RESUMO
Traditional cocoa bean fermentation is a spontaneous process and can result in heterogeneous sensory quality. For this reason, yeast-integrated starter cultures may be an option for creating consistent organoleptic profiles. This study proposes the mixture of Hanseniaspora opuntiae and Kluyveromyces marxianus (from non-cocoa fermentation) as starter culture candidates. The microorganisms and volatile compounds were analyzed during the cocoa fermentation process, and the most abundant were correlated with predominant microorganisms. Results showed that Kluyveromyces marxianus, isolated from mezcal fermentation, was identified as the dominant yeast by high-throughput DNA sequencing. A total of 63 volatile compounds identified by HS-SPME-GC-MS were correlated with the more abundant bacteria and yeast using Principal Component Analysis and Agglomerative Hierarchical Clustering. This study demonstrates that yeasts from other fermentative processes can be used as starter cultures in cocoa fermentation and lead to the formation of more aromatic esters, decrease the acetic acid content.
Assuntos
Cacau , Fermentação , Hanseniaspora , Kluyveromyces , Compostos Orgânicos Voláteis , Compostos Orgânicos Voláteis/análise , Compostos Orgânicos Voláteis/metabolismo , Kluyveromyces/metabolismo , Hanseniaspora/metabolismo , Cacau/microbiologia , Cacau/metabolismo , Cacau/química , Microbiologia de Alimentos , Cromatografia Gasosa-Espectrometria de Massas , Ácido Acético/metabolismo , Fatores de TempoRESUMO
Kluyveromyces marxianus is a non-Saccharomyces yeast that has gained importance due to its great potential to be used in the food and biotechnology industries. In general, K. marxianus is a known yeast for its ability to assimilate hexoses and pentoses; even this yeast can grow in disaccharides such as sucrose and lactose and polysaccharides such as agave fructans. Otherwise, K. marxianus is an excellent microorganism to produce metabolites of biotechnological interest, such as enzymes, ethanol, aroma compounds, organic acids, and single-cell proteins. However, several studies highlighted the metabolic trait variations among the K. marxianus strains, suggesting genetic diversity within the species that determines its metabolic functions; this diversity can be attributed to its high adaptation capacity against stressful environments. The outstanding metabolic characteristics of K. marxianus have motivated this yeast to be a study model to evaluate its easy adaptability to several environments. This chapter will discuss overview characteristics and applications of K. marxianus and recent insights into the stress response and adaptation mechanisms used by this non-Saccharomyces yeast.
Assuntos
Etanol , Kluyveromyces , Biotecnologia , Etanol/metabolismo , Fermentação , Kluyveromyces/genética , Kluyveromyces/metabolismoRESUMO
This research paper addresses the hypotheses that Kluyveromyces marxianus can be cultured with good alcohol production on different whey-derived matrices, and that the fermented product can be used in order to develop alcoholic beverages with acceptable sensory characteristics by mixtures with yeast-fermented fruit-based matrices. Growth and fermentative characteristics of Kluyveromyces marxianus LFIQK1 in different whey-derived matrices were explored by culturing (24 h, 30°C) on reconstituted whey, demineralized whey, heat-treated whey and milk permeate media. High lactose consumption, ethanol production and yield were observed. Reconstituted whey matrix was selected for mixing with orange or strawberry juices fermented using Saccharomyces cerevisiae to obtain alcoholic beverages (W-OR and W-ST, respectively). Consumer evaluation of beverages was performed using acceptability and Check-All-That-Apply (CATA) questions. Good acceptance was observed, significantly higher for W-ST than for W-OR. CATA questions gave information about organoleptic characteristics of beverages. Penalty analysis showed W-R and W-ST were positively associated with smooth/refreshing and fruity/natural, respectively. Liking was represented, accordingly with penalty analysis, by natural/refreshing. A novel alternative for utilization of whey and whey-related matrices by alcoholic beverages production with natural ingredients is presented.
Assuntos
Bebidas Alcoólicas , Fermentação , Sucos de Frutas e Vegetais , Kluyveromyces , Soro do Leite , Kluyveromyces/metabolismo , Soro do Leite/química , Bebidas Alcoólicas/análise , Sucos de Frutas e Vegetais/análise , Etanol/metabolismo , Saccharomyces cerevisiae/metabolismo , Paladar , HumanosRESUMO
A bioassay containing Kluyveromyces marxianus in microtiter plates was used to determine the inhibitory action of 28 antibiotics (aminoglycosides, beta-lactams, macrolides, quinolones, tetracyclines and sulfonamides) against this yeast in whey. For this purpose, the dose-response curve for each antibiotic was constructed using 16 replicates of 12 different concentrations of the antibiotic. The plates were incubated at 40°C until the negative samples exhibited their indicator (5-7h). Subsequently, the absorbances of the yeast cells in each plate were measured by the turbidimetric method (λ=600nm) and the logistic regression model was applied. The concentrations causing 10% (IC10) and 50% (IC50) of growth inhibition of the yeast were calculated. The results allowed to conclude that whey contaminated with cephalosporins, quinolones and tetracyclines at levels close to the Maximum Residue Limits inhibits the growth of K. marxianus. Therefore, previous inactivation treatments should be implemented in order to re-use this contaminated whey by fermentation with K. marxianus.
