Maintenance of the anaerobic beer spoilage bacteriaPectinatus andMegasphaera

Food Microbiology,

1990,7,33-41

Maintenance of the anaerobic Pectin&us and Megasphaera M.-L.

Suihko*

beer spoilage

bacteria

and A. Haikara

VTT, Biotechnical Laboratory, Received 7 June 1989

SF-02150 Espoo, Finland

The preservation of Pectinatus and Megasphaera strains by freeze-drying and freesing, using different working methods and protective agents, was studied. Pectinatus strains isolated from beer, and all the Megasphaera strains, recovered well after freezedrying and after freezing in reducing conditions. However, the recovery of Pectinatus strains isolated from yeast cultures was, in most of the conditions tested, very low, and after 2 years storage, they were almost non-viable. The only satisfactory method for these strains was rapid freezing in liquid nitrogen using5% DMSO as protective agent.

Introduction As a result of improved filling techniques in modern breweries, the dissolved oxygen content of beer, as well as the volume of air in the head space, has decreased considerably in recent years. This has led to the improved chemical stability of beer, but on the other hand it has caused a new and very serious problem. The conditions in beer bottles are now suitable for the growth of strictly anaerobic bacteria such as Pectinatus cerevisiiphilus and Megasphaera cerevisiae. These bacteria make the beer turbid and cause foul off-flavours in beer. . P. cerevisiiphilus, first isolated in 1978 (Lee et al. 1978), ‘is a gram-negative, catalase-negative, rod-shaped bacterium, having flagella on only one side of the cell (Lee et al. 1978, Back et al. 1979, Haikara et al. 1981a). The main metabolic products causing off-flavours are propionic acid and organic sulphur compounds (Haikara et al. 1981bl. M. cerevisiae, first isolated in 1979 (Weiss et al. 1979), is a gram-negative coccus, producing hydrogen sulphide and different fatty acids *Corresponding author. 0740-0020/90/010033

+ 09 $02.00/O

(Engelmann and Weiss 1985, Haikara 1985). During the past 10 years these strains have been studied in many brewing laboratories. Difficulties have been encountered in their preservation because of their sensitivity to oxygen. The Pectinatus strains, especially, isolated from yeast cultures, lose their viability rapidly after isolation. In this work we compared different working and preservation methods for Pectinatus and Megasphaera strains. Special emphasis was placed on the improvement of long term maintenance of the two very sensitive strains isolated from yeast cultures.

Materials

and Methods

Bacterial strains The 26 Pectinatus and nine Megasphaera strains studied were from the VTT Collection of Industrial Microorganisms (Suihko 1989). All Megasphaeru and most Pectin&us strains, e.g. VTT-E-79100 and VTT-E-79103, were isolated from beer. Two Pectinatus strains, VTT-E-86296 and VTT-E-36273, were isolated from sediment yeast in a brewery, one was isolated from water on the floor of a bottling hall and two from the air of a bottling hall. Q 1990

Academic

Press

Limited

34

M.-L. Suihko

and A. Haikara

Cultivation conditions The growth medium used was PYG broth (1% peptone, 1% yeast extract, 2% glucose) for Pectin&us and PYF broth (1% peptone, 1% yeast extract, 2% fructose) for Megasphaera, both containing 0.5 g 1-l cysteine hydrochloride as a reducing agent and 1 mg 1-l resazurin as an indicator. The media were reduced before use in anaerobic jars at least for 24 h, or by heating to 100°C. The strains were cultivated in anaerobic jars at 30°C for l-2 days before, and for 7 days after preservation. The suspensions used and ampoules or straws prepared were cultivated as dilution series in growth media, and the highest dilution showing growth was recorded.

placed in a freezer at -20°C or -75°C. One bead was used for each cultivation. f’;lefng

in ampoules and in Portagerm

The suspension was prepared as described above for freezing on glass beads. The plastic screw-cap 2-ml ampoules (Nunc) were filled with 1 ml of suspension and placed in a freezer at - 20°C or - 75°C. The suspension (1 ml1 was also injected into the commercial anaerobic Portagerm vials (bioM&ieux 419951 containing a solid, reduced, buffered medium. The vials were placed in a freezer at - 75°C.

