WP9159ch19.pdf

(977 KB) Pobierz
19
Brewing control systems:
microbiological analysis
Ê
E. Storgards, A. Haikara and R. Juvonen, VTT Technical
Research Centre of Finland
19.1
Introduction
The presence of inhibitors such as hop compounds, alcohol, carbon dioxide and
sulphur dioxide as well as the shortage of nutrients and oxygen and the low pH
all make beer resistant to microbial contamination. Moreover, processes such as
filtration, storage at low temperatures and possible pasteurisation reduce con-
tamination. The special environment in the brewing process restricts the range of
microorganisms likely to be encountered to relatively few species (Ingledew
1979, Haikara 1984, Back 1994, Dowhanick 1994). Although the contaminants
found may cause quality defects, pathogens have not to our knowledge been
reported to grow in standard beer products. Ensuring the well-being of the
production yeast strains is a fundamental part of brewing as in all processes
based on fermentation technology. Thus, monitoring yeast quality and quantity
is also an important part of the microbiological control carried out in breweries.
In this chapter, a summary of the microorganisms likely to be encountered in
breweries and the different possibilities to detect and identify them is given.
Different possibilities to quantify yeast mass and estimate the brewing per-
formance as well as differentiate between yeast strains is also described. Both
methods currently in use and emerging technologies are discussed. However, as
it is not possible within the framework of this book to review all the techniques
that have attracted attention among brewery microbiologists in the past, merely
those methods showing most potential for brewery applications to date are
reviewed here.
© 2006, Woodhead Publishing Limited
392
Brewing
19.1.1 Microorganisms associated with beer production
Only very few species and strains can adapt to grow in beer. On the other hand,
species adapted to the brewery environment have often not been isolated elsewhere
(Back 1994, Haikara and Helander 2002). Beer spoilage organisms such as lactic
acid bacteria, wild yeasts and even anaerobic bacteria are often present on the
equipment, in the air or in raw materials. These organisms may survive for a long
time in niches of the process, probably outside the direct product stream, without
causing signs of contamination. Then suddenly, they may contaminate the entire
process as a consequence of technological faults or insufficient cleaning. With the
introduction of modern fingerprinting methods, such as ribotyping, into brewery
microbiology, it has become evident that even the same contaminating strains can
pop up after years of absence. In addition to true beer-spoiling organisms that do
grow in finished beer there are a range of organisms that may grow at some stages
of the process, causing off-flavours in the final product if present in sufficient
numbers. There are also indicator organisms that do not cause spoilage but appear
as a consequence of insufficient cleaning or errors in production. The growth of
seemingly harmless microorganisms on brewery surfaces may facilitate the
subsequent colonisation of beer spoilage organisms by producing a more
Ê
favourable environment for their growth (Storgards
et al.
2006). The effects
caused by different spoilage organisms during fermentation and in final beer are
summarised in
Table 19.1.
The microbiological safety risks involved in beer production include
mycotoxin production by toxigenic fungi in raw materials, mainly barley and
malt (Vanne and Haikara 2001, Flannigan 2003). Some of these fungi can
proliferate on barley already in the field, such as
Fusarium,
whereas others such
as
Aspergillus
and
Penicillium
grow in too humid storage conditions. Myco-
toxins are often very stable compounds and can therefore survive throughout
processing and enter the final product. The brewing process itself can be
contaminated by
Obesumbacterium proteus
having the ability to reduce nitrate
present in wort to nitrite, which in turn reacts with amines, producing
nitrosamine compounds (ATNCs or apparent total N-nitroso compounds) (Prest
et al.
