The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Influence of wine-related physicochemical factors on the growth and metabolism of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts in mixed culture

The influence of two physicochemical factors involved in winemaking, temperature and SO₂, on the kinetics and metabolic behavior of Kloeckera apiculata and Saccharomyces cerevisiae was examined. Highest biomass was reached at 15 and 25°C for K. apiculata and S. cerevisiae, respectively. Pure cultures of K. apiculata died off early with increasing temperature, but in co-culture with S. cerevisiae it showed higher viability and a change in the death curve from exponential to linear. Statistical analysis revealed that metabolite production was significantly different for the three cultures and also at the different fermentation temperatures. Besides, the interaction between culture type and temperature was significant. At temperatures from 15 to 30°C the mixed culture showed similar ethanol and lower acetic acid production compared with a pure culture of K. apiculata. SO₂ addition slightly increased survival of the non-Saccharomyces species in pure and mixed cultures. Statistical evaluation indicated that culture type and SO₂ addition significantly affected metabolite production, but the interaction between culture and SO₂ was not significant. These results contribute to current knowledge of enological factors and their effect on prevalence and fermentative activities of the composite yeast flora and the statistical significance emphasizes the importance of the combined influence of the culture type and physicochemical factors on the production of fermentation metabolites.     

Tandem repeat-tRNA (TRtRNA) PCR method for the molecular typing of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> subspecies

There is a worldwide trend to understand the impact of non-Saccharomyces yeast species on the process of winemaking. Although the predominant species at the end of the fermentation is Saccharomyces cerevisiae, several non-Saccharomyces species present during the first days of the process can produce and/or release aromas that improve the bouquet and complexity of the final wine. Since no genomic sequences are available for the predominant non-Saccharomyces species selected from grapes or musts (Hanseniaspora uvarum, Hanseniaspora vineae, Hanseniaspora opuntiae, Metschnikowia pulcherrima, Candida zemplinina), a reproducible PCR method was devised to discriminate strains at the subspecies level. The method combines different oligonucleotides based on tandem repeats with a second oligonucleotide based on a conserved tRNA region, specific for ascomycetes. Tandem repeats are randomly dispersed in all eukaryotic genomes and tRNA genes are conserved and present in several copies in different chromosomes. As an example, the method was applied to discriminate native M. pulcherrima strains but it could be extended to differentiate strains from other non-Saccharomyces species. The biodiversity of species and strains found in the grape ecosystem is a potential source of new enzymes, fungicides and/or novel sustainable methods for biological control of phytopathogens.     

An optimized procedure for the enological selection of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> starter cultures

The apiculate yeasts are the species predominating the first stage of grape must alcoholic fermentation and are important for the production of desired volatile compounds. The aim of the present investigation was to establish a protocol for the enological selection of non-Saccharomyces strains directly isolated from a natural must fermentation during the tumultuous phase. At this scope, fifty Hanseniaspora uvarum isolates were characterized at strain level by employing a new combined PCR-based approach. One isolate representative of each identified strain was used in fermentation assays to assess strain-specific enological properties. The chemical analysis indicated that all the analyzed strains were low producers of acetic acid and hydrogen sulphide, whereas they showed fructophilic character and high glycerol production. Analysis of volatile compounds indicated that one strain could positively affect, during the alcoholic fermentation process, the taste and flavour of alcoholic beverages. The statistical evaluation of obtained results indicated that the selected autochthonous H. uvarum strain possessed physiological and technological properties which satisfy the criteria indicated for non-Saccharomyces wine yeasts selection. Our data suggest that the described protocol could be advantageously applied for the selection of non-Saccharomyces strains suitable for the formulation of mixed or sequential starters together with Saccharomyces cerevisiae.     

