Note: Descriptions are shown in the official language in which they were submitted.
-- W094/29430 2 1 6 4 ~ 0 4 PCT~P94/01793
Method for improving the properties of malted cereals
The present invention relates to improving the properties
of malted cereals, especially of malted cereals for the
brewing, distilling and bakery industry.
Since ancient ages, mankind uses fermentation in the
preparation of a number of foodstuffs, including beverages,
meat products, dairy products etcetera. With respect to
beverages, the main applications are in the field of
brewing various types of beer, in providing fermented wort
as is used in the whisky distilling industry. Further
applications of malted cereal are in providing malt
products like syrups and extracts for the bakery industry.
Before fermentation or other processing of raw vegetable
material like cereal can take place, it is generally needed
that this material is malted first to generate or activate
a complex mix of endogenous enzymes. Malting comprises the
steps of steeping of the raw cereals, germination,
subsequent drying or kilning and optionally root and
acrospire removal.
For malting, any cereal may be used depending e.g. on the
end product for which the malted matter is used, the
availability of the raw material etcetera. Such cereals are
for example wheat, barley, rye, rice, corn and sorghum. For
further processing, other starchy vegetable matter like
cassava (including the derived tapioca), arrowroot, sago
and potato may be added to the malted cereals, depending on
e.g. its end use.
In the present malting processes malted cereals are
obtained which have a quality which is far from optimal.
This implies that measures have to be taken of various
kinds, depending upon the use of the malted cereal, to
upgrade the quality of the already malted cereal. If, for
W094/29430 216 4 ~ 0 4 2 PCT~P94/01793 --
example, a malted barley is to be used for the fermentation
to beer, it is common practice to add e.g. enzymes to the
malted barley, to make up for the poor endogenous enzyme
balance formed by the traditional malting process. An
example of an enzyme used for this is ~-glucanase. Enzyme
compositions for this purpose are generally added prior to
mash preparation or fermentation. This addition of separate
enzyme mixes is more and more considered as undesirable.
Besides enzymes, there are often compounds added to speed
up germination during the malting stage. Faster germination
is of clear economical advantage, and additionally it may
lead to a higher quality malt, more fermentable sugars and
improved balance between desired and undesired products.
The most known and widely applied germination aid is
giberellic acid, or compositions comprising this plant
growth hormone (see e.g. "Cereal science and technology",
edited by G.H. Palmer, Aberdeen University Press). Use of
giberellic acid (or similar additives) is, however,
undesired.
Another disadvantage caused by known malting processes is
the relatively high loss of fermentable (poly)saccharides,
which in its turn is caused by extensive steeping of the
raw cereals. Another negative effect of extensive steeping
is that a high volume waste water stream is obtained with a
very high BOD (Biological Oxygen Demand), which is
undesirable from an environmental point of view. Still
other disadvantages related to a poor quality of malted
cereals are short shelf life of products produced by
fermentation of the malted cereals and that the malt
rootlets or sprouts obtained as waste material after
malting are of such poor quality that they are not suitable
to be used as feed stock in high concentrations. The latter
is due to the amount of toxins which might be present in
this waste material (see J. Inst. Brew., 98, 139-142,
1992).
wo 94,29430 2 1 ~ 4 1 0 4 PCT~P94/01793
Thus, there is a need for a process for improving the
quality or properties of malted cereals in a convenient,
environmentally acceptable way. Improving the quality or
properties in this respect means that one or more of the
following conditions is fulfilled:
- improving the enzyme balance in the malted cereals,
- increase in the amount of ~-glucanase in the malted
cereals,
- faster germination during malting,
- improving the flavour of the malted cereals
- better microbial quality of the malted cereals,
Which of the above conditions is to be fulfilled will
depend on the end use for which the malted cereals are
used, which may be in brewing different kinds of beer
(including low or zero percent alcohol beer), distilling,
bakery etcetera.
It has now been found that the quality or properties of
malted cereals can be improved by a process wherein starter
cultures are added to the cereals prior to or during
malting of said cereals. Such starter cultures may comprise
moulds, yeasts and bacteria.
With respect to the improved enzyme balance, this is meant
to comprise both a higher total amount of enzymes as well
as a higher concentration of desired enzymes. This better
enzyme balance might be caused by enzymes produced by the
starter cultures added as well as due to a promotion by
added microorganisms of the activity of enzymes which are
already present or developed in the cereal. The level of
enzymes like proteases, peptidases or phytase (which can
break down the undesired phytate) and tannin degrading
enzymes (including tannin-like polyfenols) may be increased
by the process according to the invention. In the case of
the preparation of beer, tannins or polyfenols present in
the malted cereals may shorten shelf life because they may
lead to haze formation and/or off flavour. Reduction of
wog4n9430 2~6 4~ PCTEP941~1793
some tannins or tannin-like polyfenols may be desired. An
increased level of carbohydrate modifying enzymes like
glucanase (and in particular of ~-glucanase) may be
obtained, which is of particular significance in the
brewing industry.
