Glucose metabolism,enzymic analysis and product formation in chemostat culture of Hanseniaspora uvarum

The physiology of Hanseniaspora uvarum K₅ was studied in glucose-limited chemostat cultures and upon glucose pulse. Up to a dilution rate of 0·28 h^?1, glucose was completely metabolized in biomass and CO₂. Above this value, increase in the dilution rate was accompanied by sequential production of metabolites (glycerol, acetate and ethanol) and decrease in cell yield. Similar results were observed upon glucose pulse. From the enzyme activities (pyruvate dehydrogenase, pyruvate decarboxylase, NAD and NADP-dependent acetaldehyde dehydrogenases, acetyl coenzyme A synthetase and alcohol dehydrogenase) and substrate affinities, the following conclusions were drawn with respect to product formation of cells: (1) pyruvate was preferentially metabolized via pyruvate dehydrogenase, when biomass and CO₂ were the only products formed; (2) acetaldehyde formed by pyruvate decarboxylase was preferentially oxidized in acetate by NADP-dependent aldehyde dehydrogenase; acetate accumulation results from insufficient activity of acetyl-CoA synthetase required for the complete oxidation of acetate; (3) acetaldehyde was oxidized in ethanol by alcohol dehydrogenase, in addition to acetate production.     

Mutants of the methylotrophic yeast Pichia pinus defective in C2 metabolism

A collection of mutants of Pichia pinus which are unable to grow on ethanol but retain the ability to grow on glucose and methanol, was obtained. Genetic and biochemical analysis of these strains revealed mutations in seven nuclear genes affecting activities of isocitrate lyase (icl1), malate synthase (mls1), phosphoenolpyruvate carboxykinase (pck1), ‘malic’ enzyme (mdd1) and acetyl-CoA synthetase (acs1, acs2 and acs3). All mutations except acs1-acs3 have no effect on the activities of other enzymes involved in C₂ metabolism. Mutations acs1, acs2 and acs3 have a pleiotropic action, leading to partial reduction in activities of isocitrate lyase and malate synthase. Ethanol-induced repression of the synthesis of the methanol oxidative enzymes, alcohol oxidase and catalase, is not impaired in these seven mutant classes. On the other hand, C₂ compound-induced inactivation of alcohol oxidase and catalase is impaired in mutants acs1, acs2, acs3 and icl1. It was suggested that glyoxylate and acetate (or acetate precursors) act as low molecular weight effectors, ‘switching on’ inactivation and repression, respectively, of alcohol oxidase and catalase in the medium containing ethanol or acetate.     

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (GGS1FDP1BYP1CIF1GLC6TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and the wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of d-glucose and an equal concentration of radiolabelled l-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total d-glucose measured (intracellular+periplasmic/extracellular) and the total l-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mm-glucose to 0·5–2 mm in the wild-type strain, ±10 mm in a hxk1Δ hxk2Δ glk1Δ and 2–3 mm in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict properly up to 50-fold higher hexokinase activity. © 1998 John Wiley & Sons, Ltd.     

Glutamate dehydrogenases in the oleaginous yeast Yarrowia lipolytica

Glutamate dehydrogenases (GDHs) are fundamental to cellular nitrogen and energy balance. Yet little is known about these enzymes in the oleaginous yeast Yarrowia lipolytica. The YALI0F17820g and YALI0E09603g genes, encoding potential GDH enzymes in this organism, were examined. Heterologous expression in gdh-null Saccharomyces cerevisiae and examination of Y. lipolytica strains carrying gene deletions demonstrate that YALI0F17820g (ylGDH1) encodes a NADP-dependent GDH whereas YALI0E09603g (ylGDH2) encodes a NAD-dependent GDH enzyme. The activity encoded by these two genes accounts for all measurable GDH activity in Y. lipolytica. Levels of the two enzyme activities are comparable during logarithmic growth on rich medium, but the NADP-ylGDH1p enzyme activity is most highly expressed in stationary and nitrogen starved cells by threefold to 12-fold. Replacement of ammonia with glutamate causes a decrease in NADP-ylGdh1p activity, whereas NAD-ylGdh2p activity is increased. When glutamate is both carbon and nitrogen sources, the activity of NAD-ylGDH2p becomes dominant up to 18-fold compared with that of NADP-ylGDH1p. Gene deletion followed by growth on different carbon and nitrogen sources shows that NADP-ylGdh1p is required for efficient nitrogen assimilation whereas NAD-ylGdh2p plays a role in nitrogen and carbon utilization from glutamate. Overexpression experiments demonstrate that ylGDH1 and ylGDH2 are not interchangeable. These studies provide a vital basis for future consideration of how these enzymes function to facilitate energy and nitrogen homeostasis in Y. lipolytica.     

