The safety of a Kluyveromyces lactis strain lineage for enzyme production.

Kluyveromyces lactis is broadly considered as a safe yeast in food and a suitable organism for the production of food enzymes. The K. lactis enzyme production strains of DSM are used to produce a variety of enzymes, for example beta-galactosidase (lactase), chymosin and esterase. All of these production strains are derived from the same lineage, meaning they all originate from the same ancestor strain after classical mutagenesis and/or genetic engineering. Four different enzyme preparations produced with strains within this lineage were toxicologically tested. These enzyme preparations were nontoxic in repeated-dose oral toxicity studies performed in rats and were non-genotoxic in vitro. These studies confirm the safety of the DSM K. lactis strains as a production platform for food enzymes, as well as the safety of the genetic modifications made to these strains through genetic engineering or classical mutagenesis. The outcome of the toxicity studies can be extended to other enzyme preparations produced by any strain from this lineage through read across. Therefore, no new toxicity studies are required for the safety evaluation, as long as the modifications made do not raise safety concerns. Consequently, this approach is in line with the public ambition to reduce animal toxicity studies.

Growth and autolysis of the kefir yeast Kluyveromyces marxianus in lactate culture.

Kluyveromyces marxianus is a yeast that could be identified from kefir and can use a broad range of substrates, such as glucose and lactate, as carbon sources. The lactate produced in kefir culture can be a substrate for K. marxianus. However, the complexity of the kefir microbiota makes the traits of K. marxianus difficult to study. In this research, we focused on K. marxianus cultured with lactate as the sole carbon source. The optimal growth and released protein in lactate culture were determined under different pH conditions, and the LC-MS/MS-identified proteins were associated with the tricarboxylic acid cycle, glycolysis pathway, and cellular stress responses in cells, indicating that autolysis of K. marxianus had occurred under the culture conditions. The abundant glyceraldehyde-3-phosphate dehydrogenase 1 (GAP1) was cocrystallized with other proteins in the cell-free fraction, and the low transcription level of the GAP1 gene indicated that the protein abundance under autolysis conditions was dependent on protein stability. These results suggest that lactate induces the growth and autolysis of K. marxianus, releasing proteins and peptides. These findings can be fundamental for K. marxianus probiotic and kefir studies in the future.

Whole-Genome Transformation Promotes tRNA Anticodon Suppressor Mutations under Stress.

tRNAs are encoded by a large gene family, usually with several isogenic tRNAs interacting with the same codon. Mutations in the anticodon region of other tRNAs can overcome specific tRNA deficiencies. Phylogenetic analysis suggests that such mutations have occurred in evolution, but the driving force is unclear. We show that in yeast suppressor mutations in other tRNAs are able to overcome deficiency of the essential TRT2-encoded tRNAThrCGU at high temperature (40°C). Surprisingly, these tRNA suppressor mutations were obtained after whole-genome transformation with DNA from thermotolerant Kluyveromyces marxianus or Ogataea polymorpha strains but from which the mutations did apparently not originate. We suggest that transient presence of donor DNA in the host facilitates proliferation at high temperature and thus increases the chances for occurrence of spontaneous mutations suppressing defective growth at high temperature. Whole-genome sequence analysis of three transformants revealed only four to five nonsynonymous mutations of which one causing TRT2 anticodon stem stabilization and two anticodon mutations in non-threonyl-tRNAs, tRNALysCUU and tRNAeMetCAU, were causative. Both anticodon mutations suppressed lethality of TRT2 deletion and apparently caused the respective tRNAs to become novel substrates for threonyl-tRNA synthetase. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) data could not detect any significant mistranslation, and reverse transcription-quantitative PCR results contradicted induction of the unfolded protein response. We suggest that stress conditions have been a driving force in evolution for the selection of anticodon-switching mutations in tRNAs as revealed by phylogenetic analysis.IMPORTANCE In this work, we have identified for the first time the causative elements in a eukaryotic organism introduced by applying whole-genome transformation and responsible for the selectable trait of interest, i.e., high temperature tolerance. Surprisingly, the whole-genome transformants contained just a few single nucleotide polymorphisms (SNPs), which were unrelated to the sequence of the donor DNA. In each of three independent transformants, we have identified a SNP in a tRNA, either stabilizing the essential tRNAThrCGU at high temperature or switching the anticodon of tRNALysCUU or tRNAeMetCAU into CGU, which is apparently enough for in vivo recognition by threonyl-tRNA synthetase. LC-MS/MS analysis indeed indicated absence of significant mistranslation. Phylogenetic analysis showed that similar mutations have occurred throughout evolution and we suggest that stress conditions may have been a driving force for their selection. The low number of SNPs introduced by whole-genome transformation may favor its application for improvement of industrial yeast strains.

