T

Yeast checked for purity

Discard 65 Hl yeast contaminated propagation yeast vessel

650 Hl fermentation vessel Fig. 2.24 Yeast propagation. After MacDonald et al.. (1984).

Laboratory

Brewery

The sugar is 'dribbled in' and the end result is a far higher yield of biomass, perhaps four-fold more than is produced when the sugar is provided in a single batch at the start of fermentation.

The majority of brewing yeasts are resistant to acid (pH 2.0-2.2) and so the addition of phosphoric acid to attain this pH is very effective in killing bacteria with which yeast may become progressively contaminated from fermentation to fermentation. Many brewers use such an acid washing of yeast between fermentations.

There are two key indices of yeast health: viability and vitality. Both should be high if a successful fermentation is to be achieved. Viability is a measure of whether a yeast culture is alive or dead. While microscopic inspection of a yeast sample is useful as a gross indicator of that culture (e.g. presence of substantial infection), quantitative evaluation of viability needs a staining test. The most common is the use of methylene blue: viable yeast decolourises it, dead cells do not. Although a yeast cell may be living, it does not necessarily mean that it is healthy. Vitality is a measure of how healthy a yeast cell is. Many techniques have been advanced as an index of vitality, but none has been accepted as definitive.

Preferably yeast is stored in a readily sanitised room that can be cleaned efficiently and which is supplied with a filtered air supply and possesses a pressure higher than the surroundings in order to impose an outwards vector of contaminants. Ideally it should be at or around 0°C. Even if storage is not in such a room, the tanks must be rigorously cleaned, chilled to 0-4°C and have the facility for gently rousing (mixing) to avoid hot spots. Yeast is stored in slurries ('barms') of 5-15% solids under 6 in. of beer, water or potassium phosphate solution. An alternative procedure is to press the yeast and store it at 4°C in a cake form (20-30% dry solids). Pressed yeast may be held for about 10 days, water slurried and beer slurried for 3-4 weeks and slurries in 2% phosphate, pH 5 for 5 weeks.

Brewers seeking to ship yeast normally transport cultures for repropagation at the destination. However, greater consistency is achieved when it is feasible to propagate centrally and ship yeast for direct pitching. Such yeast must be contaminant-free and of high viability and vitality, washed free from fermentable material and cold (0°C). The longer the distance, the greater the recommendation for low moisture pressed cake.

Apart from the importance of pitching yeast of good condition, it is also important that the amount pitched is in the correct quantity. The higher the pitching rate, the more rapid the fermentation. As the pitching rate increases, initially so too does the amount of new biomass synthesised, until at a certain rate, the amount of new yeast synthesised declines. The rate of attenuation and the amount of growth directly impacts the metabolism of yeast and the levels of its metabolic products (i.e. beer flavour), hence the need for control. Yeast can be quantified by weight or cell number. Typically some 107 cells per mL will be pitched for wort of 12°Plato (1.5-2.5 g pressed weight per L). At such a pitching rate, lager yeast will divide 4-5 times in fermentation. Yeast numbers can be measured using a haemocytometer, which is a counting chamber loaded onto a microscope slide. It is possible to weigh yeast or to centrifuge it down in pots which are calibrated to relate volume to mass, but in these cases it must be remembered that there are usually other solid materials present, for example, trub.

Another procedure that has come into vogue is the use of capacitance probes that can be inserted in-line. An intact and living yeast cell acts as a capacitor and gives a signal whereas dead ones (or insoluble materials) do not. The device is calibrated against a cell number (or weight) technique and therefore allows the direct read-out of the amount of viable yeast in a slurry. Other in-line devices quantify yeast on the basis of light scatter.

