Abstract:
Proces for the preparation of malted cereals, wherein the steeping step includes one or more wetting stages at a temperature 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 aftr 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:
FIELD OF THE INVENTION  
         [0001]    The present invention is related to an improved pxoess for te preparation of malted corealws, the improved malted cereals obtained and their use, especially In biotechnological processos for the preparaton of beverages.  
         TECHNOLOGICAL BACKGROUND OF THE INVENTION  
         [0002]    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.  
           [0003]    In traditional malting processes, the moisture content of cereals is raised either by immersion(s) andfor sproying(s), and the resulting high-moisture content cereal is allowed to germinate. After reaching the proper physological condition, it is preferably subrnitted 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).  
           [0004]    The quality of the malt obtained is, to a large extent, determined by the presence of plant endogenous enzymes genereted during the melting process. For instance, with cereals like barley used as a raw materal for the malt production. the variety, the oomposwon of the microbal 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 melting 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.  
           [0005]    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 imbalanced of specific enzymatic potential and insufficient cell wall degradation. Apart from this, problems with microbial safety can oocur. 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  
         [0006]    During the malting of cereals, the micrflora present on the cereals develops and the quality of the matt and beverages is influenced by the activity of the ister microorganisms.  
           [0007]    In analogy with other blotechnological 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 Matt 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)).  
           [0008]    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 app.  
           [0009]    The influence of  Lactobacillus plantarum  and  Pediocoscus 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.  
           [0010]    The Internatonal 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.  
           [0011]    The preferred bacteria used are lactic acid producing bacteria, such as various Lactobacilli, e.g.,  Lactobacillus casel, Lactobacillus casel  var.  rarmnosus, Lactobacillus fenmentum, Laciobacillus plantarum  and  Lactobacllus brevis , and bacteria of the genus Pediococcus, e.g.,  Pediococcus acidilactici.    
           [0012]    Preferred moulds are moulds of the genus Aspergillus and Geotrichum, like  Geotrichum candidum.    
           [0013]    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 bateria. The preferred bacteria are lactic acid bacteria belonging to genus Lactococcus, Leuconostoc, Pedlococcus or Lactobacillus.  
           [0014]    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.  
           [0015]    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 speciffcally, contamination of barley malt wfth fungi whl has led to gushing and other qualtative changes In the beer.  
           [0016]    The German patent application DE-3028360 disclosm a rnetod to make malt out of corn.  
           [0017]    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  
         [0018]    The present invention aims to provide an improved prepared process for malted cereals and improved malted cereals.  
           [0019]    A main aim of the invention is to provide an improved preparation process for matted 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.  
           [0020]    Another aim is to provide a process and improved malted cereals which vary less in quality with the raw material used.  
           [0021]    A further aim of the invention is to obtain malted cereals which improve the biotechnological production process of beverges and may improve the properties of the said obtained beverages.  
         SUMMARY OF THE INVENTION  
         [0022]    The present invention is directed to a process for the preparation of a malted cereal, the malted cereal which is the product of the proess 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 enymes, and/or protease activity, over malted cereal products which are similarly prepared with wetted cereal products with or without microorganisms.  
           [0023]    The prss 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 weted 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 acity of at least one enzyme, such as tlucanase, xyanase, amylase, naturally occurring enzymes, and/or protease activity, whith is greater than the enzymatic activity which is obtained by holding the wetted oreal without activated spores, or even with the bacteria or moulds from which the spores come.  
           [0024]    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., preferaby 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 oombinain 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 btween 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 beteen about 45° and 85° C. until the dried malted cereal has a moisture content of from about 2 to about 15 weight percent moisture, preftrably 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 matted cereal prepared without activated spores according to the invention.  
           [0025]    In another important aspect, the present invention provdesd 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.  
           [0026]    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.  
           [0027]    In another aspect, the oprals may be disinfected or may not be disinfected.  
           [0028]    Preferably, for the preparation of matted 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  Lactobacellus amylovorus  strain ATCC 33620 , Lactobacillus bavaricus, Lactobacillus bifermentans, Lactobacillus brevis  var  llndneri, Lactobacillus casei  var  casel, Lactobacillus delbrueckii, Lactobacillus delbrueckii  var  iacts, Lactobacillus delbrueckii  var  bulgaricus, Lactobacillus fermenti, Lactobacillus gasserii, Lactobactilus helveticus, Lactobacillus hilgardii, Lactobacillus renteril, Lactobacillus sake, Lactobacillus sativorius, Lactobacillus cremoris, Lactobacillus kefir, Lactobacillus pentoceticus, Lactobacillus cellobiosus, Lactobacillus bruxellensis, Lactobacillus buchneii, Lactobacillus coryneformis, Lactobacillus confusus, Lactobacillus floretinus, Lactobacillus viridescens , Corynebactrium spp., Propionibacterium spp., Bifidobaterium spp., Streptomyces spp., Bacillus spp., preferentially  Bacillus subtilis  stain ATCC 6051, preferentially  Bacillus circulans , Sporolactobacillus app., Acetobacter spp., Agrobacterlum spp., Alcaligenes spp., Peoudomonas spp., preferentially  Pseudomonas amylophilia, Pseudormonas aeruginosa, Pseudomonas cocovenenans, Pseudomonas mexdcana, Pseudomonas pudomaliel , Gluconobadler spp., Enterobacter spp., Erwinia spp., Kiebsiella spp., and Proteus spp.  
