Volatile fatty acid production during beer spoilage by Pectinatus sp.

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VOLATILEFATTYACIDPRODUCTION DURINGBEER SPOILAGE BY Pecfinatussp. J. M. Membrk, J. L. Tholozan,* Station de Technologie

G. Delattre, B. Eulalie & G. Albagnac

Alimentaire, Institut National de la Recherche Agronomique, Villeneuve d’Ascq Cedex, France

(Paper presented at ‘UnderstandingFluvour 20-23

Quality: Relating Sensmy to Chemical and Physical Data ‘,

Se$tember 1992, Bristol, UK) Production rate of lactate on glucose (mM/h) Production rate of propionate on glucose (mM/h) Production rate of propionate on lactate (mM/h) Production rate of biomass on glucose (h-l) Degradation rate of glucose (mM/h) Degradation rate of lactate (mM/h) Production rate associated to the growth rate (mM/h) Production rate associated to growth concentration

ABSTRACT The genus Pectinatus

includes strictly anaerobic Gram-

negative non-spore-forming mesophilic bacteria, often referred to (ISbeer spoilage bacteria. Pectinahrs fiisingensis was chosen as reference strain. Growth was achieved in

(mM/h) Specific growth rate (h-l)

batch cultures under stringent anaerobic conditions with synthetic medium and pH regulation.

369, rue J. Guesde, BP 39, F-59651

Various glucose

concentrations were used, and low inoculum reproduced spoilage conditions in bottled beer A lag$hase of around

INTRODUCTION

20 hours was observed in every case; glucose was converted to pqbionate, acetate, succinate and CO,, while lactate was a transient metabolite. Glucose concentrations

The

genus Pectinatus is often cited as beer spoilage bacteria. These bacteria are mesophilic, Gram-negative non-sporulated strict anaerobes. Pectinatus cereuisiiphilus (Bacteriodaceae) was first isolated and described as a new genus by Lee et al. (1978) in the United States.

typically f oun d an . beer allowed good growth of Pectinatus frisingensis, rapidly leading to cloudy culture medium. Resulting

volatile fatty

acid concentrations

were far

Those

higherJlavour thresholds. Monod-type modelling of strain time periods.

Keywords: Pectinatus; bee?; spoilage; model.

NOTATION Acetate Carbon Glucose Lactate

concentration (mM) dioxide concentration (IIIM) concentration (ITIM) concentration (mM) PrOpiOnate concentration (mM) Biomass (OD unit) Parameter which takes account the non-linear effect of lactate inhibition. Keld product on substrate Inhibition constant due to propionate (ITIM) Inhibition constant due to lactate (mM) Production rate of acetate on glucose (mM/h) Production rate of acetate on lactate (mM/h) Production rate of CO, on glucose (mM/h) Production rate of CO, on lactate (mM/h) *To whom correspondence

were isolated

later from turbid and off-

beer samples in Germany (Back et al., 1979), Scandinavia (Haikara, 1985) and Japan (Takahashi, 1983). Recently, a new species of Pectinatus, P. frisingensis (DSM 20465), was isolated in Germany. This strain fermented glucose to acetate, propionate, acetoin and minor amounts of succinate (Schleifer et al., 1990). In this study, growth kinetics and fermentation profiles of P. frisingensis will be described on glucose in synthetic culture medium. Implications of glucose levels on metabolism will be presented. Finally, a mathematical model derived from these experimental data which may be used will be proposed to forecast the development of P. ftisingensis contamination in beer.

growth allowed good forecast of both biomass and volatile fatty acid production overjiveday

bacteria

flavour

MATERIALS

AND

METHODS

Culture conditions Pectinatus ftisingensis came from the German collection

(DSM 20467). The culture medium was a basal medium supplemented with yeast extract and pancreatic hydrolysate of casein (0.5 g/litre) (Miller & Wolin, 1974), modified by Samain et al. (1982) and adjusted to pH 6.

should be addressed. 25

26

J. M. Memb&,J L. Thohzan, G. Del&w, B. Eddie

&’ G. Albagnac

Cultures were made in 1.7 litre anaerobic fermenters, under N, headspace, at 3O”C, with pH regulated at 6. Low inoculum quantities of 0.5% (v/v) of late lagphase growing cultures were used in batch processes, in order to reproduce conditions of beer contaminations in bottles.

Analytical methods Growth was quantified by optical density (OD, A = 660 nm) measurements and by protein content assays (Lowry et al, 1951) with bovine serum albumin as reference. Glucose concentration was determined enzymatitally with glucose oxidase (Sigma, SaintQuentinFallavier, France). Gas production was measured by volumic gas-meter (Moletta & Albagnac, 1982) and gas composition was routinely determined with a gas chromatograph equipped with a (2 m X i/4”) Porapak S filled column (Waters Associates, SaintQuentinenbelines, France) at a temperature of 50°C. Organic acids were analysed by HPLC equipped with UV detector (A = 210 nm) on an OA 1000 column (Alltech, Templeuve, France) with O-8 ml/min HsSO,, O-005 N as eluent at a temperature of 65°C.

