Preservation, inoculum development and quality management of yeasts in the brewing industry

English: Techniques to maintain and preserve microorganisms have become important to ensure availability of microorganisms for application in many institutions and processes. Several methods are available to preserve microorganisms and include drying, lyophilization, cryopreservation and sub-culturing. Many of these methods, may lead to population change through selection, loss of viability as well as poor survival rates when used for maintenance of yeasts. In this study, different preservation methods (cryopreservation at -196°C in liquid nitrogen, freezing at -70°C and lyophilization) were compared in the maintenance of brewing inocula over a period of two years. Interestingly, a decrease in the percentage of variants and respiratory deficient yeasts (RDs) was generally found when preserved through cryopreservation (-196°C) and freezing (-70°C). In contrast, the percentage variants when revived yeasts were grown in wort, increased in yeasts maintained through lyophilization. A high percentage viable cells (>95%) was recorded for yeast cultures maintained at -196°C and at -70°C while viability was low (<50%) when maintained through lyophilization. Consequently, the maintenance methods of choice are cryopreservation and freezing. The developed cryopreservation method was successfully implemented in the brewing process that produced the champion lager beer "Castle Lager" at Burton-upon-Trent in the UK (April; 2000). The results of the estimate of the components of variance showed that the largest source of variation in all three of the methods tested, was the error arising from the analytical test. On the basis of these results, it , subsequently became another aim of this study to explore alternative analytical tests to differentiate and characterise yeasts in the brewing industry. Consequently, PCR based RFLP, fatty acid and sterol profiles were evaluated. Using PCR based RFLP, a study to differentiate brewing yeasts from related yeasts using amplification and restriction polymorphism of the ITS region was conducted. Differentiation was dependent on the restriction enzymes used to digest the amplified rDNA. Digestion with Hae 111,Cfo 1, Sau3 A1 and Msp 1 divided representatives of the genus Saccharomyces into several unique groups. With Msp 1 the DNA patterns for two brewing strains were similar, but could be differentiated from Sacch. cerevisiae and other species tested. It was also possible to distinguish some members of the Saccharomyces sensu stricto group i.e. Sacch. bayanus and Sacch. pastorianus from Sacch. cerevisiae and Saceh. paradoxus using Hae 111as well as Sacch. paradoxus from the other sensu stricto members using Msp 1 digestion. Fatty acid and sterol analyses were evaluated as alternative quality control methods to conventional differentiation and characterisation systems in the brewing industry. The presence of linoleic acid (18:2) in brewing yeast could be used to distinguish these from closely related yeast species. Furthermore, the absence of lanosterol and stigmasterol enabled differentiation of the brewing yeast from the rest of the closely related species tested. However, both fatty acid and sterol methods were not sensitive enough to detect mutants (variants) of brewing yeast. Conventional brewing identification tests proved superior to the above researched methods.