utilisedfor the manufacture of stout and lager brews were studied. The bacterial population ... Viable counts of LAB during lager and stout brewhouse mashes in.
A Comparative Study of Malthouse and Brewhouse Microflora, pp.55-62
Volume 105 No. 1,1999
A Comparative Study of Malthouse and Brewhouse Microflora by T.F. O'SullivanH y Walsh*, A. O'Mahonyi, G.F. FitzgeraldW,3 and D. van Sinderen*
department of Microbiology, 2National Food Biotechnology Centre and department of Food Science and Technology, University College, Cork, Ireland Received 1st September 1998
The microflora of malting and mashing was investigated with emphasis on the numbers and
types of lactic acid bacteria (LAB)present during these processes. A traditional small-scale floor malthouse, a modern, pneumatic large-scale malthouse and two brewhouses, each ofwhich were utilised for the manufacture of stout and lager brews were studied. The bacterial population of dried, stored barley for malting was dominated by Gram-coliforms and pseudomonads, with LAB constituting a small minority of the total viable count. In both malthouses, the microbial
count increased dramatically during barley steeping. Although pseudomonads still dominated,
a significant increase in the LAB population was observed. Viable counts decreased slightly towards the end of germination and were reduced by >98%for all groups during kilning. Final counts of LAB on the kilned and screened malt were approximately 10s, comprising 0.5% of the total viable microbial count. While leuconostocs were the predominant LAB detected in the early stages of the process, there was a discernible shift towards homo-fermentative lactobacilli during barley germination. Viable counts of LAB during lager and stout brewhouse mashes in two breweries indicated that initial microbial counts after mashing-in were high (from 105-107 CFWg) and these decreased steadily during the mash programme. In the initial stages of mashing, the LAB population consisted of an equal mixture of lactobacilli and pediococci, but lactobacilli dominated the later stages of the mash. The pre-lauter viable count of LAB was generally ey- There is some
be up to 500 t, steeping takes place in large cylindro-
maltinS is in the numbers °f lactfc acid bacteria
basis of technology and scale. In traditional floor mailings germination of the grain takes place on the malthouse floor, with the grains spread to a depth of about 12-40 cm and the batch size rarely exceeding 30 t. In contrast, in modern, pneumatic mailings, where the batch size may conical vessels and germination occurs in large aerated
vats where the depth of the grains may be up to 1.5 m»
The microbiology of the malting process has been
described by several authorsMAU. Previous studies
have indicated that unprocessed malting barley carries a significant microbial load, encompassing a wide range of bacterial, yeast and fungal species. Although it has
evidence to suggest that the nature and extent of the mi«obial flora which develops during the process may influen« the final malt quality and its ultimate performance in the brewhouseW" One of the most significant microbiological changes which occurs during (LAB)3'4-5-". In addition to the well-documented role of
this SrouP of ™cro-organisms in food and feed
fermentations they are known to occur naturally on
many Plant ™tenals«w.«.
To our knowledge, no research has been reported on
the microflora of brewhouse mashes and, although it is well known that LAB make up a significant portion of
been claimed that some of these micro-organisms are washed off during the steeping process, the microbial
the flora on the principal raw material (i.e. malted barley), the numbers and types of bacteria present in
count was demonstrated to increase significantly during
brewhouse mashes have not been monitored in any
steeping and remain high during germination3-4'11.
significant detail.
While kilning brings about a reduction in the viable
In this study the microbiological changes which occur
count of at least 98%, the number of micro-organisms on
during malting in a traditional small-scale floor maltings
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A Comparative Study ofMalthouse and Brewhouse Microflora
Volume 105, No. 1,1999
(batch size 30 t) which has been in operation for 100
streptomycin (filter sterilised stock of 100 mg/ml in
years, were compared with that of a modem pneumatic
water). Counts on PCA, MRSA and VRBA were pour
large-scale malthouse (batch size 150 t) which has been
plated while those on PSA and MEA were spread plated
in continuous operation for less than 10 years. In
because of the requirement of these micro-organisms for
addition the microflora of stout and lager mashes
oxygen. Cycloheximide (50 ug/ml) was added to PCA
utilising these malts was examined in two brewhouses.
and MRSA prior to use. All incubations were at 30°C
Particular emphasis was placed on the development of
(since this temperature yielded the highest viable counts
the LAB and on their enumeration, species identification
for all of the media examined) for 48 h with the exception
and characterisation.
of VRBA and MEA which were for 24 h and 120 h respectively. Anaerobiosis for MRSA counts was maintained
MATERIALS AND METHODS
using the Anaerocult gas generating system (Merck).
