Cd

Fig. 7.11 Temperature profile measured at various depths in a bin containing 90 kg of pressed yeast cake. The bin was located in a cold room attemperated at c. 1 to 2°C (A.R. Jones, unpublished data).

Agitator and motor

Agitator and motor

Fig. 7.12 Schematic of vessel for storage of pitching yeast slurry.

stocks. After cleaning all mains and valves etc. should be sterilised. Greater flexibility of operation is permitted if separate CiP systems are available for tank and main cleans. These storage tank farms may often have a separate dedicated tank into which an appropriate quantity of yeast is transferred before pitching into fermenter. This separate pitching tank can be used for acid washing (Section 7.3.3). The room in which the tanks are housed must also be built and operated to high hygienic standards. Floors, walls and ceilings should be sealed and easily cleaned with a minimum of fittings. Preferably, the room should be isolated from the rest of the brewery, with access restricted to essential personnel. For a general discussion of measures which need to be taken to control the threat to brewery hygiene, see Section 8.2.

The physical conditions under which pitching yeast is stored, apart from freedom from contamination, relate to time, temperature and exposure to oxygen. The gas in the tank headspace will naturally be rich in carbon dioxide evolved by the yeast. To minimise the risk of contamination, vessels may be top-pressured. If so, an inert gas must be used to avoid pitching yeast being exposed to oxygen. A storage temperature of 2 to 4°C is suitable but care must be taken to ensure that the slurry does not freeze. Mechanical agitation maintains slurry homogeneity and promotes good attemperation. Agitation should not be too vigorous. McCaig and Bendiak (1985a) compared the effects of continuous agitation versus static storage and concluded that the former was associated with loss of viability, excessive glycogen breakdown and impaired fermentation performance. However, totally static storage is unsatisfactory, particularly with very flocculent yeast strains since it is necessary to keep the yeast in suspension to avoid localised heating. This may be achieved by intermittent agitation. Typically yeast is stored safely, under these conditions, for up to three days.

7.3.2.1 Properties of yeast slurries. Pitching yeast slurries bottom cropped from fermenters contain from 30-60% suspended solids, depending on the flocculence characteristics of the particular strain. The design of storage vessels and ancillary equipment should reflect the physical properties of yeast slurries. In fact, the literature dealing with this subject is scant. The rheological properties of yeast slurries are non-Newtonian, that is the apparent viscosity decreases with agitation rate (Lenoel et al., 1987).

Lentini et al. (1992) concluded that the rheological properties of yeast slurries best fitted the Bingham pseudoplastic fluid model which is described by the equation:

Fig. 7.13 Vessel for storage of pitching yeast slurry (kindly supplied by (a) Steph Valente. Bass Brewers and (b), (c) Ian Dobbs. Bass Brewers).

where:

(ip = Pseudoplastic viscosity (Pa x s)

The yield point describes the force required to initiate movement of the slurry. Lentini et al. (1992) reported that this parameter was strain-specific and varied between 12 to over 100 Pa. Strains with yield points of less than 30 Pa could be pumped easily, whereas those of 40 Pa or greater were more difficult to handle. By inference, ease of attemperation would also vary with this parameter. The same authors reported that slurry viscosity increased with storage time and that the effect was more pronounced where the temperature was too high. Elevated pH was also associated with increased viscosity. These effects were presumably associated with cell autolysis.

7.3.2.2 Inter-brewery transport of yeast. It may be necessary for some brewers to transport yeast in bulk form between sites. This may take the form of a newly propagated culture where this process is performed at a central facility or it may be as pitching yeast cropped from a previous fermentation. In both cases, the yeast will normally be transported as slurry.

Some precautions are needed to ensure that yeast quality does not suffer whilst it is in transit. Depending on the quantity of yeast involved, it is convenient to use small tanks of 8-16 hi capacity specially designed for this purpose (see Fig. 7.14). Tanks should be mounted on skids so that they may be moved by fork-lift truck. They should be made from unlagged stainless steel, of dished-end design with the exit main located flush at the lowest point of the vessel for good draining. All fittings must be of good hygienic design and there should be internal sprayballs for connection to a CiP system. The vessel should be steam sterilised prior to use. A gas exhaust valve mounted on the top of the vessel is required and this must be fitted with a micro-

Fig. 7.14 Tank used for transport of yeast slurries (kindly supplied by Steph Valente. Bass Brewers).

biological grade filter. It is not practicable to have mechanical stirring and wall cooling in such transportable tanks, and therefore attemperation tends to be poor. Journey times should be given careful consideration, particularly during summer months when tanks may be left in direct sunlight on open lorries. In these circumstances, some deterioration in yeast quality is inevitable.

From a microbiological standpoint, apart from cleaning, the most hazardous processes are emptying and filling the tanks. Usually this requires the use of flexible hose to connect the tank to a convenient brewery outlet main attached to the brewery yeast storage vessels. All such mains and associated fittings must be thoroughly cleaned and sterilised before use. In particular flexible hoses require careful handling since these may easily become foci for infections. The use of dried yeast, which can make easier the job of transport between breweries, is described in Section 7.2.2.3.

7.3.3 Acid washing

For many years, it has been recognised that handling of yeast in bulk in the brewery may be associated with low levels of infection with bacteria. Providing fermentation is vigorous and begins with little or no lag this low level of infection can be tolerated since in fermenter most bacteria do not compete successfully with the yeast. However, some bacteria can flourish under fermentation conditions if, for whatever reason, yeast growth is impaired. Typical bacterial contaminants include Pediococcus species, Lactobacillus species, Enterobacter agglomerans and, notably Obesumbacterium pro-tens (see Section 8.1.2). Of these, the latter has been shown to be responsible for the formation of the potential carcinogens ATNCs during fermentation (see Section 8.1.5.1). Therefore, for reasons of product quality and safety, there is a real need to reduce bacterial loading in pitching yeast slurries. This is typically performed by treating yeast with a chemical disinfectant.

