biochemical physiology of obesumbacterium ... - Wiley Online Library

0 downloads 0 Views 599KB Size Report
[J. Inst. Brew. fraction). The exceptions were NADH oxi- dase, NADH dehydrogenase (EC 1-6.99.3) and sucdnate dehydrogenase (EC 1.3.99.1), which were ...
BIOCHEMICAL PHYSIOLOGY OF OBESUMBACTERIUM PROTEUS, A COMMON BREWERY CONTAMINANT

By M. Thomas*, J. A. Cole and J. S. Hough

(Department of Biochemistry, University of Birmingham) Received 2&th January, 1972

In defined media, arginine plus either glutamate or aspartate supports growth of Obesumbacterlum protein but additional amino acids enhance yields. Glucose, fruc

tose, mannose and galactose are rapidly utilized; maltose and starch give slower

growth. Fermentation products include acetic, formic, 2-oxoglutaric, pyruvic and succinic acids. With brewer's wort as growth medium, the concentrations of argi nine, lyslne, NH,, aspartate, serine, tyrosine and leudne decrease whereas several

other amino acids increase in concentration. Fermentation products include acetoin,

ethanol, lactic acid, fusel alcohols, some volatile acids and dimethylsulphide. Meas urements of oxygen uptake by washed suspensions in the presence of a variety of substrates suggest synthesis of (i) a terminal electron transfer chain that is repressed anaeroblcally and (ii) a glucose oxidase complex. Radio-respirometry indicates that the EMP is not the sole pathway for glucose metabolism. Cytochrome b, is detected in the bacterial cells but not cytochromes a and c. Glucose represses the activities of several enrymes of the EMP and HMP, but not lactate dehydrogenase and 3 enzymes of the citric acid cycle.

Introduction

Bacterium Y,21 grew in wort to give a

80 vears ago «• but they received little

acids. H, and CO2 but little ethanol and no

Short fat rod-shaped bacteria were recognized as common brewery contaminants over attention because they were considered harmless.' One such isolate, Shimwell's

fruity odour of parsnips. Fermentation products included volatj e and non-volatile diacetyl. Optimum pH and temperature for growth were 5-0 and 30°-35°C respectively.

• Present address: Bass (Production) Limited, Burton-on-Trent.

Vol. 78, 1972]

THOMAS el al.: OBESUMBACTERIUM PROTEUS

No growth occurred below pH 4-0 or in 6% (v/v) ethanol, or following heat treat ment at 54° C for 5 minutes. Further

growth tests in tryptone media led Shimwell & Grimes23 to assign Bacterium Y to the genus Flavobacterium. Cells harvested from various media differed in both size and shape. This pleomorphism was indicated in the

specific name Flavobacterium proteus. In 1964, Shimwell expressed dissatisfaction with the inclusion of the organism in the genus Flavobacterium and introduced a new genus, Obesumbacterium, with the single species Obesumbaclerium proteus.22

The growth of O. proteus in brewer's wort has been studied after adding small inocula to the pitching yeast.27 The bacterial contamination increased during 9 or 10 brewery fermentations, but subsequently remained constant. The bacteria grew rap idly in the early stages of each fermentation, but then passed into a stationary phase coinciding with the decrease in the pH of the wort caused by yeast metabolism. Although fermentation by one strain of yeast was unaffected by the presence of the bacteria, a second strain was inhibited.29 Conversely it was shown that the growth of the bacterium was strongly influenced by

the initial seeding rate, the composition of

the wort, the strain of yeast and the pre vailing conditions of the fermentation. In other studies, Strandskov & Bockelmann28 showed that adenine, uracil and 7 or 8 amino acids (of which glutamate and arginine were the most important) were required for growth in synthetic media. The ability to use different carbon sources varied from strain to strain. It is curious that an organism with the unusual ability to grow simultaneously with yeast has not received more biochemical study. The fact that this bacterium is often present during brewery fermentations in large numbers and may influence not only the flavour and aroma of the beer but the speed of fermentation, indicates that detailed study of Obesum bacterium proteus is long overdue. Materials and Methods