In Vitro Properties of Potential Probiotic Indigenous Yeasts Originating from Fermented Food and Beverages in Taiwan.

Probiotics are live microorganisms that may be able to help prevent and treat some illnesses. Most probiotics on the market are bacterial, primarily Lactobacillus. Yeast are an inevitable part of the microbiota of various fermented foods and beverages and have several beneficial properties that bacteria do not have. In this study, yeast strains were isolated from fermented food and beverages. Various physiological features of the candidate probiotic isolates were preliminarily investigated, including bile salt and acid tolerance, cell surface hydrophobicity, autoaggregation, antioxidant activity, and ß-galactosidase activity. Several yeast strains with probiotic potential were selected. Overall, Kluyveromyces marxianus JYC2614 adapted well to the bile salt and acid tolerance test; it also had favorable autoaggregation and good cell-surface hydrophobicity. Klu. marxianus JYC2610 grew well according to the bile salt and acid tolerance test and performed well regarding cell surface hydrophobicity and ß-galactosidase activity. Selected yeast species can survive in a gastrointestinal environment and should be further evaluated in vivo as probiotics in the future. Our findings should encourage further studies on the application of the strains in this study as food and feed supplements.

Yeast communities associated with cacti in Brazil and the description of Kluyveromyces starmeri sp. nov. based on phylogenomic analyses.

Yeast communities associated with cacti were studied in three ecosystems of Southeast, Central and North Brazil. A total of 473 yeast strains belonging to 72 species were isolated from 190 samples collected. Cactophilic yeast species were prevalent in necrotic tissues, flowers, fruits and insects of cacti collected in Southeast and North Brazil. Pichia cactophila, Candida sonorensis and species of the Sporopachydermia complex were the most prevalent cactophilic species in Southeast and Central regions. Kodamaea nitidulidarum, Candida restingae and Wickerhamiella cacticola were frequently associated with cactus flowers and fruits. The diversity of yeasts associated with the substrates studied was high. Twenty-one novel species were found. One is described here as Kluyveromyces starmeri sp. nov. based on 21 isolates obtained from necrotic tissues, flowers, fruits and associated insects of the columnar cacti Cereus saddianus, Micranthocereus dolichospermaticus and Pilosocereus arrabidae in two different ecosystems in Brazil. Phylogenetic analyses of sequences encoding the gene of the small subunit (SSU) rRNA gene, the internal transcribed spacer, the 5.8S rRNA gene and the D1/D2 domains of the large subunit (LSU) rRNA showed that the species is related to Kluyveromyces dobzhanskii, Kluyveromyces lactis and Kluyveromyces marxianus. Phylogenomic analyses based on 1264 conserved genes shared among the new species and 19 other members of the Saccharomycetaceae confirmed this phylogenetic relationship. The holotype is K. starmeri sp. nov. CBS 16103T (=UFMG-CM-Y3682T ). The Mycobank number is MB 836817.

Kluyveromyces marxianus: An emerging yeast cell factory for applications in food and biotechnology.