           [0029]    Preferably, for the preparation of matted 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 DL Hawkworth, PM Kirk, BC Sutton, and DN Pegler (632 pp) Cab Intermal) of Ascomycota preferentially Dothideales, preferentially Mycosphaerellaceae preferentially Mycosphaerell spp., Ventunaoae preferentially Venturia spp. Eurotleles preferentially Monascaceae preferentially Monascus spp., Triciococcmacese preferentially Emericilla spp., Euroteum spp., Eupenicillium spp., Neosartorya spp., Talaromyces spp., Hypocreales preferentially Hypocrecase preferentially Hypocrea spp., Saccharomycetales preferentially Dipodascacae, Dipodascus spp., Galactomyos spp., Endomycetaceae preferentially Endomyces spp., Metschnkowiaceae preferentially Guilliermondella spp., Saccharomycetaceae preferentially Debaryomyces spp., Dekkera spp., Pichis spp., preferetially Pichia anomala, preferentially Pichia anomala strain ATCC 8168. Kluyveromyces spp. Saccharomyces spp., Torulaspora spp., Zygosaccharomyces spp., Saccharomycodaceae preferentially Hanseniaspora spp.; Schizosaccharomycetales preferentially Schlzosaccharomycetaceae preferentially Schizosaccharomyces spp., Sordariales preferentially Chaetomisse, Chaotomium spp., preferentially Chaetomium vireacens strain ATCC 32319, Sordariacea preferentially Neurospora spp., Zygomycota preferentially Mucorales preferentially Mucoraceae preferentially Absidia spp., Amylomyces spp., Rhizomucor spp., Actinomucor spp., Thermomucor spp., Chiamydomucor spp., Mucor spp. preferentially  Mucor circinelloides, Mucor grisecyanus, Mucor hiemalis, Mucor indicus, Muoor mucedo, Mucor piriformis, Mucor plumbous, Mucor praini, Mucor pusillus, Mucor silvaticus, Mucor javanicus, Mucor racemosus, Mucor rouxianus, Muoor rouxil, Mucor aromaticus, Mucor flevus, 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 thallandensis  strain ATCC 20344 , Rhizopus formosaensis , preferentially  Rhizopus formosaensis  strain ATCC 26612 , Rhizopus chinensis, Rhizopus cohnii, Rhimpus japonicus, Rhizopus nodosus, Rhizopus delemar, Rhizopus acetorinus, Rhizopus chimydosporus, Rhizopus circinans, Rhizopus javanicus, Rhizopus peka, Rhizopus saito, Rhizopus triticl, Rhizopus niveus, Rhizopus microsporus, Mitosporic fungi  preferentially Aureobasidium spp., Acremonium spp., Cercospom spp., Eplcobcum spp., Monlila spp. preferentially  Monilia candida, Monilia sitophila , Mycoderma spp., Candida spp., preferentially  Candida diddenslae, Candida edax, Candide etchellsii, Candida kefir, Candida krisel, Candida lactose, Candida lambica, Candida mlinil, Candida utills, Candida milleri, Candida mycoderma, Candida parpilosis, Candida obtux, Candida troploale, Candida valida, Candida verutllis, Candida guilliermondii , Rhodotmlea 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 rectanguistum, Geotrichum suaveolens, Geotrichum vanryiae, Geotrichum loubieri, Geotrichum microsporum , Ciadosporium spp., Trichoderma spp. preferentially  Trichoderma hamatum, Trichoderma harzianum, Trichoderma koningii, Trichoderma pseudokoningli, Trichoderma resei , preferentially  Trichoderma resei  strain ATCC 5875 , Trichoderma virgatm, Trichoderma viride , Oldium spp., Altemaria spp. preferentially  Alternaria alternata, Alternaria tenuis , Helminthosporium spp. preferentially  Helminthosporium gramineum, Helminrium sativum, Helminthosporium teres , Aspergilius spp. preferentially  Aspergillus ochraseus  Group,  Aspergillus nidulans  Group,  Aspergglus vemicolor  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, Penlcillium claviforme, Penicillum funiculosum, Penicillum italicum, Penicillum lanoso - viride, Penicillum emersonil, Peniclilum lilacinum, Penicillum expansum , and mixtures thereof.  
           [0030]    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., Corynebacterum 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 Ventura spp.; Eurotiales 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 Schlzosaccharomyces spp.; Sordariales preferentially Chaetomiaceae preferentially Chaetomium spp., Sordariacese 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  
         [0031]    As used herein the term “spore” refers to a dormant and highly resistant reproductive cell fomned 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.  
           [0032]    As used herein the term “activated spore” means a spore having one of the following properties:  
           [0033]    i) The spore is swollen such that its size increase by a factor of between about 1.2 and about 10 over its dormant size; and/or  
           [0034]    ii) one or more germ tubes per spore is formed.  
           [0035]    Activated spores are prepared by one or a combination of the following treatments.  
           [0036]    i) cycles of wetting and/or drying;  
           [0037]    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;  
           [0038]    iii) exposure to temperature changes, preferably within a temperature range of about oe to about 80° C..  
           [0039]    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.  
           [0040]    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.  
           [0041]    As used herein, the term “steeping” refers to weing 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.  
           [0042]    The term “specific ac” 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  
         [0043]    According to a prefrd embodiment, the preparatbn 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 temperatus 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., prefably  Pseudomonas herbicola.    
           [0044]    According to the invention, the malted cereals are selected from the group of barley, wheat, rye, corn, oats, rice, millet, trficoale, sorghum and the like.  
           [0045]    In the process according to the invention, the same or deferent 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;  
           [0046]    (a) cycles of wetting and/or drying;  
           [0047]    (b) addion of appropriate nutritional supplies (such ass a nitrogen source, preferably amino acids and/or a carbon source, preferably mono- or disaccharides) or spore elements;  
           [0048]    (c) exposure to temperaure changes, prsftmbly within a tempemwure range of 0° to 80° C.;  
           [0049]    (d) exposure to changes in pH, preferably wfthln a pH range of 2.0 to 8.0, more pmftrably between 3.0 and 6.0.  
           [0050]    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.  
           [0051]    The concentration of the spores may vary depednding 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.  
           [0052]    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 activies. 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.  
           [0053]    For instance, the malted cereals acoording to the invention may have a lower β-glucan content or a higher enzyme activw such as, for example, β-glucanase or xylanase actvy (as represented in the folowing 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.  
           [0054]    The activated spores and cereal may be combined and wetted by submersion in water to steep the combinatlon 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.  
           [0055]    An object of the present invention concerns the use of the malted cereals according to the invention for the preparation of beverages.  
           [0056]    The invention is also related to these improved beverages.  
           [0057]    The improved malted cereals according to the inventon 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.  
           [0058]    The present invention will be further described in various exaerles in view of the following drawings.  
       