Brewery fermentations

Primary fermentation is the fermentation stage proper in which yeast, through controlled growth, is allowed to ferment wort to generate alcohol and the desired spectrum of flavours. Increasingly brewery fermentations are conducted in cylindro-conical vessels (Fig. 2.25). The fermentation is regulated by control of several parameters, notably the starting strength of the wort (°Plato, which approximates to percentage sugar by weight, or Brix), the amount of viable yeast ('pitching rate'), the quantity of oxygen introduced and the temperature. Fermentation is monitored by measuring the

Pressure/vacuum release valves

Coolant in Coolant out-«— zzl Coolant in

Coolant out-«

Coolant in

Pressure/vacuum release valves

Cylindroconical Fermentor Principles

Antifoam spray supply

-CIP supply

Cooling jackets

Sample point

Inlet/outlet

Antifoam spray supply

-CIP supply

Cooling jackets

Sample point

Inlet/outlet

Fig. 2.25 A cylindro-conical fermentation vessel.

decrease in specific gravity (alcohol has a much lower specific gravity than sugar).

Ales are generally fermented at a higher temperature (15-20°C) than lagers (6-13°C) and therefore attenuation (the achievement of the finished specific gravity) is achieved more rapidly. Thus, an ale fermenting at 20°C may achieve attenuation gravity in 2 days, whereas a lager fermented at 8.5°C may take 10 days. The temperature has a substantial effect on the metabolism of yeast, and the levels of a flavour substance like iso-butanol will be 16.5 and 7mgL-1, respectively, for the ale and the lager. Some brewers add zinc (e.g. 0.2 ppm) to promote yeast action-it is a cofactor for the enzyme alcohol dehydrogenase. During fermentation, the pH falls because yeast secretes organic acids and protons. A diagram depicting the time course of fermentation can be found in Fig. 2.26.

Surplus yeast will be removed at the end of fermentation, either by a process such as 'skimming' for a traditional square fermenter employing top fermenting yeast, or from the base of a cone in a cylindro-conical vessel. This is not only to preserve the viability and vitality of the yeast, but also to circumvent the autolysis and secretory tendencies of yeast that will be to the detriment of flavour and foam. There will still be sufficient yeast in the beer to effect the secondary fermentation.

Fig. 2.26 Changes occurring during a brewery fermentation.

The 'green' beer produced by primary fermentation needs to be 'conditioned', in respect of establishment of a desired carbon dioxide content and refinement of the flavour. This is called secondary fermentation. Above all at this stage, there needs to be the removal of an undesirable butterscotch flavour character due to substances called vicinal diketones (VDKs; discussed later). Traditionally it is the lager beers fermented at lower temperatures that have needed the more prolonged maturation (storage: 'lagering') in order to refine their flavour and develop carbonation. The latter depends on the presence of sugars, either those (perhaps 10%) which the brewer ensures are residual from the primary fermentation or those introduced in the 'krausening' process, in which a proportion of freshly fermenting wort is added to the maturing beer. Many brewers are unconvinced by the need for prolonged storage periods (other than for its strong marketing appeal) and they tend to combine the primary and secondary fermentation stages. Once the target attenuation has been reached, the temperature is allowed to rise (perhaps by 4°C), which permits the yeast to deal more rapidly with the VDKs. Carbonation will be achieved downstream by the direct introduction of gas.

Once the secondary fermentation stage is complete (and the length of this varies considerably between brewers), then the temperature is dropped, ideally to — 1 °C or -2°C to enable precipitation and sedimentation of materials which would otherwise cause a haze in the beer. The sedimentation of yeast is also promoted in this 'cold conditioning' stage, perhaps with the aid of isinglass finings (Fig. 2.27). These are solutions of collagen derived from the swim bladders of certain species of fish from the South China Seas. Collagen has a net positive charge at the pH of beer, whereas yeast and other particulates have a net negative charge. Opposite charges attracting, the

C NH

II O

Fig. 2.27 A typical repeating structure in the collagen polypeptide chain that, when dissolved in partially degraded forms, represents isinglass. The amino and imino acid residues in this particular sequence are ~alanyl-prolyl-arginyl-glycyl-glutamyl-hydroxyprolyl-prolyl~.

isinglass forms a complex with these particles and the resultant large agglomerates sediment readily because of an increase in particle size. Sometimes, the isinglass finings are used alongside 'auxiliary finings' based on silicate, the combination being more effective than isinglass alone. Some brewers centrifuge to aid clarification.

