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s Zygosaccharomyces species are among the most problematic food spoilage yeasts. The two most infamous species are Zygosaccharomyces balii and Zygosaccharomyces rouxii, although they may also take a positive role during the production of some fermented foods. DNA sequence based yeast identification aided by freely available reference databases of barcoding DNA sequences has boosted the description rate of novel yeast species in the last two decades. The genus Zygosaccharomyces has been considerably expanded as well. Especially the number of the extremely osmotolerant Zygosaccharomyces species, related to Z. rouxii and regularly found in high-sugar foods, has enlarged. A brief account of recent developments in the taxonomy and biodiversity of this important food associated genus is given in this review.

Yeast is one of the organisms that grow and survive in diverse conditions. The influence of environmental factors on its growth and proliferation were evaluated. The yeast used for the study was isolated from honey using Sabouraud Dextrose Agar and pour plate methods. The effects of temperature, pH and ultraviolet radiation on the growth of Rhodotorula, Debaryomyces, Zygosaccharomyces and Candida species were determined using standard methods. The growths of all the yeast isolates were greatly affected at 80oC. Rhodotorulla species showed least growth at 50oC, but optimal growth was recorded at 30oC after 96 h of incubation. A similar trend was observed in Debaromyces species. Zygosaccharomyces species recorded high reduction in growth at 60oC after 96 h of incubation, though there were no significant differences in the growths of the isolates at 60oC and 80oC. Candida species recorded the least effect of temperature at 30oC and highest at 60oC and 80oC after 96 h of incubation. The results of the effect of pH on growth of the yeasts showed that Rhodotorula and Zygosaccharomyces had optimum growth at pH of 5 and 4 respectively. All the isolates showed increase in growth with increase in incubation time. Exposure of the isolates to UV-rays negatively affected the growths of the isolates. The growths of the yeasts decreased with increase in exposure time. This study showed that yeast can survive adverse temperature and pH and can as well survive UV-ray exposure.

The Zygosaccharomyces is notorious for its remarkable spoilage characteristics. In the present study, the visual and rapid identification of the genus Zygosaccharomyces was performed by a loop-mediated isothermal amplification (LAMP) assay using specific primers in Mini Dry Bath within 30 min at 65°C followed by a lateral flow dipstick (LFD) detection. The sensitivity evaluation revealed LAMP-LFD assay with 1.0×101 copies/μL of Zygosaccharomyces DNA as its detection limit, which was the same as the methods of real-time quantitative PCR (qPCR) and conventional polymerase chain reaction (PCR). However, qPCR or PCR methods not only need to be performed in a specialist analytical laboratory with expensive equipment but also with risk of aerosol pollution. The LAMP-LFD assay had no cross-reactivity against 10 other yeast species and its specificity was 100%. A total of 25 Qiangli loquat Dew samples (17 bottles bulged and eight normal-appearing) were detected within 40 min with 100 and 92.0% accurately and specifically, when compared with the qPCR assay and the microbiology culture method, respectively. Therefore, the simple, fast, sensitive and low-cost LAMP-LFD assay is an effective and useful tool for the on-site identification of the genus Zygosaccharomyces. Key words: Zygosaccharomyces, on-site detection, loop-mediated isothermal amplification (LAMP), lateral flow dipstick (LFD).

Analysis of plant pollen can provide valuable insights into the existing spectrum of microorganisms in the environment. When harvesting bee-collected pollen as a dietary supplement for human consumption, timely preservation of the freshly collected pollen is fundamental for product quality. Environmental microorganisms contained in freshly collected pollen can lead to spoilage by degradation of pollen components. In this study, freshly collected bee pollen was sampled at different locations and stored under various storage conditions to examine the hypothesis that storage conditions may have an effect on the composition of microorganisms in pollen samples. The samples were analyzed using 16S and 18S amplicon sequencing and characterized by palynological analysis. Interestingly, the bacterial communities between pollen samples from different locations varied only slightly, whereas for fungal community compositions, this effect was substantially increased. Further, we noticed that fungal communities in pollen are particularly sensitive to storage conditions. The fungal genera proportion Cladosporium and Mycosphaerella decreased, while Zygosaccharomyces and Aspergillus increased during storage. Aspergillus and Zygosaccharomyces fractions increased during storage at 30 °C, which could negatively impact the pollen quality if it is used as a dietary supplement.

Zygosaccharomyces sp. is an industrially important yeast for the production traditional fermented foods in Japan. At present, however, there is no easy method for mating Zygosaccharomyces sp. strains in the laboratory; furthermore, little is known about the expression of mating-type-specific genes in this yeast. Here, mating was observed when Zygosaccharomyces sp. was subjected to nitrogen-starvation conditions. The expression of mating-type-specific genes, Zygo STE6 and Zygo MFα1, was induced under nitrogen-starvation conditions, as confirmed by lacZ reporter assay. This expression was mating-type-specific: Zygo STE6 was expressed specifically for mating-type a, whereas and Zygo MFα1 was expressed specifically for mating-type α. Yeast strains Zygosaccharomyces rouxii DL2 and DA2, derived from type strain Z. rouxii CBS732, did not show mating even under nitrogen-starvation conditions. Gene sequencing revealed that the Zygo STE12 in Z. rouxii CBS732 has a frameshift mutation. Under nitrogen starvation, mating was observed in both DL2 and DA2 transformed with the wild-type Zygo STE12. The expression of Zygo STE6 in Z. rouxii DL2 transformed with wild-type Zygo STE12 under nitrogen-starvation conditions was confirmed by lacZ reporter assay. Collectively, these results revealed that, under nitrogen-starvation conditions, Zygosaccharomyces sp. can mate and mating-type-specific genes are expressed. Furthermore, Zygo Ste12 is essential for both mating and the expression of mating-type-specific genes in Zygosaccharomyces sp.

Traditional fermented cereals are a rich source of naturally derived, diverse microorganisms. Illumina MiSeq high‐throughput sequencing was used to investigate thoroughly fungal microflora in Western Inner Mongolian acidic gruel. A total of 589,495 sequences were obtained from 16 acidic gruel samples. Ascomycota was found to be the predominant phylum with a relatively abundance of 97.54%, followed by Basidiomycota (2.26%) and Chytridiomycota (0.1%). The dominant genera obtained from the acidic gruel were Candida, Galactomyces, Hanseniaspora, Guehomyces, Zygosaccharomyces, Trichosporon, Rhodosporidium, Penicillium, and Blastobotrys. Candida and Galactomyces were predominant genera, and their relative abundances were 57.59% and 34.95%, respectively. A total of 50 yeast strains were isolated and identified. Statistical analysis indicated that P kudriavzevii and Geo. silvicola affiliated with Ascomycota were the dominant yeasts in acidic gruel, accounting for 28% and 22%, respectively. This study provides an unequivocal theoretical basis for the study of fungal diversity and the identification and preservation of yeasts in traditional fermented cereals. It also provides validated strain resources for further exploration of the effect of yeasts on acidic gruel quality., Diverse fungal species were abundant in traditional fermented acidic gruel, and significant differences between different samples were seen; The core fungal genera in acidic gruel were Candida and Pichia; Pichia kudriavzevii and Geotrichum silvicola were the dominant yeasts in acidic gruel.

BACKGROUND Sugar-rich foods are of the main components of daily human meals. These foods with high sugar and low water content kill bacteria. However, osmotolerant yeasts survive and multiply. The aim of this study was to examine the occurrence of intracellular Helicobacter pylori(H. pylori) and Staphylococcus spp. in yeast isolates from sugar-rich foods. METHODS Thirty-two yeast isolates from fresh fruits, dried fruits, commercial foods, and miscellaneous foods were identified by the sequencing of amplified products of 26S rDNA. Fluorescence microscopy and LIVE/DEAD bacterial viability kit were used to examine the occurrence of live bacteria inside the yeast’s vacuole. Immunofluorescence assay was used to confirm the identity of intracellular bacteria as H. pylori and Staphylococcus. Polymerase chain reaction (PCR) was used for the detection of 16S rDNA of H. pylori and Staphylococcus in the total DNA of yeasts. RESULTS Yeasts were identified as members of seven genera; Candida, Saccharomyces, Zygosaccharomyces, Pichia, Meyerozyma, Metschnikowia, and Wickerhamomyces. Intravacuolar bacteria were stained green with a bacterial viability kit, revealing that they were alive. Immunofluorescence assay confirmed the identity of intracellular H. pylori and Staphylococcus spp. PCR results revealed that among the 32 isolated yeasts, 53% were H. pylori-positive, 6% were Staphylococcus-positive, 18.7% were positive for both, and 21.8% were negative for both. CONCLUSION Detection of H. pylori- and Staphylococcus-16S rDNA in yeast isolates from dried fruits, and commercial foods showed the occurrence of more than one kind of endosymbiotic bacterium in yeasts’ vacuoles. While the establishment of H. pylori and Staphylococcus in yeast is a sophisticated survival strategy, yeast serves as a potent bacterial reservoir.