1994). Various bacterial contaminants in the brewing process may also be
responsible for the production of biogenic amines (Donhauser
et al.
1993,
Izquierdo-Pulido
et al.
1996, Virkajarvi
et al.
2001).
È
A wider range of microorganisms can cause problems in beer-dispensing
equipment than in the brewing process or in packaged beer. This is due to the
higher oxygen levels and higher temperatures at certain points in the dispensing
system. These conditions favour contamination by microorganisms such as
acetic acid bacteria, moderate levels of coliforms and aerobic wild yeast in
addition to the oxygen-tolerant beer spoilage organisms found in the brewery
environment (Ilberg
et al.
1995, Schwill-Miedaner
et al.
1996, Taschan 1996,
Ê
Storgards 1997). The occurrence of coliforms in beer-dispensing systems is a
cause of concern due to the enteric pathogen
Escherichia coli
serotype
O157:H7. This pathogen is unusually acid-resistant and has been associated with
outbreaks of serious enteric infections after consumption of contaminated apple
© 2006, Woodhead Publishing Limited
Brewing control systems: microbiological analysis 393
Table 19.1
Effects of contaminants during fermentation and on final beer
Group or genera
Wild yeasts
Lactobacillus,
Pediococcus
Acetobacter,
Gluconobacter
Enterobacteria
Effects on
fermentation
Superattenuation
Turbidity Ropiness Off-flavours in final beer
1
Decreased
fermentation rate,
formation of ATNC
À
À
1
À
Esters, fusel alcohols,
diacetyl, phenolic
compounds, H
2
S
Lactic and acetic acids,
diacetyl, acetoin
Acetic acid
DMS, acetaldehyde,
fusel alcohols, VDK,
acetic acid, phenolic
compounds
H
2
S, acetaldehyde
H
2
S, methyl mercaptan,
propionic, acetic, lactic
and succinic acids,
acetoin
H
2
S, butyric, valeric,
caproic and acetic acids,
acetoin
Acetic, lactic and
propionic acids
Acetic and propionic
acids
À
Butyric, caproic,
propionic and valeric
acids
Zymomonas
Pectinatus
2
À
À
Megasphaera
Selenomonas
Zymophilus
Brevibacillus
Clostridium
3
À
À
À
À
À
À
ATNC: apparent total N-nitroso compounds; DMS: dimethyl sulphide; VDK: vicinal diketones; fusel
alcohols:
n-propanol,
iso-butanol, iso-pentanol, iso-amylalcohol.
1
In the presence of oxygen.
2
In primed beer.
3
At elevated pH (5±6).
cider (Semanchek and Golden 1996, Park
et al.
1999). It is infectious at a low
dose, probably due to its acid tolerance, as it can overcome the acidic barrier of
gastric juice and reach the intestinal tract at low population levels (Park
et al.
1999). Thus the possible survival in beer of acid-tolerant pathogens such as
E.
coli
O157:H7 should not be overlooked.
19.1.2 Detection of microbial contaminants in breweries
Contaminations in the brewery are usually divided into primary contaminations
originating from the yeast, wort, fermentation, maturation or the pressure tanks,
© 2006, Woodhead Publishing Limited
394
Brewing
and secondary contaminations originating from bottling, canning or kegging.
About 50% of microbiological problems can be attributed to secondary con-
taminations in the bottling section (Back 1997), but the consequences of primary
contaminations can be more comprehensive and disastrous. The spoilage
character of a particular organism depends on where in the process it is found.
After filtration, the brewing yeast should also be regarded as a contaminant
(Haikara 1984, Eidtmann
et al.
1998).
The concentration of process or product samples has always been a crucial
step in the detection of very low numbers of contaminants in beer. Filtration of
beer for the recovery of microorganisms can be improved by slightly increasing
the temperature and by the use of top pressure (Hammond
et al.
1999). A
bypass-membrane filter device for continuous sampling from product lines has
been developed which makes it possible to increase the sample volume up to 40-
fold (Back and Poschl 1998). Recently, the CellTrap
TM
device (Memteq, UK)
È
was shown to be a useful tool for isolation and recovery of cells from
contaminated beer samples prior to analysis by PCR (Whitmore and Keenan
2005).
Although breweries are still relying mainly on classical cultivation methods,
a range of alternative methods has been developed for the detection of beer
Ê