Results Freeze-drying The protective medium used was 20% skimmed milk (Difco) with or without cysteine hydrochloride. After cultivation, the cells were centrifuged and resuspended in the protective medium. The cell suspension (1 ml) was frozen on the walls of ampoules (Pyrex glass ampoules of 5 ml with long necks) in an ethanol bath at -40°C dried overnight using a one-stage drying system (dryer WKF L05, West Germany) and sealed in a vacuum. Freeze-dried ampoules were stored at +4”C. Freezing in liquid nitrogen in straws The well grown fresh culture and a protective agent were mixed in the ratio 1: 1. The polypropylene straws were filled (0.1 ml) and sealed as described by Kirsop (1984). The filled straws were packed in plastic screw-cap 2-ml ampoules (Nunc) and placed in a freezer at -20°C for 2 h before being put into liquid nitrogen. In some experiments, the straw ampoules and also the straws without insulating ampoules were put directly into liquid nitrogen. The protective agents used were 5% glycerol (normal routine), 10% glycerol, 5% dimethyl sulphoxide (DMSO) and a mixture of 5% glycerol and 5% DMSO. Freezing on glass beads After cultivation, the cells were collected by centrifugation and resuspended in fresh growth medium containing 15% glycerol, 0.5 g 1-i cysteine hydrochloride, and 1 mg 1-i resazurin. The washed, sterilized beads were rinsed vigorously with bacterial suspension to fill and wet the beads thoroughly (Jones et al. 1984). The excess suspension was removed from the bottom of the vial. The vials were

Normal lection

routine storage in the strain col-

The survival of freeze-dried Pectinatus and Megasphaera strains with and without cysteine hydrochloride is shown in Fig. 1. The reducing agent improved the recovery of strains, and the similarity of replicate ampoules, significantly. Without cysteine hydrochloride the variation among ampoules of the same batch was very high, but the mean variation during storage was low. This result indicates that the freeze-drying and rehydration were the most critical stages in this method, and not the duration of storage. With cysteine the recovery of Pectinatus strains isolated from beer and water as well as that of all Megasphaera strains was good and the number of living cells decreased only slightly during storage for 2 years. The recovery of Pectinatus strains isolated from yeast culture was very poor, varying from lo1 to lo3 cells ml-r . After storage for 2 years, one of the strains (V’IT-E-86273) was lost and the other (VTT-E-86269) contained only 10’ living cells ml-‘. The Pectinatus strains isolated from air were also rather sensitive to freeze-drying. The results of freezing of Pectinatus and Megasphaera strains, on glass beads at -75°C and in straws into liquid nitrogen at -196”C, are shown in Fig. 2. The

Anaerobic

beer spoilage

35

bacteria

A yeast

beer -

=u5

0”

water

---

lo-

8 7

P .-s>

6

5

5

$4

i 5

3

8

2

-I

k

1 I

air

u 6

9

with cysteine

without cysteine

B

12

lo9-

8765432l-

without cysteine

with cysteine

Fig. 1. Effect of cysteine hydrochloride on the recovery of Pectinatus isolates (A) and Megasphaera strains (B). The average number of cells and ranges before freeze-drying 0 and after storage for 1 week ,4 years FZJ and 6 years KI. The number of cultivated ampoules is given in the columns.

recovery and maintenance of most Pectinatus strains isolated from beer (group 1) and water as well as those of all Megasphaera strains were very good with both methods. The recovery of Pectinatus

strains isolated from yeast culture was very poor in both cases. After storage for 2 years, the strain WY’-E-86273 had died in both methods, and VTT-E-86269 contained from lo1 to lo2 living cells ml-‘.

36

M.-L. Suihko

and A. Haikara

The rather low recovery of strains isolated from beer (group 2, 4 strains) was improved to a level as high as that of

I-

IE9c

beer

group 1, when the straw ampoules were put directly into liquid nitrogen instead of being kept first at -20°C for 2 h. How-

yeast

Pectinatus

group 1 m-h

beer

yeast eve

Megasphaera

water

B1

air

group 2

8785432l-

-

Pectinatus Fig. 2. Recovery of Pectinatus

Megasphaera

isolates and Megasphaera strains after freezing on glass beads at -75°C (A) and in liquid nitrogen at -196°C (B). The average number of cells and ranges before freezing 0 and after storage for 1 week The numbers of bead cultivations and cultivated straws are given in the columns.