Summary Goals: This study addresses the increasing demand for “natural” and certified organic wines, along with the need for improved worker safety. Winemakers continue to search for alternatives to SO2 as an antioxidant and antimicrobial agent. This study compares the use of blended non-Saccharomyces cerevisiae yeasts—Torulaspora delbrueckii (Td) and Metschnikowia pulcherrima (Mp) — as antimicrobial agents to a standard addition of SO2 on Cabernet Sauvignon. This fruit possesses over 10 times the normal microbial flora typically found in California. In conjunction with this comparison study, a proof of concept prototype illustrates the use of a novel spray method for the application of these non-Saccharomyces yeasts onto a grape machine harvester for bioprotection. Key Findings: Research Winery: Overall, the blended yeasts performed better than a standard addition of SO2 at controlling wine spoilage organisms on compromised fruit. Organisms related to wine spoilage responded differently to Td/Mp than to SO2. The Td/Mp treatment exhibited an advantage over the SO2 treatment. The Td/Mp treatment appeared to work best against Zygosaccharomyces, Lactobacillus kunkeei, Hanseniaspora uvarum, and acetic acid bacteria. It was less effective against Pediococcus and other Lactobacillus species. Different stages of the trial fermentations were affected differently by Td/Mp and SO2. The Td/Mp populations performed best during prefermentation and the early stages of fermentation. Td/Mp showed an antagonistic effect on microorganisms responsible for wine spoilage. There were fewer microorganisms related to spoilage growing in the three bioreactors with non-Saccharomyces species than in the bioreactors acting as experimental controls with 60 mg/L SO2 added during processing. Td/Mp treatment increased the implantation capacity of S. cerevisiae compared to the use of SO2. Using identical inoculation rates of S. cerevisiae, we found more S. cerevisiae cells growing in the Td/Mp bioreactors than in the bioreactors treated with SO2. Furthermore, we observed greater population reduction and fewer cells/mL of S. cerevisiae at the end of fermentation. Field Trial: A reduction in spoilage microorganisms occurred when using Td/Mp directly applied to the harvester. Applying Td/Mp yeasts to the grape harvester reduced aromas related to volatile acidity coming from the machine. Impact and Significance: The use of Td/Mp yeasts provides an alternative to SO2 for controlling the growth of organisms related to wine spoilage. Incorporating these yeasts as a bioprotectant by applying them during the harvest and transport processes reduces the risk of detrimental microbial organisms in the harvested fruit, juice, and wine.

The grains fermented in Maotai-flavored liquor can be classified into two groups: high-temperature fermented grains generated by stacking and grains fermented in pits. The Maotai-flavor making process enriches a special microbiota from the fermented grains, which makes related studies more difficult. The use of modern molecular techniques to detect and analyze diversity and changes in total bacterial (16S rRNA sequencing) and fungal (internal transcribed spacer (ITS) sequencing) counts have helped to overcome the shortcomings of traditional technological research. The total RNA extracted by Fe3O4–SiO2 nanoparticles was retrieved into DNA by MagBeads Total RNA Extraction Kit. However, discrepancies exist in the microbial community structures at different fermentation periods. Dominant bacteria include Escherichia-Shigella, Lactobacillus, Clostridium, and Streptococcus species and dominant fungi include Alternaria, Ciliophora, Pyrenochaetopsis, Cyphellophora, Aspergillus, Issatchenkia, Pichia, Candida, and Zygosaccharomyces species. Analyses of the enzymatic activity of samples at different fermentation stages have revealed that some existing bacilli influence amylase activity. Here, to investigate flavor changes during each fermentation round, the liquor quality at all rounds was comprehensively assessed based on the corresponding physicochemical properties, which were summarized by sensory evaluation. These results suggested that the liquor quality in the third, fourth, and fifth fermentation rounds was superior.

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).

Kombucha, a fermented tea generated from the co-culture of yeasts and bacteria, has gained worldwide popularity in recent years due to its potential benefits to human health. As a result, many studies have attempted to characterize both its biochemical properties and microbial composition. Here, we have applied a combination of whole metagenome sequencing (WMS) and amplicon (16S rRNA and Internal Transcribed Spacer 1 [ITS1]) sequencing to investigate the microbial communities of homemade Kombucha fermentations from day 3 to day 15. We identified the dominant bacterial genus as Komagataeibacter and dominant fungal genus as Zygosaccharomyces in all samples at all time points. Furthermore, we recovered three near complete Komagataeibacter genomes and one Zygosaccharomyces bailii genome and then predicted their functional properties. Also, we determined the broad taxonomic and functional profile of plasmids found within the Kombucha microbial communities. Overall, this study provides a detailed description of the taxonomic and functional systems of the Kombucha microbial community. Based on this, we conject that the functional complementarity enables metabolic cross talks between Komagataeibacter species and Z. bailii, which helps establish the sustained a relatively low diversity ecosystem in Kombucha. Scientific Research Projects Coordination Unit of Istanbul Univ. Biotechnology and Biological Sciences Research Council (BBSRC) European Molecular Biology Laboratory

The ubiquitin-proteasome system plays an important role in metabolic regulation. In a previous study, we reported that, in Saccharomyces cerevisiae, when glucose is available, the SCFUcc1 ubiquitin ligase complex targets citrate synthase 2 (Cit2) for proteasomal degradation, thereby suppressing the glyoxylate cycle, an anabolic pathway that replenishes the TCA cycle with succinate for the activation of gluconeogenesis. However, the roles of Ucc1 in other yeast species remain unclear. Here, we cloned orthologs of the F-box protein Ucc1 from Zygosaccharomyces bailii, an aggressive food spoilage microorganism that is the most acetic acid-tolerant yeast species, and Candida glabrata, an emerging fungal pathogen. These orthologs were expressed in S. cerevisiae, and their activities were tested genetically and biochemically. The results showed that Z. bailii Ucc1 rescued the ucc1Δ phenotype, suggesting the existence of a similar mechanism regulating the glyoxylate cycle in Z. bailii. By contrast, C. glabrata Ucc1 did not complement the ucc1Δ phenotype or exhibit a dominant negative effect on Ucc1. These results suggest the importance of analysing the regulatory mechanisms of glyoxylate cycle in a broad range of yeast species.

The conversion of grape juice to wine starts with complex yeast communities consisting of strains that have colonised the harvested grape and/or reside in the winery environment. As the conditions in the fermenting juice gradually become inhibitory for most species, they are rapidly overgrown by the more adaptable Saccharomyces strains, which then complete the fermentation. However, there are environmental factors that even Saccharomyces cannot cope with. We show that when the sugar content is extremely high, osmotolerant yeasts, usually considered as &ldquo, spoilage yeasts&ldquo, ferment the must. The examination of the yeast biota of 22 botrytised Tokaj Essence wines of sugar concentrations ranging from 365 to 752 g∙L&minus, 1 identified the osmotolerant Zygosaccharomyces rouxii, Candida (Starmerella) lactis-condensi and Candida zemplinina (Starmerella bacillaris) as the dominating species. Ten additional species, mostly known as osmotolerant spoilage yeasts or biofilm-producing yeasts, were detected as minor components of the populations. The high phenotypical and molecular (karyotype, mtDNA restriction fragment length polymorphism (RFLP) and microsatellite-primed PCR (MSP-PCR)) diversity of the conspecific strains indicated that diverse clones of the species coexisted in the wines. Genetic segregation of certain clones and interactions (antagonism and crossfeeding) of the species also appeared to shape the fermenting yeast biota.