<Emphasis Type="Italic">Starmerella bombicola</Emphasis> influences the metabolism of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> at pyruvate decarboxylase and alcohol dehydrogenase level during mixed wine fermentation

Background  

The use of a multistarter fermentation process with Saccharomyces cerevisiae and non-Saccharomyces wine yeasts has been proposed to simulate natural must fermentation and to confer greater complexity and specificity to wine. In this context, the combined use of S. cerevisiae and immobilized Starmerella bombicola cells (formerly Candida stellata) was assayed to enhance glycerol concentration, reduce ethanol content and to improve the analytical composition of wine. In order to investigate yeast metabolic interaction during controlled mixed fermentation and to evaluate the influence of S. bombicola on S. cerevisiae, the gene expression and enzymatic activity of two key enzymes of the alcoholic fermentation pathway such as pyruvate decarboxylase (Pdc1) and alcohol dehydrogenase (Adh1) were studied.     

The molecular genetic differentiation of cultured <Emphasis Type="Italic">Saccharomyces</Emphasis> strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that bakers yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae × S. bayanus var. uvarum hybrids. The molecular karyotyping of bakers, brewers, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)₅ can be used to study the populations of cultured S. cerevisiae strains.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 215–223.Original Russian Text Copyright © 2005 by Naumova, Zholudeva, Martynenko, Naumov.     

Enrichment of a continuous culture of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> with the yeast <Emphasis Type="Italic">Issatchenkia orientalis</Emphasis> in the production of ethanol at increasing temperatures

A fermentation system was continuously fed with sugar-cane syrup and operated with recycling of Saccharomyces cerevisiae cells at temperatures varying from 30 to 47°C. The aim of the present work was to obtain and study the colonies of isolates showing elongated cells of yeasts which were sporadically observed at the end of this continuous process. Based on a sequence of assays involving methods of classical taxonomy and RAPD-PCR, two groups of isolates showing characteristics of non-Saccharomyces yeasts were identified in the yeast population where S. cerevisiae was the dominant yeast. The largest group of non-Saccharomyces yeasts, resulting from a slow proliferation over the 2 months, reached a final level of 29.6% at the end of the process. RAPD-PCR profiles obtained for the isolates of this dominant non-Saccharomyces yeast indicated that they were isolates of Issatchenkia orientalis. Pichia membranifaciens was the only species of non-Saccharomyces yeast detected together with I. orientalis but at a very low frequency. The optimum temperature for ethanol formation shown by the isolate 195B of I. orientalis was 42°C. This strain also showed a faster ethanol formation and biomass accumulation than the thermotolerant strain of S. cerevisiae used as the starter of this fermentation process. Some isolates of I. orientalis were also able to grow better at 40°C than at 30°C on plates containing glycerol as carbon source. Yeasts able to grow and produce ethanol at high temperatures can extend the fermentation process beyond the temperature limits tolerated by S. cerevisiae.     

Optimization and mechanism of diacetyl accumulation by <Emphasis Type="Italic">Enterobacter</Emphasis><Emphasis Type="Italic">aerogenes</Emphasis> mutant UV-3

A mutant designated as UV-3 was obtained from wild-type Enterobacter aerogenes 10293 through u.v. radiation. The activities of α-acetolactate decarboxylase (Ald), lactate dehydrogenase (Ldh) and diacetyl reductase (Dr) in UV-3 were strongly attenuated, with the lowest activities at pH 7.0–7.5, and temperature between 36 and 39°C. Compared to the wild-type, the yield of diacetyl by UV-3 was increased 18.7-fold, up to 1.05 ± 0.01 g l⁻¹. Acetoin and ethanol productions were decreased by 48.4 and 71.4%, respectively, but acetate yield was increased by 34.6%. Optimum medium for diacetyl production by UV-3 contained 10% glucose, 0.5% peptone, 0.5% yeast extract powder, 0.01% (NH₄)₂SO₄, 0.1% citric acid, 0.2% MnSO₄ and 0.2% MgSO_4, and this was determined by one-factor-at-a-time approach. Data from the five level central composite designs demonstrated that initial pH of 7.0, temperature of 37°C and rotational speed of 180 rev/min were optimum processing parameters for diacetyl production. The maximum yield of diacetyl could reach 1.35 g l⁻¹ in a 5-l bioreactor. These results showed an enhancement of the non-enzymatic oxidative decarboxylation of α-acetolactate and a decrease in the activities of Ald, Ldh and Dr as a consequence of diacetyl accumulation in UV-3.     

