Abstract:
Process for the preparation of malted cereals, wherein the steeping step includes one or more wetting stages at a temperature between 5 and 30° C. preferably between 10 and 20° C., until the material has a moisture content between 20 and 60% by weight, preferably between 38 and 47%, wherein after a germination period between 2 and 7 days, preferably between 3 to 6 days at a temperature between 10 and 30° C., preferably between 14 and 18° C., the steeped and germinated cereals are preferably kilned by increasing the temperature to values between 40 and 150° C. until the material has a moisture content between 2 and 15% by weight, and wherein one or more microbial cultures selected from the group consisting of one or more bacteria and/or one of more activated spores are added in one or more times during the process.

Description:
This application is a continuation-in-part of International application PCT/BE96/00077 filed Jul. 23, 1996 and designating the United States. 
    
    
     FIELD OF THE INVENTION 
     The present invention is related to an improved process for the preparation of malted cereals, the improved malted cereals obtained and their use, especially In biotechnological processes for the preparation of beverages. 
     TECHNOLOGICAL BACKGROUND OF THE INVENTION 
     Cereals, such as barley, wheat, rye, corn, oats, rice, millet triticale, and sorghum, are used for the production of beverages. In most cases, they have been subjected to a malting process to take advantage of their increased enzymatic potential. 
     In traditional malting processes, the moisture content of cereals is raised either by immersion(s) and/or spraying(s), and the resulting high-moisture content cereal is allowed to germinate. After reaching the proper physiological condition, it is preferably submitted to (a) drying step(s). In what follows, the term steeping refers to the increase in moisture level, while the term germination is used in the way it is in plant physiology. The drying operations are referred to as kilning and the term malting involves all operations needed to convert barley (or other cereals) to barley malts (or other cereal malts). 
     The quality of the malt obtained is, to a large extent, determined by the presence of plant endogenous enzymes generated during the melting process. For instance, with cereals like barley used as a raw material for the malt production, the variety, the composition of the microbial flora and the environmental factors, such as agricultural practice, influence the quality of the malt. During cultivation and storage, cereals are contaminated with bacteria and fungi. In the malting plant, neither the air, the water, nor the equipment are sterile, and the conditions of humidity, pH and temperature favor the growth of the microbial populations. To improve the quality of malted cereals, such as barley, enzymes have been added to the malted cereal. 
     The variable cereal quality and the lack of means to make up for deficiencies during the malting process result in variability in malt quality and enzymatic activity. In many instances, this has to do with an imbalance of specific enzymatic potential and insufficient cell wall degradation. Apart from this, problems with microbial safety can occur. As a consequence of the defects in malt, quality problems occur in the production of beer, such as a poor filtration of the wort. 
     STATE OF THE ART 
     During the malting of cereals, the microflora present on the cereals develops and the quality of the malt and beverages is influenced by the activity of the latter microorganisms. 
     In analogy with other biotechnological processes, there have been attempts to optimize malt quality aspects by the addition of starter cultures during the malting process (Boivin, P. &amp; Malanda, M., Influence of Starter Cultures in Malting on the Microflora Development and Malt Quality, EBC, Proceedings of the 24th Congress, pp. 95-102 (1993); Haikara, A. et al., Lactic Starter Cultures in Malting—A Novel Solution to Gushing Problems, European Brewery Convention, Proceedings of the 24th Congress, pp. 163-172 (1993)). 
     Addition of spores of  Geotrichum candidum  to the steeping water results in the inhibition of the development of undesirable microorganisms and in a decrease of the filtration time of wort made of the obtained malt. Treatment with  Geotrichum candidum  also inhibits the formation of mycotoxins by  Fusarium  spp. 
     The influence of  Lactobacillus plantarum  and  Pediococcus pentosaceus  has been tested on the microflora during malting, and it has been found that these cultures act as natural preservations as they restrict the growth of  Fusarium  and prevent gushing. 
     The international patent application WO 94/29430 describes a process for improving the properties of malted cereals wherein starter cultures which comprise moulds, yeasts or bacteria are added prior and/or during malting of said cereals. 
     The preferred bacteria used are lactic acid producing bacteria, such as various  Lactobacilli , e.g.,  Lactobacillus casei, Lactobacillus casei  var.  rhamnosus, Lactobacillus fermentum, Lactobacillus plantarum  and  Lactobacillus brevis , and bacteria of the genus  Pediococcus , e.g.,  Pediococcus acidilactici.    
     Preferred moulds are moulds of the genus  Aspergillus  and  Geotrichum , like  Geotrichum candidum.    
     The international patent application WO94/16053 describes a process for treating cereals for inhibiting growth of unwanted microbial species by inoculating the cereals during the germination process with a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria. The preferred bacteria are lactic acid bacteria belonging to genus  Lactococcus, Leuconostoc, Pediococcus  or  Lactobacillus.    
     The British patent application GB-1211779 provides a method for the automatic control and regulation of a malting process. It enables one to determine the parameters necessary for a successful automatically controlled and regulated malting process. 
     In the proceedings of the European Brewery Convention, volume 16, 1977, pages 245 to 254, the influence of some fungi on malt quality is described, more specifically, contamination of barley malt with fungi which has led to gushing and other qualitative changes in the beer. 
     The German patent application DE-3028360 discloses a method to make malt out of corn. 
     However, malt prepared according to the present invention is of better quality than that prepared according to WO 94/29430. This is exemplified by higher β-glucanase and xylanase activities, lower-glucan contents in malt and wort and improved European Brewery Convention analytical data. 
     AIMS OF THE INVENTION 
     The present invention aims to provide an improved preparation process for malted cereals and improved malted cereals. 
     A main aim of the invention is to provide an improved preparation process for malted cereals and improved malted cereals in terms of brewing performances, especially malted cereals having an improved quality in terms of enzymatic potential and microbial safety. 
     Another aim is to provide a process and improved malted cereals which vary less in quality with the raw material used. 
     A further aim of the invention is to obtain malted cereals which improve the biotechnological production process of beverages and may improve the properties of the said obtained beverages. 
     SUMMARY OF THE INVENTION 
     The present invention is directed to a process for the preparation of a malted cereal, the malted cereal which is the product of the process of the invention, and a combination of wetted or moistened cereal and activated spores which, when held for a sufficient time and temperature, provide a malted cereal product of enhanced enzymatic activity. The product of the invention has enhanced enzymatic activity of at least one enzyme, such as β-glucanase, xylanase, amylase, naturally occurring enzymes, and/or protease activity, over malted cereal products which are similarly prepared with wetted cereal products with or without microorganisms. 
     The process of the invention utilizes activated spores from microorganisms such as bacteria or moulds. The process generally comprises combining water, the cereal and activated spores and holding the combination until a malted cereal of enhanced enzymatic activity is formed. Generally the combination is made by inoculating the moistened cereal with the activated spores, but the activated spores and cereal may be combined before or after the moistening of the cereal. In the process of the invention, the combination of wetted cereal and activated spores has a concentration of activated spores, holding time and holding temperature which are effective for providing the malted cereal with an increase in enzymatic activty of at least one enzyme, such as β-glucanase, xylanase, amylase, naturally occurring enzymes, and/or protease activity, which is greater than the enzymatic activity which is obtained by holding the wetted cereal without activated spores, or even with the bacteria or moulds from which the spores come. 
     In an important aspect, the cereal, activated spores are combined before or after the time of wetting the cereal and the combination is held at a temperature of at least about 5° C. and not more than about 30° C., preferably between about 10° to about 20° C. and the activated spores are at a concentration in the combination to obtain an increase in enzymatic activity of the malted cereal. In another important aspect, the wetted or moistened cereal and activated spore combination is held for a time and temperature until the cereal has a moisture content of at least about 20 weight percent. In yet another aspect, after the wetted cereal has attained an increased moisture content and has started to germinate, it is dried to a moisture content of not more than about 15 weight percent. In still another important aspect, the moistened cereal and activated spores are held together until the cereal has a moisture content of between about 20 to about 60 weight percent, preferably from about 38 to about 47 weight percent, and has germinated for about 2 to about 7 days, preferably about 3 to about 6 days, at a temperature of from about 10° to about 30° C., preferably from about 14° to about 18° C. In important aspect, the germinated cereal is dried at a temperature of from about 40° to about 150° C. preferably between about 45° and 85° C. until the dried malted cereal has a moisture content of from about 2 to about 15 weight percent moisture, preferably from about 4 to abut 7 weight percent moisture. Importantly, the process of the invention may increase β-glucanase activity of a malted cereal by a factor of at least about 4 as compared to a malted cereal prepared without activated spores according to the invention. 
     In another important aspect, the present invention provides a malted cereal having a higher quantity of acrospire lengths that were significantly longer in comparison to acrospire lengths when traditional malting methods were used. 
     In an important aspect, the cereals which may be used in the invention include barley, wheat and sorghum. In one aspect of the invention the spores of moulds are used. In another aspect, the spores of bacteria are used. 
     In another aspect, the cereals may be disinfected or may not be disinfected. 
     Preferably, for the preparation of malted barley, the spores from bacteria are from gram positive bacteria or gram negative bacteria, selected from the group of  Micrococcus  spp.,  Streptococcus  spp.,  Leuconostoc  spp.,  Pediococcus  spp. preferentially  Pediococcus halophilus, Pediococcus cerevisiae, Pediococcus damnosus, Pediococccus hemophilus, Pediococccus parvulus, Pediococcus soyae, Lactococcus  spp.,  Lactobacillus  spp. preferentially  Lactobacillus acidophilus, Lactobacillus amylovorus , preferentially  Lactobacillus amylovorus  strain ATCC 33620 , Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis  var  lindneri, Lactobacillus  casei var casei,  Lactobacillus delbrueckii, Lactobacillus delbrueckii  var lactis,  Lactobacillus delbrueckii  var  bulgaricus, Lactobacillus fermenti, Lactobacillus gasserii, Lactobacillus helveticus, Lactobacillus hilgardii, Lactobacillus renterii, Lactobacillus sake, Lactobacillus sativorius, Lactobacillus cremoris, Lactobacillus kefir, Lactobacillus pentoceticus, Lactobacillus cellobiosus, Lactobacillus bruxellensis, Lactobacillus buchnerii, Lactobacillus coryneformis, Lactobacillus confusus, Lactobacillus florentinus, Lactobacillus viridescens, Corynebacterium  spp.,  Propionibacterium  spp.,  Bifidobacterium  spp.,  Streptomyces  spp.,  Bacillus  spp., preferentially  Bacillus subtilis  stain ATCC 6051, preferentially  Bacillus circulans, Sporolactobacillus  spp.,  Acetobacter  spp.,  Agrobacterium  spp.,  Alcaligenes  spp.,  Pseudomonas  spp., preferentially  Pseudomonas amylophilia, Pseudomonas aeruginosa, Pseudomonas cocovenenans, Pseudomonas mexicana, Pseudomonas pseudomallei, Gluconobacter  spp.,  Enterobacter  spp.,  Erwinia  spp.,  Klebsiella  spp., and  Proteus  spp. 