Tequila and mezcal are both traditional Mexican liquors that are produced from cooked Agave spp. must fermentation and usually rely on spontaneous or pure Saccharomyces cerevisiae strain inoculation. In order to contribute to the rational selection of yeast starters for tequila and mezcal productions, we tested a collection of 25 yeasts originally isolated from mezcal musts, spanning 10 different yeast species. These strains were first characterized in a semi synthetic medium (labeled as M2, having 90 g/L fructose and 10 g/L glucose of initial hexoses) at 48 h of culture, observing a differential pattern in the consumption of sugars and productivity. Selected Saccharomyces strains left around 10 g/L of fructose and showed higher fermentation performance. However, some non-Saccharomyces strains, specifically from Torulospora (Td), Kluyveromyces (Km), and Zygosaccharomyces (Zb) genera, consumed almost all the sugar (i.e., Km1Y9 with Saccharomyces strains presented a high production of ethyl-butyrate, ethyl-decanoate, and ethyl-hexanoate with peaks of 10, 38, and 3 μg/L, respectively. In addition, some Kluyveromyces and Torulospora strains showed a high production of phenyl ethyl acetate (i.e., Km1D5 with up to 1400 μg/L); isoamyl acetate (i.e., Km1D5 and Td1AN2 with more than 300 μg/L), and hexyl acetate (i.e., Td1AN2 with 0.3 μg/L). Representative strains of the most productive genera (Saccharomyces, Torulospora, and Kluyveromyces) were selected to evaluate their fermentative performance and survival in a mixed culture on a medium based on Agave tequilana must, and their population kinetics was characterized using specific fluorescent in situ hybridization (FISH) probes in a qualitative and semi-quantitative analysis during fermentation. We observed that the mixture ratios of 0.1:1:1 or 1:1:1 (Saccharomyces:Kluyveromyces:Torulospora), maintained good fermentation productivities, with alcohol yields above 0.45 g/g, and allowed a high survival rate of the non-Saccharomyces strains during the fermentation process. Finally, mixed inoculum fermentations on A. tequilana must medium, including different Saccharomyces strains and the finally selected Torulospora and Kluyveromyces strains, showed the best production parameters in terms of ethanol, carbon dioxide, glycerol, and acetic acid values, as well as improved volatile metabolite profiles as compared to the pure cultures. All these data were used to propose a methodology of selection of strains to be used as a pure or mixed starter for tequila and mezcal fermentations, with high primary metabolite productivity and desired aromatic profile.

The so-called nonconventional yeasts are becoming increasingly attractive in food and industrial biotechnology. Among them, Zygosaccharomyces rouxii is known to be halotolerant, osmotolerant, petite negative, and poorly Crabtree positive. These traits and the high fermentative vigour make this species very appealing for industrial and food applications. Nevertheless, the biotechnological exploitation of Z. rouxii has been biased by the low availability of genetic engineering tools and the recalcitrance of this yeast towards the most conventional transformation procedures. Centromeric and episomal Z. rouxii plasmids have been successfully constructed with prototrophic markers, which limited their usage to auxotrophic strains, mainly derived from the Z. rouxii haploid type strain Centraalbureau voor Schimmelcultures (CBS) 732T . By contrast, the majority of industrially promising Z. rouxii yeasts are prototrophic and allodiploid/aneuploid strains. In order to expand the genetic tools for manipulating these strains, we developed two centromeric and two episomal vectors harbouring KanMXR and ClonNATR as dominant drug resistance markers, respectively. We also constructed the plasmid pGRCRE that allows the Cre recombinase-mediated marker recycling during multiple gene deletions. As proof of concept, pGRCRE was successfully used to rescue the kanMX-loxP module in Z. rouxii ATCC 42981 G418-resistant mutants previously constructed by replacing the MATαP expression locus with the loxP-kanMX-loxP cassette.

The spoilage of foods caused by the growth of undesirable yeast species is a problem in the food industry. Yeast species such as Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii, Kluyveromyces lactis and Saccharomyces cerevisiae have been encountered in foods such as high sugar products, fruit juices, wine, mayonnaise, chocolate and soft drinks. The demand for new methods of preservations has increased because of the negative association attached to chemical preservatives. The sequence of a novel short peptide (KKFFRAWWAPRFLK-NH2) was modified to generate three versions of this original peptide. These peptides were tested for the inhibition of the yeasts mentioned above, allowing for the better understanding of their residue modifications. The range of the minimum inhibitory concentration was between 25 and 200 μg/mL. Zygosaccharomyces bailii was the most sensitive strain to the peptides, while Zygosaccharomyces rouxii was the most resistant. Membrane permeabilisation was found to be responsible for yeast inhibition at a level which was a two-fold increase of the MIC (400 μg/mL). The possibility of the production of reactive oxygen species was also assessed but was not recognised as a factor involved for the peptides' mode of action. Their stability in different environments was also tested, focusing on high salt, pH and thermal stability. The newly designed peptides showed good antifungal activity against some common food spoilage yeasts and has been proven effective in the application in Fanta Orange. These efficient novel peptides represent a new source of food preservation that can be used as an alternative for current controversial preservatives used in the food industry.

Soy sauce is an increasingly popular oriental fermented condiment produced through a two-step fermentation process called koji (solid-state fermentation) and moromi (brine fermentation). Complex microbial interactions play an essential role in its flavor development during the fermentation. Tetragenococcus halophilus and Zygosaccharomyces rouxii are predominant among the microbes involved in the moromi stage. Despite their importance for producing a wide range of volatile compounds, antagonism can occur due to different growth condition requirements. Furthermore, microbial interactions in moromi fermentation are affected by current efforts to reduce salt in soy sauce, in order to tackle slow fermentation due to low metabolic activity of microbes and increased health risk related to high sodium intake. Attempts to enhance and accelerate flavor formation in the presence of high salt concentration include the inoculation with mixed starter cultures, genetic modification, cell, and enzyme immobilization. Although salt reduction can accelerate the microbial growth, the flavor quality of soy sauce is compromised. Several approaches have been applied to compensate such loss in quality, including the use of salt substitutes, combination of indigenous cultures, pretreatment of raw material and starter cultures encapsulation. This review discusses the role of microorganisms in soy sauce production in relation to flavor formation and changes in production practices.

Probabilistic microbial modelling using logistic regression was used to predict the growth/no growth (G/NG) interfaces of Zygosaccharomyces bailii in simulated acid sauces as a function of natamycin, xanthan gum (XG) and sodium chloride concentrations. The growth was assessed colorimetrically by using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride and 2-methoxy-1,4-naphthoquinone as detection reagents. The logistic regression model successfully predicted G/NG probability. The detection reagents used allowed the evaluation of G/NG interfaces in opaque systems with an excellent agreement with the plate count method. Natamycin concentration of 12 mg/L was needed to inhibit Z. bailii growth independently of the presence of XG and/or NaCl. Addition of 3.00 and 6.00% of NaCl exerted an antagonistic effect on natamycin action. Furthermore, addition of 0.25 and 0.50% XG decreased natamycin and/or NaCl action. However, an increased in XG concentration to 1.00% decreased yeast growth. Mentioned results highlighted the importance of the correct selection of stress factors applied to inhibit Z. bailii growth. Fil: Zalazar, Aldana Lourdes. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Gliemmo, María Fernanda. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Químicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Químicos; Argentina Fil: Soria, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales. Universidad de Buenos Aires. Facultad de Agronomía. Instituto de Investigaciones en Biociencias Agrícolas y Ambientales; Argentina Fil: Campos, Carmen Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Industrias. Instituto de Tecnología de Alimentos y Procesos Químicos. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Tecnología de Alimentos y Procesos Químicos; Argentina

