HU220583B1 - Procedure for treatment of seed material to be germinated - Google Patents

Abstract

The present invention relates to a process for the treatment of seed to be germinated, which process comprises adding a lactic acid bacterial composition which inhibits the growth of microorganisms or lactic acid bacteria as part of a germination process to barley seeds to be used in a malting process or to seed to be germinated for human consumption. ŕ

Description

The present invention relates to a method for treating seed material for germination.

In this context, the germination process generally refers to the process step required to obtain a germinated product from a storage dry seed. For example, in the brewing and distillation industry, the germination process, i.e. the malting process, is used to make important raw materials for beer or other alcoholic beverages, namely, cereal malt, such as barley malt, rye malt or any other malt. The germination process is also used to produce various commercial germ products such as bean sprouts or any other germ intended for human consumption.

EP 0 162 805 describes a germinated / fermented seed material for animal feed, to which silicic acid bacteria are optionally added to preserve the already germinated seed material. A further purpose of the dosage is to influence the digestibility of the product. In the preparation of the above product, surfactants, gibberellins, mineral salts, vitamins, enzymes and / or biocides are optionally added to the germinable seed material. The germinated seed material (after completion or cessation of germination) is treated with lactic acid bacteria.

U.S. Patent No. 4,956,177 discloses a specific strain of fungus Lactobacillus, generally teaches preservation techniques, specific teachings only for use in fermented products (such as cucumbers or corn silage).

The germination process is usually performed under non-aseptic conditions. The seeds to be treated have microbes from the growth environment or storage site. The conditions under which the germination process is carried out are generally favorable for the microbes present on the seeds, so that the microbes generally reproduce during the process. Microbes can have a detrimental effect on the germinated product or its final product, but can also have a beneficial effect on the germinated product.

The main steps in the brewing process are malting, brewing, primary and secondary fermentation and processing. The purpose of malting is to produce enzymes in the seed that break down the endosperm material of the seed into a soluble form in the mash during the malting step. For example, the barley malt malting process is well known in three steps: soaking, germination and drying. The purified and sieved barley seeds are soaked in water until the desired moisture content is reached, for example, a moisture content of 43-44%. Part of the soaking can be done in so-called air rest. The barley is allowed to germinate under controlled conditions and the germinated barley is dried in a spinning air stream until the germination is complete. At the end of drying, the germs are removed from the malt. The control of the malting steps is based on the control of temperature, air flow and humidity / humidity.

The quality of malt is influenced by the microbial flora of the malt grain in terms of malting technique and malting conditions, which fluctuates significantly, depending on, for example, the type of grain, the weather conditions, the place of production, the length of the growing season and storage conditions.

The most common grain used in malting is barley. The natural microbial flora of barley is divided into field and storage fungi, bacteria and yeasts. The most common field fungi in barley are Fusarium, Altemaria, Cladosporium, Cephalosporium, Epicoccum and Helminthosporium. The occurrence of molds varies from country to country and from year to year. The humid weather conditions during the growing season of the grain, especially at harvest time, favor the growth of Fusarium molds. Fusarium contamination can be very severe in rainy growing season. Enterobacter agglomerans is one of the most common bacterial species in cereals. Other bacteria include Escherichia coli, bacteria of the genera Pseudomonas, Micrococcus and Bacillus, and lactic acid bacteria. The bacterial count in barley is about ΙΟ ^{5 to} 10 ⁸ colony forming units / g.

The number of molds and bacteria in barley grows during malting, peaking at the germination stage. Fusarium molds, and especially lactic acid bacteria, undergo the fastest growth. The number of yeasts also increases during malting. During the drying step, the concentration of molds, yeasts, and bacteria is legally reduced. Some of the microbes present in barley have a beneficial effect on malting and on a malted product such as beer. It is estimated that 40-50% of some of the enzymes present in malt are of microbial origin. On the other hand, some of the microbes have an adverse effect on barley and / or malt.

Among the disadvantageous microbes are the Fusarium molds, which have been found to cause a particularly harmful sputtering of beer more often than other molds, the peptides produced by these molds themselves serve to form gas bubbles which leave the bottle in the form of a strong sputtering. If more than 50% of the malted seeds are contaminated with Fusarium molds, the risk of ejection is clearly increased.

Furthermore, barley contains Gram-negative bacteria, such as species of the genera Pseudomonas and Flavobacterium, and Gram-positive bacteria of the genus Leuconostoc, which have been shown to delay the filtration of the mash during brewing. The various microorganisms present in barley can also cause other adverse effects, such as inhibiting germination, creating a by-taste or adversely affecting the analytical values of malt and beer.