Several yeasts, which are eukaryotic microorganisms, have long been used in different industries due to their potential applications, both for fermentation and for the production of specific metabolites. Kluyveromyces marxianus is one of the most auspicious nonconventional yeasts, generally isolated from wide-ranging natural habitats such as fermented traditional dairy products, kefir grain, sewage from sugar industries, sisal leaves, and plants. This is a food-grade yeast with various beneficial traits, such as rapid growth rate and thermotolerance that make it appealing for different industrial food and biotechnological applications. K. marxianus is a respiro-fermentative yeast likely to produce energy by either respiration or fermentation pathways. It generates a wide-ranging specific metabolites and could contribute to a variety of different food and biotechnological industries. Although Saccharomyces cerevisiae is the most widely used dominant representative in all aspects, many applications of K. marxianus in biotechnology, food and environment have only started to emerge nowadays; some of the most promising applications are reviewed here. The general physiology of K. marxianus is outlined, and then the different applications are discussed: first, the applications of K. marxianus in biotechnology, and then the recent advances and possible applications in food, feed and environmental industries. Finally, this review provides a discussion of the main challenges and some perspectives for targeted applications of K. marxianus in the modern food technology and applied biotechnology in order to exploit the full potential of this yeast which can be used as a cell factory with great efficiency.

γ-aminobutyric acid production by Kluyveromyces marxianus strains.

AIMS: Kluyveromyces marxianus dairy strains were tested for γ-aminobutyric acid (GABA) production. The genes involved in GABA catabolism (UGA1 and UGA2) and anabolism (GAD1) were found in K. marxianus genome. Their relative expression was evaluated with primer designed ad hoc. METHODS AND RESULTS: Strains were grouped on the basis of GAD1 gene sequence. Representative strains for each group were tested for GABA production by high-performance liquid chromatography. All strains produced it at low levels. qRT-PCR showed the absence of a relation between GABA production and GAD1 gene expression. UGA1 and UGA2 genes were not upregulated and low amounts of succinic acid were detected. CONCLUSIONS: All strains released a low amount of GABA suggesting that probably it was stored within cells. The different behaviour of strains in terms of GABA and succinic acid production as well as gene expression highlighted the genetic and phenotypic biodiversity of this species. SIGNIFICANCE AND IMPACT OF THE STUDY: GABA production and genes involved in its catabolism and anabolism were described in a population of dairy K. marxianus for the first time. The variability observed in terms of genetic and phenotypic biodiversity is important especially to exploit this non-conventional yeast as microbial platform.

Kluyveromyces osmophilus is not a synonym of Zygosaccharomyces mellis; reinstatement as Zygosaccharomyces osmophilus comb. nov.

Kluyveromyces osmophilus, a single-strain species isolated from Mozambique sugar, has been treated a synonym of Zygosaccharomyces mellis. Analyses of D1/D2 LSU rRNA gene sequences confirmed that the species belongs to the genus Zygosaccharomyces but showed it to be distinct from strains of Z. mellis. During studies of yeasts associated with stingless bees in Brazil, nine additional isolates of the species were obtained from unripe and ripe honey and pollen of Scaptotrigona cfr. bipunctata, as well as ripe honey of Tetragonisca angustula. The D1/D2 sequences of the Brazilian isolates were identical to those of the type strain of K. osmophilus CBS 5499 (=ATCC 22027), indicating that they represent the same species. Phylogenomic analyses using 4038 orthologous genes support the reinstatement of K. osmophilus as a member of the genus Zygosaccharomyces. We, therefore, propose the name Zygosaccharomyces osmophilus comb. nov. (lectotype ATCC 22027; MycoBank no. MB 833739).

Microbial characterization of an artisanal production of Robiola di Roccaverano cheese.