Saccharomyces cerevisiae is the most commonly used yeast in wine, beer, and bread fermentations. However, Torulaspora delbrueckii has attracted interest in recent years due to its properties, ranging from its ability to produce flavor- and aroma-enhanced wine to its ability to survive longer in frozen dough. In this work, publicly available genomes of T. delbrueckii were explored and their annotation was improved. A total of 32 proteins were additionally annotated for the first time in the type strain CBS1146, in comparison with the previous annotation available. In addition, the annotation of the remaining three T. delbrueckii strains was performed for the first time. eggNOG-mapper was used to perform the functional annotation of the deduced T. delbrueckii coding genes, offering insights into its biological significance, and revealing 24 clusters of orthologous groups (COGs), which were gathered in three main functional categories: information storage and processing (28% of the proteins), cellular processing and signaling (27%), and metabolism (23%). Small intraspecies variability was found when considering the functional annotation of the four available T. delbrueckii genomes. A comparative study was also conducted between the T. delbrueckii genome and those from 386 fungal species, revealing a high number of homologous genes with species from the Zygotorulaspora and Zygosaccharomyces genera, but also with Lachancea and S. cerevisiae. Lastly, the phylogenetic placement of T. delbrueckii was clarified using the core homologs that were found across 204 common protein sequences of 386 fungal species and strains., This work was supported by the UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569) and UID/BIA/04050/2019 strategic programs and by the project PTDC/BIA-MIC/32059/2017 (TODOMICS: Omics approaches towards the industrial exploitation of Torulaspora delbrueckii: Elucidation of the molecular basis underlying complex cellular traits), which is funded by national funds through the FCT-Fundação para a Ciência e Tecnologia and by the ERDF- European Regional Development Fund through the COMPETE2020–Programa Operacional Competitividade e Internacionalização (POCI) and Sistema de Apoio à Investigação Científica e Tecnológica (SAICT).

Kombucha fermentation is initiated by transferring a solid-phase cellulosic pellicle into sweetened tea and allowing the microbes that it contains to initiate the fermentation. This pellicle, commonly referred to as a symbiotic culture of bacteria and yeast (SCOBY), floats to the surface of the fermenting tea and represents an interphase environment, where embedded microbes gain access to oxygen as well as nutrients in the tea. To date, various yeast and bacteria have been reported to exist within the SCOBY, with little consensus as to which species are essential and which are incidental to Kombucha production. In this study, we used high-throughput sequencing approaches to evaluate spatial homogeneity within a single commercial SCOBY and taxonomic diversity across a large number (n = 103) of SCOBY used by Kombucha brewers, predominantly in North America. Our results show that the most prevalent and abundant SCOBY taxa were the yeast genus Brettanomyces and the bacterial genus Komagataeibacter, through careful sampling of upper and lower SCOBY layers. This sampling procedure is critical to avoid over-representation of lactic acid bacteria. K-means clustering was used on metabarcoding data of all 103 SCOBY, delineating four SCOBY archetypes based upon differences in their microbial community structures. Fungal genera Zygosaccharomyces, Lachancea and Starmerella were identified as the major compensatory taxa for SCOBY with lower relative abundance of Brettanomyces. Interestingly, while Lactobacillacae was the major compensatory taxa where Komagataeibacter abundance was lower, phylogenic heat-tree analysis infers a possible antagonistic relationship between Starmerella and the acetic acid bacterium. Our results provide the basis for further investigation of how SCOBY archetype affects Kombucha fermentation, and fundamental studies of microbial community assembly in an interphase environment.

Cheese is one of the traditional fermented dairy products in Xinjiang, China. Due to its geographical location and regional feature this type of cheese harbors certain regional characteristics. To investigate these, here Illumina MiSeq high-throughput sequencing technology was used to target the v4-v5 interval to analyze the composition of fungal flora in Xinjiang traditional cheese. Our results showed the fungal flora of this cheese is mainly composed of Pichia (65.20%), Kazachstania (9.05%), Galactomyces (7.21%), Zygosaccharomyces (6.56%), Torulaspora (3.13%), Dipodascus (2.11%) and Ogataea (1.64%) belonging to the Ascomycota. PcoA (Principal Co-ordinates Analysis) and an UPGMA (unweighted pair-group method with arithmetic means) based on the OTUs (Optical Transform Unit) horizontal-weighted UniFrac distances, revealed some differences in fungal community structure among 17 cheese samples. At the OTU level, nine dominant OTUs were found in all the samples, for which Pichia was the most important fungal group. Building on this, the moisture content (23.20–59.22%), water distribution, and salt content (1.13–4.84%) in cheese were also determined. We found that six of the seven dominant fungal genera had specific correlations with the above physical and chemical variables, with only Ogataea uncorrelated with any variables. The results provide a theoretical basis for the development and use of cheese microbial resources in Xinjiang, China. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12088-021-00967-x.

In this study, the contributions of three strains and different coculture patterns to microbial community diversity and metabolites in the high-salt liquid-state fermentation (HLF) soy sauce moromi were investigated. A comparison of two strains of Zygosaccharomyces rouxii showed that strain QH-25 had a stronger ability to contribute metabolites, including both nonvolatile and volatile types, to the moromi than strain QH-1, except for volatile acids and ketones. Of the various fortification patterns tested, the content of metabolites was significantly increased by inoculating Z. rouxii QH-25 prior to C. versatilis, especially the content of volatiles, including ketones, esters, phenols, and alcohols, which increased 1.61-fold compared with those in the control sample; the contents of these components were increased 3.07-, 1.91-, 1.36-, and 1.22-fold, respectively. In particular, characteristic components such as ethyl octanoate, 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone (HEMF), 4-ethyl-2-methoxy-phenol (4-EG), and 3-methyl-1-butanol were increased 3.99-, 3.29-, 1.63-, and 0.70-fold, respectively. Redundancy analysis (RDA) showed that Staphylococcus, Zygosaccharomyces, and Candida were positively correlated with the unique components described above. In addition, the nodes of the interaction network between Zygosaccharomyces and Candida were increased, and the positive correlation of Zygosaccharomyces with Staphylococcus was enhanced by inoculating Z. rouxii prior to C. versatilis. These results suggested that the unique flavor of soy sauce was closely related to the metabolic characteristics of strains affiliated with Z. rouxii, whether cultured singly or cocultured with C. versatilis. This study also provided a reference method for determining the differences in community structure and metabolites between traditional techniques and modern processes for soy sauce fermentation.

Non-conventional yeasts refer to a huge and still poorly explored group of species alternative to the well-known model organism Saccharomyces cerevisiae. Among them, Zygosaccharomyces rouxii and the sister species Zygosaccharomyces bailii are infamous for spoiling food and beverages even in presence of several food preservatives. On the other hand, their capability to cope with a wide range of process conditions makes these yeasts very attractive factories (the so-called "ZygoFactories") for bio-converting substrates poorly permissive for the growth of other species. In balsamic vinegar Z. rouxii is the main yeast responsible for converting highly concentrated sugars into ethanol, with a preference for fructose over glucose (a trait called fructophily). Z. rouxii has also attracted much attention for the ability to release important flavor compounds, such as fusel alcohols and the derivatives of 4-hydroxyfuranone, which markedly contribute to fragrant and smoky aroma in soy sauce. While Z. rouxii was successfully proposed in brewing for producing low ethanol beer, Z. bailii is promising for lactic acid and bioethanol production. Recently, several research efforts exploited omics tools to pinpoint the genetic bases of distinctive traits in "ZygoFactories", like fructophily, tolerance to high concentrations of sugars, lactic acid and salt. Here, I provided an overview of Zygosaccharomyces industrially relevant phenotypes and summarized the most recent findings in disclosing their genetic bases. I suggest that the increasing number of genomes available for Z. rouxii and other Zygosaccharomyces relatives, combined with recently developed genetic engineering toolkits, will boost the applications of these yeasts in biotechnology and applied microbiology.

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows β-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25–2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.

Gochujang is a Korean fermented hot pepper paste beneficial to human health by providing various nutrients. In this study, its physicochemical characteristics were identified, and its microbial communities were analyzed by high-throughput sequencing. The interrelationship between physicochemical characteristics and microbial composition was investigated to reveal the properties of gochujang before and after fermentation. After fermentation, all samples showed decreased salt concentration, pH, and reducing sugar content, while the acidity and amino-type nitrogen increased. The water content, salt concentration, amino-type nitrogen, and reducing sugar differed according to the batches of samples. Bacillus, Aerosakkonema, and Enterococcus were identified as the predominant bacterial genera. Furthermore, Aerosakkonema was the most abundant genus before fermentation; however, it was replaced by Bacillus as it decreased after fermentation. For the fungi, Aspergillus dominated before fermentation, whereas Zygosaccharomyces and Millerozyma dominated after fermentation. The high level of amino-type nitrogen in gochujang was related to the relative abundance of B. haynesii/B. licheniformis before fermentation. Additionally, the high abundance of Z. rouxii after fermentation was related to the flavor of gochujang. This comprehensive analysis of the microbial community associated with the physicochemical properties of gochujang could help in understanding the factors affecting the quality of the product.

