1

A TERM PAPER

ON

MICROBIOLOGICAL QUALITY CONTROL OF BEER IN

BREWING INDUSTRY

COMPILED BY

IMEVBORE GRACE OLUWATOSIN

ACU/570

SUBMITTED TO

MISS F. T. OJO

OF THE DEPARTMENT OF BIOLOGICAL SCIENCES, AJAYI

CROWTHER UNIVERSITY, OYO, OYO STATE.

COURSE CODE: MCB 4204

COURSE TITLE: MICROBIOLOGICAL QUALITY ASSURANCE

MAY, 2010

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CHAPTER ONE

INTRODUCTION

1.0 History of Brewing

The art of brewing is as old as civilization. Between 10,000 and 15,000 years ago, some

humans discontinued their nomadic hunting and gathering and settled down to farm. Grain

was the first domesticated crop that started that farming process. Through hieroglyphics,

cuneiform characters and written accounts, historians have traced the roots of brewing back

to ancient African, Egyptian and Sumerian tribes. The oldest proven records of brewing are

about 6,000 years old and refer to the Sumerians. Sumeria lay between the Tigris and

Euphrates rivers including Southern Mesopotamia and the ancient cities of Babylon and Ur.

It is said that the Sumerians discovered the fermentation process by chance. A seal around

4,000 years old is a Sumerian "Hymn to Ninkasi", the goddess of brewing. This "hymn" is

also a recipe for making beer. No one knows today exactly how this occurred, but it could be

that a piece of bread or grain became wet and a short time later, it began to ferment and an

inebriating pulp resulted. These early accounts, with pictograms of what is recognizably

barley, show bread being baked then crumbled into water to make a mash, which is then

made into a drink that is recorded as having made people feel "exhilarated, wonderful and

blissful!" It could be that baked bread was a convenient method of storing and transporting a

resource for making beer. The Sumerians were able to repeat this process and are assumed to

be the first civilized culture to brew beer. They had discovered a "divine drink" which

certainly was a gift from the gods.

1.1 Ingredients used in brewing of beer

The process of making beer is known as brewing. A dedicated building for the making of

beer is called a brewery, though beer can be made in the home and has been for much of its

history. A company that makes beer is called either a brewery or a brewing company. The

basic ingredients of beer are water; a starch source, such as malted barley, able to be

fermented (converted into alcohol); a brewer's yeast to produce the fermentation; and

flavouring such as hops. A mixture of starch sources may be used, with a secondary starch

source, such as maize (corn), rice or sugar, often being termed an adjunct, especially when

used as a lower-cost substitute for malted barley. Less widely used starch sources include

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millet, sorghum and cassava root in Africa, potato in Brazil, and agave in Mexico, among

others. The amount of each starch source in a beer recipe is collectively called the grain bill.

Water

Beer is composed mostly of water. Regions have water with different mineral components; as

a result, different regions were originally better suited to making certain types of beer, thus

giving them a regional character. For example, Dublin has hard water well suited to making

stout, such as Guinness; while Pilzen has soft water well suited to making pale lager, such as

Pilsner Urquell. The waters of Burton in England contain gypsum, which benefits making

pale ale to such a degree that brewers of pale ales will add gypsum to the local water in a

process known as Burtonisation.

Starch source

The starch source in a beer provides the fermentable material and is a key determinant of the

strength and flavour of the beer. The most common starch source used in beer is malted grain.

Grain is malted by soaking it in water, allowing it to begin germination, and then drying the

partially germinated grain in a kiln. Malting grain produces enzymes that convert starches in

the grain into fermentable sugars. Different roasting times and temperatures are used to

produce different colours of malt from the same grain. Darker malts will produce darker

beers. Nearly all beer includes barley malt as the majority of the starch. This is because of its

fibrous husk, which is not only important in the sparging stage of brewing (in which water is

washed over the mashed barley grains to form the wort), but also as a rich source of amylase,

a digestive enzyme which facilitates conversion of starch into sugars. Other malted and

unmalted grains (including wheat, rice, oats, and rye, and less frequently, corn and sorghum)

may be used. In recent years, a few brewers have produced gluten-free beer made with

sorghum with no barley malt for those who cannot consume gluten-containing grains like

wheat, barley, and rye.

Hops

Flavouring beer is the sole major commercial use of hops. The flower of the hop vine is used

as a flavouring and preservative agent in nearly all beer made today. The flowers themselves

are often called "hops". Hops were used by monastery breweries, such as Corvey in

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Westphalia, Germany, from 822 AD, though the date normally given for widespread

cultivation of hops for use in beer is the thirteenth century. Before the thirteenth century, and

until the sixteenth century, during which hops took over as the dominant flavouring, beer was

flavoured with other plants; for instance, Glechoma hederacea. Combinations of various

aromatic herbs, berries, and even ingredients like wormwood would be combined into a

mixture known as gruit and used as hops are now used. Some beers today, such as Fraoch' by

the Scottish Heather Ales company and Cervoise Lancelot by the French Brasserie-Lancelot

company, use plants other than hops for flavouring. Hops contain several characteristics that

brewers desire in beer. Hops contribute a bitterness that balances the sweetness of the malt;

the bitterness of beers is measured on the International Bitterness Units scale. Hops

contribute floral, citrus, and herbal aromas and flavours to beer. Hops have an antibiotic

effect that favours the activity of brewer's yeast over less desirable microorganisms, and hops

aids in "head retention", the length of time that a foamy head created by carbonation will last.

The acidity of hops is a preservative.

