Draft genomes of four Kluyveromyces marxianus isolates retrieved from the elaboration process of henequen (Agave fourcroydes) mezcal.

We report the draft genomes of four Kluyveromyces marxianus isolates obtained from the elaboration process of henequen (Agave fourcroydes) mezcal, a Mexican alcoholic beverage. The average nucleotide identity analysis revealed that isolates derived from agave plants are distinct from those from other environments, including agave fermentations.

Genotypic and Phenotypic Diversity of Kluyveromyces marxianus Isolates Obtained from the Elaboration Process of Two Traditional Mexican Alcoholic Beverages Derived from Agave: Pulque and Henequen (Agave fourcroydes) Mezcal.

Seven Kluyveromyces marxianus isolates from the elaboration process of pulque and henequen mezcal were characterized. The isolates were identified based on the sequences of the D1/D2 domain of the 26S rRNA gene and the internal transcribed spacer (ITS-5.8S) region. Genetic differences were found between pulque and henequen mezcal isolates and within henequen mezcal isolates, as shown by different branching patterns in the ITS-5.8S phylogenetic tree and (GTG)5 microsatellite profiles, suggesting that the substrate and process selective conditions may give rise to different K. marxianus populations. All the isolates fermented and assimilated inulin and lactose and some henequen isolates could also assimilate xylose and cellobiose. Henequen isolates were more thermotolerant than pulque ones, which, in contrast, presented more tolerance to the cell wall-disturbing agent calcofluor white (CFW), suggesting that they had different cell wall structures. Additionally, depending on their origin, the isolates presented different maximum specific growth rate (µmax) patterns at different temperatures. Concerning tolerance to stress factors relevant for lignocellulosic hydrolysates fermentation, their tolerance limits were lower at 42 than 30 °C, except for glucose and furfural. Pulque isolates were less tolerant to ethanol, NaCl, and Cd. Finally, all the isolates could produce ethanol by simultaneous saccharification and fermentation (SSF) of a corncob hydrolysate under laboratory conditions at 42 °C.

Biotransformation of Chromium (VI) via a Reductant Activity from the Fungal Strain Purpureocillium lilacinum.

Industrial effluents from chromium-based products lead to chromium pollution in the environment. Several technologies have been employed for the removal of chromium (Cr) from the environment, including adsorption, ion-exchange, bioremediation, etc. In this study, we isolated a Cr (VI)-resistant fungus, Purpureocillium lilacinum, from contaminated soil, which could reduce chromium. We also characterized a reductant activity of dichromate found in the cellular fraction of the fungus: optimal pH and temperature, effect of enzymatic inhibitors and enhancers, metal ions, use of electron donors, and initial Cr (VI) and protein concentration. This study also shows possible mechanisms that could be involved in the elimination of this metal. We observed an increase in the reduction of Cr (VI) activity in the presence of NADH followed by that of formate and acetate, as electron donor. This reduction was highly inhibited by EDTA followed by NaN3 and KCN, and this activity showed the highest activity at an optimal pH of 7.0 at 37 °C with a protein concentration of 3.62 µg/mL.

Traditional Fermented Beverages of Mexico: A Biocultural Unseen Foodscape.

Mexico is one of the main regions of the world where the domestication of numerous edible plant species originated. Its cuisine is considered an Intangible Cultural Heritage of Humanity and ferments are important components but have been poorly studied. Traditional fermented foods are still diverse, but some are endangered, requiring actions to promote their preservation. Our study aimed to (1) systematize information on the diversity and cultural history of traditional Mexican fermented beverages (TMFB), (2) document their spatial distribution, and (3) identify the main research trends and topics needed for their conservation and recovery. We reviewed information and constructed a database with biocultural information about TMFB prepared and consumed in Mexico, and we analyzed the information through network approaches and mapped it. We identified 16 TMFB and 143 plant species involved in their production, species of Cactaceae, Asparagaceae, and Poaceae being the most common substrates. Microbiological research has been directed to the potential biotechnological applications of Lactobacillus, Bacillus, and Saccharomyces. We identified a major gap of research on uncommon beverages and poor attention on the cultural and technological aspects. TMFB are dynamic and heterogenous foodscapes that are valuable biocultural reservoirs. Policies should include their promotion for conservation. The main needs of research and policies are discussed.