     Preferably, for the preparation of malted barley spores are from fungi which are selected from the group (genera as described by Ainsworth and Bisby&#39;s dictionary of the fungi, 8th edition, 1995, edited by D L Hawkworth, P M Kirk, B C Sutton, and D N Pegler (632 pp) Cab International) of  Ascomycota  preferentially  Dothideales, preferentially  Mycosphaerellaceae  preferentially  Mycosphaerella  spp.,  Venturiaceae  preferentially  Venturia  spp.  Eurotiales  preferentially  Monascaceae  preferentially  Monascus  spp.,  Trichocmaceae  preferentially  Emericilla  spp.,  Euroteum  spp.,  Eupenicillium  spp.,  Neosartorya  spp.,  Talaromyces  spp.,  Hypocreales  preferentially  Hypocreceae  preferentially  Hypocrea  spp.,  Saccharomycetales  preferentially  Dipodascaceae, Dipodascus  spp.,  Galactomyces  spp.,  Endomycetaceae  preferentially  Endomyces  spp.,  Metschnkowiaceae  preferentially  Guilliermondella  spp.,  Saccharomycetaceae  preferentially  Debaryomyces  spp.,  Dekkera  spp.,  Pichia  spp., preferentially  Pichia anomala , preferentially  Pichia anomala  strain ATCC 8168 . Kluyveromyces  spp.,  Saccharomyces  spp.,  Torulaspora  spp.,  Zygosaccharomyces  spp.,  Saccharomycodaceae  preferentially  Hanseniaspora  spp.;  Schizosaccharomycetales  preferentially  Schizosaccharomycetaceae  preferentially  Schizosaccharomyces  spp.,  Sordariales  preferentially  Chaetomiaceae, Chaetomium  spp., preferentially  Chaetomium virescens  strain ATCC 32319 , Sordariacea  preferentially  Neurospora  spp.,  Zygomycota  preferentially  Mucorales  preferentially  Mucoraceae  preferentially  Absidia  spp.,  Amylomyces  spp.,  Rhizomucor  spp.,  Actinomucor  spp.,  Thermomucor  spp.,  Chlamydomucor  spp.,  Mucor  spp. preferentially  Mucor circinelloides, Mucor grisecyanus, Mucor hiemalis, Mucor indicus, Mucor mucedo, Mucor piriformis, Mucor plumbeus, Mucor praini, Mucor pusillus, Mucor silvaticus, Mucor javanicus, Mucor racemosus, Mucor rouxianus, Muoor rouxil, Mucor aromaticus, Mucor flavus, Mucor miehei, Rhizopus  spp. preferentially  Rhizopus arrhizus, Rhizopus oligosporus, Rhizopus oryzae  preferentially  Rhizopus oryzae  strain ATCC 4858 , Rhizopus oryzae  strain ATCC 9363 , Rhizopus oryzae  strain NRRL 1891 , Rhizopus oryze  strain NRRL 1472 , Rhizopus stolonifer, Rhizopus thailandensis , preferentially  Rhizopus thailandensis  strain ATCC 20344 , Rhizopus formosaensis , preferentially  Rhizopus formosaensis  strain ATCC 26612 , Rhizopus chinensis, Rhizopus cohnii, Rhimpus japonicus, Rhizopus nodosus, Rhizopus delemar, Rhizopus acetorinus, Rhizopus chlamydosporus, Rhizopus circinans, Rhizopus javanicus, Rhizopus peka, Rhizopus salto, Rhizopus tritici, Rhizopus niveus, Rhizopus microsporus, Mitosporic fungi  preferentially  Aureobasidium  spp.,  Acremonium  spp.,  Cercospom  spp.,  Epicoccum  spp.,  Monilia  spp. preferentially  Monilia candida, Monilia sitophila, Mycoderma  spp.,  Candida  spp., preferentially  Candida diddensiae, Candida edax, Candide etchellsii, Candida kefir, Candida krisei, Candida lactose, Candida lambica, Candida melinii, Candida utilis, Candida milleri, Candida mycoderma, Candida parapsilosis, Candida obtux, Candida tropicalis, Candida valida, Candida versatilis, Candida guilliermondii, Rhodotorula  spp.,  Torulopsis  spp.,  Geotrichum  spp. preferentially  Geotrichum amycelium, Geotrichum armillariae, Geotrichum asteroides, Geotrichum bipunctatum, Geotrichum dulcitum, Geotrichum eriense, Geotrichum fici, Geotrichum flavo - brunneum, Geotrichum fragrans, Geotrichum gracile, Geotrichum heritum, Geotrichum kiebaknii, Geotrichum penicillatum, Geotrichum hirtum, Geotrichum pseudocandidum, Geotrichum rectangulatum, Geotrichum suaveolens, Geotrichum vanryiae, Geotrichum loubieri, Geotrichum microsporum, Cladosporium  spp.,  Trichoderma  spp. preferentially  Trichoderma hamatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma pseudokoningli, Trichoderma reesei , preferentially  Trichoderma reesei  strain ATCC 5875 , Trichoderma virgatum, Trichoderma viride, Oldium  spp.,  Alternaria  spp. preferentially  Alternaria alternata, Alternaria tenuis, Helminthosporium  spp. preferentially  Helminthosporium gramineum, Helminthosporium sativum, Helminthosporium teres, Aspergillus  spp. preferentially  Aspergillus ochraseus  Group,  Aspergillus nidulans  Group,  Aspergillus versicolor  Group,  Aspergillus wentii  Group,  Aspergillus candidus  Group,  Aspergillus flavus  Group,  Aspergillus niger  Group,  Aspergillus oryzae  strain ATCC 14156 , Penicillum  spp. preferentially  Penicillum aculeatum, Penicillum citrinum, Penicillum claviforme, Penicillum funiculosum, Penicillum italicum, Penicillum lanoso - viride, Penicillum emersonil, Peniclilum lilacinum, Penicillum expansum , and mixtures thereof. 
     Preferably, for the preparation of malted cereals other then malted barley, especially for the preparation of malted wheat, rye, corn, oats, rice, millet, triticale, and sorghum, said bacteria are gram positive or gram negative bacteria selected from the group of  Micrococcus  spp.,  Streptococcus  spp.,  Leuconostoc  spp.,  Pediococcus  spp.,  Lactococcus  spp.,  Lactobacillus  spp.,  Corynebacterium  spp.,  Propionibacterium  spp.,  Bifidobacterium  spp.,  Streptomyces  spp.,  Bacillus  spp.,  Sporolactobacillus  spp.,  Acetobacter  spp.,  Agrobacterium  spp.,  Alcaligenes  spp.,  Pseudomonas  spp.,  Gluconobacter  spp.,  Enterobacter  spp.,  Erwinia  spp.,  Klebsiella  spp.,  Proteus  spp. or a mixture thereof; and said fungi are fungi selected from the group of:  Ascomycota  preferentially  Dothideales  preferentially  Mycophaerellaceae  preferentially  Mycosphaerella  spp.,  Venturiaceae  preferentially  Venturia  spp.;  Eurotiales  is preferentially  Monascaceae  preferentially  Monascus  spp.,  Trichocomaceae  preferentially  Emericilla  spp.,  Euroteum  spp.,  Eupenicillium  spp.,  Neosartorya  spp.,  Talaromyces  spp.,  Hypocreales  preferentially  Hypocreaceae  preferentially  Hypocrea  spp.,  Saccharomycetales  preferentially  Dipodascaceae  preferentially  Dipodascus  spp.,  Galactomyces  spp.,  Endomycetaceae  preferentially  Endomyces  spp.,  Metschnikowiaceae  preferentially  Guilliermondella  spp.,  Saccharomycetaceae  preferentially  Debaryomyces  spp.,  Dekkera  spp.,  Pichia  spp.,  Kluyveromyces  spp.,  Saccharomyces  spp.,  Torulaspora  spp.,  Zygosaccharomyces  spp.,  Saccaromycodaceae  preferentially  Hanseniaspora  spp.,  Schizosaccheromycetales  preferentially  Schizosaccharomycetaceae  preferentially  Schizosaccharomyces  spp.;  Sordariales  preferentially  Chaetomiaceae  preferentially  Chaetomium  spp.,  Sordariaceae  preferentially  Neurospora  spp.,  Zygomycota  preferentially  Mucoraies  preferentially  Mucoraceae  preferentially  Absidia  spp.,  Amylomyces  spp.,  Rhizomucor  spp.,  Actinomucor  spp.,  Thermomucor  spp.,  Clamydomucor  spp.,  Mucor  spp.,  Rhizopus  spp.; preferentially  Rhizopus  oryza strain ATCC 9363 , Mitosporic fungi  preferentially  Aureobasidium  spp.,  Acremonium  spp.,  Cerocospora  spp.,  Epicoccum  spp.,  Monilia  spp.,  Mycoderma  spp.,  Candida  spp.,  Rhodotorula  spp.,  Torulopsis  spp.,  Geotrichum  spp.,  Cladosporium  spp.,  Trichoderma  spp.,  Oldium  spp.,  Alternaria  spp.,  Helminthosporium  spp.,  Aspergillus  spp.,  Penicillium  spp. 
     DEFINITIONS 
     As used herein the term “spore” refers to a dormant and highly resistant reproductive cell formed by bacteria and fungi in response to environment conditions that do not favor the growth of the organism. When exposed to favorable environmental conditions, spores are capable of developing into a viable adult organism without fusion with another cell. 
     As used herein the term “activated spore” means a spore having one of the following properties: 
     i) The spore is swollen such that its size is increased by a factor of between about 1.2 and about 10 over its dormant size; and/or 
     ii) one or more germ tubes per spore is formed. 
     Activated spores are prepared by one or a combination of the following treatments. 
     i) cycles of wetting and/or drying; 
     ii) addition of appropriate nutritional supplies (such as a nitrogen source, preferably amino acids and/or a carbon source, preferably mono- or disaccharides) or spore elements; 
     iii) exposure to temperature changes, preferably within a temperature range of about 0 to about 80° C.; 
     iv) exposure to changes in pH, preferably within a pH range of about 2.0 to about 8.0, more preferably about 3.0 to about 6.0. 
     The term “germination” as used herein means the beginning or resumption of growth by a seed. In accordance with the process of the present invention, germination begins to occur during and/or after the cereal has been steeped. Germination of cereals is generally understood to mean hydration of the seed, swelling of the cereal and inducing growth of the embryo. Environmental factors affecting germination include moisture, temperature and oxygen level. A rapid increase in cells of the root stem leads to root development, while corresponding growth sends forth a shoot. 
     As used herein, the term “steeping” refers to wetting of the cereal. Wetting may include one or more stages over a time and temperature effective for providing a moisture content of between about 20% and about 60% by weight. 
     The term “specific activity” as used herein refers to the concentration and activity of an enzyme in a preparation. The specific activity of a preparation is reported as units/mg protein. One unit of enzyme is that amount that catalyzes the formation of 1 mmole of product per minute under defined conditions. The amount of enzyme present in a preparation is measured using standard protein assay techniques and catalytic activity is determined by following the formation of product or removal of substrate over time. 
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     According to a preferred embodiment, the preparation process of malted cereals according to the invention comprises the following steps: a steeping step includes one or more wetting stages or the total time of submersion in water during steeping for physiological reasons does not exceed 30 hours (preferably 10 to 25 hours) or the kilning step includes more then two temperature steps and the microbial cultures which are added, are preferably selected from the group consisting of Rhizopus spp., preferably  Rhizopus oryzae , such as  Rhizopus oryze  strain ATCC9363 and/or Pseudomonas spp., preferably  Pseudomonas herbicola.    
     According to the invention, the malted cereals are selected from the group of barley, wheat, rye, corn, oats, rice, millet, triticale, sorghum and the like. 
     In the process according to the invention, the same or different activated spores are added in one or more time(s). The use of activated spores greatly enhances their contribution to improved malt quality, most likely because of more vigorous growth. The activated spores have one of the following properties: the treated spores are significantly more swollen than their dormant, size, more particularly, the size of the spores is increased by a factor preferably between 1.2 and 10 over their dormant size and/or one or more germ tubes per spore are formed. The activated spores are prepared by subjecting them to environmental changes, preferably by one or a combination of the following treatments;
         (a) cycles of wetting and/or drying;   (b) addition of appropriate nutritional supplies (such as a nitrogen source, preferably amino acids and/or a carbon source, preferably mono- or disaccharides) or spore elements;   (c) exposure to temperature changes, preferably within a temperature range of 0° to 80° C.;   (d) exposure to changes in pH, preferably within a pH range of 2.0 to 8.0, more preferably between 3.0 and 6.0.       