Yeast

Yeast is the microorganism that is responsible for fermentation in beer. Yeast metabolises the

sugars extracted from grains, which produces alcohol and carbon dioxide, and thereby turns

wort into beer. In addition to fermenting the beer, yeast influences the character and flavour.

The dominant types of yeast used to make beer are ale yeast (Saccharomyces cerevisiae) and

lager yeast (Saccharomyces uvarum); their use distinguishes ale and lager. Brettanomyces

ferments lambics, and Torulaspora delbrueckii ferments Bavarian weissbier. Before the role

of yeast in fermentation was understood, fermentation involved wild or airborne yeasts. A

few styles such as lambics rely on this method today, but most modern fermentation adds

pure yeast cultures.

Clarifying agent

Some brewers add one or more clarifying agents to beer, which typically precipitate (collect

as a solid) out of the beer along with protein solids and are found only in trace amounts in the

finished product. This process makes the beer appear bright and clean, rather than the cloudy

appearance of ethnic and older styles of beer such as wheat beers. Examples of clarifying

agents include isinglass, obtained from swimbladders of fish; Irish moss, a seaweed; kappa

carrageenan, from the seaweed Kappaphycus cottonii; Polyclar (artificial); and gelatin. If a

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beer is marked "suitable for Vegans", it was clarified either with seaweed or with artificial

agents.

Today modern brewing plants perform myriad analyses on their beers for quality control

purposes. Shipments of ingredients are analyzed to correct for variations. Samples are pulled

at almost every step and tested for oxygen content, unwanted microbial infections, and other

beer-aging compounds. A representative sample of the finished product often is stored for

months for comparison, when complaints are received.

1.3 The process of brewing beer

Work in the brewery is typically divided into 7 steps: Mashing, Lautering, Boiling,

Fermenting, Conditioning, Filtering, and Filling.

Mashing

Mashing is the process of mixing milled grain (typically malted grain) with water, and

heating this mixture up with rests at certain temperatures to allow enzymes in the malt to

break down the starch in the grain into sugars, typically maltose.

Lautering

Lautering is the separation of the extracts won during mashing from the spent grain to

create wort. It is achieved in either a lauter tun, a wide vessel with a false bottom, or a mash

filter, a plate-and-frame filter designed for this kind of separation. Lautering has two stages:

first wort run-off, during which the extract is separated in an undiluted state from the spent

grains, and sparging, in which extract which remains with the grains is rinsed off with hot

water.

Boiling

Boiling the wort ensures its sterility, and thus prevents infections. During the boil, hops are

added, which contribute their bitterness, aroma and flavour compounds to the beer. Along

with the heat of the boil, they cause proteins in the wort to coagulate and the pH of the wort

to fall, and they inhibit the later growth of certain bacteria. Finally, the vapours produced

during the boil volatilize off-flavours, including dimethyl sulfide precursors. The boil must be

conducted so that it is even and intense. The boil lasts between 60 and 120 minutes,

depending on its intensity, the hop addition schedule, and volume of wort the brewer expects

to evaporate.

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Fermentation

Fermentation, as a step in the brewing process, starts as soon as yeast is added to the cooled

wort. This is also the point at which the product is first called beer. It is during this stage that

fermentable sugars won from the malt (maltose, maltotriose, glucose, fructose and sucrose)

are metabolized into alcohol and carbon dioxide. Fermentation tanks come in all sorts of

forms, from enormous cylindroconical vessels which can look like storage silos, to

five gallon glass carboys in a home brewer’s closet.

Conditioning

When the sugars in the fermenting beer have been almost completely digested, the

fermentation slows down and the yeast starts to settle to the bottom of the tank. At this stage,

the beer is cooled to around freezing, which encourages settling of the yeast, and causes

proteins to coagulate and settle out with the yeast. Unpleasant flavours such as phenolic

compounds become insoluble in the cold beer, and the beer's flavour becomes smoother.

During this time pressure is maintained on the tanks to prevent the beer from going flat.

Filtering

Filtering the beer stabilizes the flavour, and gives beer its polished shine and brilliance. Not

all beer is filtered. Filters range from rough filters that remove much of the yeast and any

solids (e.g. hops, grain particles) left in the beer, to filters tight enough to strain colour and

body from the beer. Normally used filtration ratings are divided into rough, fine and sterile.

Rough filtration leaves some cloudiness in the beer, but it is noticeably clearer than unfiltered

beer. Fine filtration gives a glass of beer that you could read a newspaper through, with no

noticeable cloudiness. Finally, as its name implies, sterile filtration is fine enough that almost

all microorganisms in the beer are removed during the filtration process.

Filling

Filling (also known as "packaging") is putting the beer into the containers in which it will

leave the brewery. The containers are usually bottles, cans, or kegs; sometimes bulk tanks are

used for high-volume customers (Back, 1997).

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CHAPTER TWO

MICROBIOLOGICAL QUALITY ASSURANCE IN BREWING

Quality Assurance (Q.A) as defined by the World Health Organization (WHO) is the total

process whereby the quality of Laboratory report can be guaranteed. It has been summarized

as the right result at the right time, on the right specimen, from the right patient, with the

result interpretation based on correct reference data at the right price. Quality Control (Q.C)

on the other hand, covers that part of quality assurance which primarily concerns the control

of errors in the performance of tests and verification of test results.

All breweries need quality assurance to maintain confidence in the beer they produce. It is

required for a variety of functions from checking the quality of the raw materials, through

monitoring beer production and packaging operations, to checking final product quality.

