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Long-term storage - cryopreservation versus freezedrying

It goes without saying that the method of longterm storage is fundamental to the success of yeast supply for propagation in the brewery. As noted previously (Quain, 1995), ‘the ideal method should be genuinely long-term in that yeast can be stored for many years without compromising viability or genetic stability'. It is generally accepted that the ‘gold standard' for preservation of yeasts and other cells (e.g. blood, sperm, ova etc.) is storage in liquid nitrogen at its boiling point of -196°C. Cryopreservation is considered to be the most robust long-term approach, which maintains yeast viability without any genetic change.

There are a number of reports in the brewing literature of the use of liquid nitrogen to store production yeast strains (reviewed in Boulton and Quain, 2001; Quain, 2006b). These publications stem from a different time (1973-2003) and from brewing companies that have now been subsumed into today's global brewers. Irrespective of this, the technology supporting cryopreserva- tion remains essentially unchanged. Suspensions of the yeast ‘master culture' are stored in short, coloured polypropylene ‘straws' in boxes or tubes immersed in liquid nitrogen. The hardware consists of cryovessels, which must be kept topped up from a storage tank of liquid nitrogen. The health and safety demands of using liquid nitrogen are stringent, a point seemingly overlooked in its use in restaurants and bars!

Although long-term storage in liquid nitrogen requires little more than topping up the cryovessels, the biggest barrier to entry is the need for specialist resources to carefully manage the introduction of yeast into store. For success, there must be strict adherence to detailed methods, which define the physiology of the yeast (oxidative), its concentration (100 x 106 cells/ml) and suspension in fresh media containing a cryoprotectant (glycerol) followed by two-stage phased freezing (room temperature to -30°C and then immersion at -196°C). Recovery is a little more straightforward via removal of the straw from liquid nitrogen and transfer to water at 30°C. Bond (2007b) provides a detailed methodology for the cryopreservation of yeast as used at the National Collection of Yeast Cultures in the UK.

An alternative and older method - freeze-drying or lyophilization - has had a mixed press regarding its impact on yeast viability. Like cryopreservation, freeze-drying also demands expertise and specialist equipment (Bond, 2007a). In a companion article, Bond (2007b) notes that for freeze-drying, ‘strain viabilities are generally low, typically between 1 and 30%, as compared with more than 30% for those of yeast preserved in liquid nitrogen'. There have been a number of reports with brewing strains over the decades (Kirsop, 1955, 1974; Hall and Webb, 1975; Russell and Stewart, 1981) that paint a similar picture of a significant loss in viability post drying and then little change during storage over many years. Mindful that methods develop and improve, a similar conclusion was reported by Miyamoto-Shinohara et al. (2000). Here the viability of S. cerevisiae (95 strains) was 6-10% post drying and 4-8% (43 strains) after 10 years of storage. It is noteworthy that in this study from the Patent Microorganism Depository in Japan, Gram-positive bacteria exhibited a survival rate of around 80% and Gram-negative bacteria around 50%. However, it is clear (Bond, 2007a) that there are opportunities to improve and tune the process through the use of lyoprotectants such as a mix of skimmed milk, trehalose and sodium glutamate.

Yeast culture collections range from ‘enormous and well-known' to ‘smaller, more specialized boutique collections' (Boundy-Mills, 2012). Some of the industry-specific collections have broadened their focus to embrace other industries and to capture new fUnding sources. Table 3.1 details some commercial sources ofbrewing yeasts together with other collections that contain a myriad of diverse yeasts, some of which may have been sourced from breweries.

Although rarely explicit, it is noteworthy that culture collections are ambivalent about the mode of preservation. It would appear that both freezedrying and cryopreservation are used, with strains stored via both techniques or one or the other. A frank explanation for this is offered by the Agricultural Research Service in the USA, which maintains the biggest collection of yeasts (some 14,500) in the world. They note that ‘liquid nitrogen storage seems to cause little or no genetic change in cells. The reasons for not relying on this method exclusively for preservation of cultures are: cost and the fact that lyophilized preparations may be shipped by regular mail whereas strains preserved by liquid nitrogen must first be grown on agar or in liquid medium to avoid the expense of shipping frozen materials', (http://nrrl.ncaur.usda.gov/TheCollec- tion/AccessionMaintenance.html#Maintenance)

A different and more targeted interpretation of yeast supply is the one that feeds into the vibrant world of craft brewing. Here ale, lager and ‘speciality' brewing yeasts are marketed for direct use, either as liquid cultures or as ‘active dried' yeasts (see below). Typically, small-scale operations use the yeast culture once without any subsequent handling or recovery. The ‘speciality' segment describes a dazzling array of production strains for different beer styles. The less technically onerous approach of supplying liquid cultures is not surprisingly supportive of a greater number of yeast strains.

 
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