     The activated spores may be introduced before or during the malting process. For example, the activated spores may be introduced during the various malting or steeping stages before or after immersion of the cereal. 
     The concentration of the spores may vary depending on the conditions of the malting process and the type of active spore being utilized. Generally about 1×10 2  to about 1×10 7 , preferably about 1×10 3 to about 1×10 5  activated spores per gram air dry cereal is utilized. 
     The present invention also concerns the malted cereals obtained according to the process of the invention, which present improved European Brewery Convention analysis results. Said improvements may have to do with modification and/or increased hydrolytic enzyme activities. At the same time, a decreased level of toxins, an increased microbial safety by e.g., outcompeting undesirable microbial flora such as Fusarium and/or an increased acceptability compared to the malted cereals according to the state of the art, may be observed. 
     For instance, the malted cereals according to the invention may have a lower β-glucan content or a higher enzyme activity such as, for example, β-glucanase or xylanase activity (as represented in the following examples and figures) than the malted cereals according to the state of the art. This allows for a better processability of the malt in wort and beer production as exemplified by increased rates of filtration. 
     The activated spores and cereal may be combined and wetted by submersion in water to steep the combination which should not exceed 30 hours. The activated spores can also be sprayed on the barley during the steep period or during the germination process. The pH during this period should be from about 1.5 to about 14, preferably about 4 to about 6. β-glucanase activity of malted barley made according to the invention is higher than 700 units/kg and xylanase activity is higher than 250 units/kg. 
     An object of the present invention concerns the use of the malted cereals according to the invention for the preparation of beverages. 
     The invention is also related to these improved beverages. 
     The improved malted cereals according to the invention could also be used in other biotechnological processes well known by the Person Skilled in the Art, in which in most cases advantage is taken of their improved quality. 
     The present invention will be further described in various examples in view of the following drawings. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  represents the β-glucanase activity of malted barley obtained according to the preparation process of example 1. (legend: see example 1). 
         FIG. 2  represents the xylanese activity of malted barley obtained according to the preparation process of example 1. (legend: see example 1). 
         FIG. 3  represents the β-glucanase activity of malted barley obtained according to the preparation process of example 3. (legend: see example 3). 
         FIG. 4  represents the xylanase activity of malted barley obtained according to the preparation process of example 3. (legend: see example 3). 
         FIG. 5  represents the relative increase factory (R.I.F.) for bacterial populations (see text, malt evaluation, example 2) (legend: see example 2). 
     