HU 220 583 Bl

The quality requirements for malting barley can be set through annual cultivation contracts and delivery times. Molds are a group of microbes often mentioned in the quality definition. Many malt plants are also subject to ceilings for certain molds. If the percentage of seeds contaminated with Fusarium exceeds 65%, or if the proportion of Aspergillus and Penicillium fungi exceeds 50%, the barley may be classified as poor quality or even unsuitable for malting.

Attempts have been made to prevent mold-induced ejaculation by using high-quality barley or by mixing barley, malt or brewing lots. In the rainy years, almost all barley production can be of poor quality, in which case it may become impossible to obtain good barley. Microbicidal chemical agents have been tried to reduce the amount of molds, but no safe and generally accepted chemical has been found.

The germination process for the production of nutritional germs provides similarly suitable conditions for, for example, the growth of molds and bacteria. Such germs deteriorate rapidly. In addition, still related to germination, for example, the proliferation of pathogenic foodborne infections causing food poisoning, such as Salmonella, Yersinia and / or Listeria bacteria, can occur.

The following is a summary of the invention.

It is an object of the present invention to overcome the drawbacks discussed above.

More particularly, it is an object of the present invention to provide a method by which the quantity and quality of the microorganism flora can be carefully controlled during the germination process without, however, adversely affecting the quality of the germinated product or the final product formed from the product.

With respect to the features of the invention, reference is made to the claims.

The following is a detailed description of the invention.

The invention is based on studies in which the unexpected observation has been made that lactic acid bacteria can be used to improve the quality of germinating products. The substances involved in the process according to the invention and / or produced by the process according to the invention, i.e. microbicidal agents, inhibit the harmful microorganisms occurring in the germination process.

The use of lactic acid bacteria in the food and feed industry is well known to those skilled in the art. Lactic acid bacteria produce, under fermentation conditions, compounds that affect the composition and taste of the products, but which at the same time inhibit the growth of pathogenic microorganisms that cause the degradation of these products. Lactic acid bacteria are commonly used in dairy products, meat products, plant fermentation and bakery products, and in the preservation of animal feeds.

The addition of lactic acid bacteria or lactic acid is known in the art for the production of certain types of malt, the so-called sour malt. Addition to malt occurs during the drying step, before mashing, or during mashing. The purpose of the dosing is merely to lower the pH of the mash, and thus to influence the course of the mash process and the quality of the finished beer. However, lactic acid bacteria have not been used so far to inhibit the growth of undesired microorganisms associated with the germination process, as taught in the present invention.

The composition of the invention may be added to the product to be germinated at any stage of the germination process.

In a particularly preferred embodiment, the lactic acid bacterial composition or the lactic acid bacterial composition is added to the cereal material, such as barley seeds, during the malting process. The administered formulation inhibits the growth of molds, particularly harmful Fusarium molds and bacteria, resulting in, for example, a reduction in the risk of brewing due to Fusarium molds. However, the composition has no significant effect on the function of the beneficial microorganism flora in terms of the quality of the malt obtained or the beer produced from it. No adverse effect on the malt quality was observed with the dosage formulation, nor was it found to contain or produce compounds which are harmful to the malt or the beer being prepared.

In the malting process, the lactic acid bacterial composition or the lactic acid bacterial composition may be added to the barley seeds prior to soaking, in the soaking step, or in the germination step. The addition is preferably carried out in the soaking or germination step. In other respects, the malting process can be carried out in any manner known in the art. If desired, for example, nutrients may be added to the barley to be malted or conditions may be adjusted, for example, lactic acid may be added to provide optimal conditions for the growth of lactic acid bacteria.

Any conventionally available lactic acid bacterium which has an effect on the growth of microorganisms can be used within the scope of the invention. Suitable lactic acid bacterial genera include Lactococcus, Leuconostoc, Pediococcus and Lactobacillus. Preferred species include Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus and Lactobacillus plantarum, or any of these species, and Pactococcus or Lactobococcus is especially preferred. Genetically modified bacteria can also be used.

The lactic acid bacterial composition may consist of cell-free or cell-free culture broth or concentrated culture broth (which contains cells). A product produced by lactic acid bacteria may be cell-free

The culture broth, concentrated concentrate broth or fractionated broth filtrate may be a pure or partially purified microbicide.