Model resolution Fermentation profiles have been used to build a predictive Monod-type model, including initial glucose concentration and inoculum size, to forecast bacterial growth and final organic acids concentrations. A numerical approach was used to build model equations. Inhibition constants in the model (kid, &, y) were determined from experimental data. Parameter values qP,G), qL,G)) (kila.K,cu,, % , a~, PA, & k$’ & qA,C,, &,C), minimized global error between the model and the experimental data.

RESULTS

0

Time(h) L?%p

Lf!!f_

wbmas -

ntldmR& -+e-

lo*,. --m-

FIG. 1. Biomass, substrate and products profiles at initial glu-

cose concentration of 30 mM, with pH regulation. Experimental data (symbols) and model curves.

With pH regulation at 6, similar fermentation products were detected, but a transient lactate accumulation occurred in every case (Fig. 1) . This acid is produced in a first step of the fermentation, and consumed in a second step. In order to know the behaviour of the bacteria in the presence of both lactate and glucose, cultures on both of these compounds were performed. These showed similar average consumption rates of 2 mM/h for glucose and lactate. Moreover, preferential lactate consumption was demonstrated in every case (Fig. 2). Varying initial lactate concentrations with a fixed initial glucose concentration of 100 mM showed inhibitory effects on glucose consumption at lactate concentrations as low as 5 mh4.

Substrate and products. inhibition The effect of initial glucose concentrations on Pectinatus Jiisingensis growth was studied with pH regulation. No substrate inhibition was demonstrated for glucose concentrations over 200 mM. On the other hand, growth at glucose concentrations as low as 5 mM (1 g/litre) allowed P. fisingensis growth (Fig. 3) ; similar specific growth rates were measured whatever the glucose con5

Glucose fermentation time courses Propionate, acetate, succinate, and carbon dioxide were the main fermentation products from glucose. Lag-phase periods of 20 h and average specific growth rates of O-1 h-l were routinely determined in our batch cultures. In batch processes without pH regulation, 55 mM glucose was converted to organic acids according to the following fermentation balance: 1 Glucose + O-292 Acetate + 1.156 Propionate t 0.028 Succinate t O-364CO, with a carbon recovery of 75%. Final pH in the culture was 4, and biomass production yields were 10 g of total bacterial protein per mol of consumed glucose.

0 0

20

Tmt

“0”

LY’*

720

80

.O

e”T?s

(h) Y

‘“;p”’

“.!

FIG. 2. Kinetics profile at initial glucose concentration of 80 InM and initial lactate concentrate of 125 mM.

WA Production during Beer spoiiuge by Pectinatus sp.

27

FIG. 4. Schematic description of glucose degradation pathway.

Time(h) 0:10 ‘:* u---a--

a:.% “0

0:150 ‘:a

[Gl

r(P*G,= ap* r(x,G) *

FIG. 3. Biomass profiles at various initial glucose concentrations. Experimental data and model curves. ‘G’ and ‘L’ mean glucose and lactate, respectively.

centration between 10 and 200 mM. Lactate accumulated transiently in the medium in every case (Fig. 1). Growth inhibition by fermentation products was mainly due to proportionate accumulating in the medium. Inhibitory effects of propionate were increased with high initial glucose concentration. Propionate accumulation stopped the growth for concentrations over 110 mM; in this case the glucose consumption was not completed (Table 1) .

Model

[Gl r(A,G,= aA - r(X,G)*

(l+ [GI) [Gl

T(C,G)= QC * Y(X,G)*

(I +

(5)

[GI)

with ffr,, aA and cxc values of 2, 1 and 1 respectively. To include lactate inhibition during glucose degradation, a correcting term was added to the lactate production rate. As with propionate, acetate and CO, productions, glucose conversion to lactate depends on growth sub strate concentration, and on the amount of biomass in the culture. Equation (6) gave lactate production: (6)

T(L,G,= PL * [Xl *

Bacterial growth is well modelled by a Monod law, with a correcting term due to propionate inhibition:

(1)

P= Pmax’

withpL=2,&=10mMandy=5. In the same way, eqns. (7) to (9) describe productions of propionate, acetate and CO, from lactate (L) (way II, Fig. 4):

= 0.1 h-’ and Kcid = 60 mM. with ~max

[Ll

As strain growth rate was little or not affected by lactate concentration, biomass production from glucose was written:

[Ll = PA

* Lx1

-

[LI)

(I+

Organic acid production and glucose degradation equations involve lactate contribution to the metabolic pathway (Fig. 2). Biomass and acid productions from glucose (G) (way I, Fig. 4) are described by eqn. (3) for propionate (P), eqn (4) for acetate (A), and eqn (5) for CO,(C): TABLE 1. Final Concentration Glucose Concentrations Initialglucose (m)

Final OD (660 IlM)

and Biomass at Various Initial

r(c,L)

= PC

Propionate (-)

Finally, the consumption were written as follows:

5 6 10 45 40

9 13 40 110 180

r,=-+-+-+UP/G)

IILl Cl+ LI)

rates of glucose and lactate

r(A,G)

rec. G)

VA/G)

W/G)

r,, L)

0.35 0.8 2 3 3

[Xl *

*

with&,=1,/3A=0.3,/3c=015.

rep, G)

Acetate (-)

(7)

Cl+ &I) r(A,L)

5 10 30 150 170

(3)

(I+ [GI)

r,=-+-+W-‘/G)

r,, L)

W/G)

rcL, C)

Y(L/G) rep,L)

Y(C/G)

(IO)

(11)

In Pectinatus@ingensis batch cultures with pH regulation, glucose was nearly completely converted to fermentation products. Product yield on glucose and on

28 J M. Membrk,J L. Tholozan, G. Del&&e, B. E&lie

tnitiatgl*car*.