Robiola di Roccaverano, from the Piedmont region of Italy, is a Protected Designation of Origin soft cheese made with raw goat milk. The peculiarity of this cheese is that during the manufacturing process, a natural starter culture (NC) is added to raw milk. This study examined the viable microorganisms of technological interest, including lactic acid bacteria and fungal populations, in samples of raw milk, NC, and fresh and ripened cheese collected from one dairy using culture-dependent techniques. First, the isolated colonies were analyzed using random amplification of polymorphic DNA (RAPD) PCR, and strains with similar fingerprints were clustered together. Further, representative isolates of each group were subjected to 16S or 26S ribosomal DNA sequencing. Finally, species-specific PCR was conducted to distinguish the Lactococcus lactis ssp. lactis and Lc. lactis ssp. cremoris. Among the studied lactic acid bacteria, 13 RAPD profiles were obtained, corresponding to 9 different bacterial species or subspecies. Concerning mold and yeast isolates, 5 species were found that coincided with 5 RAPD types. Observing the strains isolated in the study, Lc. lactis was the most prevalent species in raw milk and NC samples, and Leuconostoc mesenteroides was the predominant species identified in 5- and 15-d cheese isolates. Furthermore, whereas only these 2 species were detected in NC, Enterococcus and Lactobacillus genera were found in raw milk and cheese, respectively. Concerning the mold and yeast isolates, in NC Kluyveromyces spp. was mainly found, and in cheese samples the representative species were Geotrichum candidum and Yarrowia lipolytica. Finally, raw milk and cheese safety were evaluated, and the samples complied with the standard required by European Commission regulation number 2073/2005.

Alterations in gut bacterial and fungal microbiomes are associated with bacterial Keratitis, an inflammatory disease of the human eye.

Dysbiosis, or imbalance in the gut microbiome, has been implicated in auto-immune, inflammatory, neurological diseases as well as in cancers. More recently it has also been shown to be associated with ocular diseases. In the present study, the association of gut microbiome dysbiosis with bacterial Keratitis, an inflammatory eye disease which significantly contributes to corneal blindness, was investigated. Bacterial and fungal gut microbiomes were analysed using fecal samples of healthy controls (HC, n = 21) and bacterial Keratitis patients (BK, n = 19). An increase in abundance of several antiinflammatory organisms including Dialister, Megasphaera, Faecalibacterium, Lachnospira, Ruminococcus and Mitsuokella and members of Firmicutes, Veillonellaceae, Ruminococcaceae and Lachnospiraceae was observed in HC compared to BK patients in the bacterial microbiome. In the fungal microbiome, a decrease in the abundance of Mortierella, Rhizopus, Kluyveromyces, Embellisia and Haematonectria and an increase in the abundance of pathogenic fungi Aspergillus and Malassezia were observed in BK patients compared to HC. In addition, heatmaps, PCoA plots and inferred functional profiles also indicated significant variations between the HC and BK microbiomes, which strongly suggest dysbiosis in the gut microbiome of BK patients. This is the first study demonstrating the association of gut microbiome with the pathophysiology of BK and thus supports the gut-eye axis hypothesis. Considering that Keratitis affects about 1 million people annually across the globe, the data could be the basis for developing alternate strategies for treatment like use of probiotics or fecal transplantation to restore the healthy microbiome as a treatment protocol for Keratitis.

Intraspecies polymorphisms of Kluyveromyces marxianus strains from Yaghnob valley.

In this study, 29 strains of Kluyveromyces marxianus with peculiar genetic and phenotypic traits previously isolated from a fermented goat milk of Yaghnob valley were investigated for chromosome length polymorphism (CLP) by PFGE, adhesion properties and carbon usage by Biolog analysis. Obtained data showed that strains differed in terms of number and size of chromosome bands. The number of bands ranged from 5 to 7, suggesting a probable genome size from 1.4 to 2.6 Mb. Strains showed a certain level of cell surface hydrophobicity ranging from 32% to 77.7%. Strains were also tested for their ability to form a biofilm on polystyrene plates: planktonic cells ranged from 6.3 cfu/mL to 7.95 cfu/mL, while sessile from 7.11 cfu/mL to 8.6 cfu/mL. The strains able to adhere to polystyrene plates were also able to form a mature MAT. Biolog analysis revealed that almost all strains were able to use putrescine, malic acid, α-D lactose, phenylethylamine, ß-methyl D-gucoside and xylose; 5 strains were able to grow on cellobiose and 3 were able to catabolise α-ketobutyric. The obtained data highlighted a number of interesting features underlying the peculiar capacities of these strains for industrial applications.

Candida infanticola and Candida spencermartinsiae yeasts: Possible emerging species in cancer patients.