Anaerobic ever, the poor result with yeast isolates was not improved using this procedure. Effect of different working and freezing procedures on Pectinatus strains

The effects of anaerobic working conditions on the most sensitive strain VTTE-86273 during freeze-drying and freezing at -196°C were studied in more detail. Working in an anaerobic cabinet did not improve the poor results obtained when working on the laboratory bench. Moreover, omitting of centrifugation of the culture before resuspension had no effect on the number of living cells recovered after freeze-drying. Thus the reason for the poor results cannot have been greater sensitivity to oxygen. The effects of different protective agents and freezing procedures on the yeast isolates VTT-E-86269 and VTT-E86273 were studied in more detail. The most effective protective agent was 5% DMSO, which in contrast to glycerol, penetrates the cell membrane quickly (Fig. 3). The best procedure was freezing in straws, packed in ampoules (six straws) and put directly into the liquid nitrogen. Using this procedure and 5% DMSO, the poor recovery level obtained earlier with these strains [Fig. 2(B), yeast1 was increased, even to the same level as obtained with other strains, 107-10’ living cells ml-l. Maintenance of two Pectinatus isolated from beer

strains

Maintenance of the beer isolates VTT-E79100 and VTT-E-79103 was studied in more detail, as freeze-dried preparations and after freezing, using different temperatures and procedures. When maintained frozen on glass beads or in ampoules at -20°C the number of living cells decreased strongly with both strains as a function of storage time [Fig. 4(A)]. On glass beads the effective storage time (living cells 210’ ml-‘) was only 2

beer spoilage bacteria

3’7

months for strain V’MY-E-79103 and 9 months for strain VTT-E-79100. In ampoules the storage time was longer, 2 and 3 years, respectively. When maintained frozen at -75°C on glass beads and in ampoules, the number of living cells decreased, but only slightly [Fig. 4(B)]. In an anaerobic atmosphere of Portagerm vials, the number of living cells appeared to be constant for at least 4 years of storage. When maintained frozen in liquid nitrogen at -196°C or freeze-dried, the number of living cells was constant during 4 years of storage (Fig. 5). Discussion In general, bacteria may be successfully maintained using a number of different methods. The most common preservation method has been freeze-drying using different protective agents and procedures (Bousfield 1984, Rudge 19841. Although media, special equipment, and techniques for the isolation and culturing of strict anaerobes have improved greatly during recent years, the preservation of some cultures still causes problems (Impey and Phillips 1984, Hippe 1984). However, experience of the maintenance of strict anaerobes is still limited, particularly with respect to their long-term preservation and freezing. The great difference in recovery between the Pectinatus strains isolated from beer and from yeast cultures is presumably due to the different water permeabilities of their cell membranes. The strains isolated from yeast culture will be assigned to the genus Selenomonas (Schleifer et al. 19901 in the near future. On the basis of different taxonomic positions of these strains, their different recoveries in short-term as well as in long-term maintenance is more understandable. Moreover, the poor recovery of four Pectinatus strains belonging to group 2 [Fig. 2(B)] can also be explained

38

M.-L. Suihko

and A. Haikara

Dehydration at -2O’C for 2 h

Directly into liquid nitrogen

=vi z 8 7

Et .-,)

6

6

5

8

4

e3 z

2 1

Initial

In ampoules

Without ampoules

B Dehydration at for 2 h

-20%

Initial

Directly into liquid nitrogen

In ampoules

Without ampoules

Fig. 3. Effects of different freezing procedures and protective agents on the recovery of Pectinatus yeast isolates VTT-E-86269 (A) and VTT-E-86273 (B). The average number of cells before freezing 0 and after freezing with 5% glycerol 0, 10% glycerol DMSO +5% glycerol &I as protective agent. Three straws were cultivated and the ranges are given in the columns.

by their different taxonomic position as indicated by serological and genetic differences as well as differences in the

cell wall protein patterns (Haikara Hakalehto et al. 1984, Haikara Schleifer et al. 1990).

1983, 1989,

Anaerobic

6

12

18

24

Preservation

30

beer spoilage

36

bacteria

42

39

48

time, months

B

-

I

6

12

.

18

24

Preservation

30

36

42

.

48

time, months

Fig. 4. Recovery of the Pectin&us beer isolates VTT-E-79100 (0, A, 0) and VTT-E-79103 (0, A, n ) when maintained frozen on glass beads (0, 01, in ampoules (A, A) and in Portagerm vials (0, n ) at -20°C (A) and at -75°C (B). It can be concluded that freezing in straws at -196°C and with a rapid cooling rate, with 5% DMSO as protective agent, is the most suitable method for short-term as well as for long-term maintenance of anaerobic Pectinatus and Megasphaera strains. Freeze-drying is a suitable alternative method for longterm preservation of most of these strains. Freezing at -75°C is suitable for

the maintenance of working cultures. The use of Portagerm vials prolongs the storage time. Freezing at -20°C can be recommended only for very short-term preservation of samples.