With improved microbial quality of the malted cereal or of
the products prepared thereof, it is meant that microbial
spoilage may be reduced due to a decrease in pH, formation
of acids which reduce the growth of certain microorganisms
and/or formation of other compounds which reduce the growth
of certain microorganisms, like bacteriocins. With respect
to this improved microbial quality, this means that the
shelf life of products prepared with malts prepared by the
process according to the invention may be extended. This is
especially important in beer, and more in particular in the
case of low- or zero alcohol beer.
Due to the metabolism of the added starter cultures,
desired compounds may be formed. Desired for further
processing (including mash preparation and fermentation)
may be certain specific carbohydrates and proteins. As a
result of this, an increased flavour control is achieved
and in the case the malt is used for the production of
beer, a beneficial effect on the foam stability may be
obtained.
In the case moulds or yeasts are added, suitable amounts to
be added to the cereals range from 10-3 to 104 mg moulds or
yeast (viable, based on dry weight biomass) per kg of
cereal to which such moulds or yeasts are added. A
preferred amount is O.l to lO0 mg moulds or yeast (viable,
based on dry weight biomass) per kg of cereal. In the case
of bacteria, suitable amounts to be added to the cereals
range from 10-5 to lO0 mg bacteria (viable, based on dry
weight biomass) per kg of cereal to which such bacteria are
added. A preferred amount is O.Ol to lO mg bacteria
-- WOg4/29430 ~1 6 ~ 1 0 ~ PCT~P94/01793
(viable, based on dry weight biomass) per kg of cereal.
Mixtures comprising moulds and bacteria may also be
employed. Preferred bacteria are lactic acid producing
bacteria, such as various Lactobacilli, e.g. Lactobacillus
casei, Lactobacillus casei var rhamnosus, Lactobacillus
fermentum, Lactobacillus plantarum, and Lactobacillus
brevis. Also preferred are bacteria of the genus
Pediococcus. Preferred moulds are moulds of the genus
AsPergillus and Geotrichum, like Geotrichum candidum.
Preferred cereals to which the process according to the
invention is applied to are barley and wheat. A preferred
end use of malted material obtained by a process according
to the invention is in the preparation of beer, both
alcoholic and non-alcoholic beer. The benefits of the
process according to the invention are of particular
significance when sorghum is used as cereal, since in the
conventional malting of sorghum only low levels of enzymes
are developed.
By employing the process according to the invention the
amount of undesired tannins and tannin-like polyfenols and
phytate may be reduced due to enzymes produced by the added
starter cultures which may break down these compounds.
It is preferred to add the starter cultures at some stage
of the malting process (i.e. prior to or during malting).
Most preferred is that the starter culture bacteria are
added prior to germination. Apart from the addition of
starter cultures, enzymes may still be added during some
stage of the malting process for obtaining an even better
enzyme balance.
It should also be noted that the process as disclosed above
has the additional benefit of improving germination at the
malting stage. Among such benefits is a faster germination.
This benefit reduces the need for the use of conventional
W094/2g~0 ~16 4 l0 PCT~4/01793
germination aids, like gibberellic acid.
Yet a further advantage of the present invention is that
the filterability of the mash obtained from the malt is
improved. More in particular, the time needed for
filtration can be reduced by employing the present
invention. This is a considerable advantage, since
filtration of the mash prepared from the malts is known to
cause difficulties.
A further benefit of adding starter cultures to cereals is
that the waste material obtained during or after malting is
upgraded in such a way that it becomes suitable for food or
feedstock. This is due to a reduced level of toxins in the
malt rootlets or sprouts.
The invention is illustrated by the following examples but
is in no way limited thereto.
Example 1
Preparation of microbial cultures
Before use in malting experiments strains were pre cultured
in MRS broth for 24 hours at 30C, centrifuged and washed
with physiological saline. Finally the biomass pellet was
resuspended to the original volume and the density of the
culture was measured using OD6l0 readings.
Barley
Nordeum vulgare cv Triumph (water sensitive) from 1993
harvest was used in all examples.
Application of microbial cultures
In case of Lactobacillus plantarum, 1 ml of cell suspension
was added to 200 g of barley. Additions of microbial
cultures to malting trials were made on equal OD basis,
corresponding with 107 CFU/g barley (Colony Forming Units).
-- WOg4/29430 2 t 6 ~1 0 ~ PCT~4tO1793
In case of Geotrichum candidum addition, 0.7 g cell
suspension of a full grown culture in MRS (ca 10 g dry
weight/l) was added. In this case no "colonies" are formed
therefore no CFU data can be given. These cultures were
sprayed onto the barley after the steeping operation.
