EP0678120A1 - Procedure for treatment of seed material to be germinated - Google Patents

Procedure for treatment of seed material to be germinated

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Publication number
EP0678120A1
EP0678120A1 EP93920869A EP93920869A EP0678120A1 EP 0678120 A1 EP0678120 A1 EP 0678120A1 EP 93920869 A EP93920869 A EP 93920869A EP 93920869 A EP93920869 A EP 93920869A EP 0678120 A1 EP0678120 A1 EP 0678120A1
Authority
EP
European Patent Office
Prior art keywords
lactic acid
acid bacteria
preparation
added
barley
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
EP93920869A
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German (de)
French (fr)
Inventor
Auli Haikara
Tiina Mattila-Sandholm
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PANIMOLABORATORIO - BRYGGERILABORATORIUM Oy AB
Original Assignee
PANIMOLABORATORIO - BRYGGERILABORATORIUM Oy AB
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Application filed by PANIMOLABORATORIO - BRYGGERILABORATORIUM Oy AB filed Critical PANIMOLABORATORIO - BRYGGERILABORATORIUM Oy AB
Publication of EP0678120A1 publication Critical patent/EP0678120A1/en
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C1/00—Preparation of malt
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
    • A23L7/10—Cereal-derived products
    • A23L7/152—Cereal germ products
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C1/00—Preparation of malt
    • C12C1/02—Pretreatment of grains, e.g. washing, steeping

Definitions

  • the present invention concerns a procedure for treating seed material intended to be germinated.
  • a germination process is in this context un ⁇ derstood to be a process step in general which is re ⁇ quired in order to produce a germinated product, start ⁇ ing from storage-dry seed.
  • a germination process that is the malting process, is applied in producing the important raw material of beer, or other alcoholic beverages, viz. cereal malt, such as barley malt, rye malt or any malt whatsoever.
  • the germination process is furthermore applied in producing various commercial sprouts products, e.g. bean sprouts or any sprouts for use in human nutrition.
  • Germination processes are usually carried out in non-aseptic conditions. On the seeds being treated there occur microbes originating from the growth envi ⁇ ronment or from storage. Conditions during the germina ⁇ tion process are mostly favourable to the microbes present on the seeds, such microbes usually multiplying during the course of the process. The microbes may exert a detrimental effect on the germinated product, or on the end product which may ultimately be made thereof, while they may equally have beneficial influ ⁇ ence on the product being germinated.
  • the main steps in brewing process are: mal- ting, wort production, primary and secondary fermenta ⁇ tion, and downstream processing.
  • the purpose with mal ⁇ ting is to produce in the kernel enzymes which in the mashing step decompose the substances of the kernel's endosperm to a form soluble in the wort.
  • the barley seed malting process is well known to comp ⁇ rise three steps; steeping, germination and kilning.
  • the cleaned and screened barley grains are steeped in water until desired moisture is achieved, e.g. on the order of 43 to 44%. Part of the steeping may be accom ⁇ plished in so-called air rest.
  • the barley is allowed to germinate in controlled conditions, and the germinated barley is kilned in a hot air current until the ger ⁇ mination has come to an end. On termination of kilning, the rootlets are removed from the malt. Regulation of the malting steps is based on temperature, air flow and moisture/humidity control. Malt quality is affected, on the side of malt ⁇ ing technique and malting conditions, also by the mi- crobial flora of the malt cereal, this flora varying significantly e.g. depending on cereal variety, weather conditions, growth site, length of growing season and storage conditions.
  • Barley is the cereal most often used in malt ⁇ ing.
  • the inherent, natural microbial flora of barley can be classified as field and storage fungi, bacteria and yeasts.
  • the commonest field fungi of barley are: Fusarium, Alternaria, Cladosporium, Cephalosporium, Epicoccum, and Helminthosporium.
  • the occurrence of moulds is different in different countries and differ ⁇ ent years. Wet weather conditions during the cereal's growth period, and particularly while it is being har- vested, favour the growth of Fusarium mould. Fusarium contamination may be heavy indeed in rainy growth sea ⁇ sons.
  • One of the commonest bacterium species on cereals is Enterobacter aqqlomerans.
  • bacteria which should be mentioned are; Escherichia coli, and bacteria of genera Pseudomonas, Micrococcus and Bacillus, and lactic acid bacteria.
  • the bacterial count on barley is about IO 5 to 10 8 CFU/g (colony forming units per g) .
  • the moulds and bacteria in barley increase during malting, and peak concentrations are usually reached during the germination step. Fusarium moulds, in particular, and lactic acid bacteria undergo the strongest proliferation. Yeasts also increase during malting. In the kilning step the mould, yeast and bac ⁇ terium concentrations go down again, as a rule.
  • part of the microbes present on barley have a useful effect in view of malting and of the product to be made of the malt, e.g. beer. It has been estimated that up to 40- 50% of some enzymes in the malt are of microbial ori ⁇ gin. On the other hand, part of the microbes exert a detrimental influence on the barley and/or the malts.
  • Fusarium moulds deserve to be mentioned, which have been found to cause particularly detrimental gushing of beer more often than other moulds, the peptides produced by said moulds constituting nuclei for gas bubbles discharging from the beer bottle in the form of powerful gushing. When more than 50% of malted kernels are contaminated with Fusarium moulds, the risk of gushing is clearly increased.
  • gram-negative bacteria present in the barley such as species belonging to genera Pseudomonas and Flavobacterium, and gram-positive bacteria of genus Leuconostoc have been shown to retard filtration of the mash in connection with wort production.
  • Various micro ⁇ bes present in barley may also give rise to other di ⁇ sadvantageous effects, e.g. inhibit the germination, cause off-flavours or unfavourable changes in the ana ⁇ lysis values of the wort and the beer.
  • malt barley The quality requirements of malt barley can be specified in annually established cultivation contract and delivery terms. Moulds are a group of microbes fre- quently mentioned in quality specifications. Many malt ⁇ ing plants have moreover imposed an upper limit on cer ⁇ tain moulds. If the proportion of Fusarium-contaminated kernels exceeds 65% or if the corresponding proportion of Asperqillus and Penicillium moulds exceeds 50%, the barley can be classified as poor in quality or even as unfit for use in malting. Attempts have been made to prevent gushing in ⁇ cuted by moulds, by using barley of good quality or by blending barley, malt or beer batches. In rainy years nearly the whole barley crop may be poor in quality, in which case it may be impossible to obtain good barley. Microbicidic chemicals have also been tried in order to reduce the quantity of moulds, but no safe and general ⁇ ly approved chemical could be found.
  • a germinating process for producing sprouts intended to be used for nutrition offers likewise pro- pitious conditions for proliferation e.g. of moulds and bacteria. Such sprouts products will spoil rapidly. Further still, in connection with germination increase may take place e.g. of foodstuff pathogens causing food poisoning, such as Salmonella, Yersinia and/or Listeria bacteria.
  • the object of the present invention is to eliminate the drawbacks just discussed.
  • an object of the invention is to provide a procedure by which the quantity and quality of the microbial flora can be carefully regulated du ⁇ ring the germinating process, however without dis- advantageously affecting the quality of the germinated product or of the end product potentially made of the product.
  • the invention is based on studies in connec ⁇ tion of which the unexpected observation was made that lactic acid bacteria can be used towards improving the quality of a product to be germinated.
  • the substances comprised and/or produced by the preparation of the invention i.e., the microbicidic agents, inhibit the growth of detrimental microorganisms occurring in con- nection of a germinating process.
  • lactic acid bacteria in foodstuff and animal feed industry is well known in the art. They produce in fermentative conditions such compounds which affect the composition and flavour of the products, but which also inhibit the growth of pathogenic microbes tending to spoil said products. Lactic acid bacteria have been commonly used in dairy products, meat products, vegetable fermentation and bakery products, and in fodder preservation. Addition of lactic acid bacteria or of lactic acid has been practiced in the production of a certain malt type, of so-called Sauermalz. This addition is made to the malt in the kilning step, prior to mashing or during mashing. The purpose with the addition is merely to cause lowering of the wort pH and, thus, to exert an influence on the course of the mashing process and on the quality of the finished beer. However, lactic acid bacteria have not heretofore been used as taught by the invention: to inhibit the growth of undesired microbes in connection with the germinating process.
  • the preparation of the invention can be added to the product to be germinated in any step of the ger ⁇ minating process.
  • lactic acid bacteria preparation or preparation pro ⁇ quizzed by a lactic acid bacteria
  • cereal material such as barley kernels
  • the preparation added inhibits the growth of moulds, in particular of harmful Fusarium moulds, and of bacteria, as a result of which, for in ⁇ stance, the risk of beer gushing due to Fusarium mould is reduced.
  • the preparation has no substantial effect on the action of the useful microbial flora as regards the quality of the malt that is obtained or of the beer therefrom produced. No harmful effects of the preparation added on malt quality have been observed either, nor has it been found to contain or to produce compounds which are harmful in view of the malt, or beer, being produced.
  • the lactic acid bacteri ⁇ um preparation or the preparation produced by a lactic acid bacteria, can be added to the barley kernels be ⁇ fore steeping, in the steeping step or in the germina ⁇ tion step.
  • the addition is advantageously made in the steeping or germinating step.
  • the malting process may be carried out, in other parts, in any manner known in itself in the art. If desired, e.g. nutrients may be added to the barley to be malted, or the conditions may be regulated, e.g. lactic acid added, in order to opti ⁇ mize the conditions for growth of lactic acid bacteria. It is possible in the invention to use any commonly available lactic acid bacterium whatsoever which possesses influence of inhibiting microbial growth.