In the food industry, food spoilage is a real issue that can lead to a significant amount of waste. Although current preservation techniques are being applied to reduce the occurrence of spoilage microorganisms, the problem persists. Food spoilage yeast are part of this dilemma, with common spoilers such as Zygosaccharomyces, Kluyveromyces, Debaryomyces and Saccharomyces frequently encountered. Antimicrobial peptides derived from plants have risen in popularity due to their ability to reduce spoilage. This study examines the potential application of a synthetic defensin peptide derived from barley endosperm. Its inhibitory effect against common spoilage yeasts, its mechanisms of action (membrane permeabilisation and overproduction of reactive oxygen species), and its stability in different conditions were characterised. The safety of the peptide was evaluated through a haemolysis and cytotoxicity assay, and no adverse effects were found. Both assays were performed to understand the effect of the peptide if it were to be consumed. Its ability to be degraded by a digestive enzyme was also examined for its safety. Finally, the peptide was successfully applied to different beverages and maintained the same inhibitory effects in apple juice as was observed in the antiyeast assays, providing further support for its application in food preservation.

Bactrocera tryoni (Froggatt), the Queensland fruit fly (Qfly), is a highly polyphagous tephritid fly that is widespread in Eastern Australia. Qfly physiology is closely linked with its fungal associates, with particular relationship between Qfly nutrition and yeast or yeast-like fungi. Despite animal-associated fungi typically occurring in multi-species communities, Qfly studies have predominately involved the culture and characterisation of single fungal isolates. Further, only two studies have investigated the fungal communities associated with Qfly, and both have used culture-dependant techniques that overlook non-culturable fungi and hence under-represent, and provide a biased interpretation of, the overall fungal community. In order to explore a potentially hidden fungal diversity and complexity within the Qfly mycobiome, we used culture-independent, high-throughput Illumina sequencing techniques to comprehensively, and holistically characterized the fungal community of Qfly larvae and overcome the culture bias. We collected larvae from a range of fruit hosts along the east coast of Australia, and all had a mycobiome dominated by ascomycetes. The most abundant fungal taxa belonged to the genera Pichia (43%), Candida (20%), Hanseniaspora (10%), Zygosaccharomyces (11%) and Penicillium (7%). We also characterized the fungal communities of fruit hosts, and found a strong degree of overlap between larvae and fruit host communities, suggesting that these communities are intimately inter-connected. Our data suggests that larval fungal communities are acquired from surrounding fruit flesh. It is likely that the physiological benefits of Qfly exposure to fungal communities is primarily due to consumption of these fungi, not through syntrophy/symbiosis between fungi and insect ‘host’.

Kpkt is a yeast killer toxin, naturally produced by Tetrapisispora phaffii, with possible applications in winemaking due to its antimicrobial activity on wine-related yeasts including Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. Here, Kpkt coding gene was expressed in Komagataella phaffii (formerly Pichia pastoris) and the bioreactor production of the recombinant toxin (rKpkt) was obtained. Moreover, to produce a ready-to-use preparation of rKpkt, the cell-free supernatant of the K. phaffii recombinant killer clone was 80-fold concentrated and lyophilized. The resulting preparation could be easily solubilized in sterile distilled water and maintained its killer activity for up to six months at 4 °C. When applied to grape must, it exerted an extensive killer activity on wild wine-related yeasts while proving compatible with the fermentative activity of actively growing Saccharomyces cerevisiae starter strains. Moreover, it displayed a strong microbicidal effect on a variety of bacterial species including lactic acid bacteria and food-borne pathogens. On the contrary it showed no lethal effect on filamentous fungi and on Ceratitis capitata and Musca domestica, two insect species that may serve as non-mammalian model for biomedical research. Based on these results, bioreactor production and lyophilization represent an interesting option for the exploitation of this killer toxin that, due to its spectrum of action, may find application in the control of microbial contaminations in the wine and food industries.

Humans have used high salinity for the production of bean-based fermented foods over thousands of years. Although high salinity can inhibit the growth of harmful microbes and select functional microbiota in an open environment, it also affects fermentation efficiency of bean-based fermented foods and has a negative impact on people’s health. Therefore, it is imperative to develop novel defined starter cultures for reduced-salt fermentation in a sterile environment. Here, we explored the microbial assembly and function in the fermentation of traditional Chinese broad bean paste with 12% salinity. The results revealed that the salinity and microbial interactions together drove the dynamic of community and pointed out that five dominant genera (Staphylococcus, Bacillus, Weissella, Aspergillus, and Zygosaccharomyces) may play different key roles in different fermentation stages. Then, core species were isolated from broad bean paste, and their salinity tolerance, interactions, and metabolic characteristics were evaluated. The results provided an opportunity to validate in situ predictions through in vitro dissection of microbial assembly and function. Last, we reconstructed the synthetic microbial community with five strains (Aspergillus oryzae, Bacillus subtilis, Staphylococcus gallinarum, Weissella confusa, and Zygosaccharomyces rouxii) under different salinities and realized efficient fermentation of broad bean paste for 6 weeks in a sterile environment with 6% salinity. In general, this work provided a bottom-up approach for the development of a simplified microbial community model with desired functions to improve the fermentation efficiency of bean-based fermented foods by deconstructing and reconstructing the microbial structure and function. IMPORTANCE Humans have mastered high-salinity fermentation techniques for bean-based fermented product preparation over thousands of years. High salinity was used to select the functional microbiota and conducted food fermentation production with unique flavor. Although a high-salinity environment is beneficial for suppressing harmful microbes in the open fermentation environment, the fermentation efficiency of functional microbes is partially inhibited. Therefore, application of defined starter cultures for reduced-salt fermentation in a sterile environment is an alternative approach to improve the fermentation efficiency of bean-based fermented foods and guide the transformation of traditional industry. However, the assembly and function of self-organized microbiota in an open fermentation environment are still unclear. This study provides a comprehensive understanding of microbial function and the mechanism of community succession in a high-salinity environment during the fermentation of broad bean paste so as to reconstruct the microbial community and realize efficient fermentation of broad bean paste in a sterile environment.

Though in-package water bath heat pasteurization for hard cider production is commonly employed to improve product safety and stability, there is a considerable lack of research-based guidelines to inform industry practices. In this study, fermented cider was bottled and inoculated with high populations of Saccharomyces cerevisiae and Zygosaccharomyces rouxii yeast. The bottles were then subjected to water bath pasteurization 60 °C at varying lengths of time. For both yeast species, populations were reduced to undetectable levels after just 1 min of processing time. Though validation of each individual process is recommended, cider producers may be able to sufficiently reduce the risks of spoilage organisms with minimal water bath pasteurization, especially when combined with other methods to reduce the presence of spoilage organisms.

Sonication was applied to accelerate aroma formation and shorten fermentation time of soy sauce. Effects of sonication at 68 kHz on the aroma and aroma-producing Zygosaccharomyces rouxii and Tetragenococcus halophilus in moromis were investigated using sensory evaluation, SPME–GC–olfactometry/MS, viable cell counting and scanning electron microscopy. The sensory scores of caramel-like, fruity, alcoholic, floral, malty, smoky, sour and overall aroma in sonicated moromis were enhanced by 23.4%, 23.2%, 13.6%, 12.8%, 7.6%, 6.3%, 5.6% and 14.4%, respectively. Sensory scores of samples fermented for 90–180 days were higher than those of controls fermented for 180 days, suggesting that sonication could reduce fermentation time by 90 days. Thirty-four aroma-active compounds were detected from 85 volatile compounds in soy sauces. Sonication accelerated and elevated the formation of aroma compounds by chemical reactions. It also markedly increased the reproduction and cell permeability of both microorganisms in moromis, which favored the formation of aroma compounds by both strains.

