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79, 1973] 487



By D. A. Baker and B. H. Kirsop

{Brewing Industry Research Foundation, Nutfield, RedhiU, Surrey)

Received Uth May, 1973

Beer has been produced very rapidly, with residence time of less than 1 hour,
by passing wort continuously through a plug formed from a yeast/kieselguhr
mixture. Using malt wort the total output from the fermentor is limited by
gradual blockage of the plug; nevertheless output is high per unit of yeast used.
Prolonged operation is possible using nitrogen-free sugar solutions. The
properties of beer produced from normal wort vary over the period of operation
of the fermentor and blending is required to give a constant product. The
average pH and nitrogen content of the beer is high if conventional wort is
used but can be reduced to normal levels by control of wort composition.
Details of a procedure for controlling the content of diacetyl and 2,3-pentane-
dione in the beer are described and the potential value of a yeast-plug unit for
beer conditioning is discussed.

Key words:—ale, continuous fermentation,

fermentation equipment, maturation, Saccharo-
myces cerevisiae.

The quantity of yeast produced during a
brewery fermentation is greater than is
required for repitching. The excess yeast is of
high fermentative capacity13 and the present
Support plate
paper reports the utilization of this capacity
in a high-yeast-density continuous fermentor.
It is necessary to maintain high permeability
in the yeast zone of such a fermentor so that
wort flow is not impeded. This may be
achieved by using loosely packed flocculent
yeast as in the Tower fermentor14, by stirring,
or by circulation of the yeast in some way16.
Recently the use of kieselguhr as a filter aid to
maintain wort flow was described18.19 in
connection with the production of lager, and
this especially interesting approach was
followed in the present study, which has been
particularly aimed at determining the likely
limits of applicability to ale brewing.

Fermentor operation.—-The basic design of
the fermentor is illustrated in Fig. 1. A
mixture of yeast and kieselguhr is held
between two filters and wort is passed upward
through the resultant yeast plug. Bright Fig. 1.—Diagram of a plug fcrmentor.
wort was used in order to eliminate blockage direction to simplify the problems of collect
of the lower filter with participate matter. ing the COa evolved during fermentation.
De-aerated wort was used to minimize yeast Adjunct wort was prepared by mixing malt
growth; the wort was collected with restricted wort with an equal volume of a nitrogen-free,
access to air and the absence of oxygen in maize starch hydrolysate of the same specific
solution was ensured by passing nitrogen gravity.
through the wort for some hours. It has been Analyses.—Amino nitrogen was measured
shown that appreciable yeast growth occurs by using trinitrobenzene sulphonic acid21.
in de-aerated wort if oxygen has been freely Higher alcohols, esters and acetaldehyde were
available to the yeast during propagation8. measured using gas liquid chromatography of
Accordingly, the yeast used was grown the head space vapour18. Free diacetyl and
"anaerobically" in conditions similar to those 2,3-pentanedione were measured by a similar
of a brewery fermentation, but with addi technique18 using an electron capture detector
tional precautions to prevent diffusion of and 1,3-dichloropropane as an internal
oxygen into the propagating vessel during standard; total vicinal diketones and pre
yeast growth6. A strain of Sacckaromyces cursors were assayed similarly after heating
cerevisiae, NCYC 1236, which had been used the beer (80cC for 30 min) or by distillation
in earlier studies8'18 and was known to be of followed by colorimetric analysis22. Bitter
high fermentative capacity, was used to form ness was determined using the recommended
the yeast plug. Yeast (70 g centrifuged method of the Intitute of Brewing82 and
weight) and kieselguhr (Celite 546, 80 g) were organic acids by partition chromatography on
slurried with sterile de-ionized water and silica gel12. Viability of yeast was measured
poured into a sterile glass container (QVF, using the methylene blue technique22. Yeast
5 cm diameter x 10 cm length, volume 200 growth during fermentation was assessed by
ml) closed at the lower end by a filter sheet counting the cells present before and after
(Carlson Ford 2) supported by a perforated fermentation or by measurement of the total
stainless-steel plate. Excess water was sucked content of DNA3.
away from the column and more slurry was
added until the compact yeast and kieselguhr Results and Discussion
plug completely filled the column. A second The maximum daily output of beer from
filter and a further perforated stainless-steel the fermentor was approximately 3 litres at
plate were placed on top of the column. The 20°C, when wort (specific gravity 1-040) was
interstitial volume of the plug was found to be fully attenuated (beer specific gravity, ca
approximately 120 ml by subtracting from 1-006). This amount of fermentation was that
the total volume that volume occupied by the expected on the basis of earlier results
kieselguhr particles and the centrifuged yeast, obtained using NCYC 1236, when added
the latter having been corrected for inter carbohydrate was fermented in a batch
stitial water volume0. Wort prepared as fermentor13. The quantity of beer produced
described earlier^ was passed through the diminished when wort of higher specific
plug, using a peristaltic pump, in an upward gravity (1-080) was used but the total amount