Opportunistic infections due to Candida species occur frequently in intensive care settings. We investigated the prevalence of Candida species among 65 clinical specimens obtained from 200 cancer patients by phenotypic and molecular (ITS sequencing and AFLP) methods. Among the 65 yeast isolates, Candida albicans was the most commonly isolated species (n = 34, 52.3%), whereas other Candida species comprised 47.7% (n = 31) and consisted of Candida glabrata (n = 14, 21.5%), Candida tropicalis (n = 5, 7.7%) and uncommon Candida species (n = 12, 18.5%) such as Candida pelliculosa (n = 3, 4.6%), Pichia kudriavzevii (= Candida krusei, n = 2, 3.1%), Candida orthopsilosis (n = 2, 3.1%), Candida parapsilosis (n = 1, 1.5%), Candida infanticola (n = 2, 3.1%), Candida spencermartinsiae (n = 1, 1.5%), and Kluyveromyces marxianus (=Candida kefyr, n = 1, 1.5%). Candida infanticola and Candida spencermartinsiae were recovered from oral lesions of cancer patients. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) easily confirmed these isolates as less common Candida isolates (4.6%). The in vitro antifungal susceptibilities of C. spencermartinsiae and the two strains of C. infanticola were determined according to CLSI guidelines (M27-A3). MIC results among these isolates showed they were susceptible to isavuconazole, posaconazole and voriconazole, however, fluconazole and caspofungin had high MIC values. These Candida species that may occur more commonly in infections remain unnoticed using commonly used phenotypical methods in routine microbiology laboratories. MALDI-TOF MS proved to be a more fast and robust diagnostic technique for identification of the yeasts isolated from different clinical specimens of cancer patients.

Development and implementation of multilocus sequence typing to study the diversity of the yeast Kluyveromyces marxianus in Italian cheeses.

The yeast Kluyveromyces marxianus possesses advantageous traits like rapid growth, GRAS (generally regarded as safe) status and thermotolerance that make it very suitable for diverse biotechnological applications. Although physiological studies demonstrate wide phenotypic variation within the species, there is only limited information available on the genetic diversity of K. marxianus. The aim of this work was to develop a multilocus sequence typing (MLST) method for K. marxianus to improve strain classification and selection. Analysis of housekeeping genes in a number of sequenced strains led to the selection of five genes, IPP1, TFC1, GPH1, GSY2 and SGA1, with sufficient polymorphic sites to allow MLST analysis. These loci were sequenced in an additional 76 strains and used to develop the MLST. This revealed wide diversity in the species and separation of the culture collection and wild strains into multiple distinct clades. Two subsets of strains that shared sources of origin were subjected to MLST and split decomposition analysis. The latter revealed evidence of recombination, indicating that this yeast undergoes mating in the wild. A public access web-based portal was established to allow expansion of the database and application of MLST to additional K. marxianus strains. This will aid understanding of the genetic diversity of the yeast and facilitate biotechnological exploitation.

Potential characterization of yeasts isolated from Kazak artisanal cheese to produce flavoring compounds.

Cheese is a typical handcrafted fermented food in Kazak minority from the Uighur Autonomy Region in China and Central Asia. Among the microbial community that is responsible for Kazak cheese fermentation, yeasts play important role in flavor formation during ripening. To develop ripening cultures, we isolated 123 yeasts from 25 cheese products in Kazak, and identified 87 isolates by the D1/D2 domain of the large subunit rRNA gene sequence. Pichia kudriavzevii was the dominant yeast in Kazak cheese, followed by Kluyveromyces marxianus and Kluyveromyces lactis. Of these, the ability to exhibit enzyme of dominant isolates and contribution to the typical flavor of cheeses was assessed. Enzyme producing yeast strains were inoculated in Hazak cheese-like medium and volatile compounds were identified by head space solid phase micro extraction coupled to gas chromatography and mass spectroscopy. Pichia kudriavzevii N-X displayed the strongest extracellular proteolytic and activity on skim milk agar and produced a range of aroma compounds (ethanol, ethyl acetate, 3-methylbutanol, and acetic acid) for Kazak cheese flavor, could be explored as ripening cultures in commercial production of Kazak cheeses.

