Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> juice on cell wall polysaccharides of three <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strains from different origins

In this study, a characterization of cell wall polysaccharide composition of three yeasts involved in the production of agave distilled beverages was performed. The three yeast strains were isolated from different media (tequila, mezcal and bakery) and were evaluated for the β(1,3)-glucanase lytic activity and the β-glucan/mannan ratio during the fermentation of Agave tequilana juice and in YPD media (control). Fermentations were performed in shake flasks with 30 g l⁻¹ sugar concentration of A. tequilana juice and with the control YPD using 30 g l⁻¹ of glucose. The three yeasts strains showed different levels of β-glucan and mannan when they were grown in A. tequilana juice in comparison to the YPD media. The maximum rate of cell wall lyses was 50% lower in fermentations with A. tequilana juice for yeasts isolated from tequila and mezcal than compared to the bakery yeast.     

Effect of <Emphasis Type="Italic">Agave tequilana</Emphasis> age,cultivation field location and yeast strain on tequila fermentation process

The effect of yeast strain, the agave age and the cultivation field location of agave were evaluated using kinetic parameters and volatile compound production in the tequila fermentation process. Fermentations were carried out with Agave juice obtained from two cultivation fields (CF1 and CF2), as well as two ages (4 and 8 years) and two Saccharomyces cerevisiae yeast strains (GU3 and AR5) isolated from tequila fermentation must. Sugar consumption and ethanol production varied as a function of cultivation field and agave age. The production of ethyl acetate, 1-propanol, isobutanol and amyl alcohols were influenced in varying degrees by yeast strain, agave age and cultivation field. Methanol production was only affected by the agave age and 2-phenylethanol was influenced only by yeast strain. This work showed that the use of younger Agave tequilana for tequila fermentation resulted in differences in sugar consumption, ethanol and volatile compounds production at the end of fermentation, which could affect the sensory quality of the final product.     

Fermentation behaviour and volatile compound production by agave and grape must yeasts in high sugar Agave tequilana and grape must fermentations

Few studies have been performed on the characterization of yeasts involved in the production of agave distilled beverages and their individual fermentation properties. In this study, a comparison and evaluation of yeasts of different origins in the tequila and wine industries were carried out for technological traits. Fermentations were carried out in high (300 g l⁻¹) and low (30 g l⁻¹) sugar concentrations of Agave tequilana juice, in musts obtained from Fiano (white) and Aglianico (red) grapes and in YPD medium (with 270 g l⁻¹ of glucose added) as a control. Grape yeasts exhibited a reduced performance in high-sugar agave fermentation, while both agave and grape yeasts showed similar fermentation behaviour in grape musts. Production levels of volatile compounds by grape and agave yeasts differed in both fermentations.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Fermentation of Agave tequilana juice by Kloeckera africana: influence of amino-acid supplementations

This study aimed to improve the fermentation efficiency of Kloeckera africana K1, in tequila fermentations. We investigated organic and inorganic nitrogen source requirements in continuous K. africana fermentations fed with Agave tequilana juice. The addition of a mixture of 20 amino-acids greatly improved the fermentation efficiency of this yeast, increasing the consumption of reducing sugars and production of ethanol, compared with fermentations supplemented with ammonium sulfate. The preference of K. africana for each of the 20 amino-acids was further determined in batch fermentations and we found that asparagine supplementation increased K. africana biomass production, reducing sugar consumption and ethanol production (by 30, 36.7 and 45%, respectively) over fermentations supplemented with ammonium sulfate. Therefore, asparagine appears to overcome K. africana nutritional limitation in Agave juice. Surprisingly, K. africana produced a high concentration of ethanol. This contrasts to poor ethanol productivities reported for other non-Saccharomyces yeasts indicating a relatively high ethanol tolerance for the K. africana K1 strain. Kloeckera spp. strains are known to synthesize a wide variety of volatile compounds and we have shown that amino-acid supplements influenced the synthesis by K. africana of important metabolites involved in the bouquet of tequila. The findings of this study have revealed important nutritional limitations of non-Saccharomyces yeasts fermenting Agave tequilana juice, and have highlighted the potential of K. africana in tequila production processes.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