For the most part, fermenters these days are fabricated from stainless steel and will be lagged and feature jackets that allow coolant to be circulated (the heat generated during fermentation is sufficient to effect any necessary warming - so the temperature is regulated by balancing metabolic heat with cooling afforded by the coolant in the jacket, which may be water, glycol or ammonia depending on how much refrigeration is demanded). Modern vessels tend to be enclosed, for microbiological reasons. However, across the world there remain a great many open tanks. Cylindro-conical vessels can have a capacity of up to 13 000 hL and are readily cleaned using CIP operations (see Chapter 1).

Only one company, in New Zealand, practises continuous fermentation. Many brewers nowadays maximise the output by fermenting wort at a higher gravity than necessary to give the target alcohol concentration, before diluting the beer downstream with deaerated water to 'sales gravity' (i.e. the required strength of the beer in package). This is called 'high-gravity brewing'. There are limits to the strength of wort that can be fermented. This is because yeast becomes stressed at high sugar concentrations and when the alcohol level increases beyond a certain point. Brewing is unusual amongst alcohol production industries in that it re-uses yeast for ensuing fermentations. Excluded from this are those beers in which very high alcohol levels are developed (e.g. the barley wines). The yeast is stressed in these conditions and will not be re-usable. This is the reason why wine fermentations, for instance, involve 'one trip' yeast. This is also the reason why, in the production of sweeter fortified wines (see Chapter 4), alcohol is added at the start of fermentation in order to hinder the removal of sugars.

Filtration

After a period of typically 3 days minimum in 'cold conditioning', the beer is generally filtered. Diverse types of filter are available, perhaps the most common being the plate-and-frame filter which consists of a series of plates in sequence, over each of which a cloth is hung. The beer is mixed with a filter aid - porous particles which both trap particles and prevent the system from clogging. Two major kinds of filter aid are in regular use: kieselguhr and perlite. The former comprises fossils or skeletons of primitive organisms called diatoms. These can be mined and classified to provide grades that differ in their permeability characteristics. Particles of kieselguhr contain pores into which other particles (such as those found in beer) can pass, depending on their size. Perlites are derived from volcanic glasses crushed to form microscopic flat particles. They are better to handle than kieselguhr, but are not as efficient as filter aids. Filtration starts when a pre-coat of filter aid is applied to the filter by cycling a slurry of filter aid through the plates. This pre-coat is generally of quite a coarse grade, whereas the filter-aid (the body feed) which is dosed into the beer during the filtration proper tends to be a finer grade. It is selected according to the particles within the beer that need to be removed. If a beer contains a lot of yeast, but relatively few small particles, then a relatively coarse grade is best. If the converse applies, then a fine grade with smaller pores will be used.

The stabilisation of beer

Apart from filtration, various other treatments may be applied to beer downstream, all with the aim of enhancing the shelf life of the product. A haze in beer can be due to various materials, but principally it is due to the cross-linking of certain proteins and certain polyphenols. Therefore, if one or both of these materials is removed, then the shelf life is extended. Brewhouse operations are in part designed to precipitate protein-polyphenol complexes. Thus, if these operations are performed efficiently, then much of the job of stabilisation is achieved. Good, vigorous, 'rolling' boils, for instance, will ensure precipitation. Before that, avoidance of the last runnings in the lautering operation will prevent excessive levels of polyphenol entering the wort. The cold conditioning stage also has a major role to play, by chilling out protein-polyphenol complexes, enabling them to be taken out on the filter. Control over oxygen and oxidation is important because it is particularly the oxidised polyphenols that tend to cross-link with proteins. For really long shelf lives, though, and certainly if the beer is being shipped to extremes of climate, additional stabilisation treatments will be necessary. Polyphenols can be removed with PVPP. Protein can be precipitated by adding tannic acid, hydrolysed using papain (the same enzyme from paw paw that is used as meat tenderiser) or, and most commonly, adsorbed on silica hydrogels and silica xerogels.