Varying Composition of Beer Obtained from Malt Worts of Differing Specific Gravity

Spccillc Gravity 1.040 Specific Gravity 1.080

Wort Day 1 Day 2 Day 4 Day 0 Wort Day 1 Day 2 Day 4 Day 0
Specific Gravity 1.040 1.010 1.008 1.010 1.010 1.080 1.021 1.017 1.020 1.021
pH 5.0 3.0 4 1 4 2 4 4 5.3 4.3 A ±
1.% 11
4.U A ft
Nitrogen (mg/lOOml) .. 70 40 G2 73 75 140 87 112 121 185
Anilno nitrogen (mg/lOOml) . 22 0 13 15 17 44 7 14 38 44
Bitterness (E11U) 45 IS 27 24 21 54 17 18 18 22
Dlkctonca A precursors (ppm) 0.6 0.0 0.8 2.1 >2.5 2.2 2.4 2.:t
Foam stability (half-llic, sec) _
111 112 109 110 __.

Acetoldcliyde (ppm) __

2 2
1 2 _

9 4 7 H
Iso-Butnnol (npm) 46 30 20 18 — 87 60 55 40
Iso-Amylnlcoliol (ppm) 113 85 82 61 103 128 04 119
Ethyl acetate (ppm) .. 26 20 21 18 60 58 65 58
IsO'Amylacetnte (ppm) —
2.2 1.3 1.0 1.0 2.8 2.3 2.7 2.2
Acetate (ppm) .
33 63 144 __ _

Pyruvatc (ppm).. 118 53 38 ™~ — — — — —


of fermentation, measured as the product of basis) an upper limit of 20 g dry yeast matter
the (volume of wort used) x (fall in specific (ca 100 g centrifuged weight) is produced, this
gravity) was similar with both worts. When representing 140% increase during fermenta
wort of the lower gravity was used residence tion. This is clearly a maximum value and it
time within the unit was slightly less than seems likely that the results given by other
1 hour. It seems likely that much of the methods are more realistic; on this view only
interstitial volume of the plug was occupied about half of the nitrogen removed from wort
by gas bubbles so that the true contact time is utilized for new growth. It is clear that the
between wort and yeast is probably much production of new yeast (ca 3-6 g centrifuged
less than 1 hour. yeast/litre) is substantially less than that
Output from the fermentor increased normally obtained in a batch fermentation
slightly during the period of operation, sug (ca 15 g centrifuged yeast/litre).
gesting that some yeast growth was occurring. It was noticeable that the pressure in the
Nitrogen was absorbed from the wort, tubes carrying wort to the fermentor gradu
particularly during the early stages of ally increased during the course of a run,
operation (Table I), again suggesting that de- indicating that the plug was becoming
aeration of the wort had not eliminated blocked. After about 15 litres of beer had
growth of the yeast. Some growth of oxygen- been produced, using malt-wort, resistance
requiring cells in the de-aerated wort was became so high that wort flow was greatly
expected in view of the earlier results6 with restricted and output dropped. It seems likely
this strain. Estimates of the number of cells that the increasing difficulty in passing wort
present before and after the fermentation of through the plug is due both to the precipita
15 litres of wort (S.G. 1-040) showed that the tion of wort solids in the body of the plug, as
number of cells had increased by 76% suggested earlier18, and to the growth of
indicating the production of 63 g (centrifuged yeast. The relative importance of these two
weight) of yeast; measurements showed that factors is not known, but the fact that
most of the growth occurred at the base of the resistance to flow continued to increase
plug, where cells were exposed to fresh wort during the period when yeast growth, as
(Table II). When yeast growth was assessed judged by nitrogen uptake, was diminishing,

