Influence of carbon and nitrogen source on production of volatile fragrance and flavour metabolites by the yeast Kluyveromyces marxianus

The yeast Kluyveromyces marxianus produces a range of volatile molecules with applications as fragrances or flavours. The purpose of this study was to establish how nutritional conditions influence the production of these metabolites. Four strains were grown on synthetic media, using a variety of carbon and nitrogen sources and volatile metabolites analysed using gas chromatography–mass spectrometry (GC–MS). The nitrogen source had pronounced effects on metabolite production: levels of the fusel alcohols 2‐phenylethanol and isoamyl alcohol were highest when yeast extract was the nitrogen source, and ammonium had a strong repressing effect on production of 2‐phenylethyl acetate. In contrast, the nitrogen source did not affect production of isoamyl acetate or ethyl acetate, indicating that more than one alcohol acetyl transferase activity is present in K. marxianus. Production of all acetate esters was low when cells were growing on lactose (as opposed to glucose or fructose), with a lower intracellular pool of acetyl CoA being one explanation for this observation. Bioinformatic and phylogenetic analysis of the known yeast alcohol acetyl transferases ATF1 and ATF2 suggests that the ancestral protein Atf2p may not be involved in synthesis of volatile acetate esters in K. marxianus, and raises interesting questions as to what other genes encode this activity in non‐Saccharomyces yeasts. Identification of all the genes involved in ester synthesis will be important for development of the K. marxianus platform for flavour and fragrance production. Copyright © 2014 John Wiley & Sons, Ltd.     

Potential impact of dairy yeasts on the typical flavour of traditional ewes' and goats' cheeses

The contribution of Debaryomyces hansenii, Kluyveromyces lactis and Kluyveromyces marxianus strains to the typical flavour of traditional ewes' and goats' cheeses was assessed. Fourteen yeast strains were grown in liquid medium mimicking cheese composition and volatile compounds were identified by gas chromatography-mass spectrometry. Yeasts were able to produce key volatile compounds characteristic of the cheeses from which they were isolated. Inter-species and inter-strain variations were observed. Under the conditions tested, D. hansenii produced the lowest levels of volatile compounds, with large intra-strain variations. Kluyveromyces strains primarily produced esters and alcohols. K. marxianus strains were associated with the production of acids, ethyl decanoate, 1-propanol and benzaldehyde, whereas K. lactis was correlated with the presence of ketones, ethyl acetate and secondary alcohols. In conclusion, this study shows the heterogeneous potential of dairy yeasts to contribute to final cheese flavour.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Encapsulated whey–native yeast Kluyveromyces marxianus as a feed additive for animal production

ABSTRACT

Whey is the main byproduct of the cheese industry. While the composition is variable, it retains up to 55% of milk nutrients. The beneficial features of whey indicates a promising source of new potentially probiotic strains for the development of food additives destined for animal production. The aim of this study was to identify Kluyveromyces spp. isolated from whey, to study some probiotic properties and to select the best strain to be encapsulated using derivatised chitosan. Kluyveromyces marxianus strains (VM003, VM004 and VM005) were isolated from whey and identified by phenotypic and molecular techniques. These three yeast strains were able to survive under gastrointestinal conditions. Moreover, they exhibited weak auto-aggregation and co-aggregation with pathogenic bacteria (Salmonella sp., Serratia sp., Escherichia coli and Salmonella typhimurium). In general the K. marxianus strains had a strong antimicrobial activity against pathogenic bacteria. The potential probiotic K. marxianus VM004 strain was selected for derivatised-chitosan encapsulation. Material treated with native chitosan exhibited a strong antimicrobial activity of K. marxianus, showing a total growth inhibition at 10 min exposure. However, derivatised-chitosan encapsulation showed a reduced antimicrobial activity. This is the first study to show some probiotic properties of whey–native K. marxianus, in vitro. An encapsulation strategy was applied using derivatised chitosan.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Screening for new brewing yeasts in the non‐Saccharomyces sector with Torulaspora delbrueckii as model