In a particularly preferred embodiment of the invention, the treatment is carried out with concentrated or fractionated culture broth, which may be cell-free or may contain cells. The concentration may be carried out, for example, by lyophilization or evaporation. For example, the broth may be concentrated to 2-20-40 fold.

The fractionation, i.e. purification, of the microbicidal products can be carried out in a manner known per se, for example, by chromatography or ultrafiltration.

The microbial growth inhibitory activity of a composition comprising a lactic acid bacterium or a product produced by a lactic acid bacterium in the process of the invention corresponds, for example, to 10-10,000 ml culture broth / kg of treated seed, preferably 30-7000 ml culture broth / kg treated seed, e.g. amount of seed material to be treated. The preparation of culture broth is described in the Examples. It is noted that the activity of the formulation is determined by the culture broth used. It will be apparent to those skilled in the art that the compositions of the present invention having the same antimicrobial activity may be prepared by other fermentation broths and / or processes.

According to the invention, the composition comprises microbicidal compounds and / or produces microbicidal compounds during the germination process. If a cell preparation is used, cell proliferation may be promoted, if desired, e.g., by controlling the conditions during the germination process or by adding nutrients. The preparation may also accelerate the growth of other lactic acid bacteria present in the germinating material.

Since the use of lactic acid bacteria in food is permitted and generally accepted, preparations derived from the proliferation of lactic acid bacteria can also be used safely. Lactic acid bacteria usually belong to the natural microbial flora of germinating seeds, such as barley seeds. Therefore, the process of the invention is maximally natural. It is also possible to use a strain of lactic acid bacteria which is present on the seeds.

The present invention makes it possible to reduce the adverse effects of Fusarium contamination, such as beer splashing, during malting.

Furthermore, it has been found that the process unexpectedly improves the filterability characteristics of the brewing process. This is considered to be due to the fact that the composition of the invention also reduces the number of harmful species retaining the filtration of the mash during malting, such as those of the genera Leuconostoc, Pseudomonas and Flavobacterium.

In another preferred embodiment, the lactic acid bacterial composition or the lactic acid bacterial composition is added to the core material during the preparation of the germs to be used as food.

The present invention can limit the growth of harmful microbes in the germination process. For the first time, the invention provides a biological solution that can be used in an industrial germination process to prevent the growth of harmful bacteria present on the seed to be germinated.

The process of the present invention improves the overall hygiene standard of the germination process as a whole.

The invention is further illustrated by the following non-limiting examples. The treatment of the invention can also be applied to other germination methods.

Figure 1 is a graph showing the microbicidal activity of a lactic acid bacterial culture filtrate, the results of which were determined by turbidimetry (turbidity measurement). The curves are the normal growth curve of the test organism E. agglomerans E396 and the inhibition of the growth of the test organism using culture filtrates of L. plantarum E-76 and P. pentusaceus E-390.

Figure 2 shows the effect of adding P. pentosaceus E-390 broth at different stages of malting on the total bacterial count of malting.

Figure 3 shows the effect of administration of P. pentosaceus E-390 cells at different stages of malting on the total bacterial count of malting.

Figure 4 illustrates the effect of adding P. pentosaceus E-390 and L. plantarum E76 broths or concentrated broths to total bacterial counts of different stages of laboratory malting.

Figure 5 shows the effect of adding P. pentosaceus E-390 and L. plantarum E-76 broths or concentrates and fractionated broths to the total bacterial count of different stages of laboratory malting.

Figure 6 shows the effect of lactic acid bacterial cultures added during malting on the filterability of mash (Tepral filtration).

Example 1

Microbicidal effect of various lactic acid bacterial strains on microbes occurring during malting In this study, the microbicidal activity of various lactic acid bacterial strains on microbicides occurring during malting was investigated. Sterile-filtered culture broths were used.