Yeast Growth Occurring During thb Fermentation of 15 Litres of Malt Wort

No. of cells (millions) and total D.N.A. content (mg)

Portion of plug Before fermentation After fermentation Increase

Upper Cells 60,227 72,867 21

(outlet end) D.N.A. 14-4 180 25

Middle Cells 43,740 62,000 42

D.N.A. 10-3 21-4 103

Lower Cells 53,571 142,010 167

(inlet end) D.N.A. 10-9 270 148

Total Cells 157,547 277,773 76

D.N.A. 35-6 60-4 87

by measuring the DNA content of the plug may indicate the greater importance of pre
before and after fermentation similar values cipitation as a cause of blockage. Evidence
for total reproduction to those given by cell was also obtained to show that blockage
counting were obtained (Table II). A further occurred after a particular quantity of wort
estimate was obtained by calculating the had been utilized; thus a reduction in beer
quantity of yeast likely to have been produced output increased the duration of a run.
if all the nitrogen absorbed from the wort was It was thought that use of de-aerated wort
incorporated into new yeast. If this yeast is might lead to a rapid loss of viability of the
assumed to contain 7% nitrogen (dry matter yeast in the plug. However, the increasing
output of the unit showed that the yeast
contamination by the appropriate tests11-17'24.
population as a whole remained active, A relationship between acetate production
although the growth of new cells may have and growth of yeast was noted by Nord-
masked the effects of the death of some cells. strom10 who found that inhibition of growth
Vital staining with methylene blue indicated led to acetate accumulation in the medium.
that 76% of the cells were viable on the sixth Pyruvate, which has been shown to accumu
day of operation. It was not possible, late in the medium during the period of yeast
because of blockage of the unit, to establish reproduction in a batch fermentation4, was
the longevity of the fermentor by operating it present in diminishing amount toward the
until sugar utilization ceased. However, end of a run (Table I).
experience with a similar unit which was Beer bitterness was relatively low (Table I)
supplied with de-aerated, nitrogen-free maize and study showed that this was partly due to
starch hydrolysate (specific gravity 1-040) losses during de-aeration and partly to losses
and remained unblocked, showed that fer within the plug. Head retention values were
mentation continued for a long time. Thus in the normal range (Table I).
after 27 days operation the output had fallen The variations in nitrogen utilization and
by only 50% (Fig. 2) and viability of the in growth were reflected in changing levels of
yeast, as judged by methylene blue staining, higher alcohols and esters during the opera
was 52%. It thus seems that death of the tion of the fermentor (Table I). Thus, the
yeast is unlikely to be a problem when wort levels of isobutanol and iso-amyl alcohol
is fermented.
diminished after the first day, thus paralleling
the diminution in the uptake of nitrogen.
Iso-amyl acetate levels were reduced after the
first day, paralleling the fall in the concentra
tion of iso-amyl alcohol which is required for
o its synthesis. The levels of ethyl acetate
5 20 fluctuated less markedly (Table I), pre
S sumably because the concentration of ethanol
is determined by the extent of attenuation
£15 rather than by yeast growth. The mean
reduction in the concentration of the higher
alcohols and esters was less than that noted
12 16 20 24 28
when lager was produced18"19.
Days The fresh beer had no aroma or taste of
Fig. 2. Fermentation of a nitrogen-free sugar diacetyl, and neither diacetyl nor 2,3-
solution (S.G. 1.040) over several weeks. pentanedione were found when the beers were
• Calculated as litres of sugar used x fall in analysed by gas liquid chromatography of the
specific gravity x 1000. headspace. However, fresh beer gave very
high values for vicinal diketones when the
Beer properties vary substantially during distillation procedure was used, and on
the period of operation, reflecting the storage a strong aroma and taste of diacetyl
variations in the extent of growth. Thus, developed; both diacetyl and pentanedione
nitrogen and amino nitrogen values rise were then detectable by gas liquid chromato
steadily (Table I) during the operation of the graphy.
plug with normal and high gravity worts, and In considering the value of this method of
in the later stages very little nitrogen is beer production it is clear that the beer
absorbed by yeast from either wort. Values differs from that produced conventionally in
for pH also rise, possibly because of the (a) the changing nature of the beer produced
presence of un-utUized buffer substances. during continuing operation, (6) the high
There was some variation in the nature of the content of assimilable nitrogen and the high
acids produced during the period of fermenta pH, both of which are factors which pre
tion, and, in particular, the concentration of dispose to microbiological spoilage and (c)
acetate increased after the first day (Table I). the aroma and taste of diacetyl which is
The accumulation of acetate was not due to present after the beers have been stored for
the contamination of the plugs as these were some days. Considering these problems in
shown to be free from bacterial and wild yeast turn, it seems clear that the variation in beer