Selenium-mediated differential response of β-glucosidase and β-galactosidase of germinatingTrigonella foenum-graecum

β-Glucosidase and β-galactosidase activity profile tested in different seeds during 24 h germination revealed reasonably high levels of activity inVigna radiata, Cicer arietinum, andTrigonella foenum-graecum. In all seeds tested, β-galactosidase activity was, in general, higher than that of β-glucosidase.T. foenum-graecum seedlings exhibited maximal total and specific activities for both the enzymes during 72 h germination. Se supplementation as Na₂SeO₃ up to 0.75 ppm was found to be beneficial to growth and revealed selective enhancement of β-galactosidase activity by 40% at 0.5 ppm Se. The activities of both the enzymes drastically decreased at 1.0 ppm level of Se supplementation. On the contrary, addition of Na₂SeO₃ in vitro up to 1 ppm to the enzyme extracts did not influence these activities. Hydrolytic rates of β-glucosidase in both control and Se-supplemented groups were enhanced by 20% with 0.05M glycerol in the medium and 30% at 0.1M glycerol. The rates were marginally higher in Se-supplemented seedlings than the controls, irrespective of added glycerol in the medium. In contrast, hydrolysis by β-galactosidase showed a trend of decrease in Se-supplemented seedlings compared to the control, when glycerol was present in the medium. Addition of Se in vitro in the assay medium showed no difference in the hydrolytic rate by β-galactosidase when compared to control, while the activity of β-glucosidase declined by 50%. Se-grown seedlings showed an enhancement of transglucosidation rate by 40% in the presence of 0.1M glycerol. The study reveals a differential response to Se among the β-galactosidase and β-glucosidase ofT. foenumgraecum with increase in the levels of β-galactosidase activity.     

Supercritical Extraction of Carotenoids from Rosa canina L. Hips and their Formulation with β-Cyclodextrin

The purpose of this research was to preliminary assess the suitability of a new method for the preparation of a solid formulation in form of powder composed by β-cyclodextrin and the supercritical extract of Rosa canina hips. The method implies the extraction of carotenoids, in particular β-carotene, from freeze dried fruits of R. canina with supercritical CO₂ at 70 °C and 300 bar, in the presence of varying quantity of ethanol as entrainer. The obtained supercritical solution is then expanded at ambient conditions into an aqueous solution of β-cyclodextrin to favour the interaction between β-cyclodextrin and the lipophilic components of the extract. β-carotene solubility (mole fraction) in supercritical CO₂ or in supercritical CO₂/ethanol mixtures were in the order of 1 10⁻⁷. The β-carotene extracted from R. canina fruits (nearly 10 μg/g of dry matrix), interacts almost quantitatively with β-cyclodextrin affording a solid phase, which presents a low apparent solubility in water. Finally the interaction with β-cyclodextrin results in a higher concentration of the β-carotene trans- form relative to the cis- form in the extracted product when collected in an aqueous solution of β-cyclodextrin with respect to the extract in n-hexane.     

Identification of thermostable β-xylosidase activities produced by <Emphasis Type="Italic">Aspergillus brasiliensis</Emphasis> and <Emphasis Type="Italic">Aspergillus niger</Emphasis>

Twenty Aspergillus strains were evaluated for production of extracellular cellulolytic and xylanolytic activities. Aspergillus brasiliensis, A. niger and A. japonicus produced the highest xylanase activities with the A. brasiliensis and A. niger strains producing thermostable β-xylosidases. The β-xylosidase activities of the A. brasiliensis and A. niger strains had similar temperature and pH optima at 75°C and pH 5 and retained 62% and 99%, respectively, of these activities over 1 h at 60°C. At 75°C, these values were 38 and 44%, respectively. Whereas A. niger is a well known enzyme producer, this is the first report of xylanase and thermostable β-xylosidase production from the newly identified, non-ochratoxin-producing species A. brasiliensis.     