Acetaldehyde: an intermediate in the formation of ethanol from glucose by lactic acid bacteria.

Group N streptococci formed acetaldehyde and ethanol from glucose. As the enzymes aldehyde dehydrogenase, phosphotransacetylase and acetate kinase were present this would enable these organisms to reduce acetyl-CoA to acetaldehyde and convert acetyl-CoA to acetyl phosphate and acetate. A pentose phosphate pathway which converted ribose-5-phosphate to glyceraldehyde-3-phosphate was also present. Acetaldehyde could not be formed via the hexose monophosphate shunt or by direct decarboxylation of pyruvate, as the enzymes phosphoketolase and alpha-carboxylase were absent. Phosphoketolase activity was induced in Streptococcus lactis subsp. diacetylactis after growth on D-xylose. Group N streptococci also contained an NAD-dependent alcohol dehydrogenase which reduced acetaldehyde to ethanol while both NAD- and NADP-dependent alcohol dehydrogenase activities were found in Leuconostoc cremoris.     

Comparative physiology and fermentation performance of Saaz and Frohberg lager yeast strains and the parental species Saccharomyces eubayanus

Two distinct genetic groups (Saaz and Frohberg) exist within the hybrid Saccharomyces pastorianus (S. cerevisiae × S. eubayanus) taxon. However, physiological/technological differences that exist between the two groups are not known. Fermentative capability of the parental S. eubayanus has likewise never been studied. Here, 58 lager strains were screened to determine which hybrid group they belonged to, and selected strains were characterized to determine salient characteristics. In 15 °P all‐malt wort fermentations at 22 °C, Frohberg strains showed greater growth and superior fermentation (80% apparent attenuation, 6.5% alcohol by volume in 3–4 days) compared to all other strains and maintained highest viability values (>93%). Fermentation with S. eubayanus was poor at the same temperature (33% apparent attenuation, 2.7% alcohol by volume at 6 days and viability reduced to 75%). Saaz strains and S. eubayanus were the least sensitive to cold (10 °C), though this did not translate to greater fermentation performance. Fermentation with S. eubayanus was poor at 10 °C but equal to or greater than that of the Saaz strains. Performance of Saaz yeast/S. eubayanus was limited by an inability to use wort maltotriose. [¹⁴C]‐Maltotriose transport assays also showed negligible activity in these strains (≤0.5 µmol min^?1 g^?1 dry yeast). Beers from Saaz fermentations were characterized by two‐ to sixfold lower production of the flavour compounds methyl butanol, ethyl acetate and 3‐methylbutyl acetate compared to Frohberg strains. Higher alcohol and ester production by S. eubayanus was similar to that of Frohberg strains. Copyright © 2013 John Wiley & Sons, Ltd.     