1. Production strains

The following bacterial strains were used as production strains:

Lactobacillus lactis ssp. lactis VTT-E-90414 (E-414) ssp. diacitilactis VTT-E-90423 (E-423)

I

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Leuconostoc mesenteroides ssp. mesenteroides ssp. mesenteroides ssp. mesenteroides Pediococcus damnosus Pediococcus parvulus Pediococcus pentosaceus Pediococcus pentosaceus Pediococcus pentosaceus Pediococcus pentosaceus

Lactobacillus curvatus Lactobacillus plantarum Lactobacillus plantarum

VTT-E-90389 (E-389) VTT-E-90415 (E-415) VTT-E-90466 (E-466) VTT-E-76065 (E-65) VTT-E-88315 (E-315) VTT-E-76067 (E-67) VTT-E-76068 (E-68) VTT-E-88317 (E-317) VTT-E-90390 (E-390) (DSM 7389) VTT-E-90391 ( E-391) VTT-E-78076 (E-76) (DSM 7388) VTT-E-79098 (E-98)

The strains originate from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland). The strain Lactobacillus plantarum (E-76) was deposited at the DSM deposit site (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under the number 7388. Strain E-76 (DSM 7388) was isolated from beer by known methods and analyzed / identified by known analytical methods for liquid products. Pediococcus pentosaceus (E-390) was deposited at deposit number DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under accession number 7389. Strain E-390 (DSM 7389) was isolated from homogenized samples of barley kernels and identified / analyzed by known methods [see Haikara, A. and Home, S. of EBC Congress 1991 (Quality Control). Deposits were made in accordance with the requirements of the Budapest Treaty.

2. Test strains

Test strains include, inter alia, various harmful microbial species occurring during malting and lactic acid bacterial strains serving as production strains.

Harmful molds in the studies include Fusarium molds [Gibberella avenacea (formerly Fusarium avenaceum) VTT-D-80141 (D-141) and VTTD-80147 (D-147) and Fusarium culmorum VTT-D80148 (D-148) and VTT-D -80149 (D-149), Collection of Industrial Microorganisms, Biotechnical Laboratory of VTT] and an Aspergillus species.

The harmful Gram-negative bacteria were represented by two strains of the genus Enterobacter and one strain of the genus Flavobacterium and one of the genus Pseudomonas. Lactic acid bacteria were the strains used as production strains, as well as strains of Lactococcus sp. Strain E416.

3. Cultivation of production strains and preparation of sterile filtered broths

The lactic acid bacteria were cultured in MRS medium (MRS BROTH, Oxoid). Pediococcus strains E-65, E67 and E-68 were cultured under aerobic conditions at 25 ° C, all other strains under anaerobic conditions at 30 ° C, and cultivation times vary between 2 and 5 days. The cells were then centrifuged and the supernatant was sterile filtered.

4. Breeding of test strains

Spore suspensions of Fusarium molds were prepared by culturing the mold strain in CMC (carboxymethylcellulose) solution at 25 ° C for 5 to 6 days, suspending spore formations in Tween solution, filtering the suspension and filtering.

A spore suspension of Aspergillus mold was prepared directly on PD agar (Potato dextrose, Difco) and cultured at 25 ° C for 3 days.

Gram-negative bacteria were cultured aerobically in NB medium (Nutrient broth, Difco) for 1 day, Enterobacter strains at 30 ° C, Flavobacterium and Pseudomonas strains at 25 ° C.

The lactic acid bacteria were cultured as described in section 3 above.

5. Investigation of microbicidal effect of lactic acid bacteria

The microbicidal activity of the culture broth was determined by disc or turbidimetry.

5.1. Disc method for the determination of microbicidal activity

Detect or dilute 100 μΐ sterile-filtered fact onto a 12.7 mm filter paper disk. The disks are placed in plate count agar dishes in which 0.3 ml of test organism diluted 10 ~ ^{2 is} plated. Samples were grown for 24 hours at 30 ° C and the diameter of the inhibition zone was measured in mm.

5.2. Turbidimetric assay to determine microbicidal activity

The process uses an automatic turbidimeter (Bioschreen, Labsystem).

The sample contained 10% by volume of test organism and 10% by volume of sterile filtered culture broth per sample volume and media. As a control, distilled water adjusted to the same pH as the sterile filtered formulation was used instead of the sterile filtered culture broth.

The propagation medium for each strain was the same as the medium used for culture of the test strain.

Propagation conditions for Fusarium and Aspergillus molds: 5 days, 25 ° C, vigorous shaking. For Gram-negative bacteria: Enterobacter strain, 30 ° C, all other strains: 25 ° C, two days, and shaking. Cultivation conditions for lactic acid bacteria: 3 days, 30 ° C and shaking.

The instrument measures the absorbance of the sample at 420-580 nm in visible light. After culture, the growth curve of each sample is recorded and the area under the curve is calculated.

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The microbicidal activity of the production strains on the proliferation of the test strains is expressed as a percentage of inhibition obtained by comparing the size of the control replication with a sterile filtered formulation of the production strain.