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS  
       [0059]    [0059]FIG. 1 represents the β-glucanase activity of malted barley obtained according to the preparation process of example 1. (legend: see example 1).  
         [0060]    [0060]FIG. 2 represents the xylanese activity of malted barley obtained according to the preparation process of example 1. (legend: see example 1).  
         [0061]    [0061]FIG. 3 represents the β-glucanase activity of malted barley obtained according to the preparation process of example 3. (legend: see example 3).  
         [0062]    [0062]FIG. 4 represents the xylanase activity of malted barley obtained according to the preparation prooess of example 3. (legend: see example 3).  
         [0063]    [0063]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  
       [0064]    1. Preparation of Microbial Cultures  
         [0065]    Strain  
         [0066]    S46 : Rhizopus oryzae  ATCC 9383.  
         [0067]    Preparation of the Spore Suspension  
         [0068]    the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 10 days at 28° C.;  
         [0069]    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;  
         [0070]    the spore suspension was washed twioe 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);  
         [0071]    the spore density was determined micrampically using a Thoma counting chamber.  
         [0072]    Activation of the Spore Suspension  
         [0073]    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.;  
         [0074]    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 centeugallon (3500 rpm, Soivall type SS-34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).  
         [0075]    2. Barlay  
         [0076]    Plaisant - 1994 French harvest.  
         [0077]    3. Process.  
         [0078]    Setup  
         [0079]    Malts were made by four different malting processes:  
         [0080]    A1. traditional malting  
         [0081]     (without inoculation of any spore suspension)  
         [0082]    B1. malting process using non-activated spores  
         [0083]     (inoculation of the stewp barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0084]    C1. malting process according to the invention  
         [0085]     (inoculation of fte seped barley with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0086]    D1. malting process according to the inverion  
         [0087]     (inoculation of the steeped barley during the first wet stage with a suspension of activated spores of  Rhizopus oryzme  ATCC 9363)  
         [0088]    Steeping  
         [0089]    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;  
         [0090]    use was made of 2 fermentors (Biofio III, New Brunswick Scientific), in which perforated plates were placed;  
         [0091]    temperature was only controlled during the wet stages, during the air rest stages, the system was allowed to reach room temperature (±20° C.);  
         [0092]    during the whole steeping period, the barley was serated (4 liter sterile air per minute);  
         [0093]    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                      
 