Structural comparison of the Pichia pastoris alcohol oxidase genes

In methylotrophic yeasts, alcohol oxidase is the first enzyme in the methanol-utilization pathway. The genome of one such yeast, Pichia pastoris, contains two alcohol oxidase genes, AOX1 and AOX2. Sequence analysis indicated that each gene encodes a similar protein of 663 amino acids. The protein-coding regions of the genes were 92% and 97% homologous at the nucleotide and predicted amino acid sequence levels, respectively. In contrast to homology observed within the protein-coding portions of the AOX genes, no homology was found in either the 5′ or 3′ non-coding regions. Although alcohol oxidase is found in peroxisomes of P. pastoris, the AOX amino acid sequences did not contain a peptide sequence similar to the peroxisomal transport sequence found at the C-terminus of some peroxisomally located proteins in higher eukaryotes.     

Screening of new strains of Saccharomycodes ludwigii and Zygosaccharomyces rouxii to produce low‐alcohol beer

Low‐alcohol beer (0.5–1.2% v/v ethanol) is a less common brewing industry output than standard beer but there is an increasing interest in this product, as evidenced by increased attention to health and safety and government policies on alcohol and diet. The main challenge in the production of low‐alcohol beer is the achievement of a product as similar as possible to regular beer, particularly concerning the content of the volatile compounds. These compounds can be lost during the physical removal of alcohol by dialysis, reverse osmosis and vacuum rectification. Consequently, an alternative technique is the use of biological methods, which involve the employment of non‐conventional yeasts. In this paper, 11 non‐conventional yeast strains were tested for low‐alcohol beer production. The strains used belonged to two different species: Saccharomycodes ludwigii and Zygosaccharomyces rouxii. The beer samples produced by these strains were analysed for their ethanol content and main volatile compounds. The S. ludwigii strains were more suitable for brewing low‐alcohol beer, especially strain DBVPG 3010, which also showed a higher content of esters and a lower amount of diacetyl compared with previous reports. The Z. rouxii strains produced an ethanol and diacetyl content above the taste threshold. This screening project can be considered as a first step towards the production of low‐alcohol beer by means of new selected non‐conventional yeasts. Copyright © 2015 The Institute of Brewing & Distilling     

Role of the N-terminal region of Rap1p in the transcriptional activation of glycolytic genes in Saccharomyces cerevisiae

In the yeast two-hybrid system, the N-terminal region of Rap1p was shown to interact with Gcr1p and Gcr2p. Disruption of gcr1 and/or gcr2 in the two-hybrid reporter strain demonstrated that the interaction with Gcr1p does not require Gcr2p, whereas the interaction with Gcr2p is mediated through Gcr1p. Deletion of the N-terminal region of Rap1p alone did not show a growth phenotype, but a growth defect was observed when this mutation was combined with a gcr2 deletion. The poor growth of the gcr1 null mutant was not affected further by the N-terminal deletion of Rap1p, but the growth of gcr1 strains with mutations in the DNA binding region of Gcr1p was affected by the removal of the N-terminal region of Rap1p. These results suggest that one function of the N-terminal region of Rap1p, presumably the BRCT domain, is to facilitate the binding of Gcr1p to the promoter by a protein-protein interaction.     

The shock of vacuolar PrA on glycolytic flux,oxidative phosphorylation,and cell morphology by industrial <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> WZ65

Proteinase A (PrA) is one of the most significant vacuolar proteinase in S. cerevisiae, and it plays an important role in S. cerevisiae physiology and metabolism, especially under unfavorable environment. In this study, the differences in pyruvate kinase (PYK) level under fructose-1,6-diphosphate (FDP) induction and ATP synthesis block among SC1 (the wild-type yeast that was industrial Saccharomyces cerevisiae WZ65), SC2 (PEP4 partial deletion) and SC3 (PEP4 complete deletion) were examined. Results showed that the induction caused by FDP clearly increased PYK expression no matter for which strain, but the increasing effect is more significant for SC2 (P < 0.05). The comparative results of intracellular ATP accumulation showed that the induction by FDP may be affected at the presence of PrA. The block experiment of ATP synthesis showed that PYK activities in PEP4-modified strains are lower than that of the wild type, but the intracellular ATP levels in the wild-type one are generally higher than the PEP4-modified strains after rotenone treatment (P < 0.01). This implies that the effect of PrA deficiency on intracellular ATP accumulation was much more pronounced than the effect of rotenone on oxidative phosphorylation. The cell morphology of three strains was comparatively examined by means of transmission electron microscopy (TEM). The PEP4-modified strains possessed more vacuoles, and cell structure were more integrated than the wild-type strain. Current data preliminarily indicated that the deletion of PEP4 gene in industrial S. cerevisiae WZ65 may not only affected PYK expression but also modulated the oxidative phosphorylation flux.     