Samples
Samples (200 g in duplicate) were collected from
duplicate production runs from two Irish malthouses during 1996 and 1997. Malthouse 1 was a relatively new (10 years) conventional large-scale malthouse of batch size 150 t with a gas-fired kiln. Malthouse 2 was a
traditional 30 t capacity floor maltings with an anthracite-fired kiln which has been in operation for approximately one hundred years. Samples were taken from dry barley (variety Coopers, 1995/6 harvest and
Detection of gas production
The production of gas from glucose, indicating CO2 production through hetero-fermentative metabolism, was detected by inoculating the test culture into 10 ml
sterile MRS broth (prepared without triammonium
citrate) and sealing the tube with 1.0 ml 2% sterile, molten agar. After incubation at an appropriate temperature,
gas production was indicated by a lifting of the seal. Species designation
Alexis, 1996 harvest) after the first and second steeps,
The following API systems were used, according to
green malt at 24 h intervals during the germination
the manufacturer's instructions, in the identification of
phase (5 days for Malthouse 1 and 6 days for Malthouse
bacterial isolates: 50 CHL for lactobacilli, pediococd and
2) and unscreened malt from the transfer duct after
leuconostocs, 20 Strep for streptococci and enterococci,
kilning and finished malt after screening.
20 E for coliforms and 20 NE for pseudomonads
Steep water samples were taken directly from the tank outflow. Brewhouse samples (100-200 g) were taken from the mash tun at various stages during the mash
(Biomerieux, La Balme des Grottes, France). Putative LAB
were identified as Gram +, catalase-, oxidase- prior to further characterisation by the determination of API profiles.
profile and were chilled immediately in ice cold water.
Polymerase chain reaction (PCR) of 16S ribosomal
Microbiological counts were performed within two
RNA (16S rRNA) region
hours of sampling.
The identity of representative bacterial isolates was
established by sequencing part of the 16S rRNA-
Viable counts
Malthouse samples were prepared for microbiological analysis by adding 10 g samples aseptically to 90 ml
sterile 1/4 strength Ringer's solution and incubating at
30°C for 20 min with constant agitation. This method was chosen after first comparing viable counts obtained by investigating the effect of sample homogenisation on
the viable counts. Serial decimal dilutions of samples were then prepared in Ringer's solution and appropriate dilutions were plated on selective and non-selective
media. Brewhouse samples were examined directly by serial decimal dilution. The total aerobic microflora was estimated on Standard Plate Count Agar (PCA, Oxoid). LAB were enumerated using MRS agar (Oxoid). Violet
Red Bile Agar (VRBA, Oxoid) was utilised for the enumeration of coliforms.
On this medium lactose fermenting coliforms form dark red colonies. Pseudomonads were enumerated
using Pseudomonas Selective Agar (PSA, Oxoid) with supplement SR103. Yeast and moulds were determined using
Malt
Extract
supplemented sterilised
with
stock
of
Agar
100 100
(MEA,
ug/ml mg/ml
Difco),
ampicillin in
pH
4.7
(filter-
water)
and
encoding region of the chromosome15. Genomic DNA of
isolates for PCR was prepared from 1.5 ml of overnight cultures by the method of Hoffman and Winston7.
Primers designed from conserved sequences within bacterial 16S rRNA regions were:
(i) GCGGCGCGCTAATACATG and (ii) CTGACGACAACCATGCACC (Oligo 1000M, Beckman Instruments Inc., California, USA). PCR amplification of
the 16S rRNA region was performed using the Promega Taq polymerase system (Promega, Madison, Wisconsin,
USA) according to the manufacturer's instructions. The Hybaid PCR Express system was programmed as follows: template DNA was denatured for 2 min at 94°
followed by 30 cycles of [(94°C x 30 s) + (58°C x 1 min) +
(72°C x 1 min)]. PCR products were separated by agarose gel elecrrophoresis and visualised by fluorescent labelling
with ethidium bromide prior to being cleaned using the High Pure PCR Product Purification Kit (Boehringer Mannheim). Sequencing of 16S ribosomal RNA DNA sequencing reactions were performed using an
ABI Prism™ Dye Terminator Cycle Sequencing Ready
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56
Copyright - Journal of the Institute of Brewing
Journal of The Institute of Brewing
Volume 105 No. 1,1999
A Comparative Study of Malthouse and Breivhouse Microflora
TABLE I.