Acknowledgements We thank MSc Solveig Boije-Backman and MS Helena Hakkinen for carrying out practical experiments.

40

M.-L. Suihko and A. Haikara

6

I 12

16

24

Preservation Fig. 5. Recovery when maintained

1 30

36

42

46

tine. months

0fthePectinatu.s beer isolates VTT-E-79100 in liquid nitrogen (0, 0) and as freeze-dried

(0, a) and VTT-E-79103 specimens (A, A ).

(0, A ),

References Back, W., Weiss, N. and Seidel, H. (1979) Isolierung und systematische Zuordnung bierschadlither gramnegativer Bakterien. II: Gramnegative anaerobe StfibchemAnhang: Aus Bier isolierte gramnegative fakultativ Stabchen. Brauwissenschaft 32,233-238. Bousfield, I. J. (1984) Maintenance of industrial and marine bacteria and bacteriophages. In Maintenance of microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 63-68. London, Academic Press. Engelmann, U. and Weiss, N. (1985) Megusphaeru cereuisiae sp. nov.: A new Gram-negative obligately anaerobic coccus isolated from spoiled beer. Syst. Appl. Microbial. 6,287-290. Haikara, A. (1983) Immunological characterization ofPectinatus cereuisiophilus strains. Appl. Environ. Microbial. 46, 1054-1058. Haikara, A. (1985) Detection of anaerobic, gram-negative bacteria in beer. Monatsschriff Brauwissenschaft 38, 239-243. Haikara, A. (1989) Invasion of anaerobic bacteria into pitching yeast. In Proceedings of the 22nd EBC congress, Zurich, pp. 537-544. Oxford, IRL Press. Haikara, A., Penttila, L., Enari, T.-M. and Lounatmaa, K. (1981a) Microbiological, biochemical, and electron microscopic characterization of a Pectinatus strain. Appl. Environ. Microbial. 41,511-517. Haikara, A., Enari, T.-M. and Lounatmaa, K. (1981b) The genus Pectinatus, a new group of anaerobic beer spoilage bacteria. In Proceedings of the 18th EBC congress, Copenhagen, pp. 229-240. Oxford, IRL Press. Hakalehto, E., Haikara, A., Enari, T.-M. and Lounatmaa, K. (1984) Hydrochloric acid extractable protein patterns of Pectinatus cerevisiophilus strains. Food Microbial. 1, 209216. Hippe, H. (1984) Maintenance of methanogenic bacteria. In Maintenance on microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 69-81. London, Academic Press. Impey, C. S. and Phillips, B. A. (1984) Maintenance of anaerobic bacteria. In Maintenance on microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 47-56. London, Academic Press. Jones, D., Pell, P. A. and Sneath, P. H. A. (1984) Maintenance of bacteria on glass beads at -60°C to -70°C. In Maintenance of microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 35-40. London, Academic Press.

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bacteria

41

Kirsop, B. E. (1984) Maintenance ofyeast. In Maintenance of microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 109-130. London, Academic Press. Lee, S. Y., Mabee, M. S. and Jangaard, N. 0. (1978) Pectin&us, a new genus of the family Bacteroidaceae. Int. J. Syst. Bacterial. 28, 582-594. Rudge, R. H. (1984) Maintenance of bacteria by freeze-drying. In Maintenance of microorganisms (Eds Kirsop, B. E. and Snell, J. J. S.) pp. 23-33. London, Academic Press. Schleifer, K. H., Leuteritz, M., Weiss, N., Ludwig, W., Kirchhof, G. and Seidel-Rufer, H. (1990) Taxonomic study of anaerobic, gram-negative, rod-shaped bacteria from breweries: Emended description of Pectinatus cerevisiiphilus and description of Pectinatus frisingensis sp. nov., Selenomonas lacticifex sp. nov., Zymophilus raffinosivorans gen. nov., sp. nov., and Zymophilus paucivorans gen. nov., sp. nov. Znt. J. Syst. Bacterial. 40,19-27. Suihko, M.-L. (1989) VTT collection of industrial microorganisms. Catalogue of strains (2nd edn) Espoo, V’M’ Offsetpaino. Weiss, N., Seidel, H. and Back, W. (1979) Isolierung und systematische Zuordnung bierschadlither gramnegativer Bakterien. I: Gramnegative strikt anaerobe Kokken. Bruuwissenschaft 32,189-194.