Mitochondrial damage produced by phytotoxic chromenone and chromanone derivatives from endophytic fungus Daldinia eschscholtzii strain GsE13.

Bioassay-guided fractionation of the organic extracts of the endophyte Daldinia eschscholtzii strain GsE13 led to the isolation of several phytotoxic compounds, including two chromenone and two chromanone derivatives: 5-hydroxy-8-methoxy-2-methyl-4H-chromen-4-one, 1; 5-hydroxy-2-methyl-4H-chromen-4-one, 2; 5-methoxy-2-methyl-chroman-4-one, 3; and 5-methoxy-2-methyl-chroman-4-ol, 4; as well as other aromatic compounds: 4,8-dihydroxy-1-tetralone, 5; 1,8-dimethoxynaphthalene, 6; and 4,9-dihydroxy-1,2,11,12-tetrahydroperyl-ene-3,10-quinone, 7. Compounds 1, 4, and 7 were isolated for the first time from D. eschscholtzii. The phytotoxicity of all the compounds was determined on germination, root growth, and oxygen uptake in seedlings of a monocotyledonous (Panicum miliaceum) and three dicotyledonous plants (Medicago sativa, Trifolium pratense, and Amaranthus hypochondriacus). In general, root growth was the most affected process in all four weeds, and chromenones 1 and 2 were the most phytotoxic compounds. Phytotoxins 1-4 inhibited basal oxygen consumption rate in isolated mitochondria from M. sativa seedlings and also caused serious damage to their membrane potential (ΔΨm) in percentages greater than 50% at concentrations lower than 2 mM. Based on these results, compounds 1-4 of endophytic origin could be promising for the development of new herbicides potentially useful in agriculture or for the synthesis of promising new molecules. KEY POINTS: • Endophytic fungus Daldinia eschscholtzii produces phytotoxic compounds. • Phytotoxins inhibit basal oxygen consumption rate in isolated M. sativa mitochondria. • Phytotoxins altered the mitochondrial membrane potential.

Traditional management of microorganisms in fermented beverages from cactus fruits in Mexico: an ethnobiological approach.

BACKGROUND: Fermentation is an ancient technique for preserving and improving the qualities of food and beverages throughout the world. Microbial communities, not seen by the producers of fermented goods, are the actors involved in the fermentation process and are selected upon through different management processes in order to achieve a final product with culturally accepted features. This study documented the preparation of "colonche" which is a type of traditionally fermented beverages made with the fruits from several cactus species in two main producing regions of Mexico, the Altiplano and the Tehuacán Valley. We documented the selection processes of the cactus species used and the practices that could influence microbial community composition, as well as, how the producers reach the desirable sensorial attributes of the beverages. METHODS: We conducted 53 semi-structured interviews and participatory observations with colonche producers in 7 communities of the Altiplano and the Tehuacán Valley in order to characterize the practices and processes involved in the elaboration of the beverage. Opuntia and columnar cacti species used in colonche production were collected during fieldwork and identified. Selected sensorial attributes of Opuntia colonches were characterized by a ranking table and visualized by principal component analysis in order to distinguish differences of this beverage in the Altiplano localities. RESULTS: Thirteen cactus species are used for colonche production in both regions studied. In the Altiplano, the most commonly used fruit is Opuntia streptacantha because it contributes to the preferred attributes of the beverage in this region. Selection of substrates by producers depends on their preference and the availability of fruits of O. streptacantha and other species. Fermentation is mainly conducted in clay pots which is perceived to be the best type of vessel contributing to the preferred sensorial properties of colonche. The two main differences in colonche preparation between the villages are the practice of boiling the fruit juice and the use of pulque (fermented sap of Agave species) as inoculum. The most contrasting sensorial attributes selected between localities are the alcohol content and sweetness, which might be in accordance with the practices used for obtaining the final product. Colonche is produced mainly for direct consumption and secondarily used as a commercialized good to be sold for economic gains contributing to the general subsistence of households. The preparation methods are passed on by close relatives, mainly women. CONCLUSIONS: Traditional producers of colonche use several techniques in order to reach specific sensorial attributes of the final product. The production of colonche has been upheld for generations but fermentation practices are divided into two categories; (1) the use of an inoculum (either from pulque, or from colonche saved from the previous year), and (2) the use of "spontaneous" fermentation. The differing practices documented reflect the contrasts in the preferred sensorial attributes between regions. Colonche is a beverage that contributes to regional pride, cultural identity and is appreciated because of its gastronomic value. Here, we argue that there is a clear relationship of human knowledge in the management of microbiota composition in order to produce this beverage. In-depth documentation of the microbiota composition and dynamics in colonche will contribute to the preservation of this valuable biocultural heritage.