There are essentially two approaches to microbiological testing, the conventional techniques

that involve inoculating a solid or liquid medium with a brewery sample, and after

incubation, examining for the presence of absence of growth. General-purpose media for the

cultivation and identification of microorganisms can be prepared from wort or beer but

commercial media provide for consistency and ease of use. The monitoring of all production

processes from incoming goods inspection through actual production right up to outgoing

goods inspection is of vital importance for end-to-end quality management in the brewing

industry. The complex task of ensuring a constant, high quality standard for each product can

no longer be performed by a laboratory alone. Instead, the assessment of all processes

involved requires a meaningful combination of lab analyses and in-line measurements.

2.0 Sources of microbes

Beer may contain microbial contaminants originating from a variety of sources. Primary

contaminants originate from the raw materials and the brew house vessels and secondary

contaminants are introduced to the beer during bottling, canning or kegging. While

approximately half of the documented microbiological problems can be attributed to

secondary contaminations, the consequences of primary contaminations may be more

catastrophic, with the potential loss of a complete brew.

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2.1 Primary contamination

Brewing raw materials, such as malt, hops and adjuncts (unmalted cereals used as

supplementary ingredients) carry their own microbiota as has been shown for malt. Water to

be used in a brew will be boiled during wort boiling, but later additions must be sterile to

prevent microbial contamination. For many years, it has been recognised that handling of

yeast in bulk at the brewery can result in low-level bacterial contamination of the brewing

yeast. Thus, it is imperative that the brewing yeast is handled with utmost care to avoid

contamination. Due to the nature of their contents and complex pipe work, the brew house

vessels will be sources of microbial contamination if they are not properly maintained or

cleaned. Packaging raw materials (bottles, casks and kegs) can all be heavily contaminated

with microbes when returned from trade, because they contain small amounts of beer in

which microbial growth may have occurred over an extended period of time.

2.2 Secondary contamination

All points of contact with cleaned or filled, but unsealed bottles are possible sources of

secondary contamination. Airborne microorganisms can contaminate beer in the filling

department during the transport of open bottles from the bottle washer to the filler and from

the filler to the point when the bottle is sealed (Sakamoto and Konings, 2003). Such

contamination is a significant problem in breweries that do not operate in-package

pasteurisation. A direct, proportional relationship has been observed between air humidity

and airborne microorganisms, confirming that high relative humidity leads to higher numbers

of airborne microorganisms. Persistent contamination in the filling hall can result from

sequential growth of different but interdependent microorganisms that attach to surfaces in

the form of biofilms (Back, 1994). Equipment used in the filling process is particularly prone

to biofilm formation due to the large volumes of water that are used for bottle rinsing during

filling. This creates an environment suitable for microbial attachment and accumulation on

surfaces. Biofilms are a problem in brewing primarily as a source of microbiological

contamination but they also protect microorganisms during cleaning and disinfection

operations (Kretsch, 1994).

Microbiological quality-control methods in the brewing industry fall into three principal

types:

i. To confirm sterility: no recovery of viable microorganisms from the sample.

ii. To determine that the microbiological count does not exceed the specified limit.

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iii. To examine for specific spoilage organisms.

Brewery fermentation temperatures are usually in the range 10-20oC, but result of

microbiological test are required urgently and even at 20o C growth would be too slow.

Microbial growth is accelerated by higher temperature. some brewery microorganisms are not

particularly thermotolerant , but most bacteria and yeast of the brewing industry to grow

visible colonies in 3 days with incubation at 25-27oC with incubation temperatures above or

below that range , the growth rate of most bacteria, moulds and yeasts of the malting and

brewing industries is likely to be slower . Although colonies may already be visible after

3days an additional day of incubation is advisable for samples taken after chemical

sterilization or pasteurization to be sure of recovery of viable but damaged microorganisms.

Another aspect of microbiological testing concerns Hazard Analysis Critical Control Points

(HACCPs), which have become an essential feature of quality control in the food and drink

industries, including the brewing industry, although many of the Critical Control Points

(CCPs) are not microbiological, for example to prevent fragment of broken glass in bottled

beer, microbiological aspects of HACCP are certainly important.

2.3 Microbiological Analysis of Raw Materials

Water

Water is an important raw material. malt steep water, brewing liquor high-gravity beer

dilution liquor , an even the water used for rinsing the cleaned process plan and beer

containers can all be ingested by the consumer and so it must meet the microbiological

standard for drinking water. Also the ionic composition of water is important for mashing,

hop boiling, fermentation and its contribution to beer flavour. Indeed many important

brewing centres of the world have reached that status because of the chemical properties of

the local water supply.

Any supply of water intended for human consumption, either directly as drinking water or

indirectly in foods or beverages, must be clear , odourless, and free from undesirable tastes

or dangerous chemical substance, e.g. ammonia, nitrate, haloforms, pesticides, and toxic

inorganic ions, as well as meeting international microbiological standards. It is important to

be aware that a water supply contaminated by domestic sewage or farm efffluent could

contain pathogenic intestinal bacteria. Only trained microbiologists may carry out these

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techniques in containment level 2 facilities as specified by Advisory Committee on

Dangerous Pathogens.

The purpose of routine microbiological analysis is to ensure absence of those pathogenic

bacteria that are spread by contaminated water supplies. Because the most dangerous,

Salmonella spp. (which cause food poisoning and typhoid fever), Shigella spp (dysentery),

and Vibrio cholerae (cholera), are likely to occur irregularly and in relatively small number in

contaminated water it is not normal practice to culture these pathogens directly, instead ,

Escherichia coli, which is the most numerous microorganism of the intestinal tract of warm-

blooded animal is used as an indicator organism of faecal pollution, normally E. coli is

harmless, but a few rare groups, e.g. O-55,O-111, O-157, produce dangerous toxins.