         [0094]    Addition of the Microbial Cultures  
         [0095]    ±480 g of steeped barley was immersned 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;  
         [0096]    during the steeping, 10 4  activated spores per gram air dry barley were inoculated to the water of the first wet stage (D1);  
         [0097]    the fluid was removed by draining.  
         [0098]    Germination  
         [0099]    germination was carried out in a cylindrical container with performated lids at a temperature of 16°-18° C. during 4 days;  
         [0100]    air was supplied by natural diffusion;  
         [0101]    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.  
         [0102]    Kilning  
         [0103]    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                  
 
         [0104]    4. Methods of Analysis and Results  
         [0105]    Methods for determination and units of moisture, extract, extract difference, color, total protein content, soluble protein content, Kobach index, pH, diasttic power, according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau).  
         [0106]    Metods for determination and units of turbidity, frability, homogeneity, whole grains, b-glucan content, according to Analytica-European Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau, supplement published in 1989).  
         [0107]    Postcoloration of the wort is determined after boiling the congress wort under reflux at 108° C. during 2 hours.  
         [0108]    The viscosity of the congress wort is determined with the Delta-viscosimater.  
         [0109]    For the determination of the filtration volume, the congress wort is filtered over a Schleicher and Schuell 597½folded filter. The volume (in mil) that is obtained after 1 hour of filtration is the filtration volume of the wort.  
         [0110]    Modification is determined with the Calcofluor apparatus (Haffmans) according to the Carisberg method (Analytica-Europeon Brewery Convention (Fourth Edition, 1987, Brauerei und Getränke-Rundschau).  
         [0111]    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                  
 
         [0112]    [0112]FIGS. 1 and 2 represent the β-glucanase and xcylanase 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 actvity (U/kg) was calculated as (46.8×E (590 nm)+0.9)×5).  
       EXAMPLE 2  
       [0113]    1. Preparation of Microbial Cultures  
         [0114]    Strain  
         [0115]    S46 : Rhizopus oryzae  ATCC 9363.  
         [0116]    as described in Example 1.  
         [0117]    Preparation of the Spore Suspension  
         [0118]    as described in Example 1.  
         [0119]    Activation of the Spore Suspension  
         [0120]    as described in Example 1.  
         [0121]    2. Barley  
         [0122]    Stander - 1995 North American harvest.  
         [0123]    3. Process  
         [0124]    Setup  
         [0125]    Malts were made by six different malting processes:  
         [0126]    A2, tradnonal malting process  
         [0127]     (without inoculation of any spore suspension)  
         [0128]    B2, malting process using non-activated spores  
         [0129]     (inoculation of the steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0130]    C2, malting process according to the invention  
         [0131]     (inoculation of the steeped barley during the first wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9383)  
         [0132]    D2, malting process according to the invention  
         [0133]     (inoculation of the steeped barley during the second wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0134]    E2, malting process according to the invention  
         [0135]     (inoculation of the steeped barley during the third wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0136]    F2. malting process according to the invention  
         [0137]     (inoculation of the steeped barley with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0138]    Steeping and Addition of the Microbial Cultures  
         [0139]    the steeping was carried out on a 300 g base with a total water (tap water) to air dry barley ratio of 5:3;  
         [0140]    use was made of 2000 ml flasks;  
         [0141]    a tempertre of 18° C. was maintained during the wet stages and during the air rest stages;  
         [0142]    during the whole steeping period, the barley was aerated by means of compressed air;  
         [0143]    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                      
 