Acetaldehyde kinetics of enological yeast during alcoholic fermentation in grape must

Acetaldehyde strongly binds to the wine preservative SO₂ and, on average, causes 50–70 mg l^?1 of bound SO₂ in red and white wines, respectively. Therefore, a reduction of bound and total SO₂ concentrations necessitates knowledge of the factors that affect final acetaldehyde concentrations in wines. This study provides a comprehensive analysis of the acetaldehyde production and degradation kinetics of 26 yeast strains of oenological relevance during alcoholic fermentation in must under controlled anaerobic conditions. Saccharomyces cerevisiae and non-Saccharomyces strains displayed similar metabolic kinetics where acetaldehyde reached an initial peak value at the beginning of fermentations followed by partial reutilization. Quantitatively, the range of values obtained for non-Saccharomyces strains greatly exceeded the variability among the S. cerevisiae strains tested. Non-Saccharomyces strains of the species C. vini, H. anomala, H. uvarum, and M. pulcherrima led to low acetaldehyde residues (<10 mg l^?1), while C. stellata, Z. bailii, and, especially, a S. pombe strain led to large residues (24–48 mg l^?1). Acetaldehyde residues in S. cerevisiae cultures were intermediate and less dispersed (14–34 mg l^?1). Addition of SO₂ to Chardonnay must triggered significant increases in acetaldehyde formation and residual acetaldehyde. On average, 0.33 mg of residual acetaldehyde remained per mg of SO₂ added to must, corresponding to an increase of 0.47 mg of bound SO₂ per mg of SO₂ added. This research demonstrates that certain non-Saccharomyces strains display acetaldehyde kinetics that would be suitable to reduce residual acetaldehyde, and hence, bound-SO₂ levels in grape wines. The acetaldehyde formation potential may be included as strain selection argument in view of reducing preservative SO₂ concentrations.     

Genomics and biochemistry of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine yeast strains

Saccharomyces yeasts have been used for millennia for the production of beer, wine, bread, and other fermented products. Long-term “unconscious” selection and domestication led to the selection of hundreds of strains with desired production traits having significant phenotypic and genetic differences from their wild ancestors. This review summarizes the results of recent research in deciphering the genomes of wine Saccharomyces strains, the use of comparative genomics methods to study the mechanisms of yeast genome evolution under conditions of artificial selection, and the use of genomic and postgenomic approaches to identify the molecular nature of the important characteristics of commercial wine strains of Saccharomyces. Succinctly, data concerning metagenomics of microbial communities of grapes and wine and the dynamics of yeast and bacterial flora in the course of winemaking is provided. A separate section is devoted to an overview of the physiological, genetic, and biochemical features of sherry yeast strains used to produce biologically aged wines. The goal of the review is to convince the reader of the efficacy of new genomic and postgenomic technologies as tools for developing strategies for targeted selection and creation of new strains using “classical” and modern techniques for improving wine-making technology.     

Indigenous and inoculated yeast fermentation of gabiroba (<Emphasis Type="Italic">Campomanesia pubescens</Emphasis>) pulp for fruit wine production