For the examination of chlorinated mains water, a crystal of sodium thiosulfate is added to

sample bottle in a tank, the sample should be collected from a tap. Taps must be sterilized by

blowlamp or burning ethanol before use and water must be run through the tap to cool the

metal. It may also be necessary to run for a sufficient time or water (reservoir, canals) to

avoid contamination by the sampler's hand and the open end of the sample bottle must face

into running river water. Because bacteria can grow in stored water the analysis must start as

soon as possible. If more than 3hr are likely to elapse between collection and analysis must

begin not later than the following morning. The regulations also specify the shaking routine

to resuspend bacteria that may have sedimented during transport or storage.

The test is in three parts:

1. Count of viable bacteria at 22oC and 37oC, the original method, which is still valid,

specified plate count of 1ml water in yeast extract-peptone agar, but now membrane filtration

of 100mls, may be used instead. Duplicate samples water are required, one plate is incubated

at 22oC for 3 days and the other at 37oC for 24 hours (but no longer). The natural free-living

bacteria of water grow well at 22oC but only parasitic bacteria of intestinal origin grow to

visible colonies within 1 day at 37oC therefore any substantial count at 37oC suggest faecal

pollution. The temperatures are critical and incubators accurately adjusted to these

temperatures are necessary for water analysis.

2. Coliform count at 37oC on a selective indicator medium for enteric bacteria. MacConkey's

medium is traditionally used, but other similar piped chlorinated supplies should have very

low coliform count, preferably Zero, i.e. undetectable in sample of 1, 10 and 100ml

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respectively. It is possible to membrane-filter a large volume (at least 1 litre) and

subsequently isolate and identify enteric pathogens directly on selective midis but this should

be done only in public health laboratories, never in malting or breweries, but even the

standard analysis methods could culture pathogens present in the water, therefore all positive

test must be handled as if that had actually happened (Van Vuuren and Priest, 2003).

Escherichia coli is not the only possible indicator organism for intestinal pollution:

Clostridium perfringens and Enterococus (previously Streptococcus) are also obligate

intestinal bacteria. Standard methods are available for their detection and enumeration but

result could be misleading because E. coli belongs to the same family as the intestinal

pathogens Salmonella and Shigella and has similar survival properties. Enterococcus faecalis

dies off more quickly possibly resulting in false- negative result.

Cereals

The process of worth boiling protect against contaminated raw materials but only to a limited

extent. Mould contaminants may produce heat- stable mycotoxins or the polypeptide gusting

factor particularly associated with certain Fusarium spp. although the fungi themselves are

killed by hop boiling metabolites are not entirely destroyed and can persist to the final beer. It

is theoretically possible that with high level of contamination, some of the bacteria of malt

persist through hop boiling to contaminate the wort, but a more likely situation is airborne

contamination drifting between the malt mill and fermentation vessel. Good microbiological

quality of raw materials is required for good-quality beer, and such a specification should be

included in ISO or similar accreditation. Certainly a general plate count less than 105 colony-

forming units (CFUs) per gram indicates good quality of gain. It is a debatable point whether

a general plate count is sufficient, or whether specific spoilage bacteria, e.g. lactic bacteria,

should be counted on a suitable selective medium. For distilleries, because unboiled worts are

used, the risk of contamination justifies a specified maximum count of lactic bacteria. The

case for such counts of barley destined for a brewery is less obvious, and there is no point in

carrying out an analysis unless it is intended to make use of the result.

Cereals with sufficient mould contamination to produce dangerous amounts of mycotoxin

would be obviously mouldy, by appearance and smell, and rejected for these reasons long

before results of microbiological testing could be available. Also, that level of mould

contamination of barley would inhibit germination. Therefore the only routine

microbiological laboratory test for mould contamination of malt or malting barley is an

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examination for Fusarium, the principal cause of gushing in beer. This is especially necessary

if wet weather coloured during ripening or harvest. There are two methods, although the

results are not necessarily comparable:

(a) To macerate the grain and count the number of CFUs.

(b) To incubate intact grains and determine the proportion contaminated by potentially

troublesome fungi.

Plate counts require finely divided grain samples, and much research has been devoted to the

development of methods for efficient maceration of the grain without shear damage of the

microorganisms or significant heating of the suspension. These effects are particularly

relevant in the maceration of hard unmalted cereal. Plate counts of moulds or yeast-like fungi

from such samples are notoriously difficult to interpret because a relatively small amount of

sporing fungal mycelium could be broken up to produce a high plate count of mycelia

fragments and separate spores when hyphae are chopped into individual cells or when chains

or clusters of spores are separated.

Thus, there are good practical reasons for preferring to seed grains on culture media and

observe the proportion of infected grains. It is important to distinguish fungal contaminants

that are well established on the grain from the casual superficial microbial contaminants that

are inevitable on any plant material. The former penetrate the husk, and those sections of the

fungal mycelium survive washing the grain with hypochlorite solution that kills surface

contaminants (Haikara et al., 1993). It is possible to use the grain itself as the culture medium

by laying the well-spaced grains on moist sterile filter paper in a Petri dish and incubating at

25oC. Alternately the grains are planted in microbiological culture medium, with the

advantage that different types of fungi can be selected by choice of medium. Field fungi,

including Fusarium Spp. associated with gushing, grow on the moist germinating grains,

whereas storage fungi, in particular Aspergillus and Penicillium spp. grow well in the lower

available moisture of dried grain, hence the choice of media with or without 10% salt to

select the fungi of interest. Various media are used and recommended. For field fungi,

Czapek-dox agar and potato dextrose agar, both available commercially, are useful general -

purpose mycological media. A convenient form for the storage of fungi malt salt agar:

commercial extract agar to which 10% NaCl is added before sterilization.