Sorbent Extraction and Analysis of Volatile Metabolites Synthesized by Lactic Acid Bacteria in a Synthetic Medium

Volatile metabolites synthesized by strains of Luctobacillus brevis, L. fructivorans, L. hilgardii, L. plantarum, Pediococcus parvulus, and Leuconostoc oenos, originally isolated from commercial wines, were extracted from a synthetic medium using C₁₈ sorbent cartridges and analyzed using gas-liquid chromatography. All organisms synthesized isoamyl alcohol, 1-hexanol, 3-(methylthio)-1-propanol, and 2-phenyl ethanol in varying concentrations. Several other alcohols, acids, aldehydes, and esters were produced or utilized by some species or strains and enabled differentiation of the organisms. Recoveries of metabolites were dependent on the compounds, matrix, and presence of ethanol.     

Detection of Beer‐spoilage Lactobacillus brevis strains by Reduction of Resazurin

Lactobacillus brevis is the most common beer‐spoilage bacteria found in breweries. Due to its high prevalence and biodiversity, it is necessary to differentiate between the strains based on their hop tolerance. Forcing tests are often conducted for different types of beer, which can vary in cereal base, fermentation type, ethanol‐ and hop content. These conventional tests are considered to be the safest way to determine the ability of a given strain to cause beer‐spoilage, but they are very time consuming and costly. Since NADH₂ is used as cofactor by many cellular dehydrogenases, this study used the reduced form of intracellular nicotinamide adenine dinucleotide (NADH₂) as an indicator for microbial metabolic activity and thus ability to spoil beer, in order to reduce the time required to conduct the forcing tests for beer production. Beer‐spoiling L. brevis strains were detected among other strains in beer samples within two days (of sampling).     

Allyl Alcohol Is the Sole Antiyeast Compound in Heated Garlic Extract

The principal antiyeast compound of heated garlic was isolated and identified by high‐performance liquid chromatography (HPLC) and gas chromatography‐mass spectrometry (GC‐MS) as allyl alcohol (2‐pro‐pene‐1‐ol). The generation of allyl alcohol was observed in heated garlic as well as in heated pure alliin solution. The pattern of growth inhibition of Candida utilis ATCC42416 by allyl alcohol was essentially the same as that seen with heated garlic and heated alliin solution. The minimum inhibitory concentrations of heated garlic, with an alliin content of 1.5% (w/v), and allyl alcohol were 0.6% (v/v) and 0.002% (v/v), respectively, against C. utilis ATCC42416. Yeasts were extremely sensitive to allyl alcohol, with minimum inhibitory concentration (MIC) ranging from 0.002% (v/v) to 0.014% (v/v), while bacteria were not very sensitive to allyl alcohol, with MIC ranging from 4% to 7%. Among yeasts, xerotolerant strains, including Zygosaccharomyces rouxii, were significantly less sensitive to allyl alcohol and heated garlic extract than others. Allyl alcohol is different from all other known antimicrobial compounds found in garlic in that it does not contain sulfur in its molecule.     