Assuntos
Antibacterianos , Kluyveromyces , Soro do Leite , Kluyveromyces/efeitos dos fármacos , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Relação Dose-Resposta a DrogaRESUMO
Kluyveromyces marxianus yeasts represent a valuable industry alternative due to their biotechnological potential to produce aromatic compounds. 2-phenylethanol and 2-phenylethylacetate are significant aromatic compounds widely used in food and cosmetics due to their pleasant odor. Natural obtention of these compounds increases their value, and because of this, bioprocesses such as de novo synthesis has become of great significance. However, the relationship between aromatic compound production and yeast's genetic diversity has yet to be studied. In the present study, the analysis of the genetic diversity in K. marxianus isolated from the natural fermentation of Agave duranguensis for Mezcal elaboration is presented. The results of strains in a haploid and diploid state added to the direct relationship between the mating type locus MAT with metabolic characteristics are studied. Growth rate, assimilate carbohydrates (glucose, lactose, and chicory inulin), and the production of aromatic compounds such as ethyl acetate, isoamyl acetate, isoamyl alcohol, 2-phenylethyl butyrate and phenylethyl propionate and the diversity in terms of the output of 2-phenylethanol and 2-phenylethylacetate by de novo synthesis were determinate, obtaining maximum concentrations of 51.30 and 60.39 mg/L by ITD0049 and ITD 0136 yeasts respectively.
Assuntos
Kluyveromyces , Álcool Feniletílico , Álcool Feniletílico/metabolismo , Odorantes , Kluyveromyces/genética , Leveduras/genética , Leveduras/metabolismo , Fermentação , Lactose/metabolismoRESUMO
The Inulinase from Kluyveromyces marxianus ISO3 (Inu-ISO3) is an enzyme able to hydrolyze linear fructans such as chicory inulin as well as branched fructans like agavin. This enzyme was cloned and expressed in Komagataella pastoris to study the role of selected aromatic and polar residues in the catalytic pocket by Alanine scanning. Molecular dynamics (MD) simulations and enzyme kinetics analysis were performed to study the functional consequences of these amino acid substitutions. Site-directed mutagenesis was used to construct the mutants of the enzyme after carrying out the MD simulations between Inu-ISO3 and its substrates. Mutation Trp79:Ala resulted in the total loss of activity when fructans were used as substrates, while with sucrose, the activity decreased by 98 %. In contrast, the mutations Phe113:Ala and Gln236:Ala increased the invertase activity when sucrose was used as a substrate. Although these amino acids are not part of the conserved motifs where the catalytic triad is located, they are essential for the enzyme's activity. In silico and experimental approaches corroborate the relevance of these residues for substrate binding and their influence on enzymatic activity.
Assuntos
Kluyveromyces , Simulação de Dinâmica Molecular , Glicosídeo Hidrolases/química , Kluyveromyces/genética , Frutanos/metabolismo , Aminoácidos/metabolismo , Sacarose/metabolismoRESUMO
Laccase enzyme can replace chemical additives to improve texture properties and the volume of bread. Laccase encoding gene from Phlebia brevispora, a native fungus from Misiones, Argentina, was expressed in the generally recognized as safe yeast Kluyveromyces lactis. To improve laccase activity, medium conditions were optimized. The use of iron sulfate at a concentration of 1 mM led to optimum laccase activity (1289 U·L-1 ) on the fourth day of incubation. SDS-PAGE analysis revealed that the molecular mass of purified laccase was about 180 kDa. Optimum pH for the enzyme was 4 and optimum temperature was 40°C. Laccase exhibited high stability at low pH and high temperature. The application of recombinant laccase to bread decreased hardness, gumminess, and chewiness and increased bread volume. Based on these results, recombinant laccase from P. brevispora with improved yield is a good option for application as an improver of the physicochemical quality of bread at the industrial level. Besides, it will allow us to advance toward our goal of developing healthy alternatives for the bakery industry. No previous work has been reported concerning the heterologous expression of the laccase gene native to the province of Misiones, Argentina, with an aim for application in baking. PRACTICAL APPLICATION: Healthy bakeries became a trend in recent years. The use of the laccase enzyme increases the specific volume and decreases the hardness of bread, being thus an alternative for the replacement of chemical additives in the bakery industry.