properties with time can be overcome by induce beer spoilage are amenable to adjust
blending of beers; this may perhaps be ment by control of wort composition.
achieved by utilizing a number of fermentors Diacetyl content, however, was not adequa
with plugs of different ages, so that the tely reduced by this adjustment to the
blended beer stream is of essentially constant composition of the wort.
composition. It seemed likely that reduction As diacetyl aroma developed during beer
of assimilable nitrogen levels in the beer storage and as diacetyl and 2,3-pentanedione
would be achieved by substituting a portion could not be detected in the fresh beer in
of the malt-wort with nitrogen-free adjunct. significant quantities, it seemed clear that
When this was done (Table III) the beer precursors of these substances were present
in the fresh beer and gave rise to the free
TABLE ill diketones either as a result of heating during
Influence on Some Beer Properties of analysis or of chemical breakdown during
Using Adjunct Wort Instead of Malt Wort storage. The existence of such precursors in
fresh beer is well documented ;7-9>10 no attempt
Range of values during a was made to identify the precursors in this
run of six days study. Presumably precursors were also
Malt wort +
present in the lager beer produced using the
equal part yeast kieselguhr fermentor;18-19 the remedy
sugar adopted in that case of "krausening" for
Malt Wort solution 24-48 hours would clearly overcome difficul
(SG 1-040) (SG 1-040)
ties arising from both the diketones, which are
pH 3-9-4-4 3-7-3-9 readily utilized by yeast, and their non-utiliz-
Nitrogen (mg/100 ml) 49-75 23-29 able precursors, which break down to the
Amino nitrogen diketones relatively slowly. It was desirable,
(mg/100 ml) 6-17 1-5-2-1
0-6-2-1 0-9-1-2
at least in the context of ale production, to
Diacetyl (ppm)
Acctaldchyde (ppm) 1- 2 <1- 3 accelerate this process of removing diacetyl
iso-Butanol (ppm) 46-18 39-19 and its precursors, for most of the advantages
iso-Amyl alcohol (ppm) 113-61 148-69 of rapid production of beer are eliminated if
Ethyl acetate (ppm) 26-18 27-12
the beer requires substantial further periods
iso-Amyl acetate
(ppm) 2-2-1-0 3-0-0-4 of storage to permit flavour improvement.
Accordingly, a more rapid method was
devised and a preliminary account of this has
nitrogen levels were substantially reduced been published1. Essentially the process
and, at the same time, the pH values consists of heating the beer to convert pre
decreased substantially. Fusel alcohol and cursors to free diketones followed by removal
ester levels were not substantially altered by of these by further treatment with yeast.
the presence of carbohydrate adjunct in the The precursors in beer were found to be
wort. Thus the two beer properties liable to relatively easily converted to the free dike-

Comparison of Amounts of Diketones (ppm) Formed During Treatment of Beers by Batch
and Continuous Processes

Batch treatment* Continuous treatment!

Beer Diacetyl 2,3-pentanedione Diacetyl 2,3-pentancdionc

1 1-4 1-6 0-6 0-5

2 2-0 1-6 0-8 0-5
3 0-8 M 0-5 0-6
4 1-6 1-5 00 0-7
5 11 20 10 1-4

• Batch treatment involved heating in a scaled 25-ml vessel with 5 ml air head space for the same time at
the same temperature as in the continuous process.
I Continuous treatment using the heating coil described in the text.

