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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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55

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


56



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




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


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


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