         [0144]    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;  
         [0145]    the steeped barley was immersed in 0.5 liters of tap water which contained no spores (A2), non-actvated (B2) or activated (C2, D2, E2, F2) spores;  
         [0146]    for B2, and F2, the steeped barley was inoculated with 10 4  spores per gram of air dry barley,  
         [0147]    the fluid was removed by draining.  
         [0148]    Germinaton  
         [0149]    as described in Example 1.  
         [0150]    Kilning  
         [0151]    as described in Example 1.  
         [0152]    Malt evaluation  
         [0153]    Determination of the Increase of the Bacterial Population  
         [0154]    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 pirnaricine and after incubation at 28° C. for 3 days.  
         [0155]    [0155]FIG. 5 shows the increase of the bacterial population during the malting according to the preparation process of Example 2.  
       EXAMPLE 3  
       [0156]    1. Preparation of Microbial Cultures  
         [0157]    Strain  
         [0158]    S 46 : Rhizopus oryzae  ATCC 9363  
         [0159]    Preparation of the Spore Suspension  
         [0160]    as described in Example 1  
         [0161]    Activation of the Spore Suspension  
         [0162]    as described in Example 1  
         [0163]    2. Barley  
         [0164]    Plaisant - 1994 French harvest;  
         [0165]    3. Process  
         [0166]    Setup  
         [0167]    Malts were made by three different malting processs:  
         [0168]    A3. traditional malting  
         [0169]     (without inoculavon of any spore suspension)  
         [0170]    B3. malting process using non-activited spores  
         [0171]     (inoculation of the steeped barley with a suspension of non-activated spores of  Rhizopus oryzae  ATCC 9363)  
         [0172]    C3. malting process according to the invention  
         [0173]     (inoculation of the steeped barley with a suspension of activted spores of Rhizopus oryzsa ATCC 9363)  
         [0174]    Steeping  
         [0175]    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;  
         [0176]    the pH of the steeping water was controlled at pH=5.5 by addition of lactic acid and NaOH;  
         [0177]    a fermentor (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed, was used for steeping;  
         [0178]    temperature was only controlled during the wet stages; during the air rest stages the system was allowed to reach room temperature (ca. 20° C.);  
         [0179]    during the whole steeping period the barley was aerated (4 liters startle air per minute);  
         [0180]    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                      
 
         [0181]    Addition of the Microbial Cultures  
         [0182]    460 g of steeped barley was immersed in 0.5 liters of tap water which contained no spores (A3), non-activted spores of  Rhizopus oryzae  ATCC 9363 (B3) or actvated spores of  Rhizopus oryzae  ATCC 9363 (C3 according to the invention); for B3 and C3, the steeped barley was inoculated wfth 10 4  spores per gram of air dry barley;  
         [0183]    the fluid was removed by draining.  
         [0184]    Germination  
         [0185]    as described in Example 1  
         [0186]    Kilning  
         [0187]    as described in Example 1  
         [0188]    4. Methods of analysis and results  
         [0189]    These were as described in Example 1 (4. Methods of Analysis and Results).  
         [0190]    See table on next page. In this table:  
         [0191]    A1/3: Traditional malting process  
         [0192]    B1/3: Malting process using non-activated spores  
         [0193]    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                  
 