Oiacetyl formation Diacetyl formation

0-8 at 80° C 1-6 at 60° C
• Without oxygen
■ With oxygen

0-6 1-2


0-2 04

12 16
formation at 60° C


8 12 16 10 20 30 40 50 60

Time (min) Time (min)

Fig. 3. Effect of heat on the formation of diacetyl

and 2,3-pentanedione in beers produced by plug

tones by heat. Thus, heating fresh beer emerged from the primary fermentor, so that
produced by plug fermentation for 4 min at the beer was not in contact with oxygen at
80°C, 15 min at 60°C or 45 min at 45°C was any stage. The precise conditions of heating
effective (Fig. 3). There was no apparent appeared, however, to have some effect on the
difference in the rate of breakdown of the detailed pattern of precursor breakdown, for
precursors of diacetyl and 2,3-pentanedione it was found that the continuous treatment of
(Fig. 3). The rate of conversion of precursor beer containing precursor gave rise to a lower
to diketones was not markedly affected by the yield of diacetyl and 2,3-pentanedione than did
presence or absence of oxygen (Fig. 3). heating in batch conditions (Table IV). In both
Breakdown of precursor to diacetyl also cases no precursor remained after treatment.
occurred when beer was passed continuously Effects of the method of heating on the yield
through a glass heating coil as soon as it of diacetyl have previously been reported10.

Diacetyl is rapidly removed from a medium 19 g wet weight NCYC 1236 mixed with
by Saccharomyces cerevisiae. When beer kieselguhr (Fig. 5). The combination of heat
containing added diacetyl was treated with and exposure to yeast produced beer con
brewing strains of S. cerevisiae in batch taining insignificant quantities of free dike-
conditions diacetyl levels dropped rapidly.
The rate was clearly related to temperature Beer + co2
and the rate of utilization was linear when



20 10°C


Q Wort
30° C
04 Primary Conditioning
plug fermentor plug

12 3 4 5 Fig. 5. Diagram of a primary plug fcrmentor and

Time (hours) "conditioning unit" i.e. pasteurizer and small
secondary plug.
Fig. 4. Effect of temperature on the utilization of
diacetyl in beer containing 5g (centrifuged
weight) NCYC 1230/litre.
tones (Table VI) and such beer did not
normally liberate diketones on further heating
diacetyl concentration was plotted logarith or on storage. If the beer was not fully
mically (Fig. 4). Similarly yeast contained in attenuated, and if a substantial amount of
a plug removed diacetyl from beer (Table V),
as anticipated from the studies of Tolls etal23.
Effect of a Heating Coil (60°C). and Second
TABLE V Yeast Plug on the Diketone Concentrations
Removal of Diacetyl from Beer with in a Plug-Fermented Beer
1-7 ppm Added Diacetyl by a Yeast Plug
Containing 10 g Yeast, Strain N.C.Y.C. 1236 GLC Assay

Diacetyl 2,3-Pcntanedione
Flow rate of
Beer Sample (ppm) (ppm)
beer through
plug % removal
Immediately after
Day (ml/day) of diacetyl
plug fermentation 0075 007

1 1870 85
After heating coil 0-41 0-51
2 1870 85
3 1870 88
After conditioning
4 1870 96
plug 002 None
5 1870 97
5 3760 79
6 2700 85

fermentation occurred during passage through

the second plug, further small quantities of
The fully attenuated beer emerging from precursors were produced, but these were
the primary plug was passed through a present in quantities unlikely to influence
heating coil (15min at 60°C) and then beer flavour when they decomposed to yield
through a second smaller plug containing diketones.
The control of diacetyl and 2,3-pentane- and not confined to the beer produced on the
dione levels in beer by the procedure described first day of production; this problem can be
above is rapidly achieved, for the heat simply solved by the conditioning unit which
treatment, cooling and period of exposure to has been developed.
yeast in a second plug collectively add less
than 1 hour to the time required for primary References
fermentation. In essence the procedure is, 1. Baker, D. A. & Kirsop, B. H.. Journal of the
with regard to its effects on the diketone Institute of Brewing, 1073, 79, 43.
content of beer, a conditioning unit. It was 2. Brown, M. L. & Kirsop, B. H., Journal of the
found that acetaldehyde, a common con Institute of Brewing, 1972, 78, 39.
stituent of green beer, was also removed by 3. Burton, K., Biochemical Journal, 1956, 62, 315.