    
    
     EXAMPLE 1 
     1. Preparation of Microbial Cultures 
     Strain 
     
         
         
           
             S46 : Rhizopus oryzae  ATCC 9383.
 
Preparation of the Spore Suspension
 
             the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 10 days at 28° C.; 
             the spores were harvested by flooding the cultures with sterile physiological saline (0.9% NaCl) and by rubbing the sporulated mycelium gently with a sterile spatula; 
             the spore suspension was washed twice with sterile physiological saline (0.9% NaCl) by centrifugation (5500 rpm, Sorvall type SS-34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl); 
             the spore density was determined microscopically using a Thoma counting chamber.
 
Activation of the Spore Suspension
 
             10 7  spores were transferred into 20 ml of sterile, acidifed TSB (Tryptic Soy Broth, Oxoid), pH=4.0 and incubated in a shaking water bath during 5 to 6 hours at ±42° C.; 
             The activated spores were harvested by centrifugation (3500 rpm, Sorvall type SS-34®, for 15 min.), washed once with sterile physiological saline (0.9% NaCl) by centrifugation (3500 rpm, Sorvall type SS-34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).
 
2. Barley
 
             Plaisant-1994 French harvest.
 
3. Process.
 
Setup
 
           
         
       
    
     Malts were made by four different malting processes:
         A1. traditional malting    (without inoculation of any spore suspension)   B1. malting process using non-activated spores    (inoculation of the steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)   C1. malting process according to the invention    (inoculation of the steeped barley with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)   D1. malting process according to the invention    (inoculation of the steeped barley during the first wet stage with a suspension of activated spores of  Rhizopus oryzme  ATCC 9363)
 
Steeping
   the steeping was carried out on a 2 kg base with a total water (tap water) to air dry barley ratio of 1.5:1;   use was made of 2 fermentors (Bioflo III, New Brunswick Scientific), in which perforated plates were placed;   temperature was only controlled during the wet stages, during the air rest stages, the system was allowed to reach room temperature (±20° C.);   during the whole steeping period, the barley was aerated (4 liter sterile air per minute);   steeping was carried out by immersion using the following scheme;       

                                                               Temperature (° C.)   Duration (h)                                        First wet stage   13   6:00           First air rest stage   20   17:00            Second wet stage   14   5:00           Second air rest stage   20   15:30            Third wet stage   16   2:30                        
Addition of the Microbial Cultures
         ±480 g of steeped barley was immersed in 0.5 liter of tap water which contained no spores (A1), non-activated spores of  Rhizopus oryzae  ATCC 9363 (B1) or activated spores of  Rhizopus oryzae  ATCC 9363 (C1, according to the invention); for B1 and C1, the steeped barley was inoculated with 10 4  spores per gram of air dry barley;   during the steeping, 10 4  activated spores per gram air dry barley were inoculated to the water of the first wet stage (D1);   the fluid was removed by draining.
 
Germination
   germination was carried out in a cylindrical container with perforated lids at a temperature of 16°-18° C. during 4 days;   air was supplied by natural diffusion;   the containers were slowly rotated on an electrically controlled roller system (Cellroll®, Tecnorama); i.e., every two hours the containers were rolled for 15 min. at 1 rpm.
 
Kilning
   the kilning was carried out in a Joe White malting unit (Australia),       
                                                                           Air flow   Recirc.   Temp.   Durat.           (%)   Air (%)   (° C.)   (h)                                    First kilning stage   25   0   62   3:00       Second kilning stage   25   0   65   2:00       Third kilning stage   25   0   68   2.00       Fourth kilning stage   25   25   73   2:00       Fifth kilning stage   25   50   78   1:00       Sixth kilning stage   25   75   80   2:00       Seventh kilning stage   25   100   83   6:00       Shut down air off               Time out                    
4. Methods of Analysis and Results
 
     Methods for determination and units of moisture, extract, extract difference, color, total protein content, soluble protein content, Kolbach index, pH, diastatic power, according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau). 
     Metods for determination and units of turbidity, friability, homogeneity, whole grains, b-glucan content, according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau, supplement published in 1989). 
     Postcoloration of the wort is determined after boiling the congress wort under reflux at 108° C. during 2 hours. 
     The viscosity of the congress wort is determined with the Delta-viscosimater. 
     For the determination of the filtration volume, the congress wort is filtered over a Schleicher and Schuell 597½ folded filter. The volume (in mil) that is obtained after 1 hour of filtration is the filtration volume of the wort. 
     Modification is determined with the Calcofluor apparatus (Haffmans) according to the Carisberg method (Analytica-Europeon Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau). 
     The β-glucanase and xylanase activities are determined with the β-glucazym method (Megazme (Austr.) Pty Ltd. (April, 1993) and the xylazym method ((Megazyme (Austr.) Pty Ltd. (September 1995)), respectively. 
     