         [0194]    [0194]FIG. 3 reprsents the 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 calculatd 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 inoculavon 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).  
         [0195]    These results show the increased β-glucanase activity when the pH of the steeping water is maintained at around 5.5.  
         [0196]    [0196]FIG. 4 gives the corresponding results for xylanase activity. These were measured according to xylszym method, Megazyme ((AUSTR) Pty. Ltd. (September 1995)). Malt xylanase activity was calculated as descibed in Example 1.  
         [0197]    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.  
         [0198]    In order to compare the improved results regarding β-glucenase 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                             
 
         [0199]    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.  
         [0200]    It was also calculated for the data descrbed in WO94/29430 (Example 1) where  Geotrichum candidum  was used.  
         [0201]    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(590nm)+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.  
         [0202]    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                          
 
         [0203]    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).  
         [0204]    It thus appears that it is possible to obtain malted cereals having a β-glucanase activity increasd by at least a factor 4 compared to the conventional maltng process wherein the addition of microbial culture is omitted.  
         [0205]    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  
       [0206]    1. Preparation of the microbial cultures  
         [0207]    Strain  
         [0208]    S40 : Aspergillus oryzae  ATCC 14156  
         [0209]    Preparation of the spore suspension  
         [0210]    the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 7 days at 281° C.;  
         [0211]    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;  
         [0212]    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);  
         [0213]    the spore density was determined microscopically using a Thoma counting chamber.  
         [0214]    Activation of the Spore Suspension  
         [0215]    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.,  
         [0216]    2. Cereal  
         [0217]    Clarine barley - 1995 French harvest  
         [0218]    3. Process  
         [0219]    Malts wmre made by two different malting proces:  
         [0220]    A4. traditional malting  
         [0221]     (without inoculation of any spore suspension)  
         [0222]    E4. malting process according to the invention  
         [0223]     (inoculation of the steeped barley during the first and third wet stage with a suspension of activated spores of  Aspergillus oryzae  ATCC 14156)  
         [0224]    Steeping  
         [0225]    as described in Example 1  
         [0226]    Addition of the microbial cultures  
         [0227]    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);  
         [0228]    Germination  
         [0229]    germination of ±460 g steeped barley was carried out in cylindrical containers with perfored lids at a temperature of 16°-18° C. during 4 days;  
         [0230]    air was supplied by natural diffusion;  
         [0231]    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.  
         [0232]    Kilning  
         [0233]    as described in Example 1  
         [0234]    4. Methods of the analysis and results  
         [0235]    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 Convenfton (Fourth Edtion, 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                  
 
         [0236]    [0236]                                                                     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  
       [0237]    1. Preparation of the Microbial Cultures  
         [0238]    Strains  
         [0239]    S40 : Aspergillus oryzae  ATCC 14156  
         [0240]    S46 : Rhizopus oryzae  ATCC 9363  
         [0241]    Preparation of the Spore Suspensions  
         [0242]    As described in Example 4  
         [0243]    Activiation of the Spore Suspensions  
         [0244]    S40:  
         [0245]     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.;  
         [0246]    the activated spores were harvested by cenifugation (3500 rpm, Sorvall type SS34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).  
         [0247]    S45  
         [0248]    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.;  
         [0249]    the activated spores were harvested by contrifugation (3500 rpm, Sorvall type SS34®, for 15 min.) and resuspended in sterile physiological saline (0.9% NaCl).  
         [0250]    2. Cereal  
         [0251]    Caline - 1995 French harvest  
         [0252]    3. Process  
         [0253]    Setup  
         [0254]    Malts were made by two different malting prooesm:  
         [0255]    A5. traditional malting  
         [0256]     (without inoculation of any spore suspension)  
         [0257]    F5. malting process according to the invention  
         [0258]     (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)  
         [0259]    Steeping  
         [0260]    As described in Example 1  
         [0261]    Addition of the Microbial Cultures  
         [0262]    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 inventon);  
         [0263]    ±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;  
         [0264]    the fluid was removed by draining.  
         [0265]    Germination  
         [0266]    As described in Example 4.  
         [0267]    Kilning  
         [0268]    As described in Example 1.  
         [0269]    4. Methods of Analysis and Results  
         [0270]    These were as described in Example 1 (4. Methods of Analysis and Results).  
         [0271]    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                  
 