  • lactic acid bacterium genera may be mentioned: Lactococcus, Leuconostoc, Pediococcus and Lactobacillus.
  • lactococcus lactis Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus dam- nosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus, and Lactobacillus plantarum, or any mixtures of these, among these the following being particularly advantageous: Lactobacillus plantarum and Pediococcus pentosaceus or mixtures thereof.
  • Use of genetically modified lactic acid bacteria is equally possible.
  • the lactic acid bacteria preparation may be composed of culture broth, with or without cells, or of concentrated culture broth (including cells).
  • a pre- paration produced by lactic acid bacteria may consist of cell-free culture filtrate, of concentrated culture filtrate, of fractionated culture filtrate, or of a pure or partly purified microbicidic product.
  • the treatment is carried out with concen ⁇ trated or fractionated culture broth, which may be cell-free or may contain cells. Concentration may be accomplished e.g. by lyophilization or by evaporation. The culture broth is concentrated e.g. by a factor of 2 - 20 - 40.
  • the micro ⁇ bial growth-inhibiting activity of the preparation con ⁇ taining lactic acid bacteria, or of preparation pro ⁇ quizd by lactic acid bacteria, to be added to the seed material corresponds e.g. to the culture broth quantity of about 10 to 10,000 ml/kg of seed material to be treated, suitably 30 to 7,000 ml/kg of seed material to be treated, e.g. 40 to 5,000 ml/kg of seed material to be treated.
  • the preparation of the culture broth is described in the Examples section.
  • the preparation contains microbicidic compounds, and/or the preparation produces microbicidic compounds during the course of the germination process.
  • a cell preparation When a cell preparation is employed, cell growth can be promoted, if required, e.g. by regulating the conditions during the germina ⁇ tion process, or by adding nutrients.
  • the preparation may also accelerate the growth of other lactic acid bacteria present in the material to be germinated.
  • Lactic acid bacteria belong usually to the natural microbe flora of seeds to be germinated, such as barley kernels. Therefore the procedure of the in- vention is maximally natural. It is also possible to use for lactic acid bacterium strain a strain inherent ⁇ ly occurring on the seeds.
  • the procedure has unexpectedly been found to improve the filtrability characteristic in the brewing process. This has been found to be due to the fact that the preparation of the invention also rest- ricts the counts of harmful species occurring in mal ⁇ ting and retarding the filtration of the mash, e.g. those belonging to genera Leuconostoc, Pseudomonas and Flavobacterium.
  • lactic acid bacteria preparation or preparation pro ⁇ quizzed by a lactic acid bacterium is added to the seed material when producing sprouts to be used for food.
  • the growth of harmful microbes can be restricted in connection with a germination process.
  • the invention enables biological expedients to be used in order to prevent the growth during an industrial germination process of detrimental bacteria occurring on seed which is to be germinated.
  • the procedure of the invention improves the general hygienic standard of the germination process even on the whole.
  • Fig. 1 shows a graph representing the micro ⁇ bicidic activity in a lactic acid bacteria culture filtrate, as assessed by the turbidometric method.
  • Fig. 2 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 culture broth added at different stages of the malt- ings.
  • Fig. 3 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 cells added at different stages of the maltings.
  • Fig. 4 are shown the total bacterial counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated culture broths, to the barley steeping water.
  • Fig. 5 are shown the total bacterium counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated and fractionated culture broths, to the barley steeping water.
  • Fig. 6 displays the effect of lactic acid bac ⁇ teria cultures, added to the malting, on mash filtrati- on (Tepral filtration)
  • Example 1 THE MICROBICIDIC EFFECT OF VARIOUS LACTIC ACID BACTERIA STRAINS ON MICROBES OCCURRING IN MALTING
  • the strains were obtained from the VTT, Col ⁇ lection of Industrial Microorganisms (Biotechnical Laboratory, Finland). Lactobacillus plantarum (E-76) has been deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under number 7388. The strain E-76 (DSM 7388) had been isolated from beer by known techniques used for liquid products, and analy ⁇ zed/identified using well known analysis methods. Pe ⁇ diococcus pentosaceus (E-390) has been deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkultu- ren) under number 7389.
  • DSM Deutsche Sammlung von Mikroorganismen und Zellkulturen
  • the strain E-390 (DSM 7389) had been isolated from homogenized samples of split barley kernels and identified/analyzed using techniques well known in the art [see the article of Haikara, A. and Home, S., Mash Filtration difficulties caused by split barley kernels: a Microbiological problem, in the pub ⁇ lication of EBC Congress 1991 (Quality Control)].
  • the deposits are under the provisions of the Budapest Trea ⁇ ty.
  • Test strains Various harmful microbial species occurring at malting were used for test strains, as well as lactic acid bacteria strains which served as production strains, among others.
  • Harmful moulds were represented in the tests by Fusarium moulds fGibberella avenacea (former Fusari ⁇ um avenaceum) VTT-D-80141 (D-141) and VTT-D-80147 (D- 147), and Fusarium culmorum VTT-D-80148 (D-148) and VTT-D-80149 (D-149), Collection of Industrial Microor ⁇ ganisms, Biotechnical Laboratory of VTT], and one As- perqillus species.
  • Harmful gram-negative bacteria were represent ⁇ ed by two strains from genus Enterobacter and by one species each from genus Flavobacterium and genus Pseu ⁇ domonas.
  • the lactic acid bacteria consisted of those strains which were employed as production strains, plus the strain Lactococcus sp. E-416.
  • the lactic acid bacteria were cultivated in MRS broth (MRS BROTH, Oxoid) .
  • MRS BROTH, Oxoid MRS broth
  • E-65, E-67 and E-68 were aerobically cultivated at
  • the spore suspension of Asperqillus mould was produced directly on PD agar (25°C, 3 days) (Potato dextrose, Difco) .
  • Gram-negative bacteria were aerobically cul- tivated in NB broth (Nutrient broth, Difco) for 1 day, the Enterobacter strain at 30°C, and the Flavobacterium and Pseudomonas strains at 25°C.
  • Lactic acid bacteria were cultivated as de ⁇ scribed under 3. above. 5. Examination of the microbicidic effect of the lactic acid bacteria:
  • microbicidic activity in the culture broth was assessed by the disk method, or turbidometrically.
  • the sample contained 10% by vol. of the test organism and 10% by vol. of the sterile-filtered cul ⁇ ture broth of the production strain, calculated on the sample volume, and growth medium.
  • the sterile filtrate preparation was replaced with distil ⁇ led water of which the pH had been adjusted with lactic acid to identical level with the sterile-filtered pre ⁇ paration.
  • the growth medium used was, in the case of each test strain, the same medium as in the test strain cultivation.
  • the growth conditions for Fusarium and Asper- qillus moulds were: 5 days, 25°C, powerful shaking. Those for gram-negative bacteria were: Enterobacter strain, 30°C, all others 25°C, 2 days, and shaking. The growth conditions for lactic acid bacteria were: 3 days, 30°C, and shaking.
  • the apparatus determined from the samples the absorbance at visible light wavelength 420-580 nm. After cultivation, the growth curve of each sample could be made up and the area subtended by the curve calculated.
  • microbicidic effect of a production strain on the growth of the test strain was expressed by an inhibition percentage, obtained by comparison of the growth area sizes found with the control and with the sterile-filtered preparation of the production strain, respectively. 6. Examination of the fungicidic effect of certain lactic acid bacteria:
  • Cultivation took place as a test tube culture in CMC broth, at 25°C, during 5 days. The results were visually read.
  • Tables 1 and 2, and Fig. 1 display the micro ⁇ bicidic activities determined for the different lactic acid bacteria strains by the disk method and by the turbidometric method, respectively.
  • Table 3 displays the visually determined fun ⁇ gicidic activities.
  • ND not determined; negat. number: inhibits growth; posit, number: stimulates growth
  • the results reveal that the lactic acid bac ⁇ teria strains mentioned inhibit the growth of harmful Fusarium moulds and other detrimental microbes occur- ring in the malting process, exerting substantially no influence on useful microbes.
  • the results demonstrate the usability of lactic acid bacteria in the procedure of the present invention.
  • a sterile culture broth of lactic acid bacteria prepared according to Example 1, was used. All the other test strains were cultivated for 16 to 18 hrs in Iso-Sensitest broth (Oxoid) except the Listeria strain, which was grown in tryptose/phos- phate broth. Cultivating temperature was 30°C. except for the Salmonella, Listeria and Staphylococcus strains, 37°C.
  • microbicidic activity was determined by the turbidometric method, described in Example 1. Ex ⁇ perimental conditions, as regards growth substrate and temperature, were as described in the foregoing. The incubation time was 24 hrs, except for Bacillus and Yersinia strains, 48 hrs.
  • MRS broth was used for growth medium of ino ⁇ culum.
  • the bacteria were anaerobically cultivated for 2 days in 10 ml MRS broth, temperature 30°C.
  • the inocula ⁇ tion volume was 1% of the growth solution volume.
  • Kymppi barley of the harvest year 1990 was used, in which the proportion of Fusarium mould-contaminated kernels was 55%.
  • the barley was germinated in a germination boxes for 6 days, at 14°C. In order to maintain the moisture on the 44% level, the barley batches were moistened and turned every day. The green malt thus obtained was dried in a 21-hour temperature programme. The temperature was 50°C for 4.5 hrs. During the next 4.5 hrs it was raised to 60°C, where it was held 4 hrs. The temperature was further raised uniformly during 5 hrs, up to 85°C and held there the remaining 3 hrs. The ultimate moisture content of the malt became about 4%. Finally, the rootlets were mecha ⁇ nically removed. A malting run without any additions whatsoever served as control.