the second yeast plug (Table VII) further 4. Coote, N., Kirsop, B. H. & Buckce, G. K,
Journal of the Institute of Brewing, 1973, 79,
5. David, M. H. & Kirsop, B. H., Journal of the
Removal of Acetaldehyde, Added to a
Institute of Brewing, 1973, 79, 20.
Batch Beer, by a Yeast Plug Containing
10 c N.C.Y.C. 1236 0. Eddy, A. A. in The Chemistry and Biology of
Yeasts, Ed. Cook, A. H. Academic Press,
London & New York, 1058, 228.
Flow rate Acetaldehyde in
7. Haukeli, A. D. & Lie, S., Journal ofthe Institute
of beer beer (ppm)
of Brewing, 1071, 77, 538.
Beer collected plug Before 8. Hudson. J. R. & Birtwistle, S. E., Journal of the
after day: (ml/day) plug plug Institute of Brewing, 1966, 72, 46.
9. Inoue, T. & Yamamoto, Y., Proceedings of the
1 2329 42-4 3-5 American Society of Brewing Chemists, 1070,
2 1080 42-5 3-25 198.
3 1680 420 4-5 10. Inoue, T. & Yamamoto, Y., Report of the
Research Laboratories of the Kirin Brewery
Co.. 1972, 78, 51.
indicating its potential value as a condition 11. Kato, S., Bulletin of Brewing Science, 1067. 13.
ing unit. The use of a combined heating unit 19.
and yeast-kieselguhr plug should be of value 12. Kesner, L. & Muntwyler, E., in Methods in
as a conditioning unit to treat beer produced in Enzymology, XIII, Ed. Colowick, S. P. &
Kaplan, N. O., Academic Press, London &
batch as well as in continuous fermentations. New York, 1969, 115.

Conclusions 13. Kirsop, B. H. & Brown, M. L., Journal of the

Institute of Brewing, 1972, 78, 51.
Clearly the yeast/kieselguhr plug can be 14. Klopper, W. J., Roberts, R. H., Royston,
used for the rapid production of ale, providing M. G. & Ault, R. G., Proceedings of the
that wort composition is appropriately European Brewery Convention, Stockholm
adjusted. The maximum daily output of fully 1965, 238.

attenuated beer is about 15 times the total 15. Maule, D. R., Journal of the Institute of
Brewing, 1907, 73, 351.
internal plug volume, including yeast and
10. Millin, D. J. & Pinnegar, M. A., British Patent
kieselguhr. In our experience blockage of the No. 1,079,517. 1907.
fermentor is likely to be the limiting factor 17. Morris, E. O. & Eddy, A. A., Journal of the
and this seems to occur after about 15 litres Institute of Brewing. 1057, 63, 34.
of beer have been produced using the 200-ml 18. Narziss, L. & Hellich, P., Brauwelt, 1971, 111,
fermentor described above. Design modifica 1491.
tions to the fermentor may allow the period 19. Narziss, L. & Hcllich, P., Brewers Digest, 1972,
of use to be extended. The quantity of yeast 47, 106.

which is required to produce 1 hectolitre of 20. Nordstrom. K., Journal of the Institute of
Brewing, 1984, 70, 209.
beer is of the order of 0-5 kg (centrifuged
21. Pinncgar, M. A., Journal of the Institute of
yeast), i.e. 1-8 lb yeast/barrel. The yeast Brewing, 1966, 72, 62.
harvested after the fermentation of one barrel 22. Recommended Methods of Analysis, Journal of
of wort in a conventional brewery batch the Institute of Brewing, 1071, 77, 181.
fermentation would thus ferment a further 23. Tolls, T. N., Shovers, J., Sandine, W. E. &
2-3 barrels of wort when used in a fermentor Elliker, P. R., Applied Microbiology, 1070,
of the plug type. Our experience differs from 19, 640.

that reported elsewhere18-19 in that the 24. Williamson, D. H., Journal of the Institute of
Brewing, 1060, 65, 154.
presence of diacetyl is a continuing problem