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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