Deviant Pex3p Levels affect Normal Peroxisome Formation in Hansenula polymorpha: A Sharp Increase of the Protein Level Induces the Proliferation of Numerous,Small Protein-import Competent Peroxisomes

Pex3p has been implicated in the biosynthesis of the peroxisomal membrane of the yeast Hansenula polymorpha. Here we show that in the initial stages of a sharp increase in Pex3p levels, induced in batch cultures of cells of a constructed H. polymorpha strain, which contained seven copies of PEX3 under control of the alcohol oxidase promoter (WT::P_AOX.PEX3^7x), strongly interfered with normal peroxisome proliferation. Ultrastructural studies demonstrated that in such cells numerous small peroxisomes had developed, which were absent in wild-type controls. These organelles, which contained typical peroxisomal matrix and membrane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density (1·18 g cm⁻³) after sucrose gradient centrifugation of WT::P_AOX.PEX3^7x homogenates, compared to normal peroxisomes (1·23 g cm⁻³). We furthermore demonstrated that these early induced, small peroxisomes were protected against glucose-induced proteolytic degradation and did not fuse to form larger organelles. Remarkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalization of minor amounts of alcohol oxidase protein in the cytosol. However, when the cells were subsequently placed under conditions in which the synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repression of the P_AOX), amine oxidase protein was selectively incorporated into these organelles. This indicated that the small peroxisomes had regained a normal protein import capacity. Based on these results we argue that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. © 1997 John Wiley & Sons, Ltd.     

The role of two putative nitroreductases,Frm2p and Hbn1p,in the oxidative stress response in Saccharomyces cerevisiae

The nitroreductase family is comprised of a group of FMN‐ or FAD‐dependent enzymes that are able to metabolize nitrosubstituted compounds using the reducing power of NAD(P)H. These nitroreductases can be found in bacterial species and, to a lesser extent, in eukaryotes. There is little information on the biochemical functions of nitroreductases. Some studies suggest their possible involvement in the oxidative stress response. In the yeast Saccharomyces cerevisiae, two nitroreductase proteins, Frm2p and Hbn1p, have been described. While Frm2p appears to act in the lipid signalling pathway, the function of Hbn1p is completely unknown. In order to elucidate the functions of Frm2p and Hbn1p, we evaluated the sensitivity of yeast strains, proficient and deficient in both oxidative stress proteins, for respiratory competence, antioxidant‐enzyme activities, intracellular reactive oxygen species (ROS) production and lipid peroxidation. We found reduced basal activity of superoxide dismutase (SOD), ROS production, lipid peroxidation and petite induction and higher sensitivity to 4‐nitroquinoline‐oxide (4‐NQO) and N‐nitrosodiethylamine (NDEA), as well as higher basal activity of catalase (CAT) and glutathione peroxidase (GPx) and reduced glutathione (GSH) content in the single and double mutant strains frm2Δ and frm2Δ hbn1Δ. These strains exhibited less ROS accumulation and lipid peroxidation when exposed to peroxides, H₂O₂ and t‐BOOH. In summary, the Frm1p and Hbn1p nitroreductases influence the response to oxidative stress in S. cerevisae yeast by modulating the GSH contents and antioxidant enzymatic activities, such as SOD, CAT and GPx. Copyright © 2009 John Wiley & Sons, Ltd.     

The metabolism of several carboxylic acids by lactic acid bacteria

Summary The anaerobic metabolism of citrate, fumarate, gluconate, malate, 2-oxoglutarate and pyruvate by 137 strains of 23 species of lactic acid bacteria was investigated. The bacteria were from various sources (plant material, meat and dairy products, dough and wine) and belonged to the generaLactobacillus, Leuconostoc, Pediococcus, andStreptococcus. The ability of metabolize the acids was determined by thin layer chromatography or by enzymatic analysis after growth of the strains in a glucose-containing medium. All strains metabolized pyruvate and only 12 mainly heterofermentative strains were malate negative. These strains were also unable to decompose citrate. This acid was fermented by 23 strains, all of which metabolized malate. Many lactic acid bacteria reduced 2-oxoglutarate to hydroxyglutarate. The strains ofLactobacillus plantarum did not metabolize 2-oxoglutarate whereas all strains ofLeuconostoc oenos decarboxylated this acid and formed 4-hydroxybutyrate and succinate. Gluconate was fermented by 52 mainly heterofermetative strains. No correlation was observed between the ability to ferment citrate, malate or gluconate.