Background Suansun is a traditional salt-free fermented bamboo shoot product that has been widely consumed as a cooking ingredient in south China for centuries. The aim of this study was to evaluate and compare the microbial and metabolic diversity in samples of two kinds of suansun, namely Guangdong suansun (GD) and Yunnan suansun (YN), using high-throughput sequencing (HTS) and headspace solid-phase microextraction-gas chromatograph-mass spectrometry (HS-SPME/GC-MS), respectively, and then to assess the influence of environmental factors on the microbial communities. Results The results showed that Lactobacillus and Serratia were the most abundant bacterial genera in both the GD and YN groups. For the fungi, Pichia, Candida, and Debaryomyces were the major genera in the GD group, whereas Pichia and Zygosaccharomyces were the dominant genera in the YN group. The canonical correlation analysis (CCA) results demonstrated that three environmental factors - temperature, longitude, and altitude - play a more important role in affecting the microbial community composition of suansun than physical and chemical factors. The fugal community composition was more influenced by environmental factors than the bacterial community. The volatile profile of the GD group differed from that of the YN group, and the difference was mainly reflected in the relative alcohol, aldehyde, ester, and aromatic compound content. Conclusions This study provided insights into the microbial and metabolic profiles of suansun products. The findings might be useful for the improvement and standardization of suansun production. © 2020 Society of Chemical Industry.

With the increasing popularity of soy sauce containing high amounts of amino acid nitrogen (AAN) in China and the positive correlation of biogenic amines (BAs) with AAN, reducing BAs formation during soy sauce fermentation has become an urgent task. In this work, the effect of salt-tolerant yeasts and Staphylococcus piscifermentans on BAs formation was extensively studied. Combined inoculation for different osmotolerant yeast strains indicated that Z. rouxii CICC 1417 and T. candida CICC 1019 co-inoculation produced the lowest amount of BAs (89.43 mg/L). Furthermore, co-inoculation of soy sauce mash with S. piscifermentans and salt-tolerant yeasts reduced the BAs production by 31.51% under laboratory-scale experiments. Only 133.40 g/L BAs were produced after S. piscifermentans inoculation in 50-T fermentator, and this value was 63.25% lower than that of the control. Most of the alcohols, aldehydes, phenols, and esters were increased, whereas bacteria, such as Pediococcus, Weissella, and Streptococcus, were inhibited and thus reduced BAs formation. The increased abundance in Zygosaccharomyces and Pichia may contribute to the improvement in volatile compound production. These findings reveal the high potential of the co-inoculation of S. piscifermentans and salt-tolerant yeasts for soy sauce production with limited BAs formation.

In this research, anti-yeast activity of natamycin in apple juice inoculated with both Zygosaccharomyces rouxii and Z. bailii during the storage at different temperatures was investigated. For this purpose, a response surface methodology approach was used to test and optimize effects of some processing variables; storage time (1, 21 and 41 days), storage temperature (4, 12 and 20 degrees C), sodium benzoate as a positive control (0, 0.05 and 0.1%) and natamycin concentration (0, 30 and 60 mg/L) on several physicochemical and bioactive properties of the apple juice samples. The results showed that the natamycin performed a remarkable anti-yeast effect on Z. bailii rather than on Z. rouxii. The brix levels of the samples decreased and so the turbidity values increased significantly due to the yeast activity during the storage. Bioactive properties were also significantly affected by the natamycin which was also revealed to increase the antioxidant capacity of apple juice during storage. Using multiple response optimization technique, it was calculated that minimum yeast count (YC) values would occur at storage time = 38.64 and 40.9 days, storage temperature = 19.81 and 14.4 degrees C, sodium benzoate level (fixed to 0%) and natamycin concentration = 40 and 51.9 mg/L for the samples inoculated with Z. bailii and Z. rouxii, respectively. It was concluded that the bioactive properties of apple juice could be preserved by addition of natamycin which is suggested to be a natural inhibitor during the storage.

Zygosaccharomyces rouxii produces high levels of 4-hydroxy-2,5-dimethyl-3(2H)-furanone in YPD medium supplemented with 120 g/L D-fructose and 180 g/L NaCl after 5 d. D-fructose has a stress effect on Z. rouxii, and GSH-Px is a main enzyme involved in the defense of Z. rouxii against oxygen stress according to our previous report. In order to further explore the molecular mechanism of the glutathione metabolism pathway in Z. rouxii in response to D-fructose stress, changes in the expression of genes and proteins involved in the synthesis of glutathione precursor amino acids and enzymes were observed. In addition, changes in the intermediates related to glutathione synthesis in Z. rouxii were reported. The results indicated that some gene-encoding enzymes involved in the glutamate, cysteine and glycine biosynthesis pathways and key genes involved in glutathione synthesis were upregulated. The expression levels of other genes, except SHMT, were consistent with the qRT-PCR results. The contents of γ-glutamylcysteine and glutathione amide in the D-fructose group were higher than those in the control group. In the D-fructose stress groups, the metabolic flux towards glutathione synthesis was increased. These results might provide more in-depth and detailed theoretical support for the oxidative stress defense mechanism of Z. rouxii under D-fructose stress.

Pixian broad bean paste (PBP) is a traditional Chinese condiment, famous for its distinctive flavor. Microbial communities play a vital role in producing the unique flavor of PBP, and a significant accumulation of these volatile flavors occurs during the post-fermentation stage of its production. However, little is known about the relationship between the microbes and flavor compounds in PBP. In this study, high-throughput sequencing (HTS) analysis revealed that Leuconostoc (8.30%), Lactobacillus (7.05%), Weissella (5.80%) and Staphylococcus (4.03%) were the dominant bacterial genera, while the most prevalent yeast genera were Zygosaccharomyces (41.45%) and Pichia (5.83%). Gradual accumulations of free amino acids (glutamic acid and asparagine), organic acids (malic acid and tartaric acid), and unique volatiles (aldehydes, phenols and pyrazines) were evident throughout the post-fermentation process. Analysis of the Pearson’s correlation coefficients between 66 key microbes and the key flavors was investigated. Nine core microbes were identified based on the linear discriminant analysis (LDA) scores ≥ 4 (or an average abundance >0.1%) and a high correlation with at least two flavor categories (P 0.8), namely Kosakonia, Kazachstania, Debaryomyces, Lactobacillus, Myroides, Stenotrophomonas, Ochrobactrum, Wohlfahrtiimonas, and Lactococcus genera. These results provide a clearer insight into microbial succession during PBP post-fermentation, thereby contributing to further quality improvement of PBP.

The spontaneous fermentation was carried out on Carica papaya leaf (CPL) in view of its potential improvement on antioxidant functionality and cultivation of lactic acid bacteria. The effect of the spontaneous fermentation on the total phenolic content and antioxidant activity of CPL, as well as biodiversity profiling were evaluated in this study. Total phenolic content and antioxidant capacity of the fermented CPL were 31.14 mg GAE g -1 and 405.8 mM TE g -1 respectively, higher than the unfermented CPL (5.71 mg GAE/g and 130.5 mM TE g -1 ) respectively. Microbial community was predominantly lactic acid bacteria (LAB) and yeasts, both populated at 10 4 to 10 8 CFU/mL during most part of the fermentation. Presumptive Enterobacteriaceae showed up briefly at the onset of the fermentation before disappearing. PCR-DGGE fingerprinting revealed Lactobacillus plantarum ( Lb. plantarum ) as the sole dominant bacterial species. More diverse yeasts community was detected by PCR-DGGE where succession of Zygosaccharomyces , Saccharomyces , Candida and Aspergillus genera were detected along fermentation time. Spontaneous fermentation successfully enhanced the total phenolic content and antioxidant capacity of the CPL. The cultivation of lactic acid bacteria was indicated by the presence of Lb. plantarum , whereas the disappearance of Enterobacteriaceae conferred a safe consumption of the fermented CPL.