Steeping
Immersion steeping was carried out in two stages at T =
15C using a 500 g batch of barley. Total barley to water
ratio was 1:8 with a water change after 24 hours. During
steeping there was continuous aeration of the water via
circulation of a connected stirred reactor, which was
aerated at a rate of 0.3 vvm (volume gas per volume liquid
per minute). No air rests were applied.
Germination
In this example, germination was carried out on a 150 gram
scale using an covered sieve box (type ~ 22 cm laboratory
test sieve, Endecotts Ltd, England). The boxes were placed
in a temperature controlled cabinet at 18 C. Air was
supplied to the germinating grain by natural diffusion.
Kilning
Kilning was performed in an "Aeromatic" (Muttenz,
Switzerland) drier. Three sieve boxes were put in the drier
and air was blown through at speed 10. Inlet temperature
for the first 18 hours was 500C followed by 4 hours at
80C
Malt evaluation
Filtration performance was measured using the "Tepral"
filtration system as described by M.Moll et.al.
(Am.Soc.Brew.Chem. 47 (1), 14-17, 1989). This system is
claimed to mimic large scale filtration best, one of the
reasons is that it operates at high temperatures (75C)
From a cold extract of the malt, ~-Amylase and ~-Glucanase
activities were measured using the Megazyme "Amylazyme" and
WOg4/29430 216 ~10 4 PCT~P94/01793
"Glucazyme" kits.
Results
The results of the filtration test are shown in table 1.
Commercial malt A is taken as reference example of a high
quality malt, while malt B is regarded as a low quality
malt. It can be seen that the control is a moderately good
malt. Treatment with G. candidum seems to have a negative
effect on the filtration rate at gravity, however is
improved after applying 1 bar pressure and would be rated
as a high quality malt. The L.plantarum treated malt can be
rated as a very good quality malt, both at gravity
filtration and at 1 bar pressure.
The results of the enzyme activities are described in table
2. No significant differences in ~-Amylase activities were
observed in control malts and treated malts. Important
however is that ~-Amylase activity in malt is generally
present in high levels (excess) and is therefore not the
most critical enzyme which needs to be increased. The
contrary is the case with B-Glucanase in malts. The B-
Glucanase activity from the malts that have been treated,
either with Lactics or with Geotrichum, are significantly
higher than controls and are at least as good as the high
quality of commercial malt A.
-- WOg4/29~0 2 1 6 ~1 04 PCT~4/01793
,
Table 1 ~epral filtration rates of various malt samples
malt gravity 1.0 bar
filtration pressure
~ml/min.) ~ml/min.)
Commercial malt A9.9 7.2
Commercial malt B6.5 4.6
5 control 8.0 5.0
G.candidum 5.8 12.6
treated
L.plantarum 11.0 12.5
treated
Table 2. Enzyme analysis of malts from example l
malt sample ~-Amylase ~-Glucanase
~Units/g) ~Units/g)
control 170 265
L.plantarum treated 165 397
L.plantarum treated 183 515
control 181 224
G.candidum treated182 332
G.candidum treated - 444
Commercial malt A 179 351
Example 2
Preparation of microbial cultures
Before use in malting experiments Lactobacillus casei var
rhamnosus was pre-cultured in MRS broth for 24 h at 30C,
centrifuged and washed in culture medium. Finally the
biomass pellet was resuspended to the original volume.
Alternatively, the Lactobacillus fermentum and Pediococcus
wo 94/2g430 ~16 4~ 4 PCT/EPg4l0l7g3
acidilactici used are commercial products from Quest
International, and were not pre-cultured but applied as
delivered.
Application of microbial cultures
The second set up consisted of a complete own-design 5 dm3
laboratory micromalting system with provisions for T -
controlled steeping, sparging and head space aeration,
agitation and kilning using heated air. However after
steeping, 150 g aliquots were taken and put in the same
sieve boxes and germinated and kilned as described in
example 1. Cultures (10 ml) were added to the barley which
was immersed in part of the final steeping water (1:1).
After removal of the liquid, the effective dosage was
estimated at 107 CFU/g.
Steeping
Immersion steeping was carried out in two stages at T =
15C using a 500 g batch of barley. Total barley to water
ratio was 1:8 with a water change after 24 hours. During
steeping there was continuous aeration of the water as
described in example 1. No air rests were applied.
Germination
Germination (150 g scale) was carried out as described in
example 1. Small aliquots (25 g) were taken from the steep
and separately incubated in petri-dishes. These samples
were used to monitor differences in germination rate.
Kilning
Kilning of 125 g samples was carried out as described in
example 1.