Gas control

Final adjustment will now be made to the level of gases in the beer. As we have seen, it is important that the oxygen level in the bright beer is as low as possible. Unfortunately, whenever beer is moved around and processed in a brewery, there is always the risk of oxygen pick-up. For example, oxygen can enter through leaky pumps. A check on oxygen content will be made once the bright beer tank (filtered beer is bright beer) is filled and, if the level is above specification (which most brewers will set at 0.1-0.3 ppm), oxygen will have to be removed. This is achieved by purging the tank with an inert gas, usually nitrogen, from a sinter in the base of the vessel. The level of carbon dioxide in a beer may either need to be increased or decreased. The majority of beers contain between two and three volumes of CO2, whereas most brewery fermentations generate 'naturally' no more than 1.2-1.7 volumes of the gas. The simplest and most usual procedure by which CO2 is introduced is by injection as a flow of bubbles as beer is transferred from the filter to the Bright Beer Tank. If the CO2 content needs to be dropped, this is a more formidable challenge. It may be necessary for beers that are supposed to have a relatively low carbonation and, as for oxygen, this can be achieved by purging. However, concerns about 'bit' production have stimulated the development of gentle membrane-based systems for gas control. Beer is flowed past membranes, made from polypropylene or polytetrafluoroethylene, that are water-hating and therefore do not 'wet-out'. Gases, but not liquids, will pass freely across such membranes, the rate of flux being proportional to the concentration of each individual gas and dependent also on the rate at which the beer flows past the membrane.

Packaging

The packaging operation is the most expensive stage in the brewery, in terms of raw materials and labour. Beer will be brought into specification in the Bright Beer Tank (sometimes called the Fine Ale Tank or the Package Release Tank). The carbonation level may be higher (e.g. by 0.2 volume) than that specified for the beer in package, to allow for losses during filling.

Although beer is relatively resistant to spoilage, it is by no means entirely incapable of supporting the growth of micro-organisms. For this reason, most beers are treated to eliminate any residual brewing yeast or infecting wild yeasts and bacteria before or during packaging. This can be achieved in one of two ways: pasteurisation or sterile filtration. Pasteurisation can take one of two forms in the brewery: flash pasteurisation for beer pre-package and tunnel pasteurisation for beer in can or bottle. The principle in either case, of course, is that heat kills micro-organisms. One PU is defined as exposure for 1 min at 60°C. The higher the temperature, the more rapidly the micro-organisms are destroyed. A 7°C rise in temperature leads to a ten-fold increase in the rate of cell death. The pasteurisation time required to kill organisms at different temperatures can be read off from a plot. Typically, a brewer might use 5-20 PU - but higher 'doses' may be used for some beers, for example, low alcohol beers which are more susceptible to infection. In flash pasteurisation, the beer flows through a heat exchanger (essentially like a wort cooler acting in reverse), which raises the temperature typically to 72°C. Residence times of between 30 and 60 s at this temperature are sufficient to kill off virtually all microbes. Ideally there will not be many of these to remove: good brewers will ensure low loadings of micro-organisms by attention to hygiene throughout the process and ensuring that the prior filtration operation is efficient. Tunnel pasteurisers comprise large heated chambers through which cans or glass bottles are conveyed over a period of minutes, as opposed to the seconds employed in a flash pasteuriser. Accordingly, temperatures in a tunnel pasteuriser are lower, typically 60°C for a residence time of 10-20 min. An increasingly popular mechanism for removing micro-organisms is to filter them out by passing the beer through a fine mesh filter. The rationale for selecting this procedure rather than pasteurisation is as much for marketing reasons as for any technical advantage it presents: many brands of beer these days are being sold on a claim of not being heat-treated, and therefore free from any 'cooking'. In fact, provided the oxygen level is very low, modest heating of beer does not have a major impact on the flavour of many beers, although those products with relatively subtle, lighter flavour will obviously display 'cooked' notes more readily than will beers that have a more complex flavour character. The sterile filter must be located downstream from the filter that is used to separate solids from the beer. Sterile filters may be of several types, a common variant incorporating a membrane formed from polypropylene or polytetrafluoroethylene and with pores of between 0.45 and 0.8 ^m.