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It is an unfortunate complication of such microbiological testing that the moist incubation

condition encourage germination of the grains, and kilned malted barley should be tested

rather than raw grain if possible. However even if the grain has sprouted during the test, not

only fungi growth be observed but the distinctive deep-red colour of Fusarium colonies can

be recognized.

Priming Sugar

Although much of the microbial flora of cereal raw materials will be destroyed by boiling

others are added at a late stage of beer production, either as adjuncts near the end of hop

boiling or as priming to beer for conditioning, so high microbiological quality is essential.

Concentrated sugar syrups, the form added to the hop kettle are protected by their low

water activity but the more dilute solution for priming of beer have no such protection and

must be used immediately. It is unrealistic to expect powdered syrup to be sterile but a very

low count must be specified, for example 2000 or 3000 CFUs per gram of sample. Of 1g

made up to 10ml, the maximum acceptable count would be 200 or 300 colonies from a 1ml

sample. It is not normal practice to examine for any.

Culture media for brewing microbiology

The original medium for brewery microbiology was the brewery’s own wort, solidified with

agar if necessary. The advantage of using a medium with the inhibitory effect of hops is that

only potential beer contaminants are able to grow, but the medium is much richer than

necessary for laboratory purposes and varying from brewery to brewery, prevents

standardized culture methods. Modern Malt Extract (ME) broth, available commercially in

dehydrated form, is much weaker than worth but yet of sufficient nutrient content for all yeast

and most brewery bacteria and is widely used. However, its constant composition consistency

was one advantage of the introduction.

Unfortunately there is no single medium that suppresses brewing yeast but allows the growth

of all possible contaminants. Commonly used selective media for culture and isolation of

brewery contaminants are the following:

1. Actidione agar: Any suitable bacterial culture medium could be used, supplemented

with an antifungal antibiotic to suppress the growth of yeast. After initial tests with

various antibiotics, cycloheximide, known then as Actidione, became the standard

inhibitor. Commercially available (e.g. Oxoid, Difco) Actidione agar is WLN agar

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+100 mg/ ml Actidione, sufficient to inhibit the growth of all brewery strains of S.

Cerevisiae and most other yeasts. Cycloheximide is an antifungal agent but is also

toxic to humans and so should be handled with care. When handling crystals of pure

antibiotic it is important to prevent inhalation and skin and eye contact: gloves,

spectacles, and a face mask are essential. The medium was originally intended to be

selective for brewery bacteria, and is ideal for that purpose, but some resistant yeast

are able to grow. However, at only 10mg/ ml Actidione, still sufficient to inhibit most

culture strains of S. Cerevisiae, growth of a wider range of yeasts is possible, but the

resistant strains that can be detected on Actidione will represent only a part of the

wild-yeast population. Commercial Actidione agar is safer than handling pure

cycloheximide in the laboratory; it is sold as 100 ml bottles of ready-made sterile

100µg/ml medium to be melted at 100oc for preparation of plates (higher temperature

will destroy a proportion of the antibiotic). Lower concentrations are easily and safely

prepared by prepared by mixing with the appropriate amount of melted normal (i.e.

Actidione-free) WLN agar before pouring the plates.

2. Lysine agar (also available from Difco and Oxoid) is a synthetic medium of glucose,

vitamins, inorganic salts, and L-Lysine as the sole N source. Saccharomyces spp. are

unable to grow, but all other spoilage yeast genera of beer do grow, using the –NH2

groups of lysine for all N requirements. Therefore only hose non-Saccharomyces

yeasts that may be present are able to grow to colonies of normal size. Two important

disadvantages of lysine agar are first, that its selective effect is by starvation, and

carried-over extracellular or intracellular nutrient will allow some slight growth of

Saccharomyces spp. To visible colonies and second, that Saccharomyces spp.,

biochemically adapted to grow well in brewery and distillery fermentations, are

potentially the most common contaminants of these situations but cannot be detected.

Difco yeast nitrogen base (YNB) with 0.5% dextrin or starch as carbon source is

selective for amylolytic yeasts. Alternately, ME agar supplemented with 0.5% starch

could be used, but then it is necessary to flood the plate with iodine solution after

incubation to observe the zone of starch hydrolysis around amylolytic colonies. As a

general rule, contaminants recovered on media with specific carbon nutrients other

than dextrin or starch are of general other than Saccharomyces and would be detected

anyway on lysine agar.

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Microbiological analysis of wort

Routine testing of each batch is unnecessary. Only a few heat-resistant spores of Bacillus and

Clostridium spp. survive hop boiling, and provided the wort is pitched during collection, or at

the finish of collection, the start of fermentation prevents their germination and growth.

However, in the event of contaminated fermentations, it is necessary to investigate the wort

as one possible source of the contamination. Spoilage organisms should not survive wort

boiling; the likely sources of contamination are pipe work, a contaminated or leaking heat

exchanger, the fermentation vessel, and air, either injected air or the atmosphere of the

fermentation area. Unless the wort has been left uninoculated for some time, the number of

contaminant cells will be insufficient to detect by a standard plate count or microscopic

examination. Two types of test are possible: (a) membrane filtration of at least 100 ml and

culture on a non-selective culture medium or (b) a ‘forcing test’ incubating an aseptically

collected sample in a sterile bottle to grow any contaminants that may be present. The

disadvantage of both methods is that bacteria or yeasts that are incapable of spoilage of beer

will, if present, grow in the test, so any microbial growth must be subcultured and identified.