spoilage organisms (Barney and Kot 1992, Dowhanick 1994, Storgards
et al.
1998a, Quain 1999, Russell and Stewart 2003). Examples of alternative
methods with brewery applications are presented in
Table 19.2.
Unfortunately,
many of these `rapid' techniques need a pre-enrichment step to increase the
sensitivity of the method, thus still being dependent on cultivation. Reasons for
the slow implementation of alternative methods in brewery quality control have
been lack of the speed, sensitivity and specificity required and/or the need for
advanced, expensive equipment and reagents. Therefore, classical micro-
biological methods remain to be the methods preferred by breweries, even
though the detection of beer spoilage organisms by cultivation in laboratory
media does not always provide the specificity and the sensitivity required
(Jespersen and Jakobsen 1996). However, the implementation of new available
technology into brewery microbiology has speeded up considerably during the
twenty-first century.
Detection methods can be divided into culture-dependent and culture-
independent approaches. Culture-dependent methods include traditional
cultivation in combination with phenotypic (physiological and biochemical)
and genotypic (species-specific PCR, DNA fingerprinting, sequencing)
characterisation or identification techniques of selected, isolated strains. The
advantage of this approach is that microbial cultures are available for further
characterisation and exploitation. In recent years, culture-independent
approaches have been developed to complement the culture-dependent ones.
New powerful analytical tools enable us to investigate microbial populations in
their natural environment without the need for cultivation. Direct DNA/RNA
extraction approaches coupled with PCR amplification and community profiling
techniques have become widely applied in microbiology (Muyzer 1999, Ercolini
© 2006, Woodhead Publishing Limited
Brewing control systems: microbiological analysis 395
Table 19.2
Microbiological detection methods with brewery applications
Method
Classical cultivation
methods
Fluorescence
microscopy
Flow cytometry
Detection threshold
Theoretically 1 cfu
per sample
Theoretically 1 cell
per sample, depends
on the application
10
2
±10
4
yeast cells
per ml
Detection time
1 to several days
or weeks
30±60 min, if
enrichment needed
1±3 days
0.5±1 hour, if
enrichment needed
1±3 days
Identification at the
same time
No
No
Yes (in
combination with
fluorescent
antibodies or DNA
probes)
Yes
Yes
Yes
No
No
Laser scanning
cytometry
DNA or RNA
hybridisation
including FISH
PCR
ATP bioluminescence
RMDS (Micro Star
Rapid Microbiology
System)
1 cell per filterable 2±4 hours
sample
10
3
±10
4
cells per ml 24±30 hours
including
pre-cultivation
2
3
0.5±2 hours
10 ±10 cells
10±100 yeast cells
5 min
10
3
±10
4
bacterial
cells
1 yeast cell
1 day
2±3 days
10
2
±10
3
bacterial
cells
2004). The major advantage of different culture-independent approaches is that
organisms in both a cultivable and a non-cultivable state can be analysed.
Moreover, a semi-quantitative picture of a microbial population can be obtained
without time-consuming cultivation and isolation steps.
19.1.3 Identification and characterisation
Identification can be defined as assigning an unknown microorganism to a
particular class in an existing classification (Priest 2003). Identification of
microorganisms to species level is time consuming and seldom needed in
brewery quality control. When identification is performed, it aims to be prag-
matic, searching for key properties such as beer spoilage ability rather than for
taxonomic details. This kind of characterisation of particular problem-causing
strains is an important tool in the tracing of contamination sources. Identification
and characterisation can be based on four levels of expression of genetic
information, namely on the genome, on proteins, on cell components or mor-
phology, and on behaviour (Gutteridge and Priest 1996). Identification, unlike
specific detection, generally requires a pure culture and the use of reference
© 2006, Woodhead Publishing Limited

Plik z chomika:

Inne pliki z tego folderu:

Inne foldery tego chomika:

Zgłoś jeśli naruszono regulamin