Microbiological analysis of the malting process
prior to transfer to the germination vats. At this stage the
in a conventional malthouse (Malthouse 1).
LAB flora had increased to 1 x 108 CFU/g, accounting
for 7.6% of the population, while coliforms accounted Viable Count (CFU/g)
for 0.2% (3 x 107 CFU/g) and pseudomonads 27.6% (3.6
Pseuds Coliforms Yeasts/Moulds
Stage
Aerobic
LAB
Barley
6.5xlO5
4xl0>
1.25x10*
1.5x102
4.4 x 108 CFU/g of the green malt, of which LAB comprised 14.7% (6.5 x 107), coliforms 3.75% 1.65 x 107)
Post-Steep
1.3x10"
lxlO"
3.6x10"
3xl07
8.4x103
lxlC
1.2x10"
ND"
3x107
4x10*
Germination
lxHy
ND>
2.5xl07
8x10*
4.4x10* 6.5xlO7
3.3xlO8
1.65xlO7
38x10*
Kilned
35x107 2.5x10'
1.6xl05
132x10*
6.1x10*
Screened
1.7xlO7 1.7xl05
1.7x10*
7.5xlO5
2.7x10*
Day 3 Germination
decreased slightly over the 5 day germination period to
1.3x105
Germination Dayl
1x10"
x 108 CFU/g) of the population. The total aerobic flora
Day 5
and pseudomonads 75% (3.3 x 108 CFU/g). After kilning
and screening, the total microbial load of the malt decreased to 3.5 x 107 CFU/g, representing a 92% kill. The LAB count in the finished malt was 1.72 x 105 or 0.5% of the population.
Coliforms accounted for 11.5% of the population (7.5 x 105 CFU/g) and pseudomonads 5% (1.7 x 10fc CFU/g).
aND not determined
The development of the yeast and mould population
was similar to that observed of the bacterial flora. The Reaction Kit with Amplitaq DNA polymerase (Perkin
stored barley count was 1.5 x 102 CFU/g and consisted
Elmer, Warrington, Cheshire, UK). Sequence determination
principally of yeasts, rising 56-fold to 8.4 x 103 during
was performed using an Applied Biosystems 373
steeping and continuing to increase to 8 x 106 during the
automated DNA sequencer (Applied Biosystems, Foster
5 day germination phase. After kilning and screening,
City, California, (iii)
USA) employing oligonucleotides
CTTAATAACCCGCATTTC
and
(iv)
GAATTATTGGGCGTAAAG, which were chosen as
TABLE II.
Microbiological counts of barley prior to and immediately after wetting with steeps
divergent primers within the rRNA region. Database
water (Malthouse 1).
searches were performed using the NCBI BLAST
Viable Count (CFU/g)
programme1.
Pseuds Coliiorms Yeasts/Moulds
Slage
Aerobic
LAB
Enumeration and identification of microbial flora
Barley
3.2x10*
1.6x102
5.1x10*
5x10*
3.1x102
Malthouse 1
Wetted Barley
4.0x10*
1.6x10*
3.2x105
1.6x105
1.5x10*
Steeping
1.2x10*
< 1
2.0x10'
2x10'
6.0x10*
1.3x10"
RESULTS
In Malthouse 1 the process was initiated by the treatment of 1501 of stored barley with 170 m3 of water,
Water
increasing the moisture content of the barley from 12-
Steep Water
15% towards 45%.
Over the 48 h of steeping, the water was drained twice, resulting in two dry cycles of about 8 h each and 3 wet cycles. The steeped malt, which had already commenced germination, was then transferred to germination beds
and allowed to germinate for 5 days, after which it was kilned (gas-fired kiln) for 1 day to a final moisture of 3.5-
4% and the malt rootlets and dust removed by screening. The microflora during malting was monitored for two
batches of barley (variety: Coopers, 1995 and 1996 harvest) as it passed through the malting process from steeping to kilning and screening. The results obtained are summarised in Table I. The initial aerobic bacterial count on stored barley was 6.5 x 105 CFU/g, containing
1.3 x
105 CFU/g coliforms and
1.24 x
10* CFU
pseudomonads. The LAB population at this stage was 40-50 CFU/g, comprising only 0.06% of the total bacterial population, and consisted almost entirely of
mesophilic (growth at S30°C) hetero-fermenters. During barley steeping, the total aerobic population
grew rapidly, reaching 1.3 x 109 CFU/g immediately
1.2x10" 5.1xlO7
5.7x10*
the yeast count decreased to 2.7 x 104 CFU/g. The mould count increased to a maximum of 4 x 106 during germination and was 7 x 102 after kilning and screening.