Bioconversion of Agave tequilana fructans by exo-inulinases from indigenous Aspergillus niger CH-A-2010 enhances ethanol production from raw Agave tequilana juice

Agave tequilana fructans are the source of fermentable sugars for the production of tequila. Fructans are processed by acid hydrolysis or by cooking in ovens at high temperature. Enzymatic hydrolysis is considered an alternative for the bioconversion of fructans. We previously described the isolation of Aspergillus niger CH-A-2010, an indigenous strain that produces extracellular inulinases. Here we evaluated the potential application of A. niger CH-A-2010 inulinases for the bioconversion of A. tequilana fructans, and its impact on the production of ethanol. Inulinases were analyzed by Western blotting and thin layer chromatography. Optimal pH and temperature conditions for inulinase activity were determined. The efficiency of A. niger CH-A-2010 inulinases was compared with commercial enzymes and with acid hydrolysis. The hydrolysates obtained were subsequently fermented by Saccharomyces cerevisiae to determine the efficiency of ethanol production. Results indicate that A. niger CH-A-2010 predominantly produces an exo-inulinase activity. Optimal inulinase activity occurred at pH 5.0 and 50 °C. Hydrolysis of raw agave juice by CH-A-2010 inulinases yielded 33.5 g/l reducing sugars, compared with 27.3 g/l by Fructozyme^® (Novozymes Corp, Bagsværd, Denmark) and 29.4 g/l by acid hydrolysis. After fermentation of hydrolysates, we observed that the conversion efficiency of sugars into ethanol was 97.5 % of the theoretical ethanol yield for enzymatically degraded agave juice, compared to 83.8 % for acid-hydrolyzed juice. These observations indicate that fructans from raw Agave tequilana juice can be efficiently hydrolyzed by using A. niger CH-A-2010 inulinases, and that this procedure impacts positively on the production of ethanol.     

Screening of β-fructofuranosidase-producing microorganisms and effect of pH and temperature on enzymatic rate

Seventeen different strains of filamentous fungi were grown in batch cultures to compare their abilities for the production of β-fructofuranosidase. Three of them, Aspergillus oryzae IPT-301, Aspergillus niger ATCC 20611 and strain IPT-615, showed high production with total fructosyltransferase activity higher than 12,500 units l⁻¹. In addition, the β-fructofuranosidases of those strains have a high fructosyltransferase activity-to-hydrolytic activity ratio. The temperature and pH effects on the sucrose-β-fructofuranosidase reaction rate were studied using a 2² factorial experimental design. The comparative analysis of the tested variable coefficients shows that the variable pH contributes mostly to the changes in the fructosyltransferase and hydrolytic rates and in the V _t/V _h ratio. At 40 and 50°C, there were no significant differences between the fructosyltransferase and hydrolytic velocities of these enzymes.     

Inulinase-hyperproducing strains of Kluyveromyces sp. isolated from aguamiel (Agave sap) and pulque

Summary Two strains of Kluyveromyces marxianus (A1 and A2) isolated from ‘aguamiel’ (agave sap) and one strain of K. lactis var. lactis (P7) isolated from ‘pulque’ (its fermented product), were studied to make a survey of inulinase production. The strains of K. marxianus A1 and A2 were the best producers of inulinase, giving up to 2.5 times more enzyme than the control hyperproducing strain K. marxianus CDBB-L-278, and showed lower catabolic repression than this. One strain isolated from pulque was identified as K. lactis var. lactis and was also an excellent inulinase producer, being the first strain of this species reported as such. These strains were very good inulinase producers and they had low susceptibility to catabolic repression probably because the source from which they were isolated was rich in sucrose and oligofructans. They can be used in the transformation of inulin to produce fructose and/or oligofructans.     