This study investigated the capability of near-infrared spectroscopy (NIRS) to predict the concentration of Zygosaccharomyces rouxii in apple and kiwi fruit juices. The yeast was inoculated in fresh kiwi fruit juice ( n = 68), reconstituted kiwi juice ( n = 85), and reconstituted apple juice ( n = 64), followed by NIR spectra collection and plate counting. A principal component analysis indicated direct orthogonal signal correction preprocessing was suitable to separate spectral samples. Parameter optimization algorithms increased the performance of support vector machine regression models developed in a single variety juice system and a multiple variety juice system. Single variety juice models achieved accurate prediction of Z. rouxii concentrations, with the limit of quantification at 3 to 15 CFU/mL ( R2 = 0.997 to 0.999), and the method was also feasible for Hanseniaspora uvarum and Candida tropicalis. The best multiple variety juice model obtained had a limit of quantification of 237 CFU/mL ( R2 = 0.961) for Z. rouxii. A Bland-Altman analysis indicated good agreement between the support vector machine regression model and the plate counting method. It suggests that NIRS can be a high-throughput method for prediction of Z. rouxii counts in kiwi fruit and apple juices.

Doenjang(Korean fermented soybean paste) with an improved flavor and safety was prepared by the simultaneous fermentation of autochthonous mixed starters at the pilot plan scale. First, whole soybean mejuwas fermented by coculturing safety‐verified starters Aspergillus oryzaeMJS14 and Bacillus amyloliquefacienszip6 or Bacillus subtilisD119C. These fermented whole soybean mejuwere aged in a brine solution after the additional inoculation of Tetragenococcus halophilus7BDE22 and Zygosaccharomyces rouxiiSMY045 to yield doenjang. Four doenjangbatches prepared using a combination of mold, bacilli, lactic acid bacteria, and yeast starters were free of safety issues and had the general properties of traditional doenjang, a rich flavor and taste. All doenjangbatches received a high consumer acceptability score, especially the ABsT and ABsTZ batches. This study suggests that flavor‐rich doenjangsimilar to traditional doenjangcan be manufactured safely and reproducibly in industry by mimicking the simultaneous fermentation of autochthonous mixed starters as in traditional doenjangfermentation. The development of a pilot plant process for doenjangfermentation using safety‐verified autochthonous mixed starter will facilitate the manufacture of flavor‐rich doenjangsimilar to traditional doenjangsafely and reproducibly in industry.

The use of Tetragenococcus halophilus and Zygosaccharomyces rouxii as starter cultures is essential for desirable volatiles production during moromi stage of soy sauce fermentation. In this study, the effect of simultaneous and sequential inoculation of cultures in moromi fermentation models, with respect to viability, physicochemical changes, and volatiles formation (using SPME-GC/MS) was investigated. Interestingly, an antagonism was observed as T. halophilus only proliferated (3 log increase) in the presence of Z. rouxii, while Z. rouxii growth was suppressed by 4 log in concurrence with pH increase to 7.31. Final content of reducing sugars, ethanol, acetic acid, and amino nitrogen did not differ significantly (p < 0.05) between co-inoculation and sequential inoculation. However, Z. rouxii promoted alcohols formation and produced a more complex aroma profile under suppression. According to Principal Component Analysis (PCA), the inoculation sequence (co-inoculation and sequential) has impacts on volatile compound profiles during moromi fermentation.

We report the results of research on the effect of food fibers on the fermentation process of whey – the base of beverages. By using the method of IR spectroscopy, we studied various forms of moisture bonds of apple pectin in fiber and orange fruit fibers Citri-Fi with water or whey. The results obtained show the existence of strong hydrogen bonds and high concentration of the movable proton. High sorption capacity for water is observed. This effect is important in terms of regulating the viscosity of wort. By employing mathematical modeling, we established conditions for the preparation and introduction of Citri-Fi to whey for obtaining beverages with enhanced viscosity. The visualization is provided of the transformations of dry Citri-Fi when swelling in whey – an increase in the volume of tubular fibers. For the basic indicators of fermentation (the content of carbon dioxide, the amount of formed ethyl alcohol and yeast cells, the content of reducing sugars) we defined rational conditions for the fermentation of whey-plant wort with enhanced viscosity. During fermentation using the race Zygosaccharomyces lactis 868-K, the most intensive growth of yeast cells at the level from 42.3 to 71.3 mln/cm 3 was observed over the interval of 6…24 hours of fermentation. The dynamics of accumulation of carbon dioxide in wort is correlated to the indicators of growth in yeast cells. The data obtained indicate a certain oppression of yeast development in whey-plant wort. This is due to the presence of colloidal substances of whey and the residue of insoluble components of food fibers. We established a fermentation temperature of whey-plant wort with enhanced viscosity − 30…32 °C, at which there is a sufficient accumulation of ethyl alcohol at the level of 0.64...0.69 % by volume. Further increase or decrease in temperature results in a decrease in the amount of alcohol, indicating a decrease in the activity of cell enzymes. We have proven the possibility to apply the results obtained for developing a technology for manufacturing whey-based fermented beverages with enhanced viscosity

The design of novel efficient antimicrobial peptides (AMPs) faces several issues, such as cost of synthesis, proteolytic stability or cytotoxicity. The identification of key determinants involved in the activity of AMPs, such as cationicity and amphipathicity, allowed the synthesis of short peptides with optimized properties. An ultrashort peptide made of the sequence H-Orn-Orn-Trp-Trp-NH2 (O3TR) showed antifungal activity against several contaminants from food products. This peptide inhibited the growth of the filamentous fungi Fusarium culmorum, Penicillium expansum and Aspergillus niger within a range of concentration of 12.5–50 μg/ml. In addition, O3TR inhibited the growth of the yeast Saccharomyces cerevisiae, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Debaryomyces hansenii and Kluyveromyces lactis within the range 12.5–50 μg/ml. A derivative peptide, called C12O3TR, made by the addition of lauric acid at the N-terminus of O3TR was 2- to 8-fold more active than O3TR against every species. In addition to the inhibition of conidial germination, O3TR and C12O3TR killed F. culmorum hyphae at 100 and 50 μg/ml respectively. The MIC of the two peptides against F. culmorum and Z. bailii after heat treatment at 100°C for 60 min and within the pH range 3–10, were not changed. However, the activity of O3TR against F.culmorum and Z. bailii was strongly reduced in salt solutions, whereas the lauric acid peptide kept its antifungal activity and resistance to proteolytic digestion. The conjugation with lauric acid reduced the random coiled structure and increased the α-helical content of O3TR. After conjugation with the dye tetramethylrhodamine (TMR), both peptides entered F. culmorum spores. They also both induced permeabilization of F. culmorum hyphae but only C12O3TR permeabilized Z. bailii membrane. In contrast to the lipopeptide, O3TR did not show haemolytic or cytotoxic activity when applied at the concentrations that exhibited antifungal potency. The two peptides were challenged against a yeast cocktail of S. cerevisiae and Z. bailii, and A. niger in different commercial beverages. After 7 days, O3TR was able to inhibit the yeast cocktail in a commercial lager and carbonated drink. Due to its antifungal potency, high stability and low cytotoxicity, the tetrapeptide could represent a promising starting point of a novel food preservative.

Despite the fungal abundance in honey and bee bread, little is known about the fungal gut community of the honey bee and its effect on host fitness. Using pyrosequencing of the 16S rRNA gene and ITS2 region amplicons, we analysed the bacterial and fungal gut communities of the honey bee as affected by the host social status. Both communities were significantly affected by the host social status. The bacterial gut community was similar to those characterised in previous studies. The fungal gut communities of most worker bees were highly dominated by Saccharomyces but foraging bees and queens were colonised by diverse fungal species and Zygosaccharomyces, respectively. The high fungal density and positive correlation between Saccharomyces species and Lactobacillus species, known yeast antagonists, were only observed in the nurse bee; this suggested that the conflict between Saccharomyces and Lactobacillus was compromised by the metabolism of the host and/or other gut microbes. PICRUSt analysis revealed significant differences in enriched gene clusters of the bacterial gut communities of the nurse and foraging bees, suggesting that different host social status might induce changes in the gut microbiota, and, that consequently, gut microbial community shifts to adapt to the gut environment.