6. Examination of the fungicidal action of certain lactic acid bacteria

The fungicidal activity of six lactic acid bacterial strains, E-76, E-98, E-315, 10 E-317, E-414 and E-415, was tested separately using all Fusarium molds as test strains. In the test, the turbidity of mold cultures obtained after the addition of various dilutions of the sterile filtered solution of each lactic acid bacterial culture was visually examined and the results are shown in Table 3.

Control samples were supplemented with sterilized Milli-Q water and sterilized Milli-Q water with lactic acid adjusted to pH 3.6 instead of culture broth.

Cultures were carried out in CMC medium as test tube cultures at 25 ° C for 5 days. The results are read visually.

7. Results

Tables 1 and 2 and Figure 1 show the microbicidal activity of various lactic acid bacterial strains as determined by the disc method and the turbidimetric method.

Figure 3 shows the results of visually determined fungicidal activity.

Table 1

Microbicidal activity of lactic acid bacteria broths as determined by the disc method

Lactic Acid Bacteria Cell count, CFU / ml * Tcnyészlé pH Diameter of inhibition zone, mm E-76 3.2 x ^{10 8} 3.70 19 E-98 1.2x10 " 3.72 17 E-315 1,6x10 " 3.90 16 E-317 1,0x10 " 3.86 16 E-390 1,9x108 3.92 15

The E-396 test organism has a cell number of 4 x 10 ⁷ colony forming units / ml; 1.2 1,2 10 ⁵ colony forming units / ml on the dish. * Colonizer / ml

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Table 2

Microbicidal effect of lactic acid bacteria on the growth of various microorganisms

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ND: not determined; negative numbers mean: inhibit reproduction; meaning of positive numbers; stimulates reproduction; > 55%; strong inhibition; 35-55%: fairly strong inhibition; 15-34% rather poor inhibition; 15%: weak inhibition or no inhibition.

triggering beer spills;

²⁾ hindering mash filtration.

*) Colony forming unit / ml

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Table 3

Effect of lactic acid bacteria on the broth of Fusarium fungi causing beer spout + definite reproduction; - no reproduction

D-141 Lactic acid- bacterium- tribe Tenyészléhígítás 1: 6 2: 6 3: 6 4: 6 5: 6 6: 6 E-76 + + - - - - E-98 + + - - - - E-315 + + + - - - E-317 + + + - - - E-414 + + + + - - E-415 + + + - - - control + pH control +

D-147 Lactic acid- bacterium- tribe Tenyészléhígítás 1: 6 2: 6 3: 6 4: 6 5: 6 6: 6 E-76 + + - - - - E-98 + + - - - - E-315 + + + - - - E-317 + + + - - - E-414 + Ί- + + - - E-415 + + + - - - control + pH control +

D-148 Lactic acid- bacterium- tribe Tenyészléhígítás 1: 6 2: 6 3: 6 4: 6 5: 6 6: 6 E-76 + - - - - - E-98 + + - - - - E-315 + + - - - - E-317 + + - - - - E-414 + + + - - - E-415 + + - - - - control + pH control

D-149 Lactic acid- bacterium- tribe Tenyészléhígítás 1: 6 2: 6 3: 6 4: 6 5: 6 6: 6 E-76 + + - - - - E-98 + + - - - - E-315 + + + - - - E-317 + + + - - - E-414 + + - - - E-415 + + + - - - control + pH control +

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The results reveal that said strains of lactic acid bacteria inhibit the growth of harmful Fusarium molds and other harmful microorganisms that occur during the malting process, while essentially not affecting the beneficial microorganisms. The results show that these lactic acid bacteria can be used in the process of the invention.

Example 2

Microbicidal activity of certain strains of lactic acid bacteria against food pathogens and food-borne microorganisms

Preparations of Pediococcus pentosaceus strains VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) were tested for their microbicidal activity against food pathogens and food-borne microorganisms. Strains of the genera Bacillus, Yersinia, Listeria, Pseudomonas, Salmonella and Staphylococcus were used as test organisms.

The preparation was prepared using the sterile broth of lactic acid bacteria prepared in Example 1. All other test strains were cultured in Iso-Sensitest medium (Oxoid) for 16-18 hours except Listeria strain which was cultured in tryptose / phosphate medium. The culture temperature was 30 ° C except for strains of Salmonella, Listeria and Staphylococcus which were grown at 37 ° C.

The microbicidal activity was determined by the turbidimetric method described in Example 1. Experimental conditions for propagation substrate and temperature are as described above. The incubation period was 24 hours except for the Bacillus and Yersinia strains, which were incubated for 48 hours.