This study describes a screening system for future brewing yeasts focusing on non‐Saccharomyces yeasts. The aim was to find new yeast strains that can ferment beer wort into a respectable beer. Ten Torulaspora delbrueckii strains were put through the screening system, which included sugar utilization tests, hop resistance tests, ethanol resistance tests, polymerase chain reaction fingerprinting, propagation tests, amino acid catabolism and anabolism, phenolic off‐flavour tests and trial fermentations. Trial fermentations were analysed for extract reduction, pH drop, yeast concentration in bulk fluid and fermentation by‐products. All investigated strains were able to partly ferment wort sugars and showed high tolerance to hop compounds and ethanol. One of the investigated yeast strains fermented all the wort sugars and produced a respectable fruity flavour and a beer of average ethanol content with a high volatile flavour compound concentration. Two other strains could possibly be used for pre‐fermentation as a bio‐flavouring agent for beers that have been post‐fermented by Saccharomyces strains as a consequence of their low sugar utilization but good flavour‐forming properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Investigating flavour characteristics of British ale yeasts: techniques,resources and opportunities for innovation

Five British ale yeast strains were subjected to flavour profiling under brewery fermentation conditions in which all other brewing parameters were kept constant. Significant variation was observed in the timing and quantity of flavour‐related chemicals produced. Genetic tests showed no evidence of hybrid origins in any of the strains, including one strain previously reported as a possible hybrid of Saccharomyces cerevisiae and S. bayanus. Variation maintained in historical S. cerevisiae ale yeast collections is highlighted as a potential source of novelty in innovative strain improvement for bioflavour production. Copyright © 2014 John Wiley & Sons, Ltd.     

Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products

The yeast species Saccharomyces cerevisiae and Kluyveromyces marxianus are associated with fermentation of West African indigenous foods. The aim of this study was to characterize potential probiotic properties of S. cerevisiae and K. marxianus isolates from the West African milk products lait caillé and nunu and a cereal-based product mawè. The strains (14 in total) were identified by 26S rRNA gene sequencing and characterized for survival at gastrointestinal stress (bile salts and low pH) and adhesion to Caco-2 intestinal epithelial cells. Selected yeast isolates were tested for their effect on the transepithelial electrical resistance (TEER), using the intestinal epithelial cell line Caco-2 and for maintenance of intracellular pH (pH_i) during perfusion with gastrointestinal pH (3.5 and 6.5). All tested yeasts were able to grow in bile salts in a strain-dependent manner, exhibiting a maximum specific growth rate (μ_max) of 0.58–1.50 h⁻¹. At pH 2.5, slow growth was observed for the isolates from mawè (μ_max of 0.06–0.80 h⁻¹), whereas growth of yeasts from other sources was mostly inhibited. Yeast adhesion to Caco-2 cells was strain specific and varied between 8.0% and 36.2%. Selected strains of S. cerevisiae and K. marxianus were able to maintain the pH_i homeostasis at gastrointestinal pH and to increase TEER across the Caco-2 monolayers, indicating their potential to improve intestinal barrier functions. Based on overall results, strains of K. marxianus and S. cerevisiae from mawè exhibited the highest probiotic potential and might be recommended for further development as starter cultures in West African fermented products.     

Random and targeted gene integrations through the control of non‐homologous end joining in the yeast Kluyveromyces marxianus

Kluyveromyces marxianus DMKU3‐1042 is a thermotolerant yeast strain suitable for high‐temperature ethanol fermentation and genetic engineering with linear DNA. We have developed a highly efficient random gene integration method with a frequency that exceeds 2.5 × 10⁶ transformants/µg linear DNA, a figure comparable to what is observed with autonomously replicating plasmid transformation in Saccharomyces cerevisiae. To establish the mechanism of random integration in DMKU3‐1042, we identified and deleted the K. marxianus KU70 gene, which is known to be involved in the non‐homologous end‐joining (NHEJ) pathway. In yeast lacking KU70, high‐frequency non‐homologous gene integration was abolished and the Kmku70 mutants showed 82–95% homologous gene targeting efficiencies using homologous sequences of 40–1000 bp. These results indicate that the highly efficient NHEJ pathway can be utilized with random gene disruption techniques such as transposon mutagenesis and plasmid‐free gene manipulations in K. marxianus. Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of self‐cloning bottom‐fermenting yeast with low vicinal diketone production by the homo‐integration of ILV5