Malt evaluation
Filtration performance was measured as described in
example 1.
Differences in germination rate were scored by monitoring
- WOg4/29430 11 PCT~P94/01793
acrospire length.
Results
Initial filtration rates are described in table 3. It can
be seen that filtration rates of the control malt is
relatively poor but treated malts are clearly superior to
the control. and would rate as high quality malt.
Table 3 Tepral filtration rates of various malt samples
lO malt gravity
filtration
(ml/min.)
control 5.0
L.casei v 10.6
rhamnosus
P.acidilactici11.3
15 L.fermentum 12.7
In table 4. the acrospire length of the various malts after
5 days of germination is given. The length is rated into
groups of O to 2 mm, 2 to 5 mm, 5 to lO mm, lO to 15 mm and
bigger than lS mm. From this table it can be seen that
acrospires of the treated malt(s) are longer, indicating
that they are in a further stage of development than the
control. This indicates that less time is required to reach
the point where a good quality malt would be kilned
( acrospire length should be approximately 3/4 of the grain
length).
WOg4/29~0 2 ~ 6 410 4 PCT~4/01793 --
12
Table 4 Rating of acrospire length of various malt samples
malt Length
( mm )
control 0-2
L.casei v > 15
rhamnosus
P.acidilactici 5-10
L.fermentum 10-15
Example 3
Preparation of microbial cultures
Before use in malting experiments strains were pre cultured
in MRS broth for 24 hours at 30C, centrifuged and washed
in culture medium. Finally the biomass pellet was
resuspended to the original volume and the viability of the
culture was measured, and is expressed in CFU's.
Application of microbial cultures
In this example use was made of the micro-malting facility
at TN0-Zeist (Nibem). In trial-l no cultures were added
(control). In trial 2 and 3 respectively Lactobacillus
casei var rhamnosus (4.8 105 CFU/g barley) and
Lactobacillus plantarum (1. 3 107 CFU/g barley) were added
and in trial 4, a mixture of L. casei var rhamnosus ( 2.4
105 CFU/g), L.brevis (1.6 106 CFU/g) and Pediococcus
acidilactici (1. 4 107 CFU/g) was added. These cultures
were sprayed onto the barley after the final air rest of
the steeping operation.
Steeping
Intermittent wet-dry steeping was carried out at T = 15C,
using initially a 2 hour wet and 2 hour air rest, followed
by 12 cycles of 1 hour wet - 2 hours dry. The barley to
water ratio was 1:11. After 24 hours of steeping the steep
W094/29~0 2 1 6 4 1 0 4 PCT~4/01793
water was replaced with fresh water.
Germination
Germination was carried out at T = 15C, using humidified
air at lO l/min. for the first three days and 5 l/min
afterwards.
Rilning
Kilning was done according to the following scheme: 11
hours at T = 50C, followed by 2 hours at T = 60C and 5
hours at T = 80C.
Malt evaluation
Differences in germination rate were monitored by measuring
the degree of modification according to the "Carlsberg"
method (Calcofluor staining: Aastrup S & Jorgensen K.G., in
Modern methods of plant analysis 7, 56-66, 1986).
Results
In table 5 the degree of modification is shown of malts
that were analysed with the Calcofluor method after 3 days
of germination. The increased degree of modification is a
preferred character by brewers (and thus malsters) and
reflects the increased germination rate as was observed in
example 2. Although in this experiment only "starter"
cultures were used, it should not be excluded to apply a
mixture of "starters" on their own but also mixtures with
yeasts or moulds (e.g. Geotrichum or Aspergillus species).
W094/29~0 ~16 4 ~ 4 PCT~4/01793
14
Table 5 Degree of modification of various malts after 3
day germination
malt Modification
(%)
control 68
5 L.casei v 7 5
rhamnosus
L.plantarum 78
Lactics mixture 74
The wort that was prepared from the filtration trial was
tasted by expert tasters. The flavour description is given
in table 6. The flavour of the control is relatively green
and beany, which is probably caused by the way the malt is
kilned. Despite the "mild" kilning, clear differences could
be tasted between control and especially malt treated with
the Lactics mixture. These differences will only be
enhanced on treating the malt with a more severe kilning
regime.
Table 6 Flavour description of various malts.
2 O malt Flavour description
control Raw, Green, Beany
L.plantarum More complex than control, Branny
Lactics mixture Most complex, less Green,
slightly Beany
The microorganisms used in the above experiments were:
NRRL-18.368 ( L. plantarum), CBS 2 406 2 (G.candidum) and the
following available from Quest International, the
Netherlands, with their respective Quest index no.:
L. brevis (90545), P. acidilactici (90589), L. casei var
r~amnosus (90594) and L. fermentum (90596).
