Immobilized cell technology has shown a significant promotional effect on the fermentation of alcoholic beverages such as beer, wine and cider. However, genetic, morphological and physiological alterations occurring in immobilized yeast cells impact on aroma formation during fermentation processes. The focus of this review is exploitation of existing knowledge on the biochemistry and the biological role of flavour production in yeast for the biotechnological production of aroma compounds of industrial importance, by means of immobilized yeast. Various types of carrier materials and immobilization methods proposed for application in beer, wine, fruit wine, cider and mead production are presented. Engineering aspects with special emphasis on immobilized cell bioreactor design, operation and scale-up potential are also discussed. Ultimately, examples of products with improved quality properties within the alcoholic beverages are addressed, together with identification and description of the future perspectives and scope for cell immobilization in fermentation processes.

The dynamics of two wild type strains of Saccharomyces cerevisiae (BY4741 and EGY48) that vary in the ability to produce sterols were compared in batch cultures under different aeration conditions. Poor supply of oxygen enhanced selectivity of the bioprocess in favor of squalene formation. Optimization of inoculum size and fermentation time arranged according to a central composite statistical design revealed significant differences between the strains in terms of yield and productivity. Experimental verification showed that an optimized bioprocess under semianaerobic conditions is competitive with regard to those reported in the literature. Maximum squalene yield and productivity were, respectively, 2967.6 +/- 118.7 microg/L of culture medium and 104 +/- 4.2 microg/Lh for BY4741 and 3129 +/- 109.5 microg/L of culture medium and 155.9 +/- 5.5 microg/Lh for EGY48. The prospect of developing high-purity squalene preparations that meet food safety regulation demands is expected to attract the interest of the food industry.

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The constructed strains AM63, having an extra copy of the HMG2 gene with a K6R stabilizing mutation in Hmg2p expressed under the control of the inducible galactose promoter and stably integrated into the chromosomal HO locus, and AM64, a derivative of AM63 with an additional deletion of the ERG6 gene, were used as tools to test the squalene accumulation capacity of Saccharomyces cerevisiae. Kinetic data indicated high squalene levels in the early stages of semi-anaerobic cultivation of these strains. The stable Hmg2p induced a strong increase in the squalene pool and a smaller increase in the lanosterol pool. In AM63, the squalene content was approximately 20-fold higher than in the wild-type EGY48 parental strain. In AM64, the combined Hmg2p stabilization and ERG6 deletion did not further enhance squalene accumulation, as lack of ergosterol feedback inhibition led to an elevated transfer of surplus squalene into C27 sterols. The obtained maximum capacity of the selected strains to accumulate squalene and our observations provide a further understanding of the regulation of ergosterol pathway and may also be used as a reference value for its production using food-grade strains of S. cerevisiae.

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Interest is increasing in establishing renewable sources for squalene, a functional lipid, as the conventional ones are limited. In the present study, squalene production was achieved in a wild-type laboratory Saccharomyces cerevisiae strain by two safe chemical means using terbinafine (0.05-0.55 mM) and methyl jasmonate (MJ) (0-1.00 mM). Bioprocess kinetics optimized by response surface methodology and monitored by high-performance liquid chromatography revealed a clear dependence of growth and squalene content (SQC) and yield (SQY) on the above regulators. Maximum SQC (10.02±0.53 mg/g dry biomass) and SQY (20.70±1.00 mg/L) were achieved using 0.442 mM terbinafine plus 0.044 mM MJ after 28 h and 0.300 mM terbinafine after 30 h, respectively. A 10-fold increase in SQY was achieved in comparison to that in the absence of regulator. The ruggedness of optimum conditions for SQY was verified for five industrial strains. The cellular lipid fraction (∼12% of dry biomass) was rich in squalene (12-13%). Results are encouraging toward bioprocess scale up.

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Crude olive pomace oil (COPO) and crude soybean oil (CSO), two low-cost carbon sources, were examined as cosubstrates of glucose for carotenoid production by Blakeslea trispora. Results were compared to those obtained in glucose as a sole carbon source (medium 1) and glucose plus the respective end-line refined oil counterparts. Microbial growth in the presence of oils resulted in an increase in total carotenoid production. The performance of crude oils was better than that of the respective refined forms. Carotenoid production depended on both type and added oil amount. An increase in added oil amount did not necessarily favor carotenoid accumulation. The addition of 10 g oil/L of substrate stimulated carotenoid synthesis, mainly that of beta-carotene, more than 14 (COPO) and 40 times (CSO) in comparison to that observed in medium 1. The maximum total carotenoid content (as mg beta-carotene per g of biomass dry weight) was 75 (COPO) and 235 mg (CSO), respectively. Growth, substrate assimilation, and lipid accumulation-degradation also depended on the presence of oil in the substrate.