Filling bottles and cans

Bottles entering the brewery's packaging hall are first washed and, if they are returnable bottles (i.e. they have been used previously to hold beer), they will need a much more robust cleaning and sterilisation, inside and out, involving soaking and jetting with hot caustic detergent and thorough rinsing with water. The beer coming from the Bright Beer Tanks is transferred to a bowl at the heart of the filling machine. Bottle fillers are machines based on a rotary carousel principle. They have a series of filling heads: the more the heads, the greater the capacity of the filler. The bottles enter on a conveyor and, sequentially, each is raised into position beneath the next vacant filler head, each of which comprises a filler tube. An air-tight seal is made and, in modern fillers, a specific air evacuation stage starts the filling sequence. The bottle is counter-pressured with carbon dioxide, before the beer is allowed to flow into the bottle by gravity from the bowl. The machine will have been adjusted so that the correct volume of beer is introduced into the vessel. Once filled, the 'top' pressure on the bottle is relieved, and the bottle is released from its filling head. It passes rapidly to the machine that will crimp on the crown cork but, en route, the bottle will have been either tapped or its contents 'jetted' with a minuscule amount of sterile water in order to fob the contents and drive off any air from the space in the bottle between the surface of the beer and the neck (the 'headspace'). Next stop is the tunnel pasteuriser if the beer is to be pasteurised after filling, but if sterile filtration is used, the filler and capper are likely to be enclosed in a sterile room. The bottles now head off for labelling, secondary packaging and warehousing.

Putting beer into cans has much in common with bottling. It is the container, of course, that is very different - and definitely one trip. Cans may be of aluminium or stainless steel, which will have an internal lacquer to protect the beer from the metal surface and vice versa. Cans arrive in the canning hall on vast trays, all pre-printed and instantly recognisable. They are inverted, washed and sprayed, prior to filling in a manner very similar to the bottles. Once filled, the lid is fitted to the can basically by folding the two pieces of metal together to make a secure seam past which neither beer nor gas can pass.

Filling kegs

Kegs are manufactured from either aluminium or stainless steel. They are containers generally of 100 L or less, containing a central spear. Kegs, of course, are multi-trip devices. On return to the brewery from an 'outlet', they are washed externally before transfer to the multi-head machine in which successive heads are responsible for their washing, sterilising and filling. Generally they will be inverted as this takes place. The cleaning involves high-pressure spraying of the entire internal surface of the vessel with water at approximately 70°C. After about 10 s, the keg passes to the steaming stage, the temperature reaching 105°C over a period of perhaps half a minute. Then the keg goes to the filling head, where a brief purge with carbon dioxide precedes the introduction of beer, which may take a couple of minutes. The discharged keg is weighed to ensure that it contains the correct quantity of beer and is labelled and palleted before warehousing.

The quality of beer

Flavour

The flavour of beer can be split into three separate components: taste, smell (aroma) and texture (mouthfeel).

There are only four proper tastes: sweet, sour, salt and bitter. They are detected on the tongue. A related sense is the tingle associated with high levels of carbonation in a drink: this is due to the triggering of the trigeminal nerve by carbon dioxide. This nerve responds to mild irritants, such as carbonation and capsaicin (a substance largely responsible for the 'pain delivery' of spices and peppers).

Carbon dioxide is also relevant insofar as its level influences the extent to which volatile molecules will be delivered via the foam and into the headspace above the beer in a glass.

The sweetness of a beer is due, of course, to its level of sugars, either those that have survived fermentation or those introduced as primings.

The principal contributors to sourness in beer are the organic acids that are produced by yeast during fermentation. These lower the pH: it is the H+ ion imparted by acidic solutions that causes the sour character to be perceived on the palate. Most beers have a pH between 3.9 and 4.6.

Saltiness in beer is afforded by sodium and potassium, while of the anions present in beer, chloride and sulphate are of particular importance. Chloride is said to contribute a mellowing and fullness to a palate, while sulphate is felt to elevate the dryness of beer.

Perhaps the most important taste in beer is bitterness, primarily imparted by the iso-a-acids derived from the hop resins.

