Fermentative capabilities of native yeast strains grown on juices from different Agave species used for tequila and mezcal production.

The Asparagaceae family is endemic from America, being the Agave genus the most important. The Agave species possess economic relevance and are use as raw material to produce several distilled alcoholic beverages, as bacanora, tequila, and mezcal. The fermentation process has been carry out either spontaneously or by adding a selected yeast strain. The latter is generally responsible for the production of ethanol and volatile compounds. This study comprised five Agave species (A. angustifolia, A. cupreata, A. durangensis, A. salmiana, and A. tequilana) and eight endogenous yeast strains: five of them were non-Saccharomyces (Torulaspora delbrueckii, Zygosaccharomyces bisporus, Candida ethanolica, and two Kluyveromyces marxianus) and three Saccharomyces cerevisiae strains. The results showed that the S. cerevisiae strains were not able to grow on A. durangensis and A. salmiana juices. The Kluyveromyces marxianus strains grew and fermented all the agave juices and displayed high ethanol production (48-52 g L-1) and volatile compounds. The ethanol production was higher on A. angustifolia juice (1.1-2.8-fold), whereas the volatile compound was dependent on both yeast strain and the Agave species. The use of endogenous non-Saccharomyces yeast strains is feasible, as they may outperform S. cerevisiae regarding the production of fermented beverages from agave plants with a high content of ethanol and aromatic compounds. Graphical abstract.

Growth Modeling of Aspergillus niger Strains Isolated from Citrus Fruit as a Function of Temperature on a Synthetic Medium from Lime (Citrus latifolia T.) Pericarp.

Molds are responsible for postharvest spoilage of citrus fruits. The objective of this study was to evaluate the effect of temperature on growth rate and the time to visible growth of Aspergillus niger strains isolated from citrus fruits. The growth of these strains was studied on agar lime medium (AL) at different temperatures, and growth rate was estimated using the Baranyi and Roberts model (Int. J. Food Microbiol. 23:277-294, 1994). The Rosso et al. cardinal model with inflexion (L. Rosso, J. R. Lobry, S. Bajard, and J. P. Flandrois, J. Theor. Biol. 162:447-463, 1993) was used as a secondary model to describe the effect of temperature on growth rate and the lag phase. We hypothesized that the same model could be used to calculate the time for the mycelium to become visible (tv) by substituting the lag phase (1/λ and 1/λopt) with the time to visible colony (1/tv-opt and 1/tv), respectively, in the Rosso et al. MODEL: High variability was observed at suboptimal conditions. Extremes of temperature of growth for A. niger seem to have a normal variability. For the growth rate and time tv, the model was satisfactorily compared with results of previous studies. An external validation was performed in lime fruits; the bias and accuracy factors were 1.3 and 1.5, respectively, for growth rate and 0.24 and 3.72, respectively, for the appearance time. The discrepancy may be due to the influence of external factors. A. niger grows significantly more slowly on lime fruit than in culture medium, probably because the nutrients are more easily available in medium than in fruits, where the peel consistency may be a physical barrier. These findings will help researchers understand the postharvest behavior of mold on lime fruits, host-pathogen interactions, and environmental conditions infecting fruit and also help them develop guidelines for future work in the field of predictive mycology to improve models for control of postharvest fungi.

Performance evaluation of Pichia kluyveri, Kluyveromyces marxianus and Saccharomyces cerevisiae in industrial tequila fermentation.

Traditionally, industrial tequila production has used spontaneous fermentation or Saccharomyces cerevisiae yeast strains. Despite the potential of non-Saccharomyces strains for alcoholic fermentation, few studies have been performed at industrial level with these yeasts. Therefore, in this work, Agave tequilana juice was fermented at an industrial level using two non-Saccharomyces yeasts (Pichia kluyveri and Kluyveromyces marxianus) with fermentation efficiency higher than 85 %. Pichia kluyveri (GRO3) was more efficient for alcohol and ethyl lactate production than S. cerevisiae (AR5), while Kluyveromyces marxianus (GRO6) produced more isobutanol and ethyl-acetate than S. cerevisiae (AR5). The level of volatile compounds at the end of fermentation was compared with the tequila standard regulation. All volatile compounds were within the allowed range except for methanol, which was higher for S. cerevisiae (AR5) and K. marxianus (GRO6). The variations in methanol may have been caused by the Agave tequilana used for the tests, since this compound is not synthesized by these yeasts.

Effect of Agave tequilana 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.

Effects of the addition of different nitrogen sources in the tequila fermentation process at high sugar concentration.