Polymorphisms in the LAC12 gene explain lactose utilisation variability in Kluyveromyces marxianus strains.

Kluyveromyces marxianus is a safe yeast used in the food and biotechnology sectors. One of the important traits that sets it apart from the familiar yeasts, Saccharomyces cerevisiae, is its capacity to grow using lactose as a carbon source. Like in its close relative, Kluyveromyces lactis, this requires lactose transport via a permease and intracellular hydrolysis of the disaccharide. Given the importance of the trait, it was intriguing that most, but not all, strains of K. marxianus are reported to consume lactose efficiently. In this study, primarily through heterologous expression in S. cerevisiae and K. marxianus, it was established that a single gene, LAC12, is responsible for lactose uptake in K. marxianus. Strains that failed to transport lactose showed variation in 13 amino acids in the Lac12p protein, rendering the protein non-functional for lactose transport. Genome analysis showed that the LAC12 gene is present in four copies in the subtelomeric regions of three different chromosomes but only the ancestral LAC12 gene encodes a functional lactose transporter. Other copies of LAC12 may be non-functional or have alternative substrates. The analysis raises some interesting questions regarding the evolution of sugar transporters in K. marxianus.

Kluyveromyces marxianus as a host for heterologous protein synthesis.

The preferentially respiring and thermotolerant yeast Kluyveromyces marxianus is an emerging host for heterologous protein synthesis, surpassing the traditional preferentially fermenting yeast Saccharomyces cerevisiae in some important aspects: K . marxianus can grow at temperatures 10 °C higher than S. cerevisiae, which may result in decreased costs for cooling bioreactors and reduced contamination risk; has ability to metabolize a wider variety of sugars, such as lactose and xylose; is the fastest growing eukaryote described so far; and does not require special cultivation techniques (such as fed-batch) to avoid fermentative metabolism. All these advantages exist together with a high secretory capacity, performance of eukaryotic post-translational modifications, and with a generally regarded as safe (GRAS) status. In the last years, replication origins from several Kluyveromyces spp. have been used for the construction of episomal vectors, and also integrative strategies have been developed based on the tendency for non-homologous recombination displayed by K. marxianus. The recessive URA3 auxotrophic marker and the dominant Kan(R) are mostly used for selection of transformed cells, but other markers have been made available. Homologous and heterologous promoters and secretion signals have been characterized, with the K. marxianus INU1 expression and secretion system being of remarkable functionality. The efficient synthesis of roughly 50 heterologous proteins has been demonstrated, including one thermophilic enzyme. In this mini-review, we summarize the physiological characteristics of K. marxianus relevant for its use in the efficient synthesis of heterologous proteins, the efforts performed hitherto in the development of a molecular toolbox for this purpose, and some successful examples.

Multilocus analysis reveals large genetic diversity in Kluyveromyces marxianus strains isolated from Parmigiano Reggiano and Pecorino di Farindola cheeses.

In the present study, we have analysed the genetic diversity in Kluyveromyces marxianus isolated from Parmigiano Reggiano and Pecorino di Farindola cheesemaking environment. Molecular typing methods inter-RTL fingerprint and mtDNA RFLPs, as well as, sequence diversity and heterozygosity in the intergenic region between KmSSB1 and KmRIO2 genes and analysis of the mating locus were applied to 54 K. marxianus strains. Inter-RTL fingerprint revealed a large degree of genetic heterogeneity and clustering allowed differentiation of K. marxianus strains from different geographical origins. In general, inter-LTR profiles were more discriminating than RFLPs of mtDNA; however our results also indicate that both techniques could be complementary unveiling different degrees of genetic diversity. Sequence analysis of the intergenic region between KmSSB1 and KmRIO2 genes revealed 26 variable positions in which a double peak could be observed in the sequence chromatogram. Further analysis revealed the presence of heterozygous strains in the K. marxianus population isolated from Parmigiano Reggiano. On the other hand, all strains isolated from Pecorino di Farindola were homozygous. Two very different groups of haplotypes could be observed as well as mixtures between them. Phylogenetic reconstruction divided K. marxianus dairy strains into two separate populations. A few heterozygous strains in an intermediate position between them could also be observed. Mating type locus analysis revealed a large population of diploid strains containing both MATa and MATα alleles and few haploid strains, most of them presenting the MATα allele. Different scenarios explaining the presence and maintaining of homozygous and heterozygous diploids as well as hybrids between them in the Parmigiano Reggiano K. marxianus population are proposed. A principal component analysis supported the large differences between K. marxianus isolated from Parmigiano Reggiano and Pecorino di Farindola.