Substrates’ and products’ inhibition in fructanase production by a new Kluyveromyces marxianus CF15 from Agave tequilana fructan in a batch reactor

This study focuses on fructanase production in a batch reactor by a new strain isolated from agave juice (K. marxianus var. drosophilarum) employing different Agave tequilana fructan (ATF) concentrations as substrate. The experimental data suggest that the fructanase production may be inhibited or repressed by high substrate (50 g/L) and ethanol (20.7 g/L) concentrations present in culture medium. To further analyze these phenomena an unstructured kinetic mathematical model taking into account substrate and products inhibition was proposed and fitted. The mathematical model considers six reaction kinetics and the ethanol evaporation, and predicts satisfactorily the biomass, fructan, glucose, fructose, ethanol, and fructanase behavior for different raw material initial concentrations. The proposed model is the first to satisfactorily describe the production of fructanase from branched ATF with a new strain of K. marxianus.

     

Substrate specificity of a recombinant chicken <Emphasis Type="Italic">β</Emphasis>-carotene 15,15′-monooxygenase that converts <Emphasis Type="Italic">β</Emphasis>-carotene into retinal

The recombinant β-carotene 15,15′-monooxygenase from chicken liver was purified as a single 60 kDa band by His-Trap HP and Resource Q chromatography. It had a molecular mass of 240 kDa by gel filtration indicating the native form to be tetramer. The enzyme converted β-carotene under maximal conditions (pH 8.0 and 37°C) with a k _cat of 1.65 min⁻¹ and a K _m of 26 μM and its conversion yield of β-carotene to retinal was 120% (mol mol⁻¹). The enzyme displayed catalytic efficiency and conversion yield for β-carotene, β-cryptoxanthin, β-apo-8′-carotenal, β-apo-4′-carotenal, α-carotene and γ-carotene in decreasing order but not for zeaxanthin, lutein, β-apo-12′-carotenal and lycopene, suggesting that the presence of one unsubstituted β-ionone ring in a substrate with a molecular weight greater than C₃₀ seems to be essential for enzyme activity.     

Poly-β-hydroxyalkanoate production by halotolerant <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> U7

Medium optimization for production of poly-β-hydroxyalkanoate (PHA) from Rhodobacter sphaeroides U7 cultivated in glutamate–acetate (GA) medium supplemented with 40 mM valeric acid as co-substrate under aerobic-dark condition was investigated. Studies on effect of nitrogen source and cultivation temperature by conventional and statistical methods illustrated that (NH₄)₂SO₄ (0.2 g/l) had no effect and the optimal temperature was at 30°C. The optimum environmental conditions were found to be anaerobic-light (3000 lux) cultivation with aeration rate of 1.0 vvm and agitation speed of 200 rpm for PHA production (2.5 g/l) with the highest PHA content (65.15%) at 0.5 vvm, and 200 rpm. Under this optimized medium and condition, PHA production from R. sphaeroides U7 increased 3.86-folds (from 0.69 to 2.66 g/l) (PHA content increased 1.5-folds). The biopolymer was purified and characterized by using ¹³C NMR, FTIR, DSC, X-ray diffraction and intrinsic viscosity techniques to be a copolymer poly(β-hydroxybutyrate-co-β-hydroxyvalerate) (PHBV) consisting of 84.8 mol% β-hydroxybutyric acid (HB) and 15.2 mol% β-hydroxyvaleric acid (HV).     

Effect of Azospirillum Lectins on the Activities of Wheat-root Hydrolytic Enzymes

This work studied the effect of two cell-surface lectins isolated from the nitrogen-fixing soil bacterium Azospirillum brasilense Sp7 and from its mutant defective in hemagglutinating activity, A. brasilense Sp7.2.3, on the activities of α-glucosidase, β-glucosidase and β-galactosidase in the exocomponent, membrane and apoplast fractions of wheat-seedling roots. Lectin (40 μg mL⁻¹) incubation for 1 h of the plant fractions increased the enzymes’ activities; both wild-type and mutant lectins were most stimulatory to the activities of all the exocomponent-fraction enzymes studied and to the apoplast-fraction β-glucosidase. Pretreatment of the lectins with their carbohydrate hapten, L-fucose, lowered the effect. The observed differences in the lectins’ ability to influence enzyme catalytic activity are explained by change in the antigenic properties of the mutant lectin.     