Representative CFU (20 of each) chosen randomly from each group of micro-organisms were examined in
more detail using the API systems described in the Materials and Methods, to determine more precisely the composition of the microflora. At all stages of the malting process, the LAB flora was dominated by hetero-fermentative cocci, principally Leucotwstoc lactis
and Leuc. mesenteroides. Other LAB detected
were
Enterococcus faecalis, Lb. paracasei, Lb. plantarum and other unspecified enterococd and streptococci as determined by their API profiles. The coliforms isolated from stored barley consisted of
those typically associated with plant material, including Serratia odorifera, Erwinia nigrificans and Enterobacter
agglomeratts. Following steeping and germination, a shift took place with Klebsiella pneumoniae becoming the
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Journal of The Institute of Brewing
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57
Volume 105, No. 1, 1999
TABLE III.
A Comparative Study ofMalthouse and Brewhouse Microflora
Microbiological analysis of the malting process in a traditional floor maltings (Malthouse 2).
development of the microbial population was similar to that
described
for
the
modern
malthouse,
it
is
noteworthy that (a) the counts of all groups of micro
organisms at each stage were always lower by one or
Viable Count (CFU/g)
two logs; (b) the development of the Gram - flora was
not of the same order of magnitude, and (c) the final
Stage
Total
LAB
Pseuds
Barley
2.8x10*
8xl0>
1.1x105
2.0x105
2.3x10*
Steep 1
7.3x10* 2.2x105
5.3x105
3.2x105
5.0x10*
Steep 2
1.9x108 9.0x105
3.7x10*
2.0x107
8.6x105
Germt(l)
3.3x10? 1.5xlO8
ND»
1.7x10*
3.3x10*
Germ (2)
9.0xl07 1.5xl0»
ND»
6.3x10*
4.2x10*
Germ (3)
2.5xlO8 1.2x10"
ND"
1.2xlO7
2.1x10*
Germ (4)
3JxlO» 7.4x10?
ND4
2.2xlO7
8.0x10*
and comprised 16% of the population (8 x 104) after
Germ (5)
2.1x10* 5.1xlO7
4.4x10*
1.8xlO7
7.7x10*
kilning. Among the Gram - genera detected during
Kilned
5.0x105 8.0xl(H
9.0x10'
2.0x10*
3.0x10*
floor-malting
Coliform Yeasts/Moulds
post-kiln viable count of each group is lower. Once
again, the most significant increase during malting was in the numbers of LAB. These comprised less than 0.006% (80 CFU/g) of the total viable count on the stored barley, increased to 0.45% (9 x 105) during steeping,
reached a peak of 50% (1.2 x 108) during germination
Rahnella,
*«ND not determined
were
pseudomonads,
t Germ = Germination
Klebsiella,
Chromobacter
Enterobacter,
and
principally,
a
wide
Ps.
Serratia,
range
of
fluorescens,
Ps.
cholororaphis and Ps. aureofaciens. On the starting dry
dominant species. Other isolates at this stage included
those identified as Rahnella aquitilis, Citrobacter freundii, Enterobacter aerogenes and Serratia plymuthica. After
kilning, the much reduced coliform population was again dominated by Ent. agglomerans and Serratia ficaria.
The pseudomonad population on stored barley was mixed, but consisted mainly of Ps. fluorescens, Ps. cepacia
barley, the LAB population consisted of approximately 50% Leuconostoc mesenteroides, 25% Lactococcus lactis and 25%
lactobacilli
(25
CFU examined),
including
Lb.
coprophihis and Lb. plantarinn. Leuconostocs dominated during
steeping
but
lactobacilli,
principally
Lb.
plantarum, began to dominate during germination,
comprising 70% of the LAB population at this stage, and
and Aeromonas hydrolytica. After steeping,
Ps.
putida
became
the
dominant
TABLE IV.
Microbiology of a typical lager mash from Brewhouse 1.
pseudomonad and after germination the
Viable Count (CFU/g)
population consisted of approximately a 3:2 mixture of Ps. cholororaphis and Ps.
Time (min)
Temp.
kilning and screening. Microbiological analysis of steep water
Aerobic
LAB
Coliform
Pseuds
Yeast/Moulds