Many people believe that they can taste other notes on a beer. In fact they are detecting them with the nose, the confusion arising because there is a continuum between the back of the throat and the nasal passages. The smell (or aroma) of a beer is a complex distillation of the contribution of a great many individual molecules. No beer is so simple as to have its 'nose' determined by one or even a very few substances. The perceived character is a balance between positive and negative flavour notes, each of which may be a consequence of one or a combination of many compounds of different chemical classes. The 'flavour threshold' is the lowest concentration of a substance which is detectable in beer.

The substances that contribute to the aroma of beer are diverse. They are derived from malt and hops and by yeast activity (leaving aside for the moment the contribution of contaminating microbes). In turn there are interactions between these sources, insofar as yeast converts one flavour constituent from malt or hops into a different one, for example.

Various alcohols influence the flavour of beer (Table 2.5), by far the most important of which is ethanol, which is present in most beers at levels at least 350-fold higher than any other alcohol. Ethanol contributes directly to the

Table 2.5 Some alcohols in beer.

Alcohol

Flavour threshold (mg L 1)

Perceived character

Ethanol

14000

Alcoholic

Propan-1-ol

800

Alcoholic

Butan-2-ol

16

Alcoholic

Iso-amyl alcohol

50

Alcohol, banana, vinous

Tyrosol

200

Bitter

Phenylethanol

40-100

Roses, perfume

Table 2.6 Some esters in beer.

Ester

Flavour threshold (mg L 1 )

Perceived character

Ethyl acetate

33

Solventy, fruity, sweet

Iso-amyl acetate

1.0

Banana

Ethyl octanoate

0.9

Apples, sweet, fruity

Phenylethyl acetate

3.8

Roses, honey, apple

flavour of beer, registering a warming character. It also influences the flavour contribution of other volatile substances in beer. Because it is quantitatively third only to water and carbon dioxide as the main component of beer, it is not surprising that it moderates the flavour impact of other substances. It does this by affecting the vapour pressure of other molecules (i.e. their relative tendency to remain in beer or to migrate to the headspace of the beer). The higher alcohols in beer are important as the immediate precursors of the esters, which are proportionately more flavour active (see Table 2.6). And so it is important to be able to regulate the levels of the higher alcohols produced by yeast if ester levels are also to be controlled.

The higher alcohols are produced during fermentation by two routes: catabolic and anabolic. In the catabolic route, yeast amino acids taken up from the wort by yeast are transaminated to a-keto-acids, which are decarboxylated and reduced to alcohols:

RCH(NH2)COOH + R1 COCOOH ^ RCOCOOH + R1 CH(NH2)COOH

The anabolic route starts with pyruvate (the end point of the EMP pathway proper), the higher alcohols being 'side shoots' from the synthesis of the amino acids valine and leucine (Fig. 2.28). The penultimate stage in the production of all amino acids is the formation of the relevant keto acid which is transaminated to the amino acid. Should there be conditions where the keto acids accumulate, they are then decarboxylated and reduced to the equivalent alcohol. Essentially, therefore, the only difference between the pathways is the origin of the keto acid: either the transamination product of an amino acid assimilated by the yeast from its growth medium or synthesised de novo from pyruvate.

In view of the above, it is not surprising that the levels of FAN in wort influence the levels of higher alcohols formed. Higher alcohol production is increased at both excessively high and insufficiently low levels of assimilable nitrogen available to the yeast from wort. If levels of assimilable N are low, then yeast growth is limited and there is a high incidence of the anabolic pathway. If levels of N are high, then the amino acids feedback to inhibit further synthesis of them and therefore the anabolic pathway becomes less important. However, there is a greater tendency for the catabolic pathway to 'kick in'.

HoC CHo

NADH

H3C CH H3C CH

H3C CH3 H3C CH3

ch2 ch2

Transamination

2-Ketoisocaproate

COOH

3-Methyl butan-1-ol Isovaleraldehyde OH

NADPH, acetyl-CoA CO2

CH3COCOOH

Pyruvate

CH3 Acetolactate

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