Our results are shown in Table 4, which shows that the administration of the lactic acid bacterial compositions of the invention inhibits the growth of food pathogens and food-borne microorganisms.

Table 4

Reproduction inhibition by P. pentosaceus E-390 and L. plantarum E-76

producing strains E-390 E-76 Test organism Reproduction area decrease,% Bacillus cereus ATCC91339 *) 93 80 Yersinia enterolitica ELI351 *) 85 54 Listeria monoeytogenes KTL4126 *) 41 49 Pseudomonoas fluorescens ELI97 59 97 Pseudomonas fragi ATCC4973 84 93

producing strains E-390 E-76 Test organism Reproduction area decrease,% Salmonella infantis ELI5 *) 90 98 Staphylococcus aureus ELI200 *) 73 86

*) Pathogens in food.

ATCC: American Type Culture Collection

ELI: VTT, Food Research Laboratory

KTL: National Institute of Public Health

Example 3

Effect of lactic acid bacteria and lactic acid bacteria on the microflora of malt and its quality

1. Strain used

Lactic acid bacterial strain Pediococcus pentosaceus VTT-E90390 (E-390) was used in the assay.

MRS medium was used as inoculum propagation medium. The bacteria were grown for 2 days in 10 ml MRS medium at 30 ° C under anaerobic conditions. The culture solution was inoculated with 1% by volume of inoculum.

2. Barley

We used Kymppi barley from the 1990 harvest, where the proportion of seeds contaminated with Fusarium mold was 55%.

3. Malting procedure

Batches of 1000 g of barley are rinsed in a water bath at 12 ° C for 1 hour. The rinse water is replaced with the first soak water and after 5 hours it is replaced with the second soak water. This is followed by a 16-hour air rest. The purpose of air rest is to eliminate water on the surface of the seeds. It lasts for 8 hours. During the soaking, the barley reached a moisture content of 44%. The barley is aerated throughout the soaking process.

Soaking follows germination. The barley is germinated in germination boxes at 14 ° C for 6 days. To maintain a moisture content of 44%, the barley is moistened and rotated daily. The green malt thus obtained was dried in a 21 hour temperature program. The temperature is maintained at 50 ° C for 4.5 hours and then raised to 60 ° C for the next 4.5 hours, which is maintained for 4 hours. The temperature was then raised steadily to 85 ° C over a period of 5 hours and maintained at that temperature for the remaining 3 hours. The final moisture content of the malt is about 4%. Finally, the germs are mechanically removed.

As a control, any malting process without additives is performed.

4. Lactic acid bacterial preparation and lactic acid bacterial preparation Cultivated broths containing lactic acid bacterial cells and microbicidal compounds isolated from culture broth are used in combination or separately.

HU 220 583 B l l. To 1 kg of barley, culture broth containing 100 ml of cells or cells isolated from 120 ml of broth is added.

Isolation is accomplished by centrifugation of the culture broth followed by suspension of the cells in water. In cases where the broth is added, it is used alone as it is. The number of cells administered was approximately ^{8 to} 10 ⁹ colony forming units / ml.

5. Administration of lactic acid bacterial preparations

The lactic acid bacterial preparations were added to the barley at the beginning of the first soaking, at the beginning of the first and second soaking, or at the beginning of germination.

6. Analytical tests

Samples were taken from each malting step.

6.1. The examination of molds is carried out in the following way:

The percentage of seeds contaminated with Fusarium molds was determined using CZAPEK IPRODION DICLORAL agar (CZID agar), which is selective for Fusarium molds and wet with 20 filter papers (EBC-Analytica Microbiologica, Part II, 1987). Fusarium factual fungi are determined by their characteristic colony and spore morphology and their red color.

Aspergillus and Penicillium molds were determined using selective malt salt agar (EBC-Analytica Microbiologica, Part II, 1987). The other most common molds are determined on moistened filter paper.

6.2. Lactic acid bacteria are assayed on MRS agar for both dosing cultures and malting samples.

6.3. Total bacterial count was determined using Plate Count agar (Difco).

6.4. The chemical characteristics of malt are carried out using methods known in the malting context (EBC-Analytica, 1987, 4th edition).

7. Results

Table 5 shows the number of Fusarium molds and lactic acid bacteria in the various steps of malting with the addition of E-390 culture broth.