Assuntos
Kluyveromyces , Lacase , Argentina , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Kluyveromyces/genética , Kluyveromyces/metabolismo , Lacase/genética , Lacase/metabolismo , Temperatura , CulináriaRESUMO
Lytic enzymes secreted by Kluyveromyces marxianus can lyse Saccharomyces cerevisiae cells. Their ability to hydrolyze yeast cell walls can be used in biotechnological applications, such as the production of glucans and protoplasts, as well as a biological control agent against plant pathogenic fungi. Herein, 27 proteins secreted by K. marxianus were identified by mass spectrometry analyses. Importantly, 14 out of the 27 proteins were classified as hydrolases. Indeed, the enzyme extract secreted by K. marxianus caused damage to S. cerevisiae cells and reduced yeast cell viability. Moreover, K marxianus inhibited spore germination and mycelial growth of the phytopathogenic fungus Botrytis cinerea in simultaneous cocultivation assays. We suggest that this inhibition may be partially related to the yeast's ability to secrete lytic enzymes. Consistent with the in vitro antagonistic tests, K. marxianus was able to protect strawberry fruits inoculated with B. cinerea. Therefore, these findings suggest that K. marxianus possesses potential as a biocontrol agent against strawberry gray mold during the postharvest stage and may also have potential against other phytopathogenic fungi by means of its lytic enzymatic arsenal.
Assuntos
Kluyveromyces , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Fungos , Kluyveromyces/metabolismo , BiotecnologiaRESUMO
Kluyveromyces marxianus is a non-conventional yeast with outstanding physiological characteristics and a high potential for lignocellulosic ethanol production. However, achieving high ethanol productivity requires overcoming several biotechnological challenges due to the cellular inhibition caused by the inhibitors present in the medium. In this work, K. marxianus SLP1 was adapted to increase its tolerance to a mix of inhibitory compounds using the adaptive laboratory evolution strategy to study the adaptation and stress response mechanisms used by this non-Saccharomyces yeast. The fermentative and physiological parameters demonstrated that the adapted K. marxianus P8 had a better response against the synergistic effects of multiple inhibitors because it reduced the lag phase from 12 to 4 h, increasing the biomass by 40% and improving the volumetric ethanol productivity 16-fold than the parental K. marxianus SLP1. To reveal the effect of adaptation process in P8, transcriptome analysis was carried out; the result showed that the basal gene expression in P8 changed, suggesting the biological capability of K. marxianus to activate the adaptative prediction mechanism. Similarly, we carried out physiologic and transcriptome analyses to reveal the mechanisms involved in the stress response triggered by furfural, the most potent inhibitor in K. marxianus. Stress response studies demonstrated that P8 had a better physiologic response than SLP1, since key genes related to furfural transformation (ALD4 and ALD6) and stress response (STL1) were upregulated. Our study demonstrates the rapid adaptability of K. marxianus to stressful environments, making this yeast a promising candidate to produce lignocellulosic ethanol. KEY POINTS: ⢠K. marxianus was adapted to increase its tolerance to a mix of inhibitory compounds ⢠The basal gene expression of K. marxianus changed after the adaptation process ⢠Adapted K. marxianus showed a better physiological response to stress by inhibitors ⢠Transcriptome analyses revealed key genes involved in the stress response.
Assuntos
Furaldeído , Kluyveromyces , Furaldeído/metabolismo , Kluyveromyces/genética , Kluyveromyces/metabolismo , Perfilação da Expressão Gênica , Fermentação , Etanol/metabolismoRESUMO
The beany flavor adversely influences consumer acceptance of soymilk (SM) products. Thus, in this work, the co-fermentation of isolated new yeasts (Kluyveromyces marxianus SP-1, Candida ethanolica ATW-1, and Pichia amenthionina Y) and Kluyveromyces marxianus K (a commercial yeast) along with an XPL-1 starter (including five strains of lactic acid bacteria (LAB)) was utilized to mend the beany flavor of fermented SM (FSM) beverages. Probiotic count, pH, titratable acidity, syneresis, water holding capacity, rheological characteristics, and sensory attributes were investigated. Furthermore, the free amino acids, nucleotides, and volatile compounds (VCs) were analyzed, also presenting the collected VC data by exploiting a principal component analysis (PCA) and a heatmap with a hierarchical cluster analysis. The co-fermentation with Kluyveromyces marxianus SP-1 and K remarkably enhanced the LAB strain growth and acid production, improving the rheological attributes, whereas that of yeast along with XPL-1 as a mullite starter could reduce the beany odor. PCA chart displayed that higher amounts of alcohols, ketones, acids, and esters that significantly improved the flavor quality of FSM beverages were generated throughout the co-fermentation process. The co-fermentation with Pichia amenthionina Y generated the highest acetoin (36.19%) and diacetyl (2.02%), thus improving the overall acceptance of FSM, as well as the sensory characteristics of FSM beverages with the highest umami, sweet, odorless amino acids, and umami nucleotides, and the lowest content of alcohol and inosine. Taken together, the co-fermentation of Pichia amenthionina Y along with XPL-1 within SM provides novel insights regarding the development of FSM and fermented beverages.