Consolidated Bioprocess for Bioethanol Production from Raw Flour of Brosimum alicastrum Seeds Using the Native Strain of Trametes hirsuta Bm-2.

Consolidated bioprocessing (CBP), which integrates biological pretreatment, enzyme production, saccharification, and fermentation, is a promising operational strategy for cost-effective ethanol production from biomass. In this study, the use of a native strain of Trametes hirsuta (Bm-2) was evaluated for bioethanol production from Brosimum alicastrum in a CBP. The raw seed flour obtained from the ramon tree contained 61% of starch, indicating its potential as a raw material for bioethanol production. Quantitative assays revealed that the Bm-2 strain produced the amylase enzyme with activity of 193.85 U/mL. The Bm-2 strain showed high tolerance to ethanol stress and was capable of directly producing ethanol from raw flour at a concentration of 13 g/L, with a production yield of 123.4 mL/kg flour. This study demonstrates the potential of T. hirsuta Bm-2 for starch-based ethanol production in a consolidated bioprocess to be implemented in the biofuel industry. The residual biomass after fermentation showed an average protein content of 22.5%, suggesting that it could also be considered as a valuable biorefinery co-product for animal feeding.

Volatile organic compounds from Hypoxylon anthochroum endophytic strains as postharvest mycofumigation alternative for cherry tomatoes.

The antifungal activity and chemical composition of the volatile organic compounds (VOCs) produced by four Hypoxylon anthochroum endophytic strains were analyzed. The bioactivity of the VOCs synthesized at different periods of incubation on rice medium was assessed, both in vivo and in vitro, against the phytopathogen Fusarium oxysporum. The in vivo effect was evaluated on cherry tomatoes, while the mechanism of action was determined in vitro analyzing the phytopathogen's growth, respiration and cell membrane permeability. In general, the VOCs from all strains and incubation periods significantly inhibited the growth of F. oxysporum on cherry tomatoes with percentages over 50%. They significantly inhibited the pathogen growth and respiration, and altered the cell membrane permeability and hyphal morphology. The chemical composition was analyzed after solid phase microextraction. In total, 36 VOCs were identified in the four strains, mainly sesquiterpenes and monoterpenes. Among the monoterpenes, eucalyptol had the highest fiber affinity (>60% area) in three of the four strains studied; thus, it could be considered as a chemical marker for H. antochroum. Chemical markers are important for the identification and differentiation of species. The H. anthochroum strains are potential mycofumigation agents against postharvest diseases caused by F. oxysporum.

Phytotoxic Potential of Secondary Metabolites and Semisynthetic Compounds from Endophytic Fungus Xylaria feejeensis Strain SM3e-1b Isolated from Sapium macrocarpum.

Bioactivity-directed fractionation of the combined culture medium and mycelium extract of the endophytic fungus Xylaria feejeensis strain SM3e-1b, isolated from Sapium macrocarpum, led to the isolation of three known natural products: (4S,5S,6S)-4-hydroxy-3-methoxy-5-methyl-5,6-epoxycyclohex-2-enone or coriloxine, 1; 2-hydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 2; and 2,6-dihydroxy-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione or fumiquinone B, 3. This is the first report of compound 3 being isolated from this species. Additionally, four new derivatives of coriloxine were prepared: (4R,5S,6R)-6-chloro-4,5-dihydroxy-3-methoxy-5-methylcyclohex-2-enone, 4; 6-hydroxy-5-methyl-3-(methylamino)cyclohexa-2,5- diene-1,4-dione, 5; (4R,5R,6R)-4,5-dihydroxy-3-methoxy-5-methyl-6-(phenylamino)cyclohex-2-enone, 6; and 2-((4-butylphenyl)amino)-5-methoxy-3-methylcyclohexa-2,5-diene-1,4-dione, 7. X-ray analysis allowed us to unambiguously determine the structures and absolute configuration of semisynthetic derivatives 4, 5, and 6. The phytotoxic activity of the three isolated natural products and the coriloxine derivatives is reported. Germination of the seed, root growth, and oxygen uptake of the seedlings of Trifolium pratense, Medicago sativa, Panicum miliaceum, and Amaranthus hypochondriacus were significantly inhibited by all of the tested compounds. In general, they were more effective inhibiting root elongation than suppressing the germination and seedling oxygen uptake processes as shown by their IC50 values.