Compared to bacteria, yeasts are relatively resistant to low pH, and therefore yeast slurry may be conveniently washed with acid. This is not a new suggestion; for example, Pasteur recommended the use of tartaric acid to reduce the bacterial loading of pitching yeast slurries. Other mineral acids such as hydrochloric, nitric, sulphuric and phosphoric may also be used, with the latter being the most common. Typically, the pH is reduced to a value of pH 2.2-2.5 and the yeast held under these conditions for a few hours, at a temperature of less than 4°C. The oxidising agent, ammonium persulphate (c. 0.75% w/v), either alone or in conjunction with phosphoric acid, is also used (Bruch et al., 1964). Simpson (1987) reported that ammonium persulphate and phosphoric acid used in combination at a pH of 2.8 was more effective than acid alone at the lower pH of 2.2.

Alternatives to acid treatment have been proposed. Comparatively soon after their discovery it was suggested that antibiotics could be used to free yeast from bacteria. Gray and Kazin (1946) made a preliminary study of the use of tyrothricin and Case and Lyon (1956) proposed the use of polymyxin B. Both were shown to be effective and such treatments would have the benefit of persisting and providing residual protection throughout fermentation and beyond. However, these proposals came at a time when the dangers of the profligate use of antibiotics in terms of selection of multiple resistant strains were not recognised. Such use today would be totally unacceptable. More recently, the use of the bacteriocin nisin has been suggested as a safe alternative with the advantages of antibiotics (Ogden, 1987). This polypeptide is accepted for use in the dairy and canning industries and exhibits activity against a wide spectrum of bacteria including Lactobacillus spp. and Gram positive organisms. It has no effect on yeasts, is stable at low pH, and like antibiotics confers residual protection. It has not found great utility in brewing, no doubt because of the reluctance to use additives, which persist into finished beers.

Acid washing supposedly has no effect on brewing yeast. It follows that it is ineffective in removing wild yeast contamination from pitching yeast slurries. More importantly, the suggestion that acid washing has no effect on yeast requires some qualification. Some authors have reported that acid washing improves the performance of yeast in fermentation. Jackson (1988) observed that a pre-treatment 'conditioning' with phosphoric acid at pH 2.2 for 6 hours and 4°C produced more rapid fermentation, shorter diacetyl stand times, reduced beer acetaldehyde concentration and more 'drinkable' beers. The physiological basis of these observations was not explained other than the fact that the acid caused instant de-flocculation and the time of appearance of the first yeast buds was reduced by approximately 8 hours.

More often acid washing has been associated with deleterious effects on yeast. Fernandez et al. (1993) observed a gradual deterioration in pitching yeast condition with time of acid washing, as judged by the acidification power test (see Section 7.4.2.2). The severity of the effects correlated with acidity and was to some extent strain-specific. Simpson and Hammond (1989) studied the effects of acid washing on 16 yeast strains, both ale and lager types. Using acidified ammonium persulphate (0.75% w/v; phosphoric acid at pH 2.1) no effect was observed on viability, floccu-lation and fermentation performance. However, scanning electron microscopy revealed alterations to the yeast cell surface and leakage of cellular components was evidenced by the appearance of ATP in the external medium. The potential for deleterious effects on yeast due to acid washing was exacerbated by elevated temperature, high ethanol concentrations and over-prolonged exposure to acid. Furthermore, yeast which was already in a stressed condition was more susceptible to damage during acid washing.

Simpson and Hammond (1989) have produced guidelines for acid washing and these are summarised here, together with some observations of the present authors. Yeast should be acid washed in slurry form using food grade phosphoric or citric acid. During the treatment the temperature should be maintained between 2 and 4°C with continuous gentle stirring. The pH of the slurry should be checked with a suitable probe. Care should be taken when adding the acidulant to ensure that mixing efficiency is sufficient to avoid localised high acid concentrations. Automatic systems may be used where acidulant is added via a pump under the control of a pH probe mounted in the tank. Care should be taken to ensure that the point of addition and the pH probe are located such that there is no possibility of over-shooting.

Alternatively, the acid may be dosed in-line as the yeast is transferred from storage to pitching tank. This approach ensures good mixing and in conjunction with an inline pH probe avoids over addition of acid. The total treatment time should be no longer than two hours. Preferably, the treatment is terminated by immediate pitching into wort. Alternatively, the process may be terminated by addition of food-grade sodium hydroxide to adjust the pH to 4.0-4.5. Although often used, this practice cannot be recommended because of the additional cost, increased complexity and further opportunity for error. Most importantly, the pitching yeast is subjected to an additional and unnecessary stress.

Simpson and Hammond (1989) recommend that yeast recovered from high-gravity fermentations (ethanol > 8% abv) or in a 'distressed condition' should not be acid washed. Distressed yeast was defined as being that which is derived from a previous slow fermentation or that which is heavily contaminated. In our experience, yeast with a viability of less than 80%, as judged by methylene blue staining, should not be acid washed, or better if an alternative is available, not used at all! Similarly, if possible, yeast contaminated with other micro-organisms should be disposed of and not used for brewing. Of course, it is perfectly valid to argue that acid washing represents treatment of a symptom, as opposed to the root cause of hygiene problems (see Section 8.3.1 on the philosophy of QA versus QC). In a modern well-designed and managed brewery the standards of hygiene should be sufficiently high to avoid the need for acid washing. Certainly, the move towards ever-higher-gravity fermentations as practised in many modern breweries would suggest that the opportunity should be taken to eliminate any stresses to which yeast is subject. It is perhaps fair comment that acid washing represents an unnecessary stress.

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