Figures 2 and 3 show total bacterial counts at various stages of malting using E-390 culture broth and E-390 cells.

Table 6 shows the results of the analysis of malting with E-390 broth or E-390 cells.

Table 5

Effect of P. pentosaceus E-390 broth on different stages of malting on Fusarium mold and lactic acid bacteria during malting

Dosing step Fusarium molds (% of contaminated seeds) Barley Soak germination Malt control 55 72 96 25 120 ml CB ') for barley 3 6 77 3 120 ml CB ') for soaking water 1 55 62 98 14 120 mL CB ”) for the 1st + 2. soaking water 55 42 90 9 120 ml CB ') for germination 55 78 91 3 Lactic acid bacteria (cell density, CFU / g **) control 6,0x10 ' 3.4 x 10 ² 2,2x10 " 3,0x10 ' 120 ml CB ') for barley 1.2 x ^{10 8} 1.1 x ^{10 8} 2,2x10 " 1,8x10 " 120 ml CB ') for soaking water 1 6,0x10 ' 3,1x10 ' 2,8x10 ' 1,4x10 ' 120 mL CB ”) for the 1st + 2. soaking water 6,0xl0> 7,4x10 ' 8,3x10 ' 5,3x10 ' 120 ml CB ') for germination 6,0x10 ' 1.5x10 " 2,7x10 " 2,6x10 "

*) CB: tenycszlc

Colony-forming egyscg / g

HU 220 583 B1

Table 6

Effect of P. pentosaceus E-390 broth or cells on malt quality at different stages of malting

Germ 00 00 ι / Ί m r- cn © cn cn CN 00 cn OK cn STONE cn 35 1 β-glucan, kinase activity i 1 U / kg 365 355 stone cn 360 386 359 376 353 392 α-aminoase activity 1 Sky [202 1 207 you- Οκ 186 OK »Shop 223 Γ- ΟΟ 00 OK r ~ homo- geneity SHE' OK Ό Γ- V "> m 64 OK tn OK tn OK un 67 OK STONE amended rip x® TENDON 00 © 00 stone r- © 00 OK STONE OK r- OK C- OO 00 FAN 1 mg / 1 163 164 r- 168 I- 129 r- STONE 1 1 148 viscosity inappropriate optimization 0th Q OK tendon W 1.50 OK you; 00 STONE 1.54 i ί 1.63 Cukrosí- trative time minute 1 She V <10 <10 <10 <10 <10 1 1 <10 Szűrhe- confusion fine Orlean ml / h 300 tn © cn 310/56 minute 305 310 315/61 minute 1 t 290 Purity i clean clean clean clean opalescent clean 1 1 opalescent extractions tum Differ ..... J £ • 6 χ® θ ' cn TENDON 3.0 OK_ cn cn you-' Oy TENDON 1 1 OK CN Rough Orlean 1 __-___ 1 % d. m. 77.8 77.3 77.5 OK STONE Γ- 74.9 | 1,77.4 1 1 76.6 extractions tum % d. m. © 00 80.3 1 80.5 1 80.0 79.0 80.3 1 1 79.5 Wet- ness _i X® © K- you cn you CN you cn you TENDON you cn you l l cn you β-gluká- women _i mg / 1 604 496 482 482 955 1 606 1 1 804 Λ oo «« N .5 > 2 control 120 ml CB *) for barley 120 ml CB *) for soaking water 1 120 ml CB *> 1 and 2. for soaking water 120 ml CB *> for sliding Cells (10 ⁸ CFU / g) ** for barley Cells (10 ⁸ CFU / g) ** for soaking water 1 Cells (10 ⁸ CFU / g) ** for soaking water 1 and 2 Cells (10 ⁸ CFU / g) ** for germination

-: not analyzed *>: broth **: CFU / g: colony forming unit / g

HU 220 583 Bl

The results obtained show that the treatments of the present invention particularly reduce the amount of Fusarium mold and total bacteria at various stages of malting.

The addition of the preparation does not adversely affect the quality of the malt. The opposite is true: the treatment according to the invention improves the filterability of the mash from the mash and reduces the β-glucan content of the malt.

Example 4

Preparation of preparations made by lactic acid bacteria

1. Preparation of concentrated broth

Production strains of Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) were grown in a 15-liter fermenter. The volume of the inoculum is 6-7% and culturing is carried out on MRS medium at 30 ° C for 2 days under microaerophilic conditions. The culture broths were concentrated 10-fold and 20-fold, respectively, by lyophilization or evaporation. The microbicidal activity of the concentrates is assayed by the disc method.