  • Lactic acid bacteria preparation and prepa ⁇ ration produced by lactic acid bacteria Lactic acid bacteria cells isolated from the culture broth and culture broths containing microbicidal compounds were employed for preparations, in combination as well as separately. Culture broth including cells was added 120 ml per kg of barley, or the cells were separa- ted from 120 ml of culture broth. This separation was done by centrifuging the culture broth, and the cells were suspended in water. In the cases in which culture broth was added, the culture broth was used as such. The cell counts of the preparations added were on the order of about 1100 88 ttoo 1100 99 CCFFUU//mmll..
  • Asperqillus and Penicilliummoulds were assessed using selective malt salt agar (EBC-Analytica Microbiolo ⁇ gica, Part II, 1987). Other most common moulds were assessed on moistened filter paper.
  • Lactic acid bacteria were assessed on MRS agar in the case of cultures added as well as malting samples .
  • Table 5 are given the counts of Fusarium moulds as well as lactic acid bacteria in the different malting steps on addition of E-390 culture broth.
  • Figs 2 and 3 are presented the total bac ⁇ terial counts in different malting steps for E-390 culture broths and for E-390 cells.
  • Table 6 presents the results of malt analysis on addition of E-390 culture broth or E-390 cells.
  • results here obtained reveal that treatments according to the invention reduce in particular the Fusarium mould quantity and the total bacterial count in different steps of malting. Addition of preparation had no detrimental effect on malt quality. Quite the opposite is true: treatment conforming to the invention improved the filtration of the mash derived from the wort and lowered the ⁇ -glucan content of the malt.
  • Example 5 THE EFFECT OF LACTIC ACID BACTERIA PREPARA ⁇ TIONS AND OF PREPARATIONS PRODUCED BY LACTIC ACID BAC ⁇ TERIA ON THE MICROFLORA OF MALTING AND ON MALT QUALITY 1.
  • Lactobacillus plantarum VTT-E-78076 (E-76)
  • the strains were obtained from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland) .
  • the preparations were made as described in
  • Example 1 item 3., Example 3, item 1., and Example 4, items 1. and 2.
  • Example 3 Malting was carried out as described in Example 3, however with duration of the germination step, 8 days. The final moisture content of the malt came to be less than 5%.
  • Test No. 1 Control, Kymppi barley 1991;
  • E-76 culture broth with cells is added:
  • Test No. 3 At beginning of steeping I, 120 ml tenfold concentrated E-76 culture filtrate is added;
  • Test No. 4 At beginning of steepings I and II, 120 ml tenfold concentrated E-76 culture filtrate is added; Test No. 5: At beginning of steepings I and II, 120 ml E-390 culture broth is added; Test No. 6: At beginning of steeping I, 120 ml tenfold concentrated E-390 culture filtrate is added; Test No. 7: At beginning of steepings I and II, 120 ml tenfold concentrated E-390 culture filtrate is added; Test No. 8: At beginning of steepings I and II, 60 ml E- 76 culture broth with cells and 60 ml E-390 culture broth with cells are added. 4.2. Second malting:
  • Test No. 11 At beginning of steepings I and II, 120 ml E-76 culture broth with cells is added; Test No. 12: At beginning of steepings I and II, 120 ml E-76 fractionated concentrate is added, pH 3.8; Test No. 13: At beginning of steepings I and II, 120 ml twenty-fold concentrated E-76 culture filtrate is added; Test No. 14: At beginning of steepings I and II, 120 ml E-390 culture broth with cells is added; Test No. 15: At beginning of steepings I and II, 120 ml E-390 fractionated concentrate is added, pH 3.8;
  • Test No. 16 At beginning of steepings I and II, 120 ml twenty-fold concentrated E-390 culture filtrate is added.
  • Figures 4 and 5 show the total bacterial counts at different steps in the malting process on application of lactic acid bacteria culture broths with cells or of concentrated or fractionated culture filtrates in the steeping waters.
  • Tables 11 and 12 present the results of malt analysis from both malting runs.
  • Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
  • Lactic acid bacteria content of the barley 7,5x10' CFU/g.
  • Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
  • Lactic acid bacteria content of the barley 7,5x10' CFU/g.
  • Extract content coarse % dry m. 76,8 77 76,1 75,2 77 76,4 74,8 77,1
  • Extract content coarse % dry m. 78,4 77,9 77,4 73,3 73,5 77,6 76,6 75,1
  • EXAMPLE 6 THE EFFECT ON MASH FILTRATION OF PREPARATIONS PRODUCED BY LACTIC ACID BACTERIA, ADDED TO THE MALTING
  • Fig. 6 a diagram is reproduced, showing the effect of treatment according to the invention with preparations derived from lactic acid bacteria strains (120 ml culture broth per kg of barley) on the filtration of the mash produced using malt thus treated.
  • the strains mentioned in Example 1 were used in this experiment: E-390, E-416, E-98, E-317, E-390, E-76,, and E-315.
  • the test was carried out using the Tepral filtra ⁇ tion method (BIOS. 19, 1988, Grandclerc, J. et al. , , "Simplification de la methode de filtration du brassin tepral description de la methode", p. 88-92).
  • microorganism shall be made available as provided in Rule 31F(1) of the Patent Rules only by the issue of a sample to an expert nominated by the requester.
  • the furnishing of samples to a third party may be subjected to the condition that that party indicates to the depositary institution its name and address for the purpose of information of the depositor and undertakes: a) not to make available the deposited culture or a culture derived from it to a third party; b) not to use the culture outside the purview of the law; c) to produce, in case of a dispute, evidence that the obligations under items (a) and (b) have not been violated.
  • DSM Deutsche Sammlung von Mi roorganismen und Zellkulturen
  • microorganism shall be made available as provided in Rule 31F(1) of the Patent Rules only b the issue of a sample to an expert nominated by the requester.
  • the furnishing of samples to a third party may be subjected to the condition that that party indicates to the depositary institution its name and address fo the purpose of information of the depositor and undertakes: a) not to make available the deposited culture or a culture derived from it to a third party; b) not to use the culture outside the purview of the law; c) to produce, in case of a dispute, evidence that the obligations under items (a) and (b) have not been violated.

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Abstract

The invention concerns a procedure for treating seed material which is to be germinated, to said seed material being added, in conjunction with the germination process, a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria and having an effect inhibiting microbial growth.

Description

PROCEDURE FOR TREATMENT OF SEED MATERIAL TO BE GER¬ MINATED
FIELD OF THE INVENTION
The present invention concerns a procedure for treating seed material intended to be germinated.
BACKGROUND OF THE INVENTION
A germination process is in this context un¬ derstood to be a process step in general which is re¬ quired in order to produce a germinated product, start¬ ing from storage-dry seed. In the brewery and distil- lery industry, for instance, a germination process, that is the malting process, is applied in producing the important raw material of beer, or other alcoholic beverages, viz. cereal malt, such as barley malt, rye malt or any malt whatsoever. The germination process is furthermore applied in producing various commercial sprouts products, e.g. bean sprouts or any sprouts for use in human nutrition.
Germination processes are usually carried out in non-aseptic conditions. On the seeds being treated there occur microbes originating from the growth envi¬ ronment or from storage. Conditions during the germina¬ tion process are mostly favourable to the microbes present on the seeds, such microbes usually multiplying during the course of the process. The microbes may exert a detrimental effect on the germinated product, or on the end product which may ultimately be made thereof, while they may equally have beneficial influ¬ ence on the product being germinated.
The main steps in brewing process are: mal- ting, wort production, primary and secondary fermenta¬ tion, and downstream processing. The purpose with mal¬ ting is to produce in the kernel enzymes which in the mashing step decompose the substances of the kernel's endosperm to a form soluble in the wort. For example, the barley seed malting process is well known to comp¬ rise three steps; steeping, germination and kilning. The cleaned and screened barley grains are steeped in water until desired moisture is achieved, e.g. on the order of 43 to 44%. Part of the steeping may be accom¬ plished in so-called air rest. The barley is allowed to germinate in controlled conditions, and the germinated barley is kilned in a hot air current until the ger¬ mination has come to an end. On termination of kilning, the rootlets are removed from the malt. Regulation of the malting steps is based on temperature, air flow and moisture/humidity control. Malt quality is affected, on the side of malt¬ ing technique and malting conditions, also by the mi- crobial flora of the malt cereal, this flora varying significantly e.g. depending on cereal variety, weather conditions, growth site, length of growing season and storage conditions.
Barley is the cereal most often used in malt¬ ing. The inherent, natural microbial flora of barley can be classified as field and storage fungi, bacteria and yeasts. The commonest field fungi of barley are: Fusarium, Alternaria, Cladosporium, Cephalosporium, Epicoccum, and Helminthosporium. The occurrence of moulds is different in different countries and differ¬ ent years. Wet weather conditions during the cereal's growth period, and particularly while it is being har- vested, favour the growth of Fusarium mould. Fusarium contamination may be heavy indeed in rainy growth sea¬ sons. One of the commonest bacterium species on cereals is Enterobacter aqqlomerans. Other bacteria which should be mentioned are; Escherichia coli, and bacteria of genera Pseudomonas, Micrococcus and Bacillus, and lactic acid bacteria. The bacterial count on barley is about IO5 to 108 CFU/g (colony forming units per g) . The moulds and bacteria in barley increase during malting, and peak concentrations are usually reached during the germination step. Fusarium moulds, in particular, and lactic acid bacteria undergo the strongest proliferation. Yeasts also increase during malting. In the kilning step the mould, yeast and bac¬ terium concentrations go down again, as a rule. Part of the microbes present on barley have a useful effect in view of malting and of the product to be made of the malt, e.g. beer. It has been estimated that up to 40- 50% of some enzymes in the malt are of microbial ori¬ gin. On the other hand, part of the microbes exert a detrimental influence on the barley and/or the malts. Among the disadvantageous microbes Fusarium moulds deserve to be mentioned, which have been found to cause particularly detrimental gushing of beer more often than other moulds, the peptides produced by said moulds constituting nuclei for gas bubbles discharging from the beer bottle in the form of powerful gushing. When more than 50% of malted kernels are contaminated with Fusarium moulds, the risk of gushing is clearly increased.