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Malting 
                 Malting 
                 Malting 
               
               
                   
                   
                 Process 
                 Process 
                 Process 
               
               
                   
                 Traditional 
                 using non- 
                 according 
                 according 
               
               
                   
                 Malting 
                 activated 
                 to the 
                 to the 
               
               
                   
                 Process 
                 spores 
                 Invention 
                 Invention 
               
               
                   
                 (A1) 
                 (B1) 
                 (C1) 
                 (D1) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Moisture 
                 3.9 
                 4.1 
                 3.8 
                 4.3 
               
               
                 Extract 
                 80.3 
                 80.4 
                 80.3 
                 79.8 
               
               
                 Extract 
                 0.8 
                 0.8 
                 0.4 
                 1.1 
               
               
                 Difference 
               
               
                 Color 
                 3.3 
                 3.3 
                 4.1 
                 4.1 
               
               
                 Wort Turbidity 
                 1.3 
                 1.2 
                 0.7 
                 0.8 
               
               
                 Postcoloratlon 
                 6 
                 6 
                 7.3 
                 7.5 
               
               
                 Total Protein 
                 10.1 
                 10.3 
                 10 
                 10.1 
               
               
                 Content 
               
               
                 Soluble Protein 
                 4.1 
                 4.4 
                 4.8 
                 5.2 
               
               
                 Content 
               
               
                 Kolbach Index 
                 40.6 
                 42.7 
                 48 
                 51.0 
               
               
                 Viscosity 
                 1.57 
                 1.52 
                 1.52 
                 1.54 
               
               
                 pH 
                 6.05 
                 6.3 
                 5.87 
                 5.79 
               
               
                 Diastatic Power 
                 345 
                 349 
                 352 
                 419 
               
               
                 Whole Grains 
                 0.3 
                 0.3 
                 0.1 
                 ND 
               
               
                 Friability 
                 83 
                 82 
                 83.9 
                 ND 
               
               
                 Homogeneity 
                 98.5 
                 97.9 
                 98.6 
                 ND 
               
               
                 β-glucan content 
                 122 
                 108 
                 46 
                 &lt;40 
               
               
                 Filtration Volume 
                 210 
                 265 
                 290 
                 275 
               
               
                 Modification 
                 88.2 
                 90.5 
                 93.4 
                 ND 
               
               
                 β-glucanase 
                 214 
                 371 
                 683 
                 3656 
               
               
                 Activity 
               
               
                 Xylanase Activity 
                 28 
                 34 
                 56 
                 984 
               
               
                   
               
             
          
         
       
     
       FIGS. 1 and 2  represent the β-glucanase and xylanase activity, respectively of the obtained malted barley (A1, B1, C1, D1). These malted barleys are obtained according to a traditional malting process (A1) or using non-activated spores during the malting process (B1) or according to the above-described malting process of the invention (C1, D1). The β-glucanase activity was determined with the β-glucazym method (Megazyme (Austr.) Pty Ltd. (April, 1993)). Therefore, malt β-glucanase activity (U/kg) was calculated at 380×E (590 nm)+20. The xylanase activity was determined with the endo 1-4-xylazym method (Megazyme (Austr.) Pty Ltd. (September 1995)). Therefore, malt xylanese activity (U/kg) was calculated as (46.8×E (590 nm)+0.9)×5). 
     EXAMPLE 2 
     1. Preparation of Microbial Cultures 
     Strain 
     
         
         
           
             S46 : Rhizopus oryzae  ATCC 9363.
 
Preparation of the Spore Suspension
 
             as described in Example 1.
 
Activation of the Spore Suspension
 
             as described in Example 1.
 
2. Barley
 
             Stander-1995 North American harvest.
 
3. Process
 
Setup
 
           
         
       
    
     Malts were made by six different malting processes:
         A2, traditional malting process    (without inoculation of any spore suspension)   B2, malting process using non-activated spores    (inoculation of the steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)   C2, malting process according to the invention    (inoculation of the steeped barley during the first wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9383)   D2, malting process according to the invention    (inoculation of the steeped barley during the second wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)   E2, malting process according to the invention    (inoculation of the steeped barley during the third wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)   F2. malting process according to the invention    (inoculation of the steeped barley with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)
 
Steeping and Addition of the Microbial Cultures
   the steeping was carried out on a 300 g base with a total water (tap water) to air dry barley ratio of 5:3;   use was made of 2000 ml flasks;   a temperature of 18° C. was maintained during the wet stages and during the air rest stages;   during the whole steeping period, the barley was aerated by means of compressed air;   steeping was carried out by immersion using the following schedule;       

     
       
         
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Duration (h) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 First wet stage 
                 6:00 
               
               
                   
                 First air rest stage 
                 18:00  
               
               
                   
                 Second wet stage 
                 5:00 
               
               
                   
                 Second air rest stage 
                 19:00  
               
               
                   
                 Third wet stage 
                 2:00 
               
               
                   
                   
               
             
          
         
       
         
         
           
             during the steeping, 10 4  activated spores per gram of air dry barley were inoculated to the water of the first wet stage (C2), of the second wet stage (D2) or of the third wet stage (E2) before immersion of the barley; 
             the steeped barley was immersed in 0.5 liters of tap water which contained no spores (A2), non-activated (B2) or activated (C2, D2, E2, F2) spores; 
             for B2, and F2, the steeped barley was inoculated with 10 4  spores per gram of air dry barley, 
             the fluid was removed by draining.
 
Germination
 
             as described in Example 1.
 
Kilning
 
             as described in Example 1.
 
Malt evaluation
 
Determination of the Increase of the Bacterial Population
 
           
         
       
    
     To judge the evolution of the bacterial population during the malting process, a relative increase factor (R.I.F.) was determined by dividing the total bacterial count occurring on the green malt by the total bacterial count occurring on the barley. The total bacterial count was determined after plating appropriate dilutions of an extract of the kernels on Tryptic Soy Agar (Oxoid) supplemented with 100 ppm pimaricine and after incubation at 28° C. for 3 days. 
       FIG. 5  shows the increase of the bacterial population during the malting according to the preparation process of Example 2. 
     EXAMPLE 3 
     1. Preparation of Microbial Cultures 
     Strain 
     
         
         
           
             S 46 : Rhizopus oryzae  ATCC 9363
 
Preparation of the Spore Suspension
 
             as described in Example 1
 
Activation of the Spore Suspension
 
             as described in Example 1
 
2. Barley
 
             Plaisant-1994 French harvest;
 
3. Process
 
Setup
 
           
         
       
    
     Malts were made by three different malting processs:
         A3. traditional malting    (without inoculation of any spore suspension)   B3. malting process using non-activated spores    (inoculation of the steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)   C3. malting process according to the invention    (inoculation of the steeped barley with a suspension of activted spores of Rhizopus oryzsa ATCC 9363)
 
Steeping
   the steeping was carried out on a 2 kg base air dry barley with a total water (tap water) to air dry barley ratio of 1.5:1;   the pH of the steeping water was controlled at pH=5.5 by addition of lactic acid and NaOH;   a fermentor (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed, was used for steeping;   temperature was only controlled during the wet stages; during the air rest stages the system was allowed to reach room temperature (ca. 20° C.);   during the whole steeping period the barley was aerated (4 liters startle air per minute);   steeping was carried out by immersion using the following schedule:       

                                                               Temperature (° C.)   Duration (h)                                        First wet stage   13   6:00           First air rest stage   20   17:00            Second wet stage   14   5:00           Second air rest stage   20   15:30            Third wet stage   16   2:30                        
Addition of the Microbial Cultures
         460 g of steeped barley was immersed in 0.5 liters of tap water which contained no spores (A3), non-activated spores of  Rhizopus oryzae  ATCC 9363 (B3) or activated spores of  Rhizopus oryzae  ATCC 9363 (C3 according to the invention); for B3 and C3, the steeped barley was inoculated with 10 4  spores per gram of air dry barley;   the fluid was removed by draining.
 