Antifungal Volatile Organic Compounds from the Endophyte Nodulisporium sp. Strain GS4d2II1a: a Qualitative Change in the Intraspecific and Interspecific Interactions with Pythium aphanidermatum.

This study demonstrates volatile organic compounds (VOCs) production as one of the defense mechanisms of the antagonistic endophyte Nodulisporium sp. GS4d2II1a, and the volatile changes in two times of the fungal growth; and, as result of its intra and interspecific interactions with the plant pathogen Pythium aphanidermatum. The antifungal activity of the volatile and diffusible metabolites was evaluated by means of three types of antagonism bioassays and by organic extract agar dilution. VOCs were obtained by gas chromatography coupled to mass spectrometry from 3- and 5-day Nodulisporium sp. cultures, as well as from its interspecific in vitro antagonistic interaction with the oomycete P. aphanidermatum, and its intraspecific Nodulisporium sp.-Nodulisporium sp. interaction. The GS4d2II1a strain completely inhibited the growth of two fungi and seven oomycetes by replacing their mycelia in simple antagonism bioassays and by producing in vitro volatile and diffusible metabolites that acted synergistically in multiple antagonism bioassays. Additionally, VOCs inhibited the growth of three oomycetes and one fungus in antagonism bioassays using divided plates. A total of 70 VOCs were detected, mainly including mono and sesquiterpenes, especially eucalyptol and limonene. Multiple correspondence analysis revealed four different volatile profiles, showing that volatiles changed with the fungus age and its intra and interspecific interactions. The metabolites produced by Nodulisporium sp. GS4d2II1a could be useful for biological control of fungal and oomycetes plant pathogens of economically important crops.

Genetic diversity of Clavispora lusitaniae isolated from Agave fourcroydes Lem, as revealed by DNA fingerprinting.

This study characterized Clavispora lusitaniae strains isolated from different stages of the processing and early fermentation of a henequen (Agave fourcroydes) spirit produced in Yucatan, Mexico using a molecular technique. Sixteen strains identified based on morphological features, obtained from different substrates, were typed molecularly. Nine different versions of the divergent D1/D2 domain of the large-subunit ribosomal DNA sequence were identified among the C. lusitaniae strains. The greatest degree of polymorphism was found in the 90-bp structural motif of the D2 domain. The MSP-PCR technique was able to differentiate 100% of the isolates. This study provides significant insight into the genetic diversity of the mycobiota present during the henequen fermentation process, especially that of C. lusitaniae, for which only a few studies in plants have been published. The applied MSP-PCR markers were very efficient in revealing olymorphisms between isolates of this species.

Yeasts associated with the production of Mexican alcoholic nondistilled and distilled Agave beverages.

The great variety of agaves and their multiple uses have played an important role in the cultural identification of Mexico. They have been exploited in many ways for over 10,000 years, and one of these applications is the production of alcoholic nondistilled and distilled beverages. Most of the production processes of these Mexican beverages involve a complex fermentation in which bacteria (mainly lactic and acetic acid) and yeasts (non-Saccharomyces and Saccharomyces) are present in stable mixed populations, or succeeding one another, and have a significant impact on the sensorial characteristics and nutritive value of the final product. This minireview focuses on several nondistilled and distilled Agave beverages, their production area, the Agave species used in their elaboration, the functional microbiota involved in the fermentation process, their fermentation products (when known), the biochemical changes of these unique fermentations, and their impact on the quality and sensorial characteristics of the product.