Further, gram-negative bacteria present in the barley, such as species belonging to genera Pseudomonas and Flavobacterium, and gram-positive bacteria of genus Leuconostoc have been shown to retard filtration of the mash in connection with wort production. Various micro¬ bes present in barley may also give rise to other di¬ sadvantageous effects, e.g. inhibit the germination, cause off-flavours or unfavourable changes in the ana¬ lysis values of the wort and the beer.
The quality requirements of malt barley can be specified in annually established cultivation contract and delivery terms. Moulds are a group of microbes fre- quently mentioned in quality specifications. Many malt¬ ing plants have moreover imposed an upper limit on cer¬ tain moulds. If the proportion of Fusarium-contaminated kernels exceeds 65% or if the corresponding proportion of Asperqillus and Penicillium moulds exceeds 50%, the barley can be classified as poor in quality or even as unfit for use in malting. Attempts have been made to prevent gushing in¬ duced by moulds, by using barley of good quality or by blending barley, malt or beer batches. In rainy years nearly the whole barley crop may be poor in quality, in which case it may be impossible to obtain good barley. Microbicidic chemicals have also been tried in order to reduce the quantity of moulds, but no safe and general¬ ly approved chemical could be found.
A germinating process for producing sprouts intended to be used for nutrition offers likewise pro- pitious conditions for proliferation e.g. of moulds and bacteria. Such sprouts products will spoil rapidly. Further still, in connection with germination increase may take place e.g. of foodstuff pathogens causing food poisoning, such as Salmonella, Yersinia and/or Listeria bacteria.
SUMMARY OF THE INVENTION
The object of the present invention is to eliminate the drawbacks just discussed.
Specifically, an object of the invention is to provide a procedure by which the quantity and quality of the microbial flora can be carefully regulated du¬ ring the germinating process, however without dis- advantageously affecting the quality of the germinated product or of the end product potentially made of the product.
Regarding the features characterizing the in¬ vention, reference is made to the claims. DETAILED DESCRIPTION OF THE INVENTION
The invention is based on studies in connec¬ tion of which the unexpected observation was made that lactic acid bacteria can be used towards improving the quality of a product to be germinated. The substances comprised and/or produced by the preparation of the invention, i.e., the microbicidic agents, inhibit the growth of detrimental microorganisms occurring in con- nection of a germinating process.
The use of lactic acid bacteria in foodstuff and animal feed industry is well known in the art. They produce in fermentative conditions such compounds which affect the composition and flavour of the products, but which also inhibit the growth of pathogenic microbes tending to spoil said products. Lactic acid bacteria have been commonly used in dairy products, meat products, vegetable fermentation and bakery products, and in fodder preservation. Addition of lactic acid bacteria or of lactic acid has been practiced in the production of a certain malt type, of so-called Sauermalz. This addition is made to the malt in the kilning step, prior to mashing or during mashing. The purpose with the addition is merely to cause lowering of the wort pH and, thus, to exert an influence on the course of the mashing process and on the quality of the finished beer. However, lactic acid bacteria have not heretofore been used as taught by the invention: to inhibit the growth of undesired microbes in connection with the germinating process.
The preparation of the invention can be added to the product to be germinated in any step of the ger¬ minating process.
In a particularly advantageous embodiment, lactic acid bacteria preparation, or preparation pro¬ duced by a lactic acid bacteria, is added to cereal material, such as barley kernels, during the course of the malting process. The preparation added inhibits the growth of moulds, in particular of harmful Fusarium moulds, and of bacteria, as a result of which, for in¬ stance, the risk of beer gushing due to Fusarium mould is reduced. However, the preparation has no substantial effect on the action of the useful microbial flora as regards the quality of the malt that is obtained or of the beer therefrom produced. No harmful effects of the preparation added on malt quality have been observed either, nor has it been found to contain or to produce compounds which are harmful in view of the malt, or beer, being produced.
In a malting process, the lactic acid bacteri¬ um preparation, or the preparation produced by a lactic acid bacteria, can be added to the barley kernels be¬ fore steeping, in the steeping step or in the germina¬ tion step. The addition is advantageously made in the steeping or germinating step. The malting process may be carried out, in other parts, in any manner known in itself in the art. If desired, e.g. nutrients may be added to the barley to be malted, or the conditions may be regulated, e.g. lactic acid added, in order to opti¬ mize the conditions for growth of lactic acid bacteria. It is possible in the invention to use any commonly available lactic acid bacterium whatsoever which possesses influence of inhibiting microbial growth. The following usable lactic acid bacterium genera may be mentioned: Lactococcus, Leuconostoc, Pediococcus and Lactobacillus. The following may be mentioned to present advantageous species: Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus dam- nosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus, and Lactobacillus plantarum, or any mixtures of these, among these the following being particularly advantageous: Lactobacillus plantarum and Pediococcus pentosaceus or mixtures thereof. Use of genetically modified lactic acid bacteria is equally possible.
The lactic acid bacteria preparation may be composed of culture broth, with or without cells, or of concentrated culture broth (including cells). A pre- paration produced by lactic acid bacteria may consist of cell-free culture filtrate, of concentrated culture filtrate, of fractionated culture filtrate, or of a pure or partly purified microbicidic product.
According to a particularly advantageous embodiment, the treatment is carried out with concen¬ trated or fractionated culture broth, which may be cell-free or may contain cells. Concentration may be accomplished e.g. by lyophilization or by evaporation. The culture broth is concentrated e.g. by a factor of 2 - 20 - 40.
Fractionation, i.e., purification of the mi¬ crobicidic products, can be carried out in a manner known in itself in the art, e.g. with the aid of chro- matographic methods or by ultrafiltration. In the procedure of the invention, the micro¬ bial growth-inhibiting activity of the preparation con¬ taining lactic acid bacteria, or of preparation pro¬ duced by lactic acid bacteria, to be added to the seed material corresponds e.g. to the culture broth quantity of about 10 to 10,000 ml/kg of seed material to be treated, suitably 30 to 7,000 ml/kg of seed material to be treated, e.g. 40 to 5,000 ml/kg of seed material to be treated. The preparation of the culture broth is described in the Examples section. It should be noted that in the application in hand the activity of the preparation is defined with the aid of the culture broths employed. It is obvious to a person skilled in the art that preparations according to the invention possessing equivalent microbial growth-inhibiting activity can equally be produced applying other culture broths and/or procedures.
According to the invention the preparation contains microbicidic compounds, and/or the preparation produces microbicidic compounds during the course of the germination process. When a cell preparation is employed, cell growth can be promoted, if required, e.g. by regulating the conditions during the germina¬ tion process, or by adding nutrients. The preparation may also accelerate the growth of other lactic acid bacteria present in the material to be germinated.
Since the use of lactic acid bacteria in food- stuffs is allowed and generally approved, the prepara¬ tion derived from lactic acid bacteria growing is also safe to use. Lactic acid bacteria belong usually to the natural microbe flora of seeds to be germinated, such as barley kernels. Therefore the procedure of the in- vention is maximally natural. It is also possible to use for lactic acid bacterium strain a strain inherent¬ ly occurring on the seeds.
Thanks to the invention, it becomes possible in malting to reduce the detriments arising from Fusa- rium contaminations, such as gushing of beer.
Moreover, the procedure has unexpectedly been found to improve the filtrability characteristic in the brewing process. This has been found to be due to the fact that the preparation of the invention also rest- ricts the counts of harmful species occurring in mal¬ ting and retarding the filtration of the mash, e.g. those belonging to genera Leuconostoc, Pseudomonas and Flavobacterium.
According to another advantageous embodiment, lactic acid bacteria preparation or preparation pro¬ duced by a lactic acid bacterium is added to the seed material when producing sprouts to be used for food.
Thanks to the present invention, the growth of harmful microbes can be restricted in connection with a germination process. For the first time, the invention enables biological expedients to be used in order to prevent the growth during an industrial germination process of detrimental bacteria occurring on seed which is to be germinated.
The procedure of the invention improves the general hygienic standard of the germination process even on the whole.
In the following the invention shall be illus¬ trated with embodiment examples, which are merely in¬ tended to illustrate the invention, without confining it to them. The treatment of the invention is also applicable in other germination processes.
Fig. 1 shows a graph representing the micro¬ bicidic activity in a lactic acid bacteria culture filtrate, as assessed by the turbidometric method. The normal growth curve of the test organism, E.aqqlomerans E-396, and the inhibition effect exerted on the growth of the test organism by culture filtrates of the pro¬ duction strains L. plantarum E-76 and P. pentosaceus E-390.
In Fig. 2 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 culture broth added at different stages of the malt- ings.
In Fig. 3 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 cells added at different stages of the maltings.