The vicinal diketones (VDK), such as diacetyl and 2,3‐pentandione, impart an unpleasant butter‐like flavour to beer. Typically, these are required to be reduced below the flavour thresholds during the maturation (lagering) stages of the brewing process. To shorten beer maturation time, we constructed a self‐cloning, bottom‐fermenting yeast with low VDK production by integrating ILV5, a gene encoding a protein that metabolizes α‐acetolactate and α‐aceto‐α‐hydroxybutyrate (precursors of VDK). A DNA fragment containing Saccharomyces cerevisiae‐type ILV5 was inserted upstream of S. cerevisiae‐type ILV2 in bottom‐fermenting yeast to construct self‐cloning strains with an increased copy number of ILV5. Via transformation, ILV2 was replaced with the sulfometuron methyl (SM) resistance gene SMR1B, which differs by a single nucleotide, to create SM‐resistant transformants. The wort fermentation test, using the SC‐ILV5‐homo inserted transformant, confirmed a consecutive reduction in VDK and a shortening period during which VDK was reduced to within the threshold. The concentrations of ethyl acetate, isoamyl acetate, isoamyl alcohol, 1‐propanol, isobutyl alcohol and active isoamyl alcohol (flavour components) were not changed when compared with the parent strain. We successfully constructed self‐cloning brewer's yeast in which SC‐ILV5 was homo‐inserted. Using the transformed yeast, the concentration of VDK in fermenting wort was reduced, whereas the concentrations of flavour components were not affected. This genetically stable, low VDK‐producing, self‐cloning bottom‐fermenting yeast would contribute to the shortening of beer maturation time without affecting important flavour components produced by brewer's yeast. Copyright © 2012 John Wiley & Sons, Ltd.     

The temperature dependent functionality of Brettanomyces bruxellensis strains in wort fermentations

In recent years, Brettanomyces bruxellensis has found increasing application in brewery fermentations. Indeed, B. bruxellensis contributes to the flavour profile of many Belgian beers, typically during secondary or spontaneous fermentation. In North America, the yeast is used in primary fermentation to produce beers with ‘Brett’ characteristics with ‘fruity’ and/or ‘funky’ sensory profiles associated with the production of volatile esters and phenols. However, little is understood about the factors that influence flavour metabolite production or fermentation rate in this yeast. Here, the impact of temperature is reported on fermentation efficiency, flavour metabolite production and carbon utilisation of one commonly used and eight poorly characterised B. bruxellensis strains during wort fermentation. A high degree of strain and temperature‐dependent variability was found in fermentation efficiency and metabolite production amongst B. bruxellensis strains. Further, fermentation efficiency and carbon utilisation were temperature dependent, while ester production increased at higher temperature and phenol production was strain and temperature independent. These results indicate significant strain and temperature dependent variation, suggesting the potential application of strain variability as a tool to achieve product diversity in B. bruxellensis primary fermentations. © 2019 The Institute of Brewing & Distilling     

Performance of indigenous yeasts in the processing of Chinese strong‐flavoured liquor during spontaneous mixed solid‐state or submerged fermentation

To explore the in situ metabolic characteristics of yeasts involved in the spontaneous fermentation process of Chinese strong‐flavoured liquor, a comparison was conducted between solid‐state fermentation (SSF) and submerged fermentation (SmF) when supplemented with 24 indigenous yeast strains, with a focus on the production of ethanol and a broad range of volatile compounds responsible for the characteristics of Chinese strong‐flavoured liquor. Under the various experimental conditions, the 24 indigenous yeast strains showed different influences on the mixed fermentation system. The fluctuations caused by different yeast strains in the mixed system were less than those caused by the different fermentation modes relative to the formation of flavour compounds. SSF was found to be more suitable for the production of ethanol, methanol and ethyl lactate, whereas SmF was more suitable for the production of 10 higher alcohols, four esters and four acids. This study revealed the relationships amongst the indigenous yeasts, SSF, and the distinctive flavour profiles of Chinese strong‐flavoured liquor. This work provides evidence of the existence of internal stability in spontaneous SSF, thereby facilitating a better understanding of the fermentative mechanism in the SSF process for Chinese strong‐flavoured liquor production Copyright © 2015 The Institute of Brewing & Distilling     

