A novel strategy to construct multi-strain starter cultures: an insight to evolve from natural to directed fermentation.

Some biotechnological strategies have succeeded in the attempt to imitate natural fermentation, and bioprocesses have been efficiently designed when the product is the result of a unique biological reaction. However, when the process requires more than one biological reaction, few bioprocesses have been successfully designed because the available tools to construct multi-strain starter cultures are not yet well defined. In this work, a novel experimental strategy to construct multi-strain starter cultures with selected native microorganisms from natural fermentation is proposed. The strategy analyses, selects, and defines the number and proportion of each strain that should form a starter culture to be used in directed fermentations. It was applied to evolve natural fermentation to directed fermentation in distilled agave production. The results showed that a starter culture integrated by Kluyveromyces marxianus, Clavispora lusitaniae, and Kluyveromyces marxianus var. drosophilarum in proportions of 35, 32, and 33%, respectively, allows obtaining fermented agave juice containing a 2.1% alcohol yield and a distilled product with a broad profile of aromatic compounds. Hence, the results show, for the first time, a tool that addresses the technical challenge for multi-strain starter culture construction, offering the possibility of preserving the typicity and genuineness of the original traditional product.

A validated strategy to design efficient fermentation-industrial processes: agave spirit production.

An industrial process is profitable when its individual unit operations are efficient and thus, this work shows a guideline for designing efficient fermentation-industrial processes for agave distilled production based on a sequential approach of optimization, beginning in the laboratory and followed by the adjustment of the variable values using the evolutionary operation method for successful process scaling. The results at the laboratory showed that a starter inoculum containing a 5 × 106 cells/mL mixture of Kluyveromyces marxianus, Clavispora lusitaniae, and Kluyveromyces marxianus var. drosophilarum strains in a bioreactor containing agave syrup with 120 g/L fermented sugar, processed at a constant temperature of 33 °C and 1.0 VVM aeration for 1.6 h, led to a fermented product with a 4.18% (v/v) alcohol content after 72 h of processing time. The scale-up process results showed that the best operating conditions at the pilot-plant level were a temperature of 35 °C and aeration at 1.0 VVM for 1.2 h, which led to a fermented product with a 4.22% (v/v) total alcohol content after 72 h of processing time. These represent similar performance values for both production processes, but each one worked with their specific values of process variables, which demonstrates that each level of production had its own specific values for process variables. The volatile compound analysis shows that both distilled products contained a similar profile of volatile components that provide fruity and ethereal aromatic notes pleasant to the palate. Therefore, the process design for agave spirit production at the semi-industrial level was successfully achieved.