Germination
   as described in Example 1
 
Kilning
   as described in Example 1
 
4. Methods of analysis and results
       
     These were as described in Example 1 (4. Methods of Analysis and Results). 
     See table on next page. In this table:
         A1/3: Traditional malting process   B1/3: Malting process using non-activated spores   C1/3: Malting process according to the invention       

     
       
         
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Example 3 
                 Example 1 
               
               
                   
                 pH control of the 
                 No pH control of the 
               
               
                   
                 steeping water (pH-5.5) 
                 steeping water 
               
             
          
           
               
                   
                 A3 
                 B3 
                 C3 
                 A1 
                 B1 
                 C1 
               
               
                   
                   
               
             
          
           
               
                 Moisture 
                 3.8 
                 3.6 
                 3.7 
                 3.9 
                 4.1 
                 3.8 
               
               
                 Extract 
                 78.9 
                 80.2 
                 80.7 
                 80.3 
                 80.4 
                 80.3 
               
               
                 Extract 
                 0.6 
                 0.7 
                 0.4 
                 0.8 
                 0.8 
                 0.4 
               
               
                 Difference 
               
               
                 Color 
                 3.2 
                 4.2 
                 4.4 
                 3.3 
                 3.3 
                 4.1 
               
               
                 Wort Turbidity 
                 1 
                 1 
                 0.8 
                 1.3 
                 1.2 
                 0.7 
               
               
                 Postcoloration 
                 5.1 
                 7 
                 7.2 
                 6 
                 6 
                 7.3 
               
               
                 Total Protein 
                 10.2 
                 10.1 
                 10 
                 10.1 
                 10.3 
                 10 
               
               
                 Content 
               
               
                 Soluble Protein 
                 4 
                 4.4 
                 4.8 
                 4.1 
                 4.4 
                 4.8 
               
               
                 Content 
               
               
                 Kolbach Index 
                 39.2 
                 43.6 
                 48 
                 40.6 
                 42.7 
                 48 
               
               
                 Viscosity 
                 1.52 
                 1.53 
                 1.52 
                 1.57 
                 1.52 
                 1.52 
               
               
                 pH 
                 6.02 
                 5.97 
                 5.91 
                 6.05 
                 6.03 
                 5.87 
               
               
                 Diastatic Power 
                 348 
                 333 
                 355 
                 345 
                 349 
                 352 
               
               
                 Whole Grains 
                 0.2 
                 0.2 
                 0.1 
                 0.3 
                 0.3 
                 0.1 
               
               
                 Friability 
                 81 
                 81 
                 85 
                 83 
                 82 
                 83.9 
               
               
                 Homogeneity 
                 97.6 
                 97.8 
                 98.9 
                 98.5 
                 97.9 
                 98.6 
               
               
                 β-glucan 
                 190 
                 57 
                 40 
                 122 
                 108 
                 46 
               
               
                 content 
               
               
                 Filtration 
                 210 
                 215 
                 200 
                 210 
                 265 
                 290 
               
               
                 Volume 
               
               
                 Modification 
                 84.1 
                 85.5 
                 87.4 
                 88.2 
                 90.5 
                 93.4 
               
               
                 β-glucanase 
                 202 
                 931 
                 1322 
                 214 
                 371 
                 683 
               
               
                 Activity 
               
               
                 Xylanase 
                 43 
                 65 
                 71 
                 28 
                 34 
                 56 
               
               
                 Activity 
               
               
                   
               
             
          
         
       
     
       FIG. 3  represents the β-glucanase activity, measured according to β-Glucazym method [Megazyme (AUSTR) Pty. Ltd.] of the malted cereals A3, B3 and C3. Malt β-glucanase activity (U/kg) was calculated as described in example 1. A3 was obtained by the traditional malting process with pH control of the steeping water (pH=5.5). B3 resulted from the malting process according to the invention with the inoculation of steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363 and with pH control of the steeping water (pH=5.6). C3 was obtained by the malting process according to the invention with the inoculation of the steeped barley with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363 and with pH control of the steeping water (pH=5.5). 
     These results show the increased β-glucanase activity when the pH of the steeping water is maintained at around 5.5. 
       FIG. 4  gives the corresponding results for xylanase activity. These were measured according to xylazym method, Megazyme ((AUSTR) Pty. Ltd. (September 1995)). Malt xylanase activity was calculated as described in Example 1.
     Comparison of the β-glucanase activity obtained according to examples 1 and 3 with the β-glucanase activity according to the state of the art as described in WO94/29430.   
     In order to compare the improved results regarding β-glucanase activity by the present invention, we defined the factor m as follows: 
     
       
         
           
             m 
             = 
             
               
                 β 
                 ⁢ 
                 
                   -glucanase  activity  of  the  treated  malt 
                 
               
               
                 β 
                 ⁢ 
                 
                   -glucanase  activity  of  the  controlled  malt 
                 
               
             
           
         
       
     
     This factor was calculated for control malt and malted treated with  Rhizopus oryzae  ATCC 9363 as described in Examples 1 and 3 of the present invention. 
     It was also calculated for the data described in WO94/29430 (Example 1) where  Geotrichum candidum  was used. 
     Both as described in WO94/29430, and in the present application, β-glucanase activity was determined with the beta-glucazyme method [Megazyme (AUSTR) Pty. Ltd. (April 1993)]. Therefore, malt β-glucanase activity (U/kg) was calculated as 380×E(590 nm)+20 and one unit of activity was defined as the amount of enzyme required to release one micromole of reducing sugar equivalents per minute under the defined above conditions. 
     Comparison of the Results: 
     
       
         
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
               
               
                 State of the Art 
                 Invention 
               
             
          
           
               
                   
                 m 
                   
                 m 
                 Ex. 1 
                 m 
                 Ex. 3 
                 m 
               
               
                   
                   
               
             
          
           
               
                 Gc* 
                 1.48 
                 Gc* 
                 198 
                 B1/A1 
                 1.73 
                 B3/A3 
                 4.61 
               
               
                   
                   
                   
                   
                 C1/A1 
                 3.19 
                 C3/A3 
                 6.54 
               
               
                   
                   
                   
                   
                 D1/A1 
                 18.02 
               
               
                   
               
               
                 *Gc:  Geotrichum candidum   
               
             
          
         
       
     
     The results clearly show that the present invention provides for a more drastic increase in malt β-glucanase activity than that described earlier (WO 94/29430). 
     It thus appears that it is possible to obtain malted cereals having a β-glucanase activity increased by at least a factor 4 compared to the conventional malting process wherein the addition of microbial culture is omitted. 
     From  FIGS. 2 and 4 , it also appears that it is possible to obtain malted cereals having a xylanase activity increased by at least a factor 4 compared to conventional malting process wherein the addition of microbial culture is omitted. 
     EXAMPLE 4 
     1. Preparation of the microbial cultures 
     
         
         Strain
       S40 : Aspergillus oryzae  ATCC 14156   
     
         Preparation of the spore suspension
       the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 7 days at 281° C.;   the spores were harvested by flooding the culture with sterile physiological saline (0.9%/NaCl) and by rubbing the sporulated mycelium gently with a sterile spatula;   the spore suspension was washed once with sterile physiological saline (0.9% NaCl) by centrifugation (5500 rpm. Sorval type SS-34®, for 15 min) and resuspended in sterile physiological saline (0.9% NaCl);   the spore density was determined microscopically using a Thoma counting chamber.   
     