It is important to cover the filter during operation to exclude airborne contaminants.

Sterility of plant

Test during fermentation

Although regular sampling during fermentation is an important part of the brewer’s duties,

normally the only useful (but certainly not essential) microbiological test would be a check

on yeast numbers. Simple tests of specific gravity, pH, and flavour assessment are normally

adequate. Unusual flavour could be a warning of a developing microbiological problem, in

which case a cultural test would be justified. With the enormous excess of pitching yeast,

microscopic examination is pointless. The sample is plated on the selective media used for

examination of pitching yeast and again, the result is available only after 3 days incubation.

The main advantage of a test of a suspect fermentation is the advance warning of whether the

pitching yeast could be reused or must be acid washed or discarded.

Fermentation vessel

Confirmation of sterility of cleaned and sterilized FVs and associated pipe work and

accessories is a CCP. Incidentally, although microbiological, CCP is understood in the

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present context, it is also a’ chemical’ CCP: to guarantee sufficient rinsing for complete

removal of detergent and sterilant compounds. Traditionally, swabbing of a known area of

cleaned surface was used to detect residual contamination of plant after sterilization. The

method is not suitable for enclosed vessels, because opening a sterilized vessel and learning

in to take the sample is not good microbiology (and dangerous, with residual CO2 after acid

sterilization). However, because traditional fermentation vessels are open anyway, it is still

acceptable to reach as far in as possible to swab an estimated 30cm x 30cm area (about

1000cm2, or 1square foot) with the sterile cotton wool swab, most conveniently the medical

type for throat swabbing. A sterile bottle with 5ml is also required. The swab is also taking

from its container, moistened with the sterile water (or preferably Ringer’s saline solution,

believed to improve survival of containments between the sample point and the laboratory),

rubbed over test surface, and returned to the bottle, breaking off the wooden handle below the

part handled by the operator. The specimen should be analyzed immediately, certainly within

2hrs; in the laboratory the swab is shaken vigorously in the bottle to dislodge the micro-

organisms into the water. A 1ml sample is counted in ME agar and the colony count

multiplied by 5 shows the number of organisms over the swabbed area. For enclosed vessels,

a sample of the final rinse water an indication of any residual contamination after

sanitization. At least 250ml is filtered through a 0.45µm membrane and counted on ME or

WLN after 3 days

2.4 Problems associated with contaminating microorganisms at various stages of the

brewing process

Growth of microorganisms can occur during the brewing process because of the nutrient-rich

environment of wort, supplemented by growth factors that are produced by the brewing yeast.

Nevertheless, the microorganisms that grow in wort and beer are restricted to relatively few

species. The spoilage character of a particular organism will depend on the stage at which it

is found in the brewing process. For example, the brewing yeast should be regarded as a

contaminant if it is detected after filtration of the beer. However, microbial growth in beer is

restricted by the presence of inhibitors, such as hop compounds, alcohol and carbon dioxide,

and inhibitory conditions such as low levels of nutrients and oxygen, and low pH. Relatively

few microorganisms are able to grow and thus spoil beer under such adverse environmental

conditions.

17

2.4.1 Barley and malt.

Although moulds are not spoilers of wort or beer, their presence in barley may negatively

impact on the quality of the malt, wort and beer. The nature and magnitude of the barley-

associated microbiota will depend on both the field conditions under which the barley crop

was grown and the post-harvest history of the grain. The expected barley microbiota consists

of moulds that contaminate and colonise the grain in the field, and moulds that grow on the

grains during storage. Species of Alternaria, Cladosporium, Epicoccum and Fusarium are all

examples of field fungi. Fusarium is one of the most important genera of plant pathogenic

fungi and Fusarium spp. are responsible for Fusarium head blight, or scab which caused

losses of more than 2.6 billion dollars to US agriculture during epidemic infections in the

1990s. In addition to causing grain yield and quality reduction in cereals, Fusarium spp.

cause food safety problems. Fusarium graminearum and Fusarium culmorum can produce

toxic secondary metabolites called mycotoxins, which are not removed or degraded by the

brewing process. These mycotoxins, particularly deoxynivalenol (DON) and zearalenone

(ZEA), can be produced during the malting of barley and persist in the final beer. In addition

to being potential mycotoxin producers, some Fusarium species are known to be active

gushing inducers, a phenomenon that describes the uncontrolled spontaneous overfoaming of

beer on opening of bottles, cans or kegs. Species of Aspergillus and Penicillium, which are

fungi that grow during barley storage, also produce toxins called aflatoxins.

2.4.2 Mashing and Wort separation.

Growth of thermophilic bacteria can present problems during mashing if mash temperatures

fall or unhopped wort is held at temperatures below 60°C. While most LAB are sensitive to

hop compounds and do not survive in wort after hop addition during wort boiling,

uncontrolled growth of LAB before hop addition may lead to the wort having undesirable off-

flavours. Aerobic, endospore-forming bacteria belonging to the genus Bacillus have on

occasion caused problems in breweries. Bacillus spores are present in malt and cereal

adjuncts and will survive wort boiling but are unable to germinate during the ensuing stages

of brewing due to their sensitivity to hop compounds and the low pH of the fermenting wort

and beer. Bacillus coagulans has been reported to produce copious amounts of lactic acid in

sweet wort held at 55–70°C for more than two hours, and has been linked to the formation of

nitrosamine at concentrations far exceeding the recommended limit of 20 μg L–1 by

reduction of nitrate to nitrite during anaerobic respiration. Coliform bacteria can be

18

introduced into wort from a contaminated water supply or by allowing seepage of fluid into

the wort from leaking pipe connections.