In Fig. 4 are shown the total bacterial counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated culture broths, to the barley steeping water.
In Fig. 5 are shown the total bacterium counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated and fractionated culture broths, to the barley steeping water.
Fig. 6 displays the effect of lactic acid bac¬ teria cultures, added to the malting, on mash filtrati- on (Tepral filtration)
Example 1: THE MICROBICIDIC EFFECT OF VARIOUS LACTIC ACID BACTERIA STRAINS ON MICROBES OCCURRING IN MALTING
In the experiment, the microbicidic effect on microbes occurring in malting exerted by preparations produced by various lactic acid bacteria strains were studied. The sterile-filtrated culture broth was used for preparations.
1. Production strains:
The following lactic acid bacteria strains were used for production strains:
Lactobacillus lactis ssp. lactis VTT-E-90414 (E-414) ssp. diacitilactis VTT-E-90423 (E-423)
Leuconostoc mesenteroides ssp. mesenteroides VTT-E-90389 (E-389) ssp. mesenteroides VTT-E-90415 (E-415) ssp. mesenteroides VTT-E-90466 (E-466)
Pediococcus damnosus VTT-E-76065 .E-65)
Pediococcus parvulus VTT-E-88315 E-315)
Pediococcus pentosaceus VTT-E-76067 | E-67)
Pediococcus pentosaceus VTT-E-76068 [E-68)
Pediococcus pentosaceus VTT-E-88317 E-317)
Pediococcus pentosaceus VTT-E-90390 | E-390) DSM 7389)
Lactobacillus curvatus VTT-E-90391 ( E-391)
Lactobacillus plantarum VTT-E-78076 ( E-76) DSM 7388)
Lactobacillus plantarum VTT-E-79098 ( E-98)
The strains were obtained from the VTT, Col¬ lection of Industrial Microorganisms (Biotechnical Laboratory, Finland). Lactobacillus plantarum (E-76) has been deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under number 7388. The strain E-76 (DSM 7388) had been isolated from beer by known techniques used for liquid products, and analy¬ zed/identified using well known analysis methods. Pe¬ diococcus pentosaceus (E-390) has been deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkultu- ren) under number 7389. The strain E-390 (DSM 7389) had been isolated from homogenized samples of split barley kernels and identified/analyzed using techniques well known in the art [see the article of Haikara, A. and Home, S., Mash Filtration difficulties caused by split barley kernels: a Microbiological problem, in the pub¬ lication of EBC Congress 1991 (Quality Control)]. The deposits are under the provisions of the Budapest Trea¬ ty.
2. Test strains: Various harmful microbial species occurring at malting were used for test strains, as well as lactic acid bacteria strains which served as production strains, among others.
Harmful moulds were represented in the tests by Fusarium moulds fGibberella avenacea (former Fusari¬ um avenaceum) VTT-D-80141 (D-141) and VTT-D-80147 (D- 147), and Fusarium culmorum VTT-D-80148 (D-148) and VTT-D-80149 (D-149), Collection of Industrial Microor¬ ganisms, Biotechnical Laboratory of VTT], and one As- perqillus species.
Harmful gram-negative bacteria were represent¬ ed by two strains from genus Enterobacter and by one species each from genus Flavobacterium and genus Pseu¬ domonas. The lactic acid bacteria consisted of those strains which were employed as production strains, plus the strain Lactococcus sp. E-416.
3. Cultivation of production strains, and preparation of sterile filtered culture broth:
The lactic acid bacteria were cultivated in MRS broth (MRS BROTH, Oxoid) . The Pediococcus strains
E-65, E-67 and E-68 were aerobically cultivated at
25°C, all other production strains anaerobically at 30°C, cultivation period varying from 2 to 5 days. The cells were then centrifuged and the supernatant was sterile-filtered.
4. Cultivation of test strains: Spore suspensions were produced from the Fusa¬ rium moulds by cultivating the mould strain in CMC (carboxymethyl cellulose) solution at 25°C, 5 to 6 days as a shaking culture, dispersing the spore formations in TWEEN solution, filtering the suspension and recov- ering the filtrate.
The spore suspension of Asperqillus mould was produced directly on PD agar (25°C, 3 days) (Potato dextrose, Difco) .
Gram-negative bacteria were aerobically cul- tivated in NB broth (Nutrient broth, Difco) for 1 day, the Enterobacter strain at 30°C, and the Flavobacterium and Pseudomonas strains at 25°C.
Lactic acid bacteria were cultivated as de¬ scribed under 3. above. 5. Examination of the microbicidic effect of the lactic acid bacteria:
The microbicidic activity in the culture broth was assessed by the disk method, or turbidometrically.
5.1. Disk method for assessment of microbicid¬ ic activity:
Sterile-filtered culture broth, or a dilution thereof, was pipetted onto a filter paper disk (12.7 mm dia.), 100 μl. The disks were placed on Plate Count agar dishes into which 0.3 ml of test organism diluti¬ on, 10"2, had been pour plated. The specimens were cul¬ tivated for 24 hrs at 30°C, whereupon the diameter in mm of the inhibition zone that had formed was measured.
5.2. Turbidometric method for assessment of microbicidic activity:
An automatic turbidometer (Bioscreen, Labsys- terns) was employed in the procedure.
The sample contained 10% by vol. of the test organism and 10% by vol. of the sterile-filtered cul¬ ture broth of the production strain, calculated on the sample volume, and growth medium. For controls, the sterile filtrate preparation was replaced with distil¬ led water of which the pH had been adjusted with lactic acid to identical level with the sterile-filtered pre¬ paration. The growth medium used was, in the case of each test strain, the same medium as in the test strain cultivation.
The growth conditions for Fusarium and Asper- qillus moulds were: 5 days, 25°C, powerful shaking. Those for gram-negative bacteria were: Enterobacter strain, 30°C, all others 25°C, 2 days, and shaking. The growth conditions for lactic acid bacteria were: 3 days, 30°C, and shaking.
The apparatus determined from the samples the absorbance at visible light wavelength 420-580 nm. After cultivation, the growth curve of each sample could be made up and the area subtended by the curve calculated.
The microbicidic effect of a production strain on the growth of the test strain was expressed by an inhibition percentage, obtained by comparison of the growth area sizes found with the control and with the sterile-filtered preparation of the production strain, respectively. 6. Examination of the fungicidic effect of certain lactic acid bacteria:
The fungicidic effect of six lactic acid bac¬ teria strains, E-76, E-98, E-315, E-317, E-414 and E-415, separately on all Fusarium moulds employed as test strains was studied. In the test, a visual exami¬ nation was made of turbidity formation in mould cul¬ tures to which sterile-filtered preparation from each lactic acid bacteria cultivation had been added at various dilutions, stated in Table 3.
In the controls, sterilized Milli-Q water and Milli-Q water adjusted with lactic acid to pH 3.6 was used instead of culture broth.
Cultivation took place as a test tube culture in CMC broth, at 25°C, during 5 days. The results were visually read.
7. Results:
Tables 1 and 2, and Fig. 1, display the micro¬ bicidic activities determined for the different lactic acid bacteria strains by the disk method and by the turbidometric method, respectively.
Table 3 displays the visually determined fun¬ gicidic activities.
TABLE 1. Microbicidic activities of lactic acid bacteria culture broths, by the disk method.
Cell count of test organism E-396 - 4.0xl07 CFU/ml; on dish - 1.2xl05 CFU/ml.
TABLE 2 . Microbisidic effe ct of lact ic acid bacteria on the growth of various microbes .
ND: not determined; negat. number: inhibits growth; posit, number: stimulates growth
>55% strong inhibition; 35-55% fairly strong inhibition; 15-34% fairly weak inhibition; 15% weak or no inhibition
-'-'inducing beer gushing; 2'retards mash filtration
TABLE 3. Effect of lactic acid bacteria culture broth Fusarium fungi causing gushing of beer.
+ distinct growth; - no growth
The results reveal that the lactic acid bac¬ teria strains mentioned inhibit the growth of harmful Fusarium moulds and other detrimental microbes occur- ring in the malting process, exerting substantially no influence on useful microbes. The results demonstrate the usability of lactic acid bacteria in the procedure of the present invention.
Example 2: THE MICROCIDIC EFFECT OF CERTAIN LACTIC ACID BACTERIA STRAINS ON FOODSTUFF PATHOGENS AND ON MICROBES DETRIMENTAL TO FOODSTUFFS
In the experiment a study was made of the microcidal effect on foodstuff pathogens and on micro- bes detrimental to foodstuffs of preparations produced with the aid of Pediococcus pentosaceus VTT-E-90390 (E- 390) and Lactobacillus plantarum VTT-E-78076 (E-76) strains. As test organisms, strains belonging to genera Bacillus, Yersinia, Listeria, Pseudomonas, Salmonella and Staphylococcus were employed.
As a preparation, a sterile culture broth of lactic acid bacteria, prepared according to Example 1, was used. All the other test strains were cultivated for 16 to 18 hrs in Iso-Sensitest broth (Oxoid) except the Listeria strain, which was grown in tryptose/phos- phate broth. Cultivating temperature was 30°C. except for the Salmonella, Listeria and Staphylococcus strains, 37°C.
The microbicidic activity was determined by the turbidometric method, described in Example 1. Ex¬ perimental conditions, as regards growth substrate and temperature, were as described in the foregoing. The incubation time was 24 hrs, except for Bacillus and Yersinia strains, 48 hrs.
The results are shown in Table 4, which reveals that addition of the lactic acid bacteria preparation of the invention causes inhibition of the growth of food¬ stuff pathogens and microbes detrimental to foodstuffs.