+

&’ G. Albagnac

wncentration (mh4) /+Il+W. ._-&-_

FIG. 5. Final acetate and propionate concentrations at various

initial glucose concentrations. Experimental data (symbols) and model curves.

lactate were in good agreement with experimental data for glucose concentrations typically found in lager beer (Fig. 3), i.e. less than 5 g/litre (Hough et al, 1982). Experimental data led to Y(A/G) = ‘(C/G) = ‘(L/G) = 1 M/M while YCP,cj= 1.5 M/M. From model equations, fermentations product levels were forecast after 5 days of culture, in particular volatile fatty acids (VFA) concentrations, for a wide range of glucose concentrations. In Fig. 5, data and model predictions have been plotted for glucose concentrations between 5 and 200 mM: very low quantities of glucose (
DISCUSSION The above results provide a better knowledge of P. capabilities of growth on glucose. This last compound is a common constituent in beer with reported concentrations from 3.3 to 27.2 mM (0.6-4-g g/litre) in lagers, and concentrations up to 66.7 mM (12 g/litre) for stouts (Hough et aZ., 1982). Experimental data, and model simulations for end-product levels prediction, explained easily the high volatile fatty acids concentrations expected with these glucose amounts, and therefore the negative effect of these VFA productions in final beer quality. Indeed, current thresholds of offflavours due to VFA contents in beer were described as 2.9 mM (0.17 g/litre) and 2 mM (0.15 g/litre) of acetate and propionate, respectively (Hough et al, 1982). Moreover, model predictions and experimental data were in very good agreement: we calculated the glucose concentrations necessary to produce detectable off-flavours in beer due to growth of P. j-isingensis. Glucose levels as low as 3.5 mm (0.6 g/litre) would be sufficient for beer alteration by bacterial growth.

ftisingensis

On the other hand, glucose consumption by ihe strain allowed good concomitant biomass production, even at low substrate concentrations, rapidly leading to cloudy culture medium. Besides off-flavours, P. jiisingensis growth produced an important haze affecting beer brightness. Model predictions of 4 mM of glucose (0.7 g/litre) consumed during bacterial growth led to a final optical density in the medium of 0.2 OD units. This low modification of medium culture transparency is already high enough to modify the brightness of filtered beer. P. frisingensis batch cultures excreted significant amounts of VFA during glucose fermentation, leading to low pH in the culture medium. When the pH was regulated, lactate accumulated transiently in the culture. Similar metabolic features have already been described in ecosystems (Lema et aL, 1988), enrichment cultures (Tholozan et aZ., 1988), or pure cultures (Tholozan et aZ., 1992). This does not necessarily imply that lactate is a metabolic intermediate during glucose conversion to VFA. Haikara et al. (1981) postulated that propionate was produced from glucose by the succinate pathway in Pectinatus sp. This way of conversion was also described in Propionibacterium (Dollle, 1975), and in other propionic bacteria, such as Pelobacterpropionicus (Schink et al., 1987) or Desulfobulbus propionicus (Stams et al., 1984). This pathway did not involve lactate dehydrogenase, and methylmalonyl-Coi\:pyruvate transcarboxylase activity was the key enzyme of the pathway, with pyruvate as substrate. However, lactate dehydrogenase activity was also detected in Desulfobulbus popionicus, and good growth lactate was described in this strain (Stams et al, 1984). As the latter enzyme catalyses an easily reversible reaction, converting lactate to pyruvate, pyruvate is thus probably the common metabolic intermediate between glucose and lactate metabolism in P. ftisingensis (way I and way II in Fig. 4). Acetate production during glucose fermentation indicates that phosphate acetyltransferase and acetate kinase are probably the main mechanisms of ATP synthesis used by P. jiisingensis. Propionate and succinate would then be excreted as electron sink by the strain.

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VFA Production

Hough, J. S., Briggs, D. E., Stevens, R. & Young, T. W. (1982). Malting and Brezuing Science, Vol. 2 Hopped Wart and Beer, 2nd edition. Chapman & Hall, London. Lee, S. Y., Mabee, M. S. & Jangaard, N. D. (1978). Pectinatus, a new genus of the family Bacteroidaceae. Znt.J Syst. Bacterial., 28,582-94.

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association

Beer Spoilage

by

Pectinatus sp.

with a Gram-negative

29

homoacetogen.

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