Brewing characteristics of haploid strains isolated from sake yeast Kyokai No. 7

Sake yeast exhibit various characteristics that make them more suitable for sake brewing compared to other yeast strains. Since sake yeast strains are Saccharomyces cerevisiae heterothallic diploid strains, it is likely that they have heterozygous alleles on homologous chromosomes (heterozygosity) due to spontaneous mutations. If this is the case, segregation of phenotypic traits in haploid strains after sporulation and concomitant meiosis of sake yeast strains would be expected to occur. To examine this hypothesis, we isolated 100 haploid strains from Kyokai No. 7 (K7), a typical sake yeast strain in Japan, and compared their brewing characteristics in small‐scale sake‐brewing tests. Analyses of the resultant sake samples showed a smooth and continuous distribution of analytical values for brewing characteristics, suggesting that K7 has multiple heterozygosities that affect brewing characteristics and that these heterozygous alleles do segregate after sporulation. Correlation and principal component analyses suggested that the analytical parameters could be classified into two groups, indicating fermentation ability and sake flavour. Copyright © 2008 John Wiley & Sons, Ltd.     

Review: Pure non‐Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications

Recently there has been increased interest in using non‐Saccharomyces yeasts to ferment beer. The worldwide growth of craft beer and microbreweries has revitalised the use of different yeast strains with a pronounced impact on aroma and flavour. Using non‐conventional yeast gives brewers a unique selling point to differentiate themselves. Belgian brewers have been very successful in using wild yeasts and mixed fermentations that often contain non‐Saccharomyces yeasts. Historically, ancient beers and beers produced before the domestication of commonly used Saccharomyces strains most likely included non‐Saccharomyces species. Given the renewed interest in using non‐Saccharomyces yeasts to brew traditional beers and their potential application to produce low‐alcohol or alcohol‐free beer, the fermentation and flavour characteristics of different species of non‐Saccharomyces pure culture yeast were screened for brewing potential (Brettanomyces anomalus and bruxellensis, Candida tropicalis and shehatae, Saccharomycodes ludwigii, Torulaspora delbrueckii, Pichia kluyveri, Zygosaccharomyces rouxii). Alcohol‐free beer is already industrially produced using S. ludwigii, a maltose‐negative species, which is a good example of the introduction of non‐Saccharomyces yeast to breweries. Overall, non‐Saccharomyces yeasts represent a large resource of biodiversity for the production of new beers and have the potential for wider application to other beverage and industrial applications. Almost all of the trials reviewed were conducted with varying fermentation parameters, which plays an important role in the outcome of the studies. To understand these impacts all trials were described with their major fermentation parameters. Copyright © 2016 The Institute of Brewing & Distilling     

Expression of GFP using Pichia pastoris vectors with zeocin or G‐418 sulphate as the primary selectable marker

Pichia pastoris is a popular host organism for expressing heterologous proteins, and various expression vectors for this yeast are currently available. Recently, vectors containing novel dominant antibiotic resistance markers have become a strong and developing field of research for this methylotropic yeast strain. We have developed new P. pastoris expression vectors, the pPICKanMX6 and pPICKanMX6α series. These vectors were constructed by replacing the zeocin resistance gene of the pPICZA, B, C and pPICZαA, B and C vectors with the Tn903 kan^R marker from pFA6a KanMX6, which confers G‐418 sulphate resistance in P. pastoris. The limits of antibiotic resistance in two transformant yeast strains were investigated, and the selection marker was shown to be stably retained. To demonstrate their usefulness, a gene encoding hexa‐histidine‐tagged green fluorescent protein (GFPH6) was cloned into one of the new vectors and GFP expression examined in P. pastoris cells. The protein expression levels using the pPICKanMX6B vector were comparable with that using the original plasmid, based on zeocin resistance as seen by yeast cell fluorescence. Moreover, GFPH6 was able to be isolated by immobilized metal ion affinity chromatography (IMAC) from lysates of both yeast strains. A model reporter construct has been used to demonstrate successful recombinant protein expression and its subsequent purification using these new vectors. Corresponding vectors can now also be engineered with foreign gene expression under the control of various different promoters, to increase the flexibility of P. pastoris as a cellular factory for heterologous protein production. Copyright © 2009 John Wiley & Sons, Ltd.     