AIMS: To study the effect of the addition of different nitrogen sources at high sugar concentration in the tequila fermentation process. METHODS AND RESULTS: Fermentations were performed at high sugar concentration (170 g l(-1)) using Agave tequilana Weber blue variety with and without added nitrogen from different sources (ammonium sulfate; glutamic acid; a mixture of ammonium sulfate and amino acids) during the exponential phase of growth. All the additions increased the fermentation rate and alcohol efficiency. The level of synthesis of volatile compounds depended on the source added. The concentration of amyl alcohols and isobutanol were decreased while propanol and acetaldehyde concentration increased. CONCLUSIONS: The most efficient nitrogen sources for fermentation rate were ammonium sulfate and the mixture of ammonium sulfate and amino acids. The level of volatile compounds produced depended upon types of nitrogen. The synthesis of some volatile compounds increased while others decreased with nitrogen addition. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of nitrogen could be a strategy for improving the fermentation rate and efficiency in the tequila fermentation process at high sugar Agave tequilana concentration. Furthermore, the sensory quality of the final product may change because the synthesis of the volatile compounds is modified.

Optimization of astaxanthin production by Phaffia rhodozyma through factorial design and response surface methodology.

Sequential methodology based on the application of three types of experimental designs was used to optimize the astaxanthin production of the mutant strain 25-2 of Phaffia rhodozyma in shake flask cultures. The first design employed was a factorial design 2(5), where the factors studied were: pH, temperature, percent of inoculum, carbon and nitrogen concentrations, each one at two levels. This design was performed in two medium types: rich YM medium and minimal medium, based on date juice (Yucca medium). With this first design the most important factors were determined (carbon concentration and temperature) that were used in the second experimental strategy: the method of steepest ascent was applied in order to rapidly approach the optimum. Finally, a second-order response surface design was applied using temperature and carbon concentration as factors. The optimal conditions stimulating the highest astaxanthin production were: 19.7 degrees C temperature; 11.25 g l(-1) carbon concentration; 6.0 pH; 5% inoculum and 0.5 g l(-1) nitrogen concentration. Under these conditions the astaxanthin production was 8100 microg l(-1), 92% higher than the production under the initial conditions.

Effects of pulse addition of carbon sources on continuous cultivation of Escherichia coli containing a recombinant E. coli gapA gene.

At high glucose concentrations, Escherichia coli produces acetate (Crabtree effect). To look for the influence of glucose and/or acetate in the medium on the expression of a recombinant gene in E. coli, the effect of a pulse addition of glucose, on transcription of a cloned E. coli gapA gene and the resulting glyceraldehyde-3P-dehydrogenase activity (GAPDH), was tested during continuous cultivation of E. coli HB101 transformed with the plasmid pBR::EcogapA. Stable continuous cultures were established in a semi-synthetic medium supplemented with 5 g/L of glucose. After the addition of 7 g of glucose within a few seconds, gapA gene expression was strongly and very rapidly induced. As shown by primer-extension analysis, promoter P1, one of the four transcriptional promoters of the gapA gene, was strongly activated, and GAPDH activity increased. However, after rapid glucose consumption, acetate was produced and acetate concentrations above 2 g/L induced stress conditions. This is shown by a strong activation of promoter P2, that is recognized by the stress specific Esigma32 RNA polymerase. During this period, the total cellular RNA content was strongly diminished. Later, when acetate was partially consumed a high level of total RNA was restored, translation was efficient and a regular increase of the GAPDH-specific activity was observed. The transitions between glucose metabolism, acetate production and the end of acetate consumption, were marked by large increases in RNase and protease activities. For comparison, pulse-addition experiments were also performed with serine and alanine. A transient increase of GAPDH production associated with an increase in biomass was also found for serine that can be utilized as an energy source, whereas the addition of alanine, which is only incorporated into newly synthesized proteins, did not increase GAPDH production. The implication of these data for overproduction of recombinant proteins in E. coli is discussed.

Glucose and acetate influences on the behavior of the recombinant strain Escherichia coli HB 101 (GAPDH).

This study highlights data about the production of a recombinant protein (glyceraldehyde-3-phosphate dehydrogenase) by E. coli HB 101 (GAPDH) during batch and fed-batch fermentations in a complex medium. From a small number of experiments, this strain has been characterized in terms of protein production performance and glucose and acetate influences on growth and recombinant protein production. The present results show that this strain is suitable for recombinant protein production, in fed-batch culture 55 g L-1 of biomass and 6 g L-1 of GAPDH are obtained. However this strain, and especially GAPDH overproduction is sensitive to glucose availability. During fermentations, maximum yields of GAPDH production have been obtained in batch experiments for glucose concentration of 10 g L-1, and in fed-batch experiments for glucose availability of 10 g h-1 (initial volume 1.5 L). The growth of the strain and GAPDH overproduction are also inhibited by acetate. Moreover acetate has been noted as an activator of its own formation.