2.4.3 Fermentation.

After hop addition and boiling, the wort is clarified, cooled and aerated so that it provides an

ideal medium for yeast growth and fermentation. However, this wort is also an ideal medium

for the growth of certain contaminants, which may be introduced with the brewing yeast

during wort aeration or as a result of inadequate cleaning and disinfection of equipment.

Yeasts that are not deliberately used in the brewery but are found in brewing materials are

designated wild yeasts. Such yeasts have been isolated from all stages of brewing and can

cause problems for brewing operations, especially at the fermentation stage. Growth of wild

yeasts during fermentation may lead to defects, including turbidity, off-flavours and aromas.

Other unwanted effects include difficulties with yeast fermentations (e.g. slow or stuck

fermentations) and super attenuation of the finished beer, a phenomenon resulting in a beer

with atypically low terminal gravity and undesirably high alcohol content. Since wild yeasts

are a diverse group, they have been divided into non-Saccharomyces and Saccharomyces

wild yeast. The non-Saccharomyces wild yeasts include organisms of the genera

Brettanomyces, Candida, Debaryomyces, Dekkera, Filobasidium, Hanseniaspora,

Kluyveromyces, Pichia, Torulaspora, Zygosaccharomyces and others. Some bacteria can

contaminate the brewing yeast and may pose a serious threat to the success of the

fermentation process. Pediococcus damnosus, a homofermentative coccoid-shaped

bacterium, is the most frequently encountered Pediococcus found in brewing yeast and beer.

Pediococcus inopinatus is detected in brewing yeast but rarely at other stages of beer

fermentations.

2.4.4 Biological stabilisation and packaging.

The flavour, aroma, and keeping qualities of the beer are modified by aging, filtration and

pasteurisation. There are two types of beer pasteurisation regimes in use, tunnel

pasteurisation and flash pasteurisation, which meet different needs and differ substantially in

both duration and the maximum temperature applied. Tunnel pasteurisation is the method

most widely used to assure the commercial sterility of brewing products packaged in cans or

bottles. It is a lengthy process where cans or bottles move through a ‘tunnel’ of fixed

temperatures. Flash or plate pasteurisation is a rapid, bulk pasteurisation process involving

passage of beer through a plate heat exchanger. It is typically used to treat beer before

19

packaging in kegs or other containers that cannot be tunnel pasteurised. However, this

reduction of oxygen levels has resulted in a higher frequency of spoilage by anaerobic

bacteria such as species of Pectinatus and Megasphaera. Pectinatus cerevisiiphilus was first

described by Lee et al. Currently, Pectinatus species have been implicated in 20–30% of

bacterial spoilage incidents encountered in finished product, usually in non-pasteurised rather

than pasteurised beers. The organisms grow under anaerobic conditions in the packaged

product, between 15 and 40°C with an optimum at 32°C and at a pH of 4.5. Pectinatus spp

produce considerable amounts of acetic and propionic acid as well as acetoin in wort and

packaged beers (Haikara et al., 1981). Beer spoilage by Pectinatus spp is characterised by

extensive turbidity and an offensive ‘rotten egg’ smell as a result of the production of

hydrogen sulphide and methyl mercaptan. Megasphaera has been linked up to 7% of

bacterial beer spoilage incidents (Haikara and Lounatmaa, 1987). Only one species of the

genus, M. cerevisiae, is known to cause such beer spoilage. It is strictly anaerobic. Growth of

the organisms is usually inhibited at pH values below 4.1 and at ethanol concentrations above

2.8% (w/v) although its growth at 5.5% (w/v) ethanol has been reported. Most bacterial

spoilage incidents are caused by the Gram-positive LAB. Spoilage may also arise from the

growth of wild yeasts. Uncontrolled growth of wild yeasts within a beer bottle may even

result in a health risk as it can lead to a build up of carbon dioxide and explosion of the bottle

(Jack et al., 1995).

2.4.5 Contamination of finished product.

Lactobacillus brevis is the most prevalent beer spoiling Lactobacillus species. More than

half of the reported incidents of beer spoilage were due to this bacterium. Lb. Brevis is an

obligate heterofermentative bacterium, which grows optimally at 30°C and pH 4 to 6. Beer

spoiling strains of this species are generally resistant to hop compounds. Lb. brevis

contaminations can cause various problems in beer such as turbidity and super-attenuation,

due to its ability to ferment dextrins and starch (Hayashi et al., 2001). Lactobacillus lindneri

has been implicated in 15–20% of beer spoilage incidents (Lawrence, 1988). It is highly

resistant to hop compounds, grows optimally at 19 to 23°C and survives more extreme

thermal treatments than other LAB. Other beer spoiling lactobacilli are Lactobacillus

buchneri, Lactobacillus casei, Lactobacillus collinoides, Lactobacillus coryneformis, and

Lactobacillus plantarum although such heterofermentative LAB are less common than Lb.