The evaluation of kefir pure culture starter: Liquid-core capsule entrapping microorganisms isolated from kefir grains.

The main purpose of this study was to develop a pure culture starter for producing kefir. In order to accomplish starter recycling, yeasts (Kluyveromyces marxianus strain, Pichia kudriavzevii clone), lactic acid bacteria (Lactobacillus kefiri strain F4Aa, Lactobacillus kefiri strain NM131-7, Lactobacillus kefiri strain NM132-3, Lactobacillus kefiri strain NM180-3, respectively), and acetic acid bacteria (Acetobacter lovaniensis strain) were entrapped in liquid core capsules based on the distribution ratio in kefir grains. The microbiological, antimicrobial, and chemical properties of kefir made with capsules (M) and kefir grains (K) were measured and compared. According to the results of plate counts in different selective medium, the number of yeasts and bacteria in the liquid core capsules gradually increased and stabilized after eight fermentation cycles. The results of gas chromatography-mass spectrometry showed that almost all the aroma components existed in the two type of kefir, except the ethyl lactate. There was no significant difference in alcohol content, protein content, and fat content, except the acidity and sugar content. Water holding capacity of kefir K was higher than kefir M. There were 14 same free amino acids in kefir M and kefir K, and the content of most free amino acids was similar. In antimicrobial test, there was no significant difference in both kefirs.

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.

Genome-wide metabolic re-annotation of Ashbya gossypii: new insights into its metabolism through a comparative analysis with Saccharomyces cerevisiae and Kluyveromyces lactis.

BACKGROUND: Ashbya gossypii is an industrially relevant microorganism traditionally used for riboflavin production. Despite the high gene homology and gene order conservation comparatively with Saccharomyces cerevisiae, it presents a lower level of genomic complexity. Its type of growth, placing it among filamentous fungi, questions how close it really is from the budding yeast, namely in terms of metabolism, therefore raising the need for an extensive and thorough study of its entire metabolism. This work reports the first manual enzymatic genome-wide re-annotation of A. gossypii as well as the first annotation of membrane transport proteins. RESULTS: After applying a developed enzymatic re-annotation pipeline, 847 genes were assigned with metabolic functions. Comparatively to KEGG's annotation, these data corrected the function for 14% of the common genes and increased the information for 52 genes, either completing existing partial EC numbers or adding new ones. Furthermore, 22 unreported enzymatic functions were found, corresponding to a significant increase in the knowledge of the metabolism of this organism. The information retrieved from the metabolic re-annotation and transport annotation was used for a comprehensive analysis of A. gossypii's metabolism in comparison to the one of S. cerevisiae (post-WGD - whole genome duplication) and Kluyveromyces lactis (pre-WGD), suggesting some relevant differences in several parts of their metabolism, with the majority being found for the metabolism of purines, pyrimidines, nitrogen and lipids. A considerable number of enzymes were found exclusively in A. gossypii comparatively with K. lactis (90) and S. cerevisiae (13). In a similar way, 176 and 123 enzymatic functions were absent on A. gossypii comparatively to K. lactis and S. cerevisiae, respectively, confirming some of the well-known phenotypes of this organism. CONCLUSIONS: This high quality metabolic re-annotation, together with the first membrane transporters annotation and the metabolic comparative analysis, represents a new important tool for the study and better understanding of A. gossypii's metabolism.