         Activation of the Spore Suspension
       5×10 7  spores were transferred into 20 ml of sterile, acidified TSB (Tryptic Soy Broth, Oxoid), pH=5.0 and incubated in a shaking water bath during 3 hours (1) or 1 hour (2) at 35° C.,
 
2. Cereal
   Clarine barley-1995 French harvest
 
3. Process
   
     
       
    
     Malts were made by two different malting process:
         A4. traditional malting    (without inoculation of any spore suspension)   E4. malting process according to the invention    (inoculation of the steeped barley during the first and third wet stage with a suspension of activated spores of  Aspergillus oryzae  ATCC 14156)       Steeping   as described in Example 1   Addition of the microbial cultures
       during the steeping, 5×10 3  activated spores (1) per gram air dry barley were inoculated to the water of the first wet stage and 10 4  activated spores (2) per gram air dry barley were inoculated to the water of the third wet stage (E4);   
       Germination
       germination of ±460 g steeped barley was carried out in cylindrical containers with perforated lids at a temperature of 16°-18° C. during 4 days;   air was supplied by natural diffusion;   the containers were slowly rotated on an electronically controlled roller system (Cellroll®, Tecnorama); i.e., every two hours the containers were rolled for 15 min at 1 rpm.   
       Kilning   as described in Example 1
 
4. Methods of the analysis and results
   

     These were described in Example 1 (4, Methods of Analysis and Results) Method for the determination of the acrospire length according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brsuerel und Getränke-Rundschau). 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 0 
                 0-¼ 
                 ¼-½ 
                 ½-¾ 
                 ¾-1 
                 &gt;1 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 day germination 
                 A4 
                 0 
                 1 
                 60 
                 39 
                 0 
                 0 
               
               
                 1 day germination 
                 E4 
                 0 
                 0 
                 11 
                 77 
                 12 
                 0 
               
               
                 4 days germination 
                 A4 
                 1 
                 1 
                 31 
                 64 
                 3 
                 0 
               
               
                 4 days germination 
                 E4 
                 1 
                 0 
                 1 
                 42 
                 49 
                 7 
               
               
                   
               
             
          
         
       
     
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Malting 
               
               
                   
                 Traditional Malting 
                 Process According 
               
               
                   
                 Process (A4) 
                 to the Invention (E4) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Moisture 
                 4.3 
                 4.0 
               
               
                 Extract 
                 80.9 
                 81.1 
               
               
                 Extract Difference 
                 1.0 
                 0.3 
               
               
                 Color 
                 2.8 
                 3.2 
               
               
                 Wort Turbidity 
                 1.6 
                 1.0 
               
               
                 Postcoloration 
                 4.8 
                 5.4 
               
               
                 Total Protein Content 
                 10.1 
                 10.0 
               
               
                 Soluble Protein Content 
                 3.9 
                 4.5 
               
               
                 Kolbach Index 
                 38.6 
                 44.7 
               
               
                 Viscosity 
                 1.57 
                 1.48 
               
               
                 pH 
                 5.98 
                 5.89 
               
               
                 Diastatic Power 
                 197 
                 201 
               
               
                 Whole Grains 
                 1.3 
                 0.6 
               
               
                 Friability 
                 81 
                 89 
               
               
                 Homogeneity 
                 95.0 
                 98.4 
               
               
                 β-glucan Content 
                 378 
                 132 
               
               
                 Filtration Volume 
                 300 
                 310 
               
               
                 Modification 
                 83.9 
                 89.8 
               
               
                 β-glucanase Activity 
                 309 
                 392 
               
               
                 Xylanase Activity 
                 27.82 
                 17.62 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 5 
     1. Preparation of the Microbial Cultures 
     
         
         Strains
       S40 : Aspergillus oryzae  ATCC 14156   S46 : Rhizopus oryzae  ATCC 9363   
     
         Preparation of the Spore Suspensions 
         As described in Example 4 
         Activiation of the Spore Suspensions 
         S40:
        5×10 7  spores were transferred into 20 ml of sterile, acidified TSB (Tryptic Soy Broth, Oxoid) pH=5.0 and incubated in a shaking water during 1 hour at 35° C.;   the activated spores were harvested by centrifugation (3500 rpm, Sorvall type SS34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).   
     
       
    
     S45
         5×10 7  spores were transferred into 20 ml of sterile, activated TSB (Tryptic Soy Broth, Oxoid) pH=4.0 and incubated in a shaking water bath during 5 hours at 42° C.;   the activated spores were harvested by centrifugation (3500 rpm, Sorvall type SS34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).
 
2. Cereal
   Clarine-1995 French harvest
 
3. Process
       Setup   

     Malts were made by two different malting processes:
         A5. traditional malting    (without inoculation of any spore suspension)   F5. malting process according to the invention    (inoculation of the steeped barley during the first wet stage with a suspension of activated spores of  Aspergillus oryzae  ATCC14156 and after steeping with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)       Steeping   As described in Example 1   Addition of the Microbial Cultures
       during steeping, 10 4  activated spores of  Aspergillus oryzae  ATCC 14156 per gram air dry barley were inoculated to the water of the first wet stage (F5, according to the invention);   ±460 g of steeped barley was immersed in 0.5 liters of tap water which contained no spores (A5) or activated spores of  Rhizopus oryzae  ATCC 9363 (F5, according to the invention); for F5 the steeped barley was inoculated with 10 4  activated spores per gram air dry barley;   the fluid was removed by draining.   
       Germination   As described in Example 4.   Kilning   As described in Example 1.
 
4. Methods of Analysis and Results
   

     These were as described in Example 1 (4. Methods of Analysis and Results). 
     Method for the determination of the acrospire length according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau). 
     
       
         
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 0 
                 0-¼ 
                 ¼-½ 
                 ½-¾ 
                 ¾-1 
                 &gt;1 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 1 day germination 
                 A5 
                 1 
                 1 
                 53 
                 44 
                 1 
                 0 
               
               
                 1 day germination 
                 F5 
                 0 
                 1 
                 21 
                 73 
                 5 
                 0 
               
               
                 4 days germination 
                 A5 
                 0 
                 0 
                 0 
                 29 
                 63 
                 8 
               
               
                 4 days germination 
                 F5 
                 0 
                 0 
                 0 
                 13 
                 63 
                 24 
               
               
                   
               
             
          
         
       
     
     It was noted that the use of activated spores of  Aspergillus oryzae  ATCC improved the malt analytical specifications. 
     Furthermore, it was found that during the malting process, the barley acrospire lengths were significantly longer using the process according to the invention in comparison to the traditional malting process. 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Melting Process 
               
               
                   
                 Traditional Malting 
                 According to the 
               
               
                   
                 Process (A5) 
                 Invention (F5) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Moisture 
                 3.9 
                 4.2 
               