Brevis or Lb. lindneri. Lb. casei is particularly problematic because it may give rise to high

levels of diacetyl in finished beer. Pediococci are homofermentative coccoid-shaped bacteria,

20

which grow in pairs or tetrads, and are found in wort fermentations and finished beers. The

most common beer spoiler belonging to this genus is the hop-resistant, prolific diacetyl

producer Pediococcus damnosus. Other Gram-positive bacteria are widely distributed in beer

and brewing materials and occasionally are responsible for beer spoilage (Priest, 1996).

Kocuria kristinae (previously known as Micrococcus kristinae) is atypical in that it is

facultatively anaerobic. It can grow in beer with low levels of alcohol and hop compounds

and at pH values above 4.5, producing a fruity atypical aroma in the beer4. Zymomonas spp.

are Gram-negative aerotolerant anaerobes, which are found in ales and in primed beer, i.e.

beer to which sugar is added. Zymomonas mobilis produces high levels of acetaldehyde and

hydrogen sulphide. Acetobacter spp. thrive in alcohol-enriched niches whereas

Gluconobacter spp. prefer sugars as carbon sources. These Gram-negative acetic acid

bacteria are resistant to hop compounds, acids and ethanol, and are aerobic or

microaerophilic. They can contaminate beer if air is present in the headspaces of bottles and

cans as a result of faulty packaging (Suihko et al., 2003).

2.4.6 Contamination of beer dispensing systems.

Kegged beers shown to be free from contaminants when delivered to retail outlets, are often

spoiled following the coupling of the keg to a dispense system. The dispensing system is

exposed to microbes present in the environment of the draught beer outlet via the open tap

and during the changing of kegs (Casson, 1985). Draught beer from the tap has been found to

contain various types of microorganisms other than those commonly present in the brewery,

which indicates that the organisms originated from the dispensing environment. Acetic acid

bacteria are particularly prevalent in ineffectively cleaned dispensing equipment and are

known to cause haze and surface film (Harper et al., 1980). Biofilms can form within a beer

dispensing line by tight microbial attachment and adhesion. The slime matrix of the biofilm

cells affords protection against line cleaning procedures and the different microbial species

throughout the dispense system may cause more than one type of spoilage microorganism to

adversely affect the beer (Back, 1981).

2.5 Controlling contamination

Prevention of microbial spoilage of beer is best achieved by controlling access of

contaminants to materials within the brewery. However, the brewing process is not aseptic

and complete elimination of spoilage microorganisms from all brewing materials is

technically not possible. A number of strategies can be adopted to minimise the risk of

21

contamination of worts and beers (Storgårds, 2000). The first step in controlling the

microbiota in the brewery is to source raw materials that carry low or harmless microbial

loads. The beer production and packaging facilities should be hygienically designed to

eliminate contamination problems that may arise in equipment such as tanks, pipelines, joints

and accessories. A suitable choice of equipment and materials, and elimination or

minimization of dead spaces and rough surfaces, correct construction, process layout and

automation is crucial to minimise contamination risks. Furthermore, the filler is a critical

point, especially in the aseptic packaging operation, where product contamination may occur,

and so it is crucial to maintain high standards in filler hygiene. The importance of cleaning

and disinfection procedures for both small and large breweries has increased significantly due

to the microbiological vulnerability of certain products such as non-pasteurised beers and

beers that are low in alcohol and bitterness. One of the most important aspects of beer

production management is keeping all equipment that comes into contact with beer or its

precursors in prime condition by regular cleaning with approved detergents, not just within

the brewery, but also at the point of dispensing in the case of draught beer. Hammond et al.

(1999) suggested that by exploiting the antimicrobial activities of beer components such as

dissolved carbon dioxide and phenolic compounds, susceptibility of the beer to spoilage

could be reduced and the effects of chance contamination could be minimised. With the

current negative attitude of consumers towards chemical preservation of foods and stricter

regulations regarding their use, the exploitation of naturally derived inhibitory components

offers an alternative and attractive means to enhance the microbiological stability of beer.

CONCLUSION

Food safety is a major concern for consumers and food producers, the latter must comply

with stringent safety regulations enforced by the legislative authorities. The brewing industry

complies with regulations that limit the presence of mycotoxins and other contaminants such

as biogenic amines, acrylamides, heavy metals, plant toxins and pesticides. The European

Community has implemented the first legislative measures relating to mycotoxins in malt and

cereals, while further legislation on beer is pending. Every effort to minimise the level of

these contaminants would be of benefit to consumer and brewer alike. The application of the

inhibitory components derived from raw materials in malt and beer could be a natural

solution for the brewing industry. However, good brewing practices from barley to beer are

22

important for the production of a high quality beverage. Malt must be stored in a dry, cool

place to prevent mould growth and a LAB malt inoculum can be used as an additional

safeguard. Bacteriocins produced by LAB and hop compounds can offer some protection to

wort from microbes but the wort must be produced with water of a suitable microbiological

standard, boiled and held at appropriate temperatures to ensure that it is not at risk from

contamination. Hop compounds must be fully identified and characterised so that their

inhibitory activities can be optimised. Good yeast management during fermentation (even if a

modified zymocin-immune yeast is used), followed by sufficient filtration and biological

stabilisation, are essential for a microbiologically stable beer. It is also important that beer is

filled in airtight packages in a clean environment where product residues and water are not

allowed to accumulate. High standards of hygiene and regular maintenance regimes are also

necessary throughout the entire brewing process to guarantee that the product is not

microbiologically compromised (Buggey et al., 2002). While exploitation of the

antimicrobial activities of components derived from beer raw materials can contribute to

enhancing the microbiological stability of the finished product, they are only effective if used

in conjunction with good brewing practices.

23

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