The objectives of this study were to evaluate the potential of gabiroba Campomanesia pubescens (DC) O. Berg in the production of a beverage fermented using selected and wild yeasts from indigenous fermentation, analyze the volatile compounds profile present during the process of fermentation, and evaluate the sensory quality of the final beverage produced. Throughout the process of fermentation, when Saccharomyces cerevisiae UFLA CA 1162 was inoculated, there were stable viable populations around 9 log cells ml⁻¹. During indigenous fermentation, yeast population increased from 3.7 log CFU ml⁻¹ to 8.1 log CFU ml⁻¹ after 14 days. The diversity and dynamics of the yeast population during indigenous fermentation observed by PFGE analysis showed five different karyotyping profiles in the first days of fermentation. After the seventh day, there was a higher frequency of a similar S. cerevisiae profile. The yeast non-Saccharomyces were identified by sequencing of the ITS region as Candida quercitrusa and Issatchenkia terricola. Inoculated fermentations yielded a higher amount of alcohol than indigenous ones, indicating the efficiency of selected strains. There was also a greater concentration of higher alcohols, which are usually responsible for the flavor found in alcoholic beverages. Based on the characteristics of the pulp and acceptance in the sensory analysis, gabiroba fruits showed good potential for use in the production of fermented beverage.     

Kinetics and metabolic behavior of a composite culture of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine related strains

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.     

Characterization of glycolytic activities from non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts isolated from Bobal musts

A large number of non-Saccharomyces yeasts were isolated from grapes of Bobal variety and identified according to their physiological and molecular characteristics. The yeasts were tested to determine the presence of β-glucosidase, β-xylosidase, α-arabinosidase, and α-rhamnosidase activities and five isolates were selected. All enzymatic activities were induced by the presence of glycosides as the only carbon source in the medium, which seems to be a characteristic of the yeast isolate, and were characterized according to different parameters of enological interest.     

Effect of cultivation conditions on invertase production by hyperproducing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolates

Invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) finds major uses in confectionery and in the production of invert syrup. In the present study, we report on invertase production by wild cultures of Saccharomyces cerevisiae. The yeast strains were isolated from dates available in a local market. Five hyperproducing yeast strains (>100- fold higher invertase activity) were kinetically analysed for invertase production. Saccharomyces cerevisiae strain GCA-II was found to be a better invertase-yielding strain than all the other isolates. The values of Q_p and Y_p/s for GCA-II were economical as compared to other Saccharomyces cultures. The effect of sucrose concentration, rate of invertase synthesis, initial pH of fermentation medium and different organic nitrogen sources on the production of invertase under submerged culture conditions was investigated. Optimum concentrations of sucrose, urea and pH were 3, 0.2 (w/v), and 6 respectively. The increase in the enzyme yield obtained after optimization of the cultural conditions was 47.7%.     

Physiological diversity within the <Emphasis Type="Italic">kluyveromyces marxianus</Emphasis> species

The Kluyveromyces marxianus strains CBS 6556, CBS 397 and CBS 712^T were cultivated on a defined medium with either glucose, lactose or sucrose as the sole carbon source, at 30 and 37°C. The aim of this work was to evaluate the diversity within this species, in terms of the macroscopic physiology. The main properties evaluated were: intensity of the Crabtree effect, specific growth rate, biomass yield on substrate, metabolite excretion and protein secretion capacity, inferred by measuring extracellular inulinase activity. The strain Kluyveromyces lactis CBS 2359 was evaluated in parallel, since it is the best described Kluyveromyces yeast and thus can be used as a control for the experimental setup. K. marxianus CBS 6556 presented the highest specific growth rate (0.70 h⁻¹) and the highest specific inulinase activity (1.65 U mg⁻¹ dry cell weight) among all strains investigated, when grown at 37°C with sucrose as the sole carbon source. The lowest metabolite formation and highest biomass yield on substrate (0.59 g dry cell weight g sucrose⁻¹) was achieved by K. marxianus CBS 712^T at 37°C. Taken together, the results show a systematic comparison of carbon and energy metabolism among three of the best known K. marxianus strains, in parallel to K. lactis CBS 2359.     

