Preservation, inoculum development and quality management of yeasts in the brewing industry
Abstract
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. Afrikaans: Tegnieke vir die bewaring van mikrobes is belangrik ten einde die
beskikbaarheid van mikrobes vir aanwending in verskeie instansies en
prosesse te verseker. Verskeie metodes vir die bewaring van mikrobes is
beskikbaar en sluit in droging, vriesdroging, 'vries en die maak van
subkulture. As hierdie metodes gebruik word vir die bewaring van giste,
mag van hulle lei tot veranderinge in die populasie a.g.v. seleksie, verlies
aan lewensvatbaarheid en lae oorlewingsvlakke. In hierdie studie is
verskillende bewaringsmetodes (vries by (-196°Cin vloeibare stikstof, vries
by -70°C en vriesdroging) vergelyk oor 'n tydperk van twee jaar in die
bewaring van brou-inokula. Oor die algemeen is 'n afname in variante en
respiratories-gebrekkige giste gevind as bewaring d.m.v. vriesing (-196°C en -70°C) gedoen is. In teenstelling, het die persentasie variante
toegeneem as gevriesdroogde giste weer in moutekstrak opgegroei is. 'n
Hoë persentasie lewensvatbare selle (>95%) is waargeneem in giskulture
bewaar by -196°C en -70°C, terwyl lewensvatbaarheid laag was <50%) in
gevriesdroogde kulture. Gevolglik is die beste bewaringsmetodes vriesing.
Die ontwikkelde vriesingsmetodes is suksesvol geïmplementeer in die
brouproses wat gelei het tot die produksie en kroning van die kampioen
lager "Castle lager" by Burton-upon-Trent in die VK (April, 2000). Die
resultate van die berekening van variasiekomponente het getoon dat die
grootste bron van variasie in al drie die getoetste metodes, die fout a.g.v.
die analitiese metode was. Op grond van hierdie resultate, het dit 'n
verdere doel van die studie geword om ondersoek in te stel na alternatiewe
analitiese metodes om giste in die brou-industrie te differensieer en te
karakteriseer. Gevolglik is PKR gebaseerde RFLP, vetsuur- en
sterolprofiele geëvalueer. Met behulp van 'n PKR gebaseerde RFLP, is 'n
studie uitgevoer om brouersgiste van verwante giste te onderskei m.b.V.
amplifisering en beperkings-polimorfisme van die ITS areas.
Onderskeiding was afhanklik van die beperkingsensieme wat gebruik is om
die geamplifiseerde rDNS te verteer. Vertering met Hae 111, Cfo 1,Sau 3A1
en Msp 1 het die verteenwoordigers van die genus Saccharomyces verdeel
in verskeie unieke groepe. Met Msp 1 was die ONS patrone vir twee
brouerstamme dieselfde, maar hulle kon onderskei ,word van Sacch.
cerevisiae en ander getoetste spesies. Daar kon ook onderskei word
tussen sommige lede van die Saccharomyces sensu stricto groep, nl.
Sacch. bayanus en Sacch. pastorianus van Sacch. cerevisiae en Sacch.
paradoxus m.b.v. Hae 111 en tussen Sacch. paradoxus en die ander lede
van die sensu stricto groep m.b.v. Msp 1. Vetsuur" en sterolanalises is
geëvalueer as alternatiewe kwaliteitsbeheermetodes vir konvensionele
onderskeidings- en karakteriseringsisteme in die brou-industrie. Die
teenwoordigheid van linoleïensuur (18:2) in brouersgiste is gebruik om
hulle te onderskei van naby verwante spesies. Verder het die afwesigheid
van lanosterol en stigmasterol dit moontlik gemaak om te. onderskei tussen
brouersgiste en die ander getoetste naby verwante spesies. Beide die
vetsuur- en sterolmetodes was egter nie sensitief genoeg om mutante
(variante) van die brouersgiste waar te neem nie. Konvensionele
brouersgis-identifikasietoetse het geblyk beter te wees as bogenoemde
metodes.