               
                 Extract 
                 81.4 
                 81.8 
               
               
                 Extract Difference 
                 0.9 
                 1.1 
               
               
                 Color 
                 38 
                 3.8 
               
               
                 Wort Turbidity 
                 1.4 
                 1.0 
               
               
                 Postcoloration 
                 6.9 
                 6.4 
               
               
                 Total Protein Content 
                 10.1 
                 10.2 
               
               
                 Soluble Protein Content 
                 4.8 
                 5.2 
               
               
                 Kolbach Index 
                 48.0 
                 51.3 
               
               
                 Viscosity 
                 1.51 
                 1.50 
               
               
                 pH 
                 5.88 
                 5.82 
               
               
                 Diastatic Power 
                 199 
                 214 
               
               
                 Whole Grains 
                 0.8 
                 1.1 
               
               
                 Friability 
                 89 
                 95 
               
               
                 Homogeneity 
                 98.3 
                 98.3 
               
               
                 β-glucan Content 
                 120 
                 51 
               
               
                 Filtration Volume 
                 270 
                 220 
               
               
                 Modification 
                 96.8 
                 98.6 
               
               
                 β-glucanase Activity 
                 263 
                 907 
               
               
                 Xylanase Activity 
                 28.86 
                 57.76 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 6 
     1. Preparation of the Microbial Cultures 
     
         
         Strains
       S46 : Rhizopus oryzae  ATCC 9363   
     
         Preparation of the Spore Suspensions 
         As described in Example 4 
         Activation of the Spore Suspensions
       5×10 7  spores were transferred into 20 ml of sterile, acidified TSB (Tryptic Soy Broth, Oxoid) pH=4.0 and incubated in a shaking water both during 5 hours at 42° C.;   the activated spores were harvested by centrifugation (3500 rpm, Sorvall type SS-34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).
 
2. Cereal
   Wheat: Mobil-1996 Belgian harvest
 
3. Process
   
     
         Setup 
       
    
     Malts were made by two different malting processes:
         A6. traditional malting    (without inoculation of any spore suspension)   D6. malting process according to the invention    (inoculation of the steeped wheat during the first wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)       Steeping
       the steeping was carried out in a 2 kg base with a total water (tap water) to air ratio of 1.5:1;   use was made of 2 fermentors (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed;   temperature was only controlled during the wet stages; during the air rest stages, the system was allowed to reach room temperature (±20° C.);   during the whole steeping period the wheat was aerated (4 liter sterile air per minute);   steeping was carried out by immersion using the following scheme:   
       

     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Temperature (° C.) 
                 Duration (h) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 First wet stage 
                 13 
                 6:00 
               
               
                   
                 First air rest stage 
                 20 
                 16:00  
               
               
                   
                 Second wet stage 
                 14 
                 4:00 
               
               
                   
                 Second air rest stage 
                 20 
                 16:00  
               
               
                   
                 Third wet stage 
                 16 
                 2:00 
               
               
                   
                   
               
             
          
         
       
         
         Addition of the Microbial Culture
       during steeping, 10 4  activated spores of per gram air dry wheat were inoculated to the water of the first wet stage (D6);   
     
         Germination 
         As described in Example 4. 
         Kilning 
         As described in Example 1.
 
4. Methods of Analysis and Results
 
       
    
     These were as dscribed in Example 1 (4. Methods of Analysis and Result). 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 Malting Process 
               
               
                   
                 Traditional Malting 
                 According to the 
               
               
                   
                 Process (A6) 
                 Invention (D6) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 Moisture 
                 5.5 
                 5.4 
               
               
                 Extract 
                 83.6 
                 85.5 
               
               
                 Extract Difference 
                 1.0 
                 0.6 
               
               
                 Color 
                 3.9 
                 7.6 
               
               
                 Wort Turbidity 
                 1.4 
                 1.4 
               
               
                 Postcoloratlon 
                 5.8 
                 11.5 
               
               
                 Total Protein Content 
                 14.0 
                 14.8 
               
               
                 Soluble Protein Content 
                 4.9 
                 9.7 
               
               
                 Kolbach Index 
                 35.0 
                 65.5 
               
               
                 Viscosity 
                 1.99 
                 1.79 
               
               
                 pH 
                 6.02 
                 5.63 
               
               
                 Diastatic Power 
                 183 
                 193 
               
               
                 Whole Grains 
                 19.4 
                 20.2 
               
               
                 Friability 
                 35 
                 42 
               
               
                 Homogeneity 
                 79.4 
                 78.7 
               
               
                 Filtration Volume 
                 220 
                 295 
               
               
                 β-glucanase Activity 
                 10.9 
                 16,640 
               
               
                 Xylanase Activity 
                 16.85 
                 1,620.1 
               
               
                   
               
             
          
         
       
     
     EXAMPLE 7 
     1. Preparation of the Microbial Cultures 
     
         
         Strain
       S46 : Rhizopus oryze  ATCC 9363   
     
         Preparation of the Spore Suspension
       the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 7 days at 28° C.;   the spores were harvested by flooding the culture with sterile physiological saline (0.9% NaCl) and by rubbing the sporulated mycelium gently with a sterile spatula;   the spore suspension was washed once with sterile physiological saline (0.9% NaCl) by centrifugation (3500 rpm, Jouan C312, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl);   the spore density was determined microscopically using a Thoma counting chamber.   
     
         Activation of the Spore Suspension
       5×10 7  spores were transferred into 20 ml of sterile, acidified TSB (Tryptic Soy Broth, Oxoid) pH=4.0 and incubated in a shaking water bath during 5 hours at 42° C.
 
2. Cereal
   Sorghum (S14)
 
3. Process
   
     
         Setup 
       
    
     Malts were made by two different malting processes:
         A7. traditional malting    (without inoculation of any spore suspension)   D7. malting process according to the invention    (inoculation of the sorghum during the first wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363).       Cleaning
       washing of the sorghum is performed by using 6 liters tap water per kilogram sorghum and by removing the excess water.   
       Steeping
       the steeping was carried out in a 2 kg base with a total water (top water) to air ratio of 1.5:1;   use was made of 2 fermentors (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed;   temperature was only controlled during the wet stages; during the air rest stages, the system was allowed to reach room temperature (±20° C.);   during the whole steeping period the barley was aerated (2 liter sterile air per minute);   steeping was carried out by immersion using the following scheme:   
       

     
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
                   
               
               
                   
                 Temperature (° C.) 
                 Duration (h) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 First wet stage 
                 28 
                 10:00 
               
               
                   
                 First air rest stage 
                 20 
                  4:00 
               
               
                   
                 Second wet stage 
                 28 
                 10:00 
               
               
                   
                 Second air rest stage 
                 20 
                  4:00 
               
               
                   
                 Third wet stage 
                 28 
                 10:00 
               
               
                   
                 Third air rest stage 
                 20 
                  4:00 
               
               
                   
                   
               
             
          
         
       
         
         Addition of the Microbial Cultures
       during steeping, 10 4  activated spores (1) per gram air dry sorghum were inoculated to the water of the first wet stage (D7).   
     
         Germination
       germination of ±460 g steeped sorghum was carried out in cylindrical container with perforated lids at a temperature of 28° C. during 4 days;   air was supplied by natural diffusion;   the containers were slowly rotated on an electronically controlled roller system (Cellroll®, Tecnorama); i.e., every two hours the containers were rolled for 15 min. at 1 rpm.   
     
         Kilning
       As described in Example 1.
 
4. Method of Analysis and Results
   
     
       
    
     These were as described in Example 1 (4. Methods of Analysis and Results). 
     
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Traditional Malting 
                 Malting Process According 
               
               
                   
                 Process (A7) 
                 to the invention (D7) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                 β-glucanase Activity 
                 98 
                 991 
               
               
                 Xylanase Activity 
                 524.72 
                 413.43