Gene cloning,expression, and characterization of a novel acetaldehyde dehydrogenase from <Emphasis Type="Italic">Issatchenkia terricola</Emphasis> strain XJ-2

Acetaldehyde is a known mutagen and carcinogen. Active aldehyde dehydrogenase (ALDH) represents an important mechanism for acetaldehyde detoxification. A yeast strain XJ-2 isolated from grape samples was found to produce acetaldehyde dehydrogenase with a high activity of 2.28 U/mg and identified as Issatchenkia terricola. The enzyme activity was validated by oxidizing acetaldehyde to acetate with NAD⁺ as coenzyme based on the headspace gas chromatography analysis. A novel acetaldehyde dehydrogenase gene (ist-ALD) was cloned by combining SiteFinding-PCR and self-formed adaptor PCR. The ist-ALD gene comprised an open reading frame of 1,578 bp and encoded a protein of 525 amino acids. The predicted protein of ist-ALD showed the highest identity (73%) to ALDH from Pichia angusta. The ist-ALD gene was expressed in Escherichia coli, and the gene product (ist-ALDH) presented a productivity of 442.3 U/mL cells. The purified ist-ALDH was a homotetramer of 232 kDa consisting of 57 kDa-subunit according to the SDS-PAGE and native PAGE analysis. Ist-ALDH exhibited the optimal activity at pH 9.0 and 40°C, respectively. The activity of ist-ALDH was enhanced by K⁺, NH4⁺, dithiothreitol, and 2-mercaptoethanol but strongly inhibited by Ag⁺, Hg²⁺, Cu²⁺, and phenylmethyl sulfonylfluoride. In the presence of NAD⁺, ist-ALDH could oxidize many aliphatic, aromatic, and heterocyclic aldehydes, preferably acetaldehyde. Kinetic study revealed that ist-ALDH had a k _cat value of 27.71/s and a k _cat/K _m value of 26.80 × 10³/(mol s) on acetaldehyde, demonstrating ist-ALDH, a catalytically active enzyme by comparing with other ALDHs. These studies indicated that ist-ALDH was a potential enzymatic product for acetaldehyde detoxification.     

Physiological characterization of non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from agro-industrial and environmental origins with possible probiotic function

Probiotics are defined as live microorganisms, which when administered in adequate amount confer a health benefit to the host. Most of studied or commercialized probiotics contain bacteria and very few of them present yeast in its composition. In this last case, the microorganisms almost always belong to Saccharomyces genus. In the present study, it was of interest to screen among 103 non-Saccharomyces yeasts a candidate for probiotic by using in vitro and in vivo criteria. In vitro assays included growth at 37°C and production of antagonistic compounds against enteropathogenic indicators, and the in vivo assays evaluated the colonization ability of mouse gastrointestinal tract without pathologic consequences and the protective ability in mice experimentally challenged with Clostridium difficile. In conclusion, Pichia kluyveri strain 898 showed to be a potential candidate for probiotic use, based on the criteria cited above, particularly as demonstrated by its protective effect against experimental infection in mice. Interestingly, an in vivo inhibition against C. difficile observed in the animal models did not correlate with the results obtained with the in vitro assays.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-phenylalanine from glycerol by a recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

The production of l-phenylalanine is conventionally carried out by fermentations that use glucose or sucrose as the carbon source. This work reports on the use of glycerol as an inexpensive and abundant sole carbon source for producing l-phenylalanine using the genetically modified bacterium Escherichia coli BL21(DE3). Fermentations were carried out at 37°C, pH 7.4, using a defined medium in a stirred tank bioreactor at various intensities of impeller agitation speeds (300–500 rpm corresponding to 0.97–1.62 m s⁻¹ impeller tip speed) and aeration rates (2–8 L min⁻¹, or 1–4 vvm). This highly aerobic fermentation required a good supply of oxygen, but intense agitation (impeller tip speed ~1.62 m s⁻¹) reduced the biomass and l-phenylalanine productivity, possibly because of shear sensitivity of the recombinant bacterium. Production of l-phenylalanine was apparently strongly associated with growth. Under the best operating conditions (1.30 m s⁻¹ impeller tip speed, 4 vvm aeration rate), the yield of l-phenylalanine on glycerol was 0.58 g g⁻¹, or more than twice the best yield attainable on sucrose (0.25 g g⁻¹). In the best case, the peak concentration of l-phenylalanine was 5.6 g L⁻¹, or comparable to values attained in batch fermentations that use glucose or sucrose. The use of glycerol for the commercial production of l-phenylalanine with E. coli BL21(DE3) has the potential to substantially reduce the cost of production compared to sucrose- and glucose-based fermentations.     