Fermentative capability and aroma compound production by yeast strains isolated from Agave tequilana Weber juice

Five yeast strains isolated from agave juice were studied for their fermentative and aromatic capacity. The experiments were performed using agave juice supplemented with ammonium sulphate, as is commonly done in tequila distilleries. Three strains classified as Saccharomyces cerevisiae showed high biomass and ethanol production, as well as higher ethanol tolerance than those classified as Kloeckera africana and Kloeckera apiculata, which showed scarce growth. The results suggest that Kloeckera strains were affected by nutritional limitation and/or toxic compounds present in agave juice. Agave juice analyses showed a lower amino acid content than those reported in grape juice. S. cerevisiae strains produced predominantly amyl and isoamyl alcohols, n-propanol, 2-phenyl ethanol, succinic acid, glycerol, methanol, isoamyl acetate, ethyl hexanoate, acetaldehyde and isobutanol, whereas Kloeckera strains showed a high production of acetic acid, 2-phenyl ethyl acetate and ethyl acetate. The methanol concentration was significantly different among the yeasts studied. The diversity between three S. cerevisiae strains were higher for the aromatic profile than for genetic level and kinetic parameter. On the other hand, the diversity of Kloeckera yeasts were lower than Saccharomyces yeasts even when belonging to two different species.     

Influence of the curing of the killer phenotype inSaccharomyces cerevisiae wine strains on their fermentative behaviour

Fermentative behaviour and cell growth have been studied in grape juice inoculated either with two killerSaccharomyces cerevisiae wild strains or with their Acridine Orange-cured isogenic counterparts. The number of viable cells/ml at the beginning of the fermentation, as well as during exponential growth, were higher in grape juices inoculated with the cured strains. The CO₂ production, fermentative rate and ethanol and acetic acid production were also higher in the cured strains, particularly during the stage of active fermentation. These differences, however, were minimal at the end of the fermentations.     

Heterologous expression of Fusarium oxysporum tomatinase in Saccharomyces cerevisiae increases its resistance to saponins and improves ethanol production during the fermentation of Agave tequilana Weber var. azul and Agave salmiana must

This paper describes the effect of the heterologous expression of tomatinase from Fusarium oxysporum f. sp lycopersici in Saccharomyces cerevisiae. The gene FoTom1 under the control of the S. cerevisiae phosphoglycerate kinase (PGK1) promoter was cloned into pYES2. S. cerevisiae strain Y45 was transformed with this vector and URA3 transformant strains were selected for resistance to α-tomatine. Two transformants were randomly selected for further study (designated Y45-1 and Y45-2). Control strain Y45 was inhibited at 50 μM α-tomatine, in contrast, transformants Y45-1 and Y45-2 did not show inhibition at 200 μM. Tomatinase activity was detected by HPLC monitoring tomatine disappearance and tomatidine appearance in the supernatants of culture medium. Maximum tomatinase activity was observed in the transformants after 6 h, remaining constant during the following 24 h. No tomatinase activity was detected in the parental strain. Moreover, the transformants were able to grow and produce ethanol in a mix of Agave tequilana Weber var. azul and Agave salmiana must, contrary to the Y45 strain which was unable to grow and ferment under these conditions.     

Characterization of the carbohydrase productions of an ectomycorrhizal fungus, Tricholoma matsutake

To evaluate the potential of the production of the ectomycorrhizal fungus Tricholoma matsutake to produce carbohydrases, (1) the distribution of carbohydrase activities among the different strains (18 strains) was investigated and (2) the abilities of T. matsutake and saprophytic fungi to produce β-glucosidase were compared. The results showed that the carbohydrase productions patterns of T. matsutake still resemble one another. Moreover, this fungus exhibited markedly higher β-glucosidase than did the saprophytic mushrooms. Tricholoma matsutake showed weak production of α-amylase and α-glucosidase in a static cultur filtrate. On the other hand, glucoamylase activity was not observed. Surprisingly, we discovered that β-glucosidase demonstrated strong activity. This finding suggests that this fungus has saprotrophic abilities. The carbohydrase production systems in T. matsutake were characterized from our experimental results. Also, we point out some weak points in the carbohydrase production systems of T. matsutake.