TABLE 4. Growth inhibition caused by P. pentosaceus E-390 and L. plantarum E-76.
Production strains E-390 E-76 Test organisms Reduction of growth area, %
Bacillus cereus 93 80 ATCC91339 *)
Yersinia enterolitica 85 54 ELI351 *)
Listeria monocytogenes 41 49 KTL4126 *)
Pseudomonas fluorescens 59 97 ELI97
Pseudomonas fragi 84 93 ATCC4973
Salmonella infantis 90 98 ELI5 *)
Staphylococcus aureus 73 86 EL1200 ")
) Pathogens occurring in foodstuffs
ATCC: American Type Culture Collection ELI: VTT, Food Research Laboratory KTL: National Public Health Institute Example 3: THE EFFECT OF LACTIC ACID BACTERIA PREPARA¬ TIONS AND OF PREPARATIONS PRODUCED BY LACTIC ACID BAC¬ TERIA ON THE MICROFLORA OF MALTING AND ON MALT QUALITY
1. Strain employed: The lactic acid bacteria strain Pediococcus pentosaceus VTT-E-90390 (E-390) was used in the experi¬ ment.
MRS broth was used for growth medium of ino¬ culum. The bacteria were anaerobically cultivated for 2 days in 10 ml MRS broth, temperature 30°C. The inocula¬ tion volume was 1% of the growth solution volume.
2 . Barley:
Kymppi barley of the harvest year 1990 was used, in which the proportion of Fusarium mould-contaminated kernels was 55%.
3. Malting process:
1000 g barley batches were rinsed in a water bath at 12°C for 1 hr. The rinsing water was replaced with the first steeping water, and this was replaced with the second steeping water after 5 hrs. Air rest was commenced 16 hrs thereafter. The purpose with air rest was to eliminate the water on the surface of the kernels. Its duration was 8 hrs. During the steepings, the barley attained 44% moisture. The barley was aerated throughout the steeping process.
Steeping was succeeded by germination. The barley was germinated in a germination boxes for 6 days, at 14°C. In order to maintain the moisture on the 44% level, the barley batches were moistened and turned every day. The green malt thus obtained was dried in a 21-hour temperature programme. The temperature was 50°C for 4.5 hrs. During the next 4.5 hrs it was raised to 60°C, where it was held 4 hrs. The temperature was further raised uniformly during 5 hrs, up to 85°C and held there the remaining 3 hrs. The ultimate moisture content of the malt became about 4%. Finally, the rootlets were mecha¬ nically removed. A malting run without any additions whatsoever served as control.
4. Lactic acid bacteria preparation and prepa¬ ration produced by lactic acid bacteria: Lactic acid bacteria cells isolated from the culture broth and culture broths containing microbicidal compounds were employed for preparations, in combination as well as separately. Culture broth including cells was added 120 ml per kg of barley, or the cells were separa- ted from 120 ml of culture broth. This separation was done by centrifuging the culture broth, and the cells were suspended in water. In the cases in which culture broth was added, the culture broth was used as such. The cell counts of the preparations added were on the order of about 110088 ttoo 110099 CCFFUU//mmll..
5. Additions of lactic acid bacteria prepara- tion:
The additions of lactic acid bacteria prepara¬ tion were made either to the barley, to the beginning of steeping I, to the beginning of steepings I and II, or to the beginning of germination.
6. Analyses performed:
Samples were drawn from each malting step.
6.1 Moulds were assessed as follows. The per- centage of kernels contaminated with Fusarium moulds was determined by means of CZAPEK IPRODION DICLORAL agar (CZID agar), which is selective to Fusarium moulds, and a moist filter paper (EBC-Analytica Microbiologica, Part II, 1987). Fusarium moulds were identified by their typical colony and spore morphology and by the red colour.
Asperqillus and Penicilliummoulds were assessed using selective malt salt agar (EBC-Analytica Microbiolo¬ gica, Part II, 1987). Other most common moulds were assessed on moistened filter paper.
6.2 Lactic acid bacteria were assessed on MRS agar in the case of cultures added as well as malting samples .
6.3 The total bacterial counts were assessed on Plate Count agar (Difco).
6.4 Chemical characteristics of the malt were determined employing methods known from the context of malting (EBC-Analytica, 1987, 4th Ed.).
7. Results:
In Table 5 are given the counts of Fusarium moulds as well as lactic acid bacteria in the different malting steps on addition of E-390 culture broth.
In Figs 2 and 3 are presented the total bac¬ terial counts in different malting steps for E-390 culture broths and for E-390 cells. Table 6 presents the results of malt analysis on addition of E-390 culture broth or E-390 cells.
TABLE 5. Effect of P. pentosaceus E-390 culture broth added at different salting steps, on Fusarius and lactic acid bacteria counts during salting.
CB; culture broth
TABLE 6.Effect of culture broth or cells of P. pentosaceus E-390 added at various steps of salting o salt quality.
Analysis β- Mois¬ Ext¬ Coar¬ Ext¬ Cla¬ Filt¬ Sac- Vis¬ FAN Modi¬ Homo¬ α-amy- β-glu- Root¬ glu- ture ract se ract rity ration char- cos¬ fica¬ gene¬ lase canase lets cans grind dif¬ fine ific. ity tion ity acti¬ acti¬ fer. grind time vity vity Sample mg/1 % %d.m. %d.m. %d.m. ml/h min. cP mg/1 % % U/g U/kg g/kg
Control 604 4,1 80,1 77,8 2,3 clear 300 <10 1,59 163 82 69 202 365 35
120 ml CB*' to 496 4,3 80,3 77,3 3,0 clear 305 <10 1,51 164 80 57 207 355 37 barley
120 ml CB*' to 482 4,2 80,5 77,5 2,9 clear 310/56 <10 1,50 171 76 51 194 316 30 1st st. water min
120 ml CB*' to 482 4,3 80,0 76,9 3,1 clear 305 <10 1,49 168 80 64 186 360 31 1+2 st. water
120 ml CB*' to 955 4,2 79,0 74,9 4,1 opal. 310 <10 1,68 129 69 59 159 386 21 germination
Cells (IO8 CFU/g) 606 4,3 80,3 77,4 2,9 clear 315/61 <10 1,54 167 79 59 223 359 38 to barley min
Cells (IO8 CFU/g) - - - - - - - - - 79 59 187 376 39 to 1st st. water
Cells (IO8 CFU/g) - - - - - - - - - - 81 67 178 353 36 to 1+2 st. water
Cells (IO8 CFU/g) 804 4,3 79,5 76,6 2,9 opal . 290 <10 1,63 148 81 69 179 392 35 to germination
- not analysed
CB; culture broth
The results here obtained reveal that treatments according to the invention reduce in particular the Fusarium mould quantity and the total bacterial count in different steps of malting. Addition of preparation had no detrimental effect on malt quality. Quite the opposite is true: treatment conforming to the invention improved the filtration of the mash derived from the wort and lowered the β-glucan content of the malt.
Example 4: PRODUCTION OF PREPARATIONS PRODUCED BY LACTIC ACID BACTERIA
1. Preparation of concentrated culture broths: The production strains, Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E- 78076 (E-76) were cultivated in a fermentor in a 15 1 volume. Inoculum volume was 6% to 7%, culture was made on MRS medium, at 30°C, 2 days in microaerophilic condi¬ tions. The culture broths were concentrated tenfold and twenty-fold by lyophilizing and evaporating procedure. The microbicidic activity of the concentrates was ascer¬ tained by the disk method.
2. Preparation of a purified solution containing microbicidic compounds: The lactic acid bacteria culture broth was purified by gel chromatographic fractionation according to molecule size. The fractions found by disk method and by turbidometry to be active were collected and rerun through the gel column.
Example 5: THE EFFECT OF LACTIC ACID BACTERIA PREPARA¬ TIONS AND OF PREPARATIONS PRODUCED BY LACTIC ACID BAC¬ TERIA ON THE MICROFLORA OF MALTING AND ON MALT QUALITY 1. Strains employed and preparations produced from the strains:
The following bacterial strains were used: Lactobacillus plantarum VTT-E-78076 (E-76) Pediococcus pentosaceus VTT-E-90390 (E-390)
The strains were obtained from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland) . The preparations were made as described in
Example 1, item 3., Example 3, item 1., and Example 4, items 1. and 2.
2. Barley:
The barley employed was Kymppi barley of the harvest year 1991.
3. Malting process:
Malting was carried out as described in Example 3, however with duration of the germination step, 8 days. The final moisture content of the malt came to be less than 5%.
4. Malting:
Two laboratory maltings were made. Malting without any additions served as control. 4.1 First malting: Culture broths without cells, concentrated tenfold, and untreated culture broth including cells were used for preparations in the laboratory malting. Prepa¬ ration was added at the beginning of steeping I or at the beginning of steepings I and II. Malting trials 1 to 8 were carried out as follows.