Metabolic engineering of <Emphasis Type="Italic">Escherichia coli</Emphasis> for improving <Emphasis Type="SmallCaps">l</Emphasis>-3,4-dihydroxyphenylalanine (<Emphasis Type="SmallCaps">l</Emphasis>-DOPA) synthesis from glucose

l-3,4-dihydroxyphenylalanine (l-DOPA) is an aromatic compound employed for the treatment of Parkinson's disease. Metabolic engineering was applied to generate Escherichia coli strains for the production of l-DOPA from glucose by modifying the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and aromatic biosynthetic pathways. Carbon flow was directed to the biosynthesis of l-tyrosine (l-Tyr), an l-DOPA precursor, by transforming strains with compatible plasmids carrying genes encoding a feedback-inhibition resistant version of 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase, transketolase, the chorismate mutase domain from chorismate mutase-prephenate dehydratase from E. coli and cyclohexadienyl dehydrogenase from Zymomonas mobilis. The effects on l-Tyr production of PTS inactivation (PTS⁻ gluc⁺ phenotype), as well as inactivation of the regulatory protein TyrR, were evaluated. PTS inactivation caused a threefold increase in the specific rate of l-Tyr production (q _l-Tyr), whereas inactivation of TyrR caused 1.7- and 1.9-fold increases in q _l-Tyr in the PTS⁺ and the PTS⁻ gluc⁺ strains, respectively. An 8.6-fold increase in l-Tyr yield from glucose was observed in the PTS⁻ gluc⁺ tyrR ⁻ strain. Expression of hpaBC genes encoding the enzyme 4-hydroxyphenylacetate 3-hydroxylase from E. coli W in the strains modified for l-Tyr production caused the synthesis of l-DOPA. One of such strains, having the PTS⁻ gluc⁺ tyrR ⁻ phenotype, displayed the best production parameters in minimal medium, with a specific rate of l-DOPA production of 13.6 mg/g/h, l-DOPA yield from glucose of 51.7 mg/g and a final l-DOPA titer of 320 mg/l. In a batch fermentor culture in rich medium this strain produced 1.51 g/l of l-DOPA in 50 h.     

Medium Selection and Effect of Higher Oxygen Concentration Pulses on <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> var<Emphasis Type="Italic">. lepidiotum</Emphasis> Conidial Production and Quality

Rice and oat flours were analyzed as media for the production of conidia by M. anisopliae var. lepidiotum. The presence of peptone increased conidia yield regardless of the substrate used; however, the highest yield was achieved on oat flour media. The effect of oxygen on conidia production using oat-peptone medium was also studied at two levels: Normal atmosphere (21% O₂) and Oxygen-rich pulses (26% O₂). Maximum conidia production (4.25 × 10⁷ conidia cm⁻²) was achieved using 26% O₂ pulses after 156 h of culture, which was higher than 100% relative to conidial levels under normal atmosphere. Conidia yield per gram of biomass was 2.6 times higher with 26% O₂ (1.12 × 10⁷ conidia mg⁻¹). Conidia quality parameters, such as germination and hydrophobicity, did not show significant differences (P < 0.05) between those treatments. Bioassays parameters, using Tenebrio molitor adults, were analyzed for conidia obtained in both atmospheres and data were fitted to an exponential model. The specific mortality rates were 2.22 and 1.26 days⁻¹, whereas lethal times for 50% mortality were 3.90 and 4.31 days, for 26% O₂ pulses and 21% O₂ atmosphere, respectively. These results are relevant for production processes since an oxygen increase allowed superior levels of conidia by M. anisopliae without altering quality parameters and virulence toward Tenebrio molitor adults.