METHOD FOR IMPROVING THE PROPERTIES OF MALTED CEREALS

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, subseguent 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 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 0-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) . 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 /3-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 some tannins or tannin-like polyfenols may be desired. An increased level of carbohydrate modifying enzymes like glucanase (and in particular of β-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 0.1 to 100 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 100 mg bacteria (viable, based on dry weight biomass) per kg of cereal to which such bacteria are added. A preferred amount is 0.01 to 10 mg bacteria (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 Asper illus 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 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 30°C, 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 OD₆₁₀ readings.

Barley

Hordeum 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) . In case of Geotrichum candidum addition, 0.7 g cell suspension of a full grown culture in MRS (ca 10 g dry weight/1) 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 = 15°C 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 ø 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 50°C followed by 4 hours at 80°C.

Malt evaluation

Filtration performance was measured using the "Tepral" filtration system as described by M.Moll et.al. (Am.Soc.Brew.Chem. 4 (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 (75°C)

From a cold extract of the malt, α-Amylase and β-Glucanase activities were measured using the Megazyme "Amylazyme" and "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 α-Amylase activities were observed in control malts and treated malts. Important however is that α-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 β-Glucanase in malts. The β-

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.

Table 1 Tepral filtration rates of various malt samples malt gravity 1.0 bar filtration pressure

(ml/min. ) (ml/min. )

Commercial malt A 9.9 7.2

Commercial malt B 6.5 4.6 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 1 malt sample α-Amylase β-Glucanase (Units/g) (ϋnits/g) control 170 265

L.plantarum treated 165 397

L.plantarum treated 183 515 control 181 224

G.candidum treated 182 332

G.candidum treated - 444

Commercial malt A 179 351

Example 2

Preparation of microbial cultures

Before use in malting experiments Lactobacillus casei var rha nosuε was pre-cultured in MRS broth for 24 h at 30°C, centrifuged and washed in culture medium. Finally the biomass pellet was resuspended to the original volume.

Alternatively, the Lactobacillus fermentum and Pediococcus 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 = 15°C 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 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 malt gravity filtration

(ml/min.) control 5.0 . casei v 10.6 rhamnosus

P. acidilactici 11.3

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 0 to 2 mm, 2 to 5 mm, 5 to 10 mm, 10 to 15 mm and bigger than 15 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) . 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 30°C, 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 CPU's.

Application of microbial cultures

In this example use was made of the micro-malting facility at TNO-Zeist (Nibem) . In trial-1 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.jrevis (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 = 15°C, 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 water was replaced with fresh water.

Germination

Germination was carried out at T = 15°C, using humidified air at 10 1/min. for the first three days and 5 1/min afterwards.

Kilning

Kilning was done according to the following scheme: 11 hours at T = 50°C, followed by 2 hours at T = 60°C and 5 hours at T = 80°C.

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 1_, 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) .

Table 5 Degree of modification of various malts after 3 day germination malt Modification (%) control 68

L. casei v 75 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. 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 24062 (G. candidum) and the following available from Quest International, the Netherlands, with their respective Quest index no.: L . breviε (90545) , P. acidilactici (90589) , L . casei var rhamnosus (90594) and L. fermentum (90596).