         [0272]    It was noted that the use of activated spores of  Aspergillus oryzae  ATCC improved the malt analytical specifications.  
         [0273]    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  
       [0274]    1. Preparation of the Microbial Cultures  
         [0275]    Strains  
         [0276]    S46 : Rhizopus oryzae  ATCC 9363  
         [0277]    Preparation of the Spore Suspensions  
         [0278]    As described in Example 4  
         [0279]    Activation of the Spore Suspensions  
         [0280]    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.;  
         [0281]    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).  
         [0282]    2. Cereal  
         [0283]    Wheat: Mobil - 1996 Belgian harvest  
         [0284]    3. Process  
         [0285]    Setup  
         [0286]    Malts were made by two different malting processes:  
         [0287]    A6. traditional malting  
         [0288]     (without inoculaton of any spore suspension)  
         [0289]    D6. malting process according to the invention  
         [0290]     (inoculation of the steeped wheat during the first wet stage with a suspension of activated spors of  Rhizopus oryzae  ATCC 9363)  
         [0291]    Steeping  
         [0292]    the steeping was carried out in a 2 kg base wfth a total water (tap water) to air ratio of 1.5:1;  
         [0293]    use was made of 2 femnentors (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed;  
         [0294]    temperature was only controlled during the wet stages; during the air rest stages, the system was allowed to reach room temperature (±20° C.);  
         [0295]    during the whole steeping period the wheat was aerated (4 liter sterile air per minute);  
         [0296]    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                      
 
         [0297]    Addition of the Microbial Culture  
         [0298]    during steeping, 10 4  activated spores of per gram air dry wheat were inoculated to the water of the first wet stage (D6);  
         [0299]    Germination  
         [0300]    As described in Example 4.  
         [0301]    Kilning  
         [0302]    As described in Example 1.  
         [0303]    4. Methods of Analysis and Results  
         [0304]    These were as dscribod 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  
       [0305]    1. Preparation of the Microbial Cultures  
         [0306]    Strain  
         [0307]    S46 : Rhizopus oryze  ATCC 9363  
         [0308]    Preparation of the Spore Suspension  
         [0309]    the strain was grown on PDA (Potato Dextrose Agar, Oxoid) for approximately 7 days at 28° C.;  
         [0310]    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;  
         [0311]    the spore suspension was washed once with sterile physiological saline (0.9% NaCl) by centifugation (3500 rpm, Jouan C312, for 15 min.) and resuspended in serile physiological saline (0.9% NaCl);  
         [0312]    the spore density was determined microscopically using a Thoma counting chamber.  
         [0313]    Activation of the Spore Suspension  
         [0314]    5×10 7  spores were transforred 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.  
         [0315]    2. Cereal  
         [0316]    Sorghum (S14)  
         [0317]    3. Process  
         [0318]    Setup  
         [0319]    Malts were made by two difftrent malting processes:  
         [0320]    A7. traditional malting  
         [0321]     (without inoculation of any spore suspension)  
         [0322]    D7. malting process accordng to the invention  
         [0323]     (inoculation of the sorghum during the first wet stage with a suspension of activated spores of  Rhizopus oryzae  ATCC 9363).  
         [0324]    Cleaning  
         [0325]    washing of the sorghum is perforrned by using 6 liters tap water per kilogram sorghum and by mvng the excess water.  
         [0326]    Steeping  
         [0327]    the steeping was carried out in a 2 kg base with a total water (top water) to air ratio of 1.5:1;  
         [0328]    use was made of 2 fermentors (Bioflo III, New Brunswick Scientific), in which a perforated plate was placed;  
         [0329]    temperature was only controlled during the wet stages; during the air rest stages, the system was allowed to reach room temperature (±20° C.);  
         [0330]    during the whole steeping period the barley was serated (2 liter sterile air per minute);  
         [0331]    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                      
 
         [0332]    Addition of the Microbial Cultures  
         [0333]    during steeping, 10 4  activated spores (1) per gram air dry sorghum were inoculated to the water of the frst wet stage (D7).  
         [0334]    Germination  
         [0335]    germination of ±460 g steeped sorghum was carried out in cylindrical container with perforated lids at a temperature of 28° C. during 4 days;  
         [0336]    air was supplied by natural diffusion;  
         [0337]    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.  
         [0338]    Kilning  
         [0339]    As described in Example 1.  
         [0340]    4. Method of Analysis and Results  
         [0341]    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