Test No. 1: Control, Kymppi barley 1991;
Test No. 2: At beginning of steepings I and II, 120 ml
E-76 culture broth with cells is added:
Test No. 3: At beginning of steeping I, 120 ml tenfold concentrated E-76 culture filtrate is added;
Test No. 4: At beginning of steepings I and II, 120 ml tenfold concentrated E-76 culture filtrate is added; Test No. 5: At beginning of steepings I and II, 120 ml E-390 culture broth is added; Test No. 6: At beginning of steeping I, 120 ml tenfold concentrated E-390 culture filtrate is added; Test No. 7: At beginning of steepings I and II, 120 ml tenfold concentrated E-390 culture filtrate is added; Test No. 8: At beginning of steepings I and II, 60 ml E- 76 culture broth with cells and 60 ml E-390 culture broth with cells are added. 4.2. Second malting:
In the laboratory malting, twenty-fold con¬ centrated culture filtrates without cells, purified microbicidic fractions without cells and untreated culture broths with cells were used for preparations. The steeping water pH was controlled. Preparation was added at the beginning of steepings I and II. Malting trials 9 to 16 were carried out as follows. Test No. 9: Control, Kymppi barley 1991; Test No. 10: At beginning of steepings I and II, 120 ml water is added, pH 3.8;
Test No. 11: At beginning of steepings I and II, 120 ml E-76 culture broth with cells is added; Test No. 12: At beginning of steepings I and II, 120 ml E-76 fractionated concentrate is added, pH 3.8; Test No. 13: At beginning of steepings I and II, 120 ml twenty-fold concentrated E-76 culture filtrate is added; Test No. 14: At beginning of steepings I and II, 120 ml E-390 culture broth with cells is added; Test No. 15: At beginning of steepings I and II, 120 ml E-390 fractionated concentrate is added, pH 3.8;
Test No. 16: At beginning of steepings I and II, 120 ml twenty-fold concentrated E-390 culture filtrate is added.
5. Analyses performed: Samples were drawn from each malting step.
Moulds, lactic acid bacteria, total bacterial counts and physical and chemical quality characteristics of the malt were determined as described in Example 3.
6. Results:
Figures 4 and 5 show the total bacterial counts at different steps in the malting process on application of lactic acid bacteria culture broths with cells or of concentrated or fractionated culture filtrates in the steeping waters.
In Tables 7, 8, 9 and 10 are presented the concentrations of Fusarium moulds and other moulds, and of lactic acid bacteria in different malting steps, in both malting runs.
Tables 11 and 12 present the results of malt analysis from both malting runs.
TABLE 7. Myco flora at malting on addition of P. pentosaceus E-390 and L- plantarum E-76 preparation to the barley steeping water (results given as % contaminated kernels) . Numbering of samples in Exampl 5.
Sample Bar¬ Steeping Germination Malt Mould genus ley 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8
Fusarium FP*' 35 80 64 60 22 76 76 48 68 72 88 88 94 96 80 90 94 26 22 2 3 25 17 7 24
CZID 36 74 72 46 38 84 54 32 78 84 82 80 82 84 78 80 96 36 22 10 2 24 20 8 24
Alternaria 26 6 18 22 20 18 22 12 24 18 10 8 2 10 2 2 10 18 8 17 5 18 5 11 16
Cephalosporium 37 22 38 34 36 42 12 28 24 72 20 42 80 42 32 36 24 2 3 5 4 7 2 6 2
Cladosporium 17 4 20 6 56 34 10 44 20 6 6 30 52 14 10 36 4 2 0 4 1 1 1 1 0
Rhitzopus 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 0 48 13 0 11 0
Mucor 0 2 4 0 2 8 14 0 4 0 10 28 64 24 44 34 14 65 79 78 88 68 83 96 73
Stemphylium 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 6 0 1
Epicoccum 2 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 2 1 1 0 0 3 0 1 3
Helminthosporium 10 26 46 10 10 36 16 14 30 48 64 60 42 50 54 38 58 28 26 14 3 11 23 8 20
Acremoniella 0 0 0 0 0 0 0 0 0 100 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Aspergillus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0
Penicillium 3 0 2 2 2 0 0 2 2 4 2 2 0 2 2 2 0 1 0 0 0 0 0 1 0
Other fungi 1 8 0 0 o 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0
FP; filter paper
TABLE 8. Lactic acid bacteria counts. (CFU/ml) at malting on addition of P. pentosaceus E-390 and L. plantarum E-76 preparations to the barley steeping water. Numbering of samples in Example 5.
Example Steeping Germination Malt
1 5,5xl03 3,3xl06 2,5xl02
2 7,3xl06 l,2xl07 5,5xl05
3 l,4xl03 4,4xl03 l,lxl03
4 3,2xl02 l,4xl04 9,5xl02
5 l,3xl07 7,5xl06 2,6xl06
6 4,8xl03 5,0xl05 4,0xl04
7 7,3xl02 2,4x10* 2,6xl03
8 l,2xl07 l,4xl07 2,3xl06
Lactic acid bacteria content of the barley: 7,5x10' CFU/g.
TABLE 9. Myco flora at malting on addition of P. pentosaceus E-390 and L- plantarum E-76 preparation to the barley steeping water (results given as % contaminated kernels) . Numbering of samples in Exampl 5.
Sample Bar¬ Steeping Germination Malt Mould genus ley 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16
Fusarium FP*' 35 78 84 32 30 0 44 36 28 98 86 94 98 59 96 94 72 23 28 37 14 1 16 65 13
CZID 36 78 78 30 14 0 26 28 22 98 98 92 66 12 92 92 54 38 31 36 7 1 24 44 11
Alternaria 26 6 2 34 10 10 24 20 14 8 2 14 24 0 0 14 6 17 16 25 13 2 19 27 3
Cephalosporium 37 18 18 34 24 0 36 44 44 52 18 56 52 4 36 36 58 5 3 6 11 1 14 13 20
Cladosporium 17 6 8 14 16 70 18 24 42 14 6 2 16 54 2 6 20 1 0 0 1 7 1 0 1
Rhitzopus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 20 0 0 0 0 11 0
Mucor 0 0 2 2 2 4 2 0 2 36 14 52 40 76 22 56 66 80 78 78 91 98 93 84 90
Stemphylium 0 0 0 0 0 2 0 0 0 0 0 0 2 2 0 2 0 0 4 4 1 0 3 2 0 n Epicoccum 2 0 0 2 0 0 0 0 0 0 0 0 0 0 0 2 0 0 1 0 0 0 0 0 0 ) Helminthosporium 10 32 28 44 18 10 26 28 26 66 50 66 82 6 78 66 58 46 58 46 52 7 76 55 17
Acremoniella 0 0 0 0 0 0 0 0 0 44 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 i
Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0
Aspergillus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
Penicillium 3 2 2 0 2 0 0 2 0 0 0 10 16 30 4 4 10 0 1 0 0 0 0 0 0
Other fungi 1 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 |o 1 0 0 0 0 0 0
FP ; filter paper
TABLE 10. Lactic acid bacteria counts. (CFU/ml) at malting on addition of P. pentosaceus E-390 and L. plantarum E-76 preparations to the barley steeping water. Numbering of samples in Example 5.
Example Steeping Germination Malt
9 8,0xl02 4,2xl06 8,0xl03
10 8,0xl02 5,0xl06 5,6x10*
11 2,7xl07 2,lxl07 8,5xl05
12 2,2xl03 3,4xl05 2,5xl03
13 l,2xl03 l,4xl07 2,3xl03
14 4,lxl07 3,5xl07 1,1x10*
15 4,8xl03 4,8xl06 3,9xl05
16 3,5xl02 2,lxl05 1,7x10*
Lactic acid bacteria content of the barley: 7,5x10' CFU/g.
TABLE 11. Effect of P. pentosaceus E-390 and L. plantarum E-76 preparations added to the barle steeping water on malt quality. Numbering of samples in Example 5.
Sample
Analysis 1 2 3 4 5 6 7 8
Moisture % 3,6 3,6 3,5 3,7 3,7 3,6 3,9 3,9
Extract content, fine % dry m. 81 80,8 80,6 80 81,2 81 80,7 81,2
Extract content, coarse % dry m. 76,8 77 76,1 75,2 77 76,4 74,8 77,1
Extract difference % 4,2 3,8 4,5 4,9 4,3 4,7 5,9 4,1
Saccharification min. <10 <10 <10 <10 <10 <10 <10 <10
Wort clarity clear clear clear clear clear clear clear clear
Filtration, fine grind ml/min 320/45 320/44 315/48 315/40 320/47 315/66 320/49 315/52
Filtration, coarse grind ml/min 260/86 265/64 270/59 255/73 260/68 260/76 255/90 275/120
Wort pH 5,93 5,88 5,86 5,86 5,86 5,86 5,97 5,99
Viscosity cP 1,69 1,57 1,59 1,57 1,55 1,58 1,59 1,58 β-glucans mg/1 784 659 705 736 610 696 822 657
Free amino nitrogen mg/1 154 156 155 139 158 150 130 154
Modification % 74 72 73 73 72 75 77 76
Homogeneity % 61 65 63 58 67 66 62 68 α-amylase U/g 178 182 148 128 179 156 144 174 β-glucanase U/kg 434 420 360 360 442 412 361 425
TABLE 12. Effect of P. pentosaceus E-390 and L. plantarum E-76 preparations added to the barle steeping water on malt quality. Numbering of samples in Example 5.