Although the microbial diversity and structure in bean-based fermented foods have been widely studied, systematic studies on functional microbiota and mechanism of community forms in multi-microbial fermentation systems were still lacking. In this work, the metabolic pathway and functional potential of microbial community in broad bean paste (BBP) were investigated by metagenomics approach, and Staphylococcus, Bacillus, Weissella, Aspergillus and Zygosaccharomyces were found to be the potential predominant populations responsible for substrate alteration and flavor biosynthesis. Among them, Staphylococcus was the most abundant and widespread functional microbe, and closely related Staphylococcus species were diverse and ubiquitously distributed, with the opportunistic pathogen S. gallinarum being the most abundant Staphylococcus specie isolated from BBP. To explain the dominance status of S. gallinarum and species distributions of Staphylococcus genus, we tested the effects of abiotic and biotic factors on three Staphylococcus species using a tractable BBP model, demonstrating that adaptation to environmental conditions (environmental parameters and other functional microbes) led to the dominant position and species coexistence of Staphylococcus, and congeneric competition among Staphylococcus species further shaped ecological distributions of closely related Staphylococcus species. In general, this work revealed the metabolic potential of microbial community and distribution mechanism of Staphylococcus species during BBP fermentation, which could help traditional factories to more precisely control the safety and quality of bean-based fermented foods.

‘Bask in daytime and dewed at night’ technique is traditionally used in doubanjiang fermentation, however, the underlying mechanism behind this technique is still unrevealed. This study aimed to elaborate the roles of three key indexes in this technique, that are air, sunlight, and mash turnover, on doubanjiang quality through the design of doubanjiang fermentation. The results showed that the exposure to air or sunlight was beneficial for doubanjiang to yield reddish-brown luster color, less acidity and balanced flavor. On the contrary, blocking of air and sunlight could accumulate higher acidity (4.75 ± 0.01 g/100gdw), amino nitrogen (2.08 ± 0.07 g/100gdw), alcohols (36.9 g/kg) and esters (26.9 g/kg). Regular mash turnover was mainly involved in making different mash layers exposure to air or sunlight equally through homogenizing mash layers. Different conditions also have been suggested to affect the microbiota dynamics by amplicon sequencing and RT-qPCR. Based on RDA analysis, Pediococcus, Meyerozyma, Bacillus and Zygosaccharomyces were predicted to be related to the changes of physicochemical properties or volatile compounds in different conditions, and their function was validated by simulated fermentation. This study would unravel the underlying scientific meaning of ‘bask in daytime and dewed at night’ technique in doubanjiang fermentation.

Pixian broad-bean paste (PXDB) is a traditional Chinese fermented condiment with a distinct flavor. The flavors stems from its strong relationship to local microorganisms. This study analyzed changes in key aroma compounds, organic acids, free amino acids (FAA), and the microbial community during different fermentation stages in order to study the correlation between flavors and microbes. Results indicated that the presence of key aroma compounds and organic acids initially increased, then decreased. Changes in total FAA content were divided into two stages due to the addition of chili pepper, however, both stages displayed an increase in FAA levels as fermentation time increased. High-throughput sequencing was used to study the microbial community and succession. Lactobacillus, Prevotella 9, Ruminococcaceae ucg-014, Lysinibacillus, Enterococcus, Weissella, Escherichia-Shigella, Sphingomonas, Aspergillus, Zygosaccharomyces, Candida, and Pichia presented as the primary microorganism during fermentation process. Simultaneously, it was found that bacteria genera indicated a stronger potential correlation with flavors, versus fungi genera. Escherichia-Shigella, Leuconostoc, Acetobacter, Chromohalobacter, Candida, Mortierella, Plectosphaerella, Podoscypha, and Podospora displayed a significant correlation with flavors (isoamyl alcohol, 2,5-dimethylpyrazine, phenethyl alcohol, 2,3,5,6-tetramethylpyrazine, citric acid, serine, asparagine, glutamate, etc.). This study provides an improved understanding on formation mechanisms of flavors, and guidance for screening PXDB fermentation strains.

Kombucha is a drink produced by spontaneous fermentation, and several studies have been conducted to unveil its microbiological and physicochemical aspects with numerous human health claims. The integration of these results is fundamental to understand and discuss the biological activities attributed to kombucha. In the present study, we isolated bacteria and yeasts involved in the fermentation of kombucha produced with green (GK) and black (BK) teas, as well as the amplicon metagenomic of the microbial communities (16S and ITS) during 0, 3, 5, 10, and 15 days of fermentation, at 28 °C. Microbial communities were linked to key biochemical parameters monitored during fermentation such as pH, total titratable acidity, total reducing sugars, polyphenols, acetic acid, and ethanol production. Moreover, ordination analysis (principal component analysis, PCA) revealed clear GK and BK separation groups during the fermentation process. Caffein, gallic acid, and chlorogenic acids majorly influenced the separation of GK and BK. Furthermore, the presence of Komagataeibacter spp. and catechins exerted selective pressure against microbial contamination. This study essentially contributes to the knowledge about the effects of integrated microbiota to the chemical results of the kombucha fermented in GK and BK teas.

Spoilage caused by microbial contamination in dried persimmon fruits processed by traditional frosting often leads to considerable economic losses. Artificial frosting has recently been considered as an alternative method to reduce the risk of spoilage, however, in this study, expansion of packaging and mildew formation were still observed. The dynamics of bacteria and fungi structure in different processes were determined. Results showed that Zygosaccharomyces and Aspergillus were the predominant genera in spoiled fruits in the packaging phase. During the artificial frosting, Wickerhamomyces (15.91%) and Candida (6.82%) were the predominant gas-generated yeasts and Aspergillus (20.45%) and Penicillium (15.91%) were consistently the dominant genera causing mildew. To reduce the contamination problem, pulsed light (24 J/cm2) were adopted on Day 0 of frosting and no fungi were detected on Day 3 post-frosting, which indicated to decrease significantly the amounts of Wickerhamomyces, Candida, Aspergillus, and Penicillium. Additionally, the results of the high-throughput sequencing suggested that the proportions of these predominant microorganisms were also decreased significantly during frosting. These results showed that pulsed light treatment in the artificial technique could almost overcome the problems of packaging expansion and mildew formation.

The physicochemical characteristics and yeasts diversity in honey samples from 17 species of stingless bees of the genera Nannotrigona, Melipona, Plebeia, Scraptotrigona, and Tetragonisca cultivated in Southern Brazil were determined. The sugar content, moisture, water activity, pH, reducing sugars/total sugar ratio, and total yeast population varied significantly among the honey from the different bee species. The highest yeast population was found in the Plebeia's honey samples and correlated with their high water-activity. Sixteen yeast species were identified based on the nuclear large subunit (26S) ribosomal RNA partial sequences. The genera Starmerella and Zygosaccharomyces were found predominant, with a high prevalence of Starmerella sp., S. etchellsii, and S. apicola. Some yeast species were only identified in honey samples from specific bee species indicating a close relationship between the yeasts and the insects. For the first time, Wickerhamomyces sydowiorum in honey is being reported. In general, the yeast species isolated from stingless bee honey samples demonstrated high osmotolerance and low sugar assimilation.

Zygosaccharomyces bisporus is a moderately halotolerant yeast isolated from highly sugary and salty foods. We performed various evident biochemical and in vivo experiments as first of its kind to sketch out the possible overlay of salt tolerance mechanism in this model organism. The growth and survival curve analysis revealed that 1.0 M NaCl concentration (sublethal) enacts growth inhibitory effects with prompting immediate delay in cell division cycle ; however, yeast cells adopted modified stress physiologically with further stretched stress spans which was accompanied by an upsurge in the level of cellular metabolites such as trehalose (reserve carbohydrate) and chiefly glycerol (polyols) as major compatible osmolytes, suggesting their role in defense mechanism against osmotic stress . To further elucidate the relation of osmotic stress cell physiology to salinity, thiobarbituric acid reactive substances, protein carbonyl, and reduced glutathione content were measured in salt-stressed cells demonstrating positive correlation of reactive oxygen species generation in Z. bisporus with an elevated concentration of lipid and protein oxidation, thereby damaging cell membrane and eventually causing cell death. We assessed NaCl exposure sourcing increased intracellular reactive oxygen species concentration, by an electron transfer-based colorimetric cupric-reducing antioxidant capacity assay justifying that cellular total antioxidant capacity which uses all the combined antioxidant activities present within vitamins, proteins, lipids, and glutathione reverses these deleterious stress effects. Henceforth, performance of Z. bisporus MTCC 4801 mounted because of stress regime seems to be multifactorial.