The Influence of Yeast Strains on the Volatile Flavour Compounds of Chinese Rice Wine

In order to assess the influence of yeast strains on volatile flavour profiles of Chinese rice wine, small‐scale Chinese rice wine brewing was carried out with eight yeast strains from three different typical Chinese rice wine brewing regions. The volatile flavour compounds were extracted by headspace solid phase microextraction (HS‐SPME) and analyzed by gas chromatography‐mass spectrometry (GC‐MS). The volatile flavour profiles of the different Chinese rice wines showed statistically significant differences depending on the yeast strains used. Yeast strains from the Shaoxing region showed a higher capacity to produce the esters 2‐phenylethanol and 3‐methylthiopropanol, while yeast strains from the Shanghai region stood out for their production of branched‐chain higher alcohols. Chinese rice wine fermented with a yeast strain from the Jiangsu region had the highest levels of organic acids. Using principal component analysis of the Chinese rice wine volatile flavour compounds, the eight yeast strains could be classified into three groups according to their origins. This is the first report about the volatile flavour characteristics of Chinese rice wine yeast. The data obtained in this work shows that the yeast strains contributed significantly to the flavour differences of the Chinese rice wines from the different regions.     

Alleviation of stuck wine fermentations using salt‐preconditioned yeast

The influence of salt (sodium chloride) on the cell physiology of wine yeast was investigated. Cellular viability and population growth of three wine‐making yeast strains of Saccharomyces cerevisiae, and two non‐Saccharomyces yeast strains associated with wine must microflora (Kluyveromyces thermotolerans and K. marxianus) were evaluated following salt pre‐treatments. Yeast cells growing in glucose defined media exposed to different sodium chloride concentrations (4, 6 and 10% w/v) exhibited enhanced viabilities compared with nontreated cultures in subsequent trial fermentations. Salt ‘preconditioning’ of wine yeast seed cultures was also shown to alleviate stuck and sluggish fermentations at the winery scale, indicating potential benefits for industrial fermentation processes. It is hypothesized that salt induces specific osmostress response genes to enable yeast cells to better tolerate the rigours of fermentation, particularly in high sugar and alcohol concentrations. Copyright © 2014 The Institute of Brewing & Distilling     

Construction of a brewing yeast expressing the glucoamylase gene STA1 by mating

Standard brewing yeast cannot utilize larger oligomers or dextrins, which represent about 25% of wort sugars. A brewing yeast strain that could ferment these additional sugars to ethanol would be useful for producing low‐carbohydrate diabetic or low‐calorie beers. In this study, a brewing yeast strain that secretes glucoamylase was constructed by mating. The resulting Saccharomyces cerevisiae 278/113371 yeast was MAT a/α diploid, but expressed the glucoamylase gene STA1 . At the early phase of the fermentation test in malt extract medium, the fermentation rate of the diploid STA1 strain was slower than those of both the parent strain S. cerevisiae MAFF113371 and the reference strain bottom‐fermenting yeast Weihenstephan 34/70. At the later phase of the fermentation test, however, the fermentation rate of the STA1 yeast strain was faster than those of the other strains. The concentration of ethanol in the culture supernatant of the STA1 yeast strain after the fermentation test was higher than those of the others. The concentration of all maltooligosaccharides in the culture supernatant of the STA1 yeast strain after the fermentation test was lower than those of the parent and reference strains, whereas the concentrations of flavour compounds in the culture supernatant were higher. These effects are due to the glucoamylase secreted by the constructed STA1 yeast strain. In summary, a glucoamylase‐secreting diploid yeast has been constructed by mating that will be useful for producing novel types of beer owing to its different fermentation pattern and concentrations of ethanol and flavour compounds. Copyright © 2017 The Institute of Brewing & Distilling     

Evolution of aroma volatiles during storage of sourdough breads made by mixed cultures of Kluyveromyces marxianus and Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus

Two mixed starter cultures were used for sourdough bread making to evaluate their ability to improve quality and increase bread shelf-life: Lactobacillus delbrueckii ssp. bulgaricus or Lactobacillus helveticus mixed with the lactose fermenting yeast Kluyveromyces marxianus as alternative baker’s yeast. Control sourdough breads (K. marxianus) without the addition of bacteria, were also prepared. The changes on the headspace aroma volatiles during storage were assessed using solid-phase microextraction (SPME) GC–MS analysis. The effect of these changes on bread flavour was evaluated by consumer preference evaluations and the results were co-evaluated with those from the GC–MS analysis. The obtained results showed differences in the volatile composition of the different types of breads examined, as well as dramatic decreases of the number and the amount of volatiles after five days of storage. The sourdough breads made with K. marxianus and L. bulgaricus, had a more complex aroma profile, longer shelf-life and achieved the highest scores in the sensory tests.