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Der Stoffwechsel verschiedener Carbonsäuren durch Milchsäurebakterien

Zusammenfassung Durch Dünnschichtchromatographie oder enzymatische Analyse der glucosehaltigen Kulturlösung wurde der Abbau von Citrat, Fumarat, Gluconat, Malat, 2-Oxoglutarat und Pyruvat bei 137 Stämmen von Milchsäurebakterien der GattungenLactobacillus, Leuconostoc, Pediococcus undStreptococcus aus Pflanzenmaterial, Milch- und Fleischprodukten, Teig und Wein untersucht. Pyruvat wurde von allen Stämmen abgebaut, und nur 12 Stämme waren Malat-negativ. Diese Stämme setzten Citrat nicht um, während alle 23 Citrat-abbauenden Bakterien auch Malat verwerteten. 2-Oxoglutarat wurde nur von den Stämmen vonLactobacillus plantarum nicht umgesetzt, während es von den meisten Organismen zu Hydroxyglutarat reduziert wurde. Aber alle Stämme vonLeuconostoc oenos bildeten CO₂, 4-Hydroxybutyrat und Succinat aus 2-Oxoglutarat. Gluconat wurde von 52 meist heterofermentativen Organismen vergoren, aber eine Beziehung zum Stoffwechsel von Malat oder Citrat wurde nicht beobachtet.

     

Assembly of amine oxidase and D-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or D-alanine as the sole nitrogen source

We have studied growth of two peroxisome-deficient mutant strains of Hansenula polymorpha on glucose in the presence of different organic nitrogen sources (methylamine, ethylamine and D -alanine), the metabolism of which is mediated by peroxisome-borne oxidases in wild-type (WT) cells. Both strains grew well on each of these substrates with growth rates comparable to WT cells. Growth on both methylamine and ethylamine was associated with enhanced levels of catalase and amine oxidase in the cells; in D -alanine-grown cells D -amino acid oxidase activity and increased. In WT cells of H-polymorpha the activities of these enzymes were confined to the peroxisomal matrix; however, in both peroxisome-deficient strains their activities were localized in the cytosol. Electron microscopy indicated that, dependent on the stage of growth, the enzymes may form large protein aggregates. The molecular masses of both amine oxidase and D -amino acid oxidase in the mutant strains were identical to their respective counterparts in WT cells, indicating that both proteins were correctly assembled and active in the cytosol.     

Microbial ecology studies of spontaneous fermentation: starter culture selection for prickly pear wine production

Abstract: A procedure for designing starter cultures for fermentation is illustrated for prickly pear wine production. The illustration includes kinetic studies on inoculated and spontaneous fermentation, microorganism identification studies based on molecular biology tools, and microbial ecology studies, which led to the selection of strains that are capable of synthesizing alcohol and desirable volatile compounds. Results show that a mixed starter inoculum containing Pichia fermentans and Saccharomyces cerevisiae leads to a fermented product that contains 8.37% alcohol (v/v). The gas chromatography and mass spectrometry (GC‐MS) analysis shows the presence of 9 major volatile compounds (Isobutanol, Isopentanol, Ethyl acetate, Isoamyl acetate, Ethyl octanoate, Ethyl decanoate, Ethyl 9‐decanoate, β‐Phenylethyl acetate, and Phenylethyl alcohol) that have ethereal, fruity, aromatic notes that are considered to be essential for a fine wine flavor. These compounds harmonically synergize with the alcohol to produce a fermented product with a unique flavor and taste. Several assays using the mixed culture show that the process is stable, predictable, controllable, and reproducible. Moreover, the results show that a mixed culture leads to a broader range of aromatic products than that produced by a single, pure culture. Therefore, we conclude that combinations of Saccharomyces strains and non‐Saccharomyces strains can be used to obtain high‐quality fermented beverages from prickly pear juice.