packaged properly and placed to dry (36 °C), followed by preparation for the analysis of volatile compounds by GC-MS technique. In addition to the control fermentation, a high capacity for the formation of desirable compounds in chocolate by the inoculants with P. kudriavzevii was observed, which was confirmed through multivariate analyses, classifying these almonds with the highest content of aldehydes, esters, ketones and alcohols and low concentration of off-flavours. We conclude that the addition of mixed culture starter can be an excellent alternative for cocoa producers, suggesting obtaining cocoa beans with desirable characteristics for chocolate production, as well as creating a product identity for the producing region.

Screening of Indigenous Yeast From Different Ecological Region of Kathmandu Valley and Its Application in Wine Production

Objectives: The aim of the study was to isolate and screen the potent yeast from the air for implementing new yeast in wine fermentation. Methods: In this study, 35 air samples collected in sterile grape juice in glass jar and left over for four days exposure for the growth of yeast from different locations around the Kathmandu Valley. Yeasts were screened by culturing on selective Ethanol Sulfite Agar (ESA) media at 30°C for 2-3 days in Microbiology Lab of Pinnacle College. Yeast isolates were characterized based on colony morphology, microscopic characteristics, Fermentative capacity, Hydrogen sulfide production. Selected yeast isolates were subjected to ethanol fermentation and tested for alcohol tolerance capacity. Wine quality was assessed by sensory evaluation. Results: Of 35 samples, only 20 yeast isolates were isolated. Among these isolates, the variation in colony characteristics along with oval and ellipsoidal microscopic appearance was observed. All the isolates were able to ferment major sugars such as glucose, fructose and sucrose, but few could not ferment galactose and maltose, while none-fermented lactose and xylose. Here, isolates showing no H2S (L29, L34) and mild H2S producer (isolate L31) were subjected to ethanol fermentation. Also, Comparative analysis was made by using commercial standard wine yeast (STAN). Rapid fermentation of grape juice with initial 21 0Brix was observed in L31 isolate, which produced 12.99% v/v alcohol with titratable acidity (TA) 5.25 g/L, followed by L29 strain with 11.99%v/v alcohol and 4.5 g/L TA which were higher than STAN (10.99% alcohol). These isolates specified as Ethanol tolerance up to 13%v/v, while none of them were able to grow at 15% v/v ethanol concentration and 45°C temperature. However, significant growth was observed at pH 3 along with sugar tolerance capacity at 30 0Brix. The wine produced by these isolates was found to be remarkably different among each other. While the sensory analysis of wine led to isolate L31 being congenial to tasters. Conclusion: L31 isolate was found to be efficient and advantageous for wine production indicating its industrial application.

The composition of sacarine substrates for ethanol production and the fermentative capacity Saccharomyces cerevisiae Pedra-2

The production of ethanol in Brazil is based on sugarcane juice, however other biomasses can be used for this process, such as sweet sorghum. However, some nutrients can interfere with fermentation, such as the presence of metals, carbon and nitrogen sources, which can affect the fermentation capacity of yeasts. Thus, this study aims to analyze the presence of fundamental nutrients present in saccharine substrates, as well as their assimilation and conversion of ethanol by the yeast Pedra-2. Samples of sugarcane and sorghum juice were obtained, in which analysis of the presence of metals was carried out using acid digestion and the levels determined by atomic flame absorption spectroscopy. The amino acid analysis was performed on the saccharine substrates at a concentration of 22 ºBrix, before and after fermentation, and analyzed by high-performance liquid chromatography and the concentration of ethanol by gas chromatography. The sorghum broth showed higher amounts of available amino acid metals. The yeast Pedra-2 showed better fermentative performance in the sorghum broth. We can conclude that the sorghum broth represents an important substrate to be used to increase the sustainability and production of ethanol in Brazil.

Renewable Sources and their Applications in Biotechnological Processes

Brazil has edaphoclimatic conditions to produce a diversity of crops with energetic potential. Thus, the study aims to correlate the technological parameters of biomasses suitable for fermentation, quantify metabolites produced by yeast FT-858, as well as assess the yield and fermentative efficiency for bioethanol production. It was performed studies of technological qualities of the biomasses and fermentative capacity test. For metabolites production, the yeast FT-858 was pre-grown in liquid medium (YPD 2%), recovered and inoculated in the substrates, and aliquots were collected for analysis of ethanol concentration by gas chromatography and glycerol accumulation by enzymatic kit of triglycerides. The yield and fermentative efficiency were assessed by consumption of sugar using DNS method, and ethanol density using digital densimeter. According to the results, the yeast presented better performance in saccharine sorghum, which also presented more expressive values of fermentative efficiency and yield. Saccharine sorghum has great bioenergetic potential and can be used as complement to sugarcane to increase ethanol production.

Comparison of the Glycolytic and Alcoholic Fermentation Pathways of Hanseniaspora vineae with Saccharomyces cerevisiae Wine Yeasts

Hanseniaspora species can be isolated from grapes and grape musts, but after the initiation of spontaneous fermentation, they are displaced by Saccharomyces cerevisiae. Hanseniaspora vineae is particularly valuable since this species improves the flavour of wines and has an increased capacity to ferment relative to other apiculate yeasts. Genomic, transcriptomic, and metabolomic studies in H. vineae have enhanced our understanding of its potential utility within the wine industry. Here, we compared gene sequences of 12 glycolytic and fermentation pathway enzymes from five sequenced Hanseniaspora species and S. cerevisiae with the corresponding enzymes encoded within the two sequenced H. vineae genomes. Increased levels of protein similarity were observed for enzymes of H. vineae and S. cerevisiae, relative to the remaining Hanseniaspora species. Key differences between H. vineae and H. uvarum pyruvate kinase enzymes might explain observed differences in fermentative capacity. Further, the presence of eight putative alcohol dehydrogenases, invertase activity, and sulfite tolerance are distinctive characteristics of H. vineae, compared to other Hanseniaspora species. The definition of two clear technological groups within the Hanseniaspora genus is discussed within the slow and fast evolution concept framework previously discovered in these apiculate yeasts.

Evaluation of the fermentative capacity of an indigenous Hanseniaspora sp. strain isolated from Lebanese apples for cider production

The present work studied the fermentative potential and carbon metabolism of an indigenous yeast isolated from Lebanese apples for cider production. The indigenous yeast strain was isolated from a spontaneous fermented juice of the Lebanese apple variety ‘Ace spur’. The sequencing of the Internal Transcribed Spacer (ITS) domain of rRNA identified the isolated yeast strain as a member of the Hanseniaspora genus. These results suggest an intragenomic ITS sequence heterogeneity in the isolated yeast strain specifically in its ITS1 domain. The different investigations on the yeast carbon metabolism revealed that the isolated yeast is ‘Crabtree positive’ and can produce and accumulate ethanol from the first hours of fermentation. Thus, our findings highlight the possibility of using the isolated indigenous Hanseniaspora strain as a sole fermentative agent during cider production.