A strategy to optimize biotechnological process design is illustrated for the production of fructose-rich syrups via enzymatic hydrolysis of agave fructo-oligosaccharides. The optimization process includes ecological studies from natural fermentations leading to the selection of a strain with capacity for inulinase synthesis, and variable optimization for the synthesis, and enzymatic hydrolysis using the response surface methodology. The results lead to the selection of Kluyveromyces marxianus , endogenous strains isolated from aguamiel (natural fermented sugary sap from agave plants), as the main strain with high capacity for enzyme synthesis with inulinase activity. Production optimization at bioreactor level revealed that operation at 30.6 degrees C, 152 rpm, 1.3 VVM of aeration, and pH 6.3 leads to maximum inulinase synthesis, whereas 31 degrees C, 50 rpm, and pH 6.2 leads to maximum hydrolysis of agave fructo-oligosaccharides. HPLC analysis of the fructose-rich syrups obtained at these optimal conditions showed an average composition of 95% of fructose and 5% of glucose and the absence of sucrose. The analysis also revealed that the syrups are free of residues and toxic compounds, an undesirable occurrence often present when traditional methods based on thermal or acid hydrolysis are applied for their obtainment. Therefore, the product may be suitable for use as additive in many applications in the food and beverage industries.

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.

Enzymatic treatments using noncommercial enzymes as a means to the improve the extraction of carotenoids and capsaicinoids from chili fruits are explored in this study. The results show that it is possible to obtain chili fruit powder with a higher concentration of both capsaicinoids and carotenoids than previously reported for similar processes. Furthermore, extraction yields above 96% for carotenoids and 85% for capsaicinoids as separate fractions can be achieved using a sequential and selective two-stage extraction. Evidence is presented demonstrating that the content and extraction yield depend directly on the extent of the enzymatic hydrolysis of chili cell walls, and higher yields are obtained when the sample is completely hydrolyzed. The enzymatic treatment described here is a promising alternative to current industrial practices, and it improves the extraction of carotenoids and capsaicinoids from chili fruits.

Endogenous microorganisms isolated from the marigold flower (Tagetes erecta) were studied to understand the events taking place during its ensilage. Studies of the cellulase enzymatic activity and the ensilage process were undertaken. In both studies, the use of approximate second-order models and multiple lineal regression, within the context of an experimental mixture design using the response surface methodology as optimization strategy, determined that the microorganisms Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans are the most significant in marigold flower ensilage and exhibit high cellulase activity. A mixed culture comprised of 9.8% Flavobacterium IIb, 41% A. anitratus, and 49.2% R. nigricans used during ensilage resulted in an increased yield of total xanthophylls extracted of 24.94 g/kg of dry weight compared with 12.92 for the uninoculated control ensilage.

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In this work is studied the effect of a noncommercial enzyme preparation on xanthophyll extraction from marigold flower (Tagetes erecta). The enzymatic extract was synthesized by endogenous microorganisms previously isolated and identified as Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans. The results show that the extraction yield depends directly on the extent of the enzymatic hydrolysis of cell walls in the flower petals and that it is possible to reach yields in excess of those previously reported for treatments with commercially available enzymes (29.3 g/kg of dry weight). HPLC analysis of the product indicates that the original xanthophyll profile is not altered. The enhanced extraction system appears to be very competitive when compared to the traditional process and current alternatives.

Marigold flowers are the main natural source of xanthophylls, and marigold saponified extract is used as an additive in several food and pharmaceutical industries. In this work, the use of a solid-state fermentation (ensilage) process for increasing the yield of xanthophylls extracted from fermented marigold flowers was examined. The process consisted of a mixed culture of three microorganisms (Flavobacterium IIb, Acinetobacter anitratus, and Rhizopus nigricans), part of the normal microbiota associated with the marigold flower. These microorganisms had been previously isolated, and were identified as relevant for the ensilage process due to their capacity to produce cellulolytic enzymes. Based on experimental design strategies, optimum operation values were determined for aeration, moisture, agitation, and marigold-to-inoculum ratio in the proposed solid-state fermentation equipment, leading to a xanthophylls yield of 17.8-g/kg dry weight. The optimum achieved represents a 65% increase with respect to the control. HPLC analysis indicated conservation of extracted oleoresin. Based on the experimental results, interactions were identified that could be associated with the heat and mass-transfer reactions taking place within the bioreactor. The insight gained allows conditions that limit growth and metabolic activity to be avoided.

The design of many separation processes is often hindered by the lack of distribution data between two immiscible phases. Inappropriate or partial designs result in inefficiencies that translate into poor recovery of the products, overdesigned separation equipment, or additional purification and separation stages. In recent years, statistically based experimental design has been successfully used to quantify relevant factors in many biological and chemical processes. In this study, we illustrate the capabilities of one of these approaches, the response surface methodology (RSM) for the optimization of lixiviation processes of marigold flower flour for xanthophyll extraction (oleoresin) using hexane. The method leads to conditions for the maximum recovery of the pigment, allowing the determination of the optimal residence time (14.7 min) and flour-to-solvent ratio (1: 5) at 35 °C. These values can be used in the lixiviation process in any operation mode. In this study, we illustrate the use of a countercurrent process that allows recovery of 97.5% of the oleoresin.