Obesumbacterium proteus strain 41, used by Strandskov & Bockelmann,29 was obtained

from the National Collection of Industrial

Bacteria, Aberdeen, as Culture No. 8770. It was mainatined on nutrient agar and subcultured monthly. For experimental

333

work, inocula were prepared by transferring cells to the appropriate liquid medium and culturing for 24 h at 30° C. The minimal medium buffered by phos phate contained: KH2PO4,4 g; MgSO4-7H2O, lg; CaCls-6H2O. 0-25 g; L-glutamic acid, 2-5 g; L-arginine hydrochloride, 1 g; adenine, 10 mg; uracil, 10 mg; trace elements, lml; and distilled water to 1 litre. After steriliza tion and cooling, 3 ml 30% (w/w) NaOH was added to adjust the pH to 6-5. Sugars were then added to a final cone, of 1% (w/v). 7>aMsaconitate (9-6 g/litre) was added with the potassium dihydrogen orthophosphate in the aconitate buffered medium. The trace element solution contained, in 1 litre of distilled water, 250 mg FeSCy7HaO; 100 mg boric acid; 45 mg CuSO45H2O; 40 mg ZnSO4-7HsO; 40 mg MnSO4-

4HSO; 20mg (NH4)2 MoO^HjO; 20 N-HC1

to pH 6-0. Bacteria for enzymic studies were grown

in 2-litre conical flasks. For aerated cultures, each flask contained 750 ml medium, and was incubated on a rotary shaker operating at 100-120 cycles/min. For micro-aerobic cultures, flasks were completely filled and incubated without shaking. Growth was assayed turbidimetrically by measuring the absorbance at 625 nm with a Unicam SP 500 spectrophotometer: under the conditions employed, optical density was proportional to the bacterial dry weight. Cells in the logarithmic phase of growth were harvested by centrifugation for 30 min in a refrigerated M.S.E. Major centrifuge. After two washes with 50 msi phosphate (pH 7-4), the bacteria

were resuspended in phosphate buffer to a

density of 50-100 mg dry wt/ml. They were then disrupted with a Mullard ultrasonicator at 18,000 cycles/sec. Cell-free

extracts were prepared by the standard

techniques described in detail by Gray, Wimpenny, Hughes & Mossman.9 Rates of oxygen uptake by bacterial suspensions at 30° C were determined polarographically in an apparatus similar to that described by Lloyd & Brookman.16 Lactic dehydrogenase, alcohol dehydrogenase, enzymes of the Embden-Meyerhof pathway,

the hexose monophosphate shunt and the citric acid cycle were assayed by standard methods with only minor modifications.8 Most of the enzymes were concentrated in the supernatant fraction following centri fugation for 1 h at 100,000 X g (HSS

334

thomas et al.: obesumbacterium proteos

fraction).

The exceptions were NADH oxi-

dase, NADH dehydrogenase (EC 1-6.99.3) and sucdnate dehydrogenase (EC 1.3.99.1), which were sedimented with the cell mem branes (CWM fraction).

Standard methods for estimating concen trations of various chemicals are listed in Table I. The amino acid composition of wort before and after fermentation by O. proteus was determined by the methods of Moore & Stein,10 and Spackman, Stein & Moore (1958).20 Samples were centrifuged and passed through a membrane filter (0-22/u. pore size; Millipore Ltd., Wembley,

Middlesex) before hydrolysis with cone. HC1 under vacuum at 110° C for 24 h. TABLE I

List of Chemical Assays Employed Chemical

Reference for assay

Etlmnol Lactic add

Burlier * Rcdotskl, 1051' Hohorst, 1903" Wotllcld. 1945" Sinclair, Hall & Thorbom Hums,

Acetoin

Dimethyl sulphide

11170"

Volatile organosulplmr Total fusel alcohols Volatile fatty acids Protein

Volatilo esters Ammonia Urea Amino acids In synthetic media Glucose Total carbohydrate

Sinclair, Hall & Tliorlnirn Hunts, 1070" PfennlnKer, 1004" Leu In and Klsdcn, 1055" Lonry, Roseboroueli, Farr & Randall. 1051" Encbo