Sample
Analysis 9 10 11 12 13 14 15 16
Moisture % 3,5 3,6 3,6 3,5 3,8 3,5 3,5 3,6
Extract content, fine % dry m. 80,5 80,5 81,2 80,5 80,3 81 81,1 80,4
Extract content, coarse % dry m. 78,4 77,9 77,4 73,3 73,5 77,6 76,6 75,1
Extract difference % 2,1 2,6 3,7 7,2 6,8 3,4 4,5 5,4
Saccharification min. <10 <10 <10 <10 <10 <10 <10 <10
Wort clarity clear clear clear clear clear clear clear clear
Filtration, fine grind ml/min 320/40 315/47 320/44 315/42 320/55 315/49 330/40 320/40
Filtration, coarse grind ml/min 290/120 270/76 260/51 265/120 245/120 285/68 305/120 265/12
Wort PH 6,04 6,05 5,99 5,97 5,94 5,95 5,98 5,99
Viscosity cP 1,54 1,65 1,51 1,53 1,5 1,49 1,49 1,52 β-glucans mg/1 442 522 397 553 612 381 463 576
Free amino nitrogen mg/1 149 145 149 128 117 149 139 120
Modification % 85 85 86 82 74 81 75 71
Homogeneity % 63 66 76 57 57 68 51 47 α-amylase U/g 161 172 161 123 72 148 125 101 β-glucanase U/kg 365 375 365 282 282 371 342 308
The results now obtained reveal that treatments according to the invention reduce, in particular, the Fusarium mould quantity and the total bacterial count in the different malting steps. It is further noted that, in addition to culture broths, concentrated and frac¬ tionated culture filtrates in particular have a favoura¬ ble effect e.g. as regards Fusarium moulds.
From the malt analyses the inference can be drawn that culture broth additions of strains E-76 and E-390 had an effect of improving the filtrability of the wort made from the malt; the β-glucan contents are also lower in the respective malts than in the control malt.
EXAMPLE 6: THE EFFECT ON MASH FILTRATION OF PREPARATIONS PRODUCED BY LACTIC ACID BACTERIA, ADDED TO THE MALTING
In Fig. 6 a diagram is reproduced, showing the effect of treatment according to the invention with preparations derived from lactic acid bacteria strains (120 ml culture broth per kg of barley) on the filtration of the mash produced using malt thus treated. The strains mentioned in Example 1 were used in this experiment: E-390, E-416, E-98, E-317, E-390, E-76,, and E-315. The test was carried out using the Tepral filtra¬ tion method (BIOS. 19, 1988, Grandclerc, J. et al. , , "Simplification de la methode de filtration du brassin tepral description de la methode", p. 88-92).
It is evident from the results that treatment according to the invention improves the filtration of the mash. INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13tøs)
A. Th. indications made below relate to the microorganism referred to in the description on page 1 0 , line 29
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet [
Name of depositary institution
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM)
Address of depositary institution (including postal code and country)
Mascheroder eg 1B D-3300 Braunschweig Germany
Date of deposit Accession Number
15 January 1993 7388
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This infomution is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the intemational Bureau later {specify the general nature of the indications eg., 'Accessio Number of Deposit')
For international Bureau use only
I I This sheet was received by the intemational Bureau on
Authorized officer
Additional sheet for PCT/RO/134
Accession Number: 7388
C. ADDITIONAL INDICATIONS
In respect of Australia, the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or prior to the lapsing, refusal or withdrawal of the application, to a person who is skilled addressee without an interest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).
In respect of Netherlands, the microorganism shall be made available as provided in Rule 31F(1) of the Patent Rules only by the issue of a sample to an expert nominated by the requester.
In respect of Switzerland, the furnishing of samples to a third party may be subjected to the condition that that party indicates to the depositary institution its name and address for the purpose of information of the depositor and undertakes: a) not to make available the deposited culture or a culture derived from it to a third party; b) not to use the culture outside the purview of the law; c) to produce, in case of a dispute, evidence that the obligations under items (a) and (b) have not been violated.
In respect of Denmark, Finland, Norway, Sweden and United Kingdom, the furnishing of a sample shall only be effected to an expert in the art.
In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, only by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4) EPC) . INDICATIONS RELATING TO A DEPOSITED MICROORGANISM
(PCT Rule 13bis)
A. The indications made below relate to the microorganism referred to in the description on page 1 0 , line 26
B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet |
Name of depositary institution
Deutsche Sammlung von Mi roorganismen und Zellkulturen (DSM).
Address of depositary institution (including postal code and country)
Mascheroder eg 1B D-3300 Braunschweig Germany
Date of deposit Accession Number
1 5 January 1 993 7389
C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet
D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not f r all deήgttated States)
E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)
The indications listed below will be submitted to the international Bureau later {specify the general nature of the indications eg., 'Accession Number of Deposit")
For receiving Office use only For international Bureau use only ly' | This sheet was received with the intemational application | | This sheet was received by the International Bureau on:
Authorized officer Authorized officer
Additional sheet for PCT/RO/134
Accession Number: 7389
C. ADDITIONAL INDICATIONS
In respect of Australia, the furnishing of a sample of a microorganism shall only be effected prior to the grant of a patent, or prior to the lapsing, refusal or withdrawal of the application, to a person who is skilled addressee without an interest in the invention (Regulation 3.25(3) of the Australian Patents Regulations).
In respect of Netherlands, the microorganism shall be made available as provided in Rule 31F(1) of the Patent Rules only b the issue of a sample to an expert nominated by the requester.
In respect of Switzerland, the furnishing of samples to a third party may be subjected to the condition that that party indicates to the depositary institution its name and address fo the purpose of information of the depositor and undertakes: a) not to make available the deposited culture or a culture derived from it to a third party; b) not to use the culture outside the purview of the law; c) to produce, in case of a dispute, evidence that the obligations under items (a) and (b) have not been violated.
In respect of Denmark, Finland, Norway, Sweden and United Kingdom, the furnishing of a sample shall only be effected to a expert in the art.
In respect of those designations in which a European patent is sought, a sample of the deposited microorganism will be made available until the publication of the mention of the grant of the European patent or until the date on which the application has been refused or withdrawn or is deemed to be withdrawn, onl by the issue of such a sample to an expert nominated by the person requesting the sample. (Rule 28(4) EPC) .

Claims

1. A procedure for treatment of seed material to be germinated, characterized in that to the seed material is added, in connection with the germination process, a lactic acid bacteria preparation or a prepara¬ tion produced by lactic acid bacteria which has an effect inhibiting microbial growth.
2. Procedure according to claim 1, character¬ ized in that the lactic acid bacterium belongs to genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus.
3. Procedure according to claim 1 or 2, char¬ acterized in that the lactic acid bacteria preparation or the preparation produced by lactic acid bacteria has been derived from species Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvu- lus, Pediococcus pentosaceus, Lactobacillus curvatus or Lactobacillus plantarum, or from a mixture of these, advantageously from species Lactobacillus plantarum or Pediococcus pentosaceus or from a mixture of these.
4. Procedure according to any one of claims 1- 3, characterized in that to barley kernels is added in connection with a malting process, a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria having an effect inhibiting the growth of Fusarium moulds.
5. Procedure according to claim 4, character¬ ized in that the lactic acid bacteria preparation or the preparation produced by lactic acid bacteria is added in the steeping or germination step.
6. Procedure according to any one of claims 1- 5, characterized in that the lactic acid bacteria prepa¬ ration or the preparation produced by lactic acid bacte¬ ria is added to seed material going to be converted to sprouts intended to serve as nutrition.
EP93920869A 1993-01-15 1993-09-27 Procedure for treatment of seed material to be germinated Ceased EP0678120A1 (en)

Applications Claiming Priority (3)

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FI930182A FI94875C (en) 1993-01-15 1993-01-15 Process for processing industrial germinated seed material for food use
PCT/FI1993/000388 WO1994016053A1 (en) 1993-01-15 1993-09-27 Procedure for treatment of seed material to be germinated

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FI97148C (en) * 1994-07-14 1996-10-25 Lahden Polttimo Ab Oy Process for the treatment of plants to improve the quality properties of seeds
FR2733121A1 (en) * 1995-04-24 1996-10-25 Inst Francais Des Boissons De APPLICATION OF SELECTED STRAINS OF GEOTRICHUM CANDIDUM IN THE PROCESS OF MALTING OF CEREALS OR OTHER PLANTS
KR100525860B1 (en) * 1996-07-23 2005-11-02 까르질 프랑스 엔.브이. Process for the preparation of malted cereals
US6613371B2 (en) 1997-07-23 2003-09-02 Cargill, Incorporated Method for malting seeds
FI109964B (en) * 1998-11-02 2002-11-15 Lp Tutkimuskeskus Oy Method and apparatus for the treatment of cereal grains
FI991435A (en) * 1999-06-24 2000-12-25 Valtion Teknillinen Method for selection of strain of lactic acid bacteria for preservation of fresh feed and preservation of fresh feed
WO2002010331A2 (en) * 2000-07-28 2002-02-07 Grain Processing Corporation Root retardant
JP5816439B2 (en) * 2011-02-21 2015-11-18 サッポロビール株式会社 Sparkling beverage and method for producing the same
CA2852456A1 (en) * 2011-10-18 2013-04-25 Institute For Environmental Health, Inc. Improved method and apparatus for growing sprouts
RU2644345C2 (en) * 2012-04-24 2018-02-08 Карджилл, Инкорпорейтед Method to increase the malt output in the malting process
WO2015021025A1 (en) 2013-08-07 2015-02-12 Cargill, Incorporated Processes for making sprouted whole grains and products comprising sprouted whole grains
WO2018106700A1 (en) * 2016-12-05 2018-06-14 Glanbia Nutritionals (Ireland) Ltd. Method for producing food-safe sprouted seed products
EA202091571A1 (en) * 2017-12-28 2020-10-12 Карлсберг А/С FAST WAYS OF OBTAINING EXTRACTS OF GRAIN CROPS

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EP0162805A1 (en) * 1984-04-19 1985-11-27 Pierre-André Schmutz Complementary food as milk replacement and germ grains
US4956177A (en) * 1985-11-04 1990-09-11 Microlife Technics, Inc. Method for inhibiting fungi
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