2. Preparation of purified solutions containing microbicidal compounds

The lactic acid bacterial culture broth was purified by gel-size fractionation by gel chromatography. Fractions found to be active by disk and turbidimetry are collected and run again on the gel column.

Example 5

Effect of lactic acid bacteria and lactic acid bacteria on the microflora of malt and its quality

1. Strains used and preparations produced by the strains

The strains used are:

Lactobacillus plantarum VTT-E-78076 (E-76) Pediococcus pentosaceus VTT-E-90390 (E-390)

The strains originated from [VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland)].

The formulations were prepared as described in Example 1, point 3, Example 3, point 1, and Example 4, points 1 and 2.

2. Barley

Barley is the Kymppi barley from the 1991 harvest.

3. Malting procedure

The malting procedure was carried out as described in Example 3, but the germination step was continued for 8 days. The final moisture content of the malt is below 5%.

4. Malting

Two laboratory malting procedures were performed and the control was prepared without any additives.

4.1. First malting

In laboratory malting, the preparations used were ten-fold concentrated cell-free culture broth and untreated cell-containing broth. The compositions were administered at the beginning of the first soaking step or at the beginning of the first and second soaking steps. 1-8. malting tests were performed as follows:

Test 1: control, Kymppi barley harvested in 1991;

Test 2: At the start of the first and second soaking steps, 120 ml of cell-containing E-76 culture broth is added;

Test 3: At the beginning of the first soaking step, 120 ml of 10-fold concentrated E-76 culture broth filtrate was added;

Test 4: At the beginning of the first and second soaking steps, 120 ml of 10-fold concentrated E-76 culture filtrate was added;

Test 5: At the beginning of the first and second soaking steps, 120 ml of E-390 broth was added;

Test 6: At the beginning of the first soaking step, 120 ml of 10-fold concentrated E-390 culture filtrate was added;

Test 7: At the beginning of the first and second soaking steps, 120 ml of 10-fold concentrated E-390 culture filtrate was added;

Test 8: At the start of the first and second soaking steps, 60 ml of E-76 cell culture broth and 60 ml of E-390 cell culture broth are added.

4.2. Second malting

For laboratory malting, the formulations used were 20-fold concentrated cell-free culture filtrates, cell-free purified microbicide fractions, and cell-containing untreated culture broth. The pH of the soaking water was controlled. The formulations were added at the beginning of the first and second soaking steps. 9-16. malting tests were performed as follows:

Test 9: control, 1991 Kymppi barley harvest;

Test 10: At the beginning of the first and second soaking steps, 120 ml of water, pH 3.8;

Test 11: At the beginning of the first and second soaking steps, 120 ml of E-76 cell culture broth was added;

Test 12: At the beginning of the first and second soaking steps, 120 ml of E-76 fractionated concentrate, pH 3.8;

Test 13: At the beginning of the first and second soaking steps, 120 ml of E-76 broth concentrated twice;

Test 14: At the beginning of the first and second soaking steps, 120 ml of cell culture medium E-390 was added;

Test 15: At the beginning of the first and second soaking steps, 120 ml of E-390 fractionated concentrate, pH 3.8;

Test 16: At the beginning of the first and second soaking steps, 120 ml of E-390 Concentrated Culture Fertilizer filtrate is added.

5. The analyzes performed are as follows

Samples were taken from each malting step.

The number of molds, lactic acid bacteria as well as the total bacterial count and the physical and chemical quality characteristics of the malt were determined as described in Example 3.

6. Our results

Figures 4 and 5 show malt in the cell soaked by the addition of a cellular culture of lactic acid bacteria to either concentrated or fractionated culture filtrate.

The total bacterial count measured in the various steps of the staining procedure is shown.

Tables 7, 8, 9 and 10 show the concentrations of Fusarium mold and lactic acid bacteria in the samples from the different malting steps of the two previous malting processes.

Tables 11 and 12 show the results of the analysis of the two malting processes.

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HU 220 583 Bl

Table 8

Number of lactic acid bacteria. Colony forming units / ml during malting with addition of P. pentosaceus E-390 and L. plantarum E-76 to barley soaking water. The numbering is as in Example 5.

Example Soak germination malting First 5,5x10 ' 3,3x106 2,5x10 ' Second 7,3x10 ^ 1.2x10 ' 5.5 x ^{10 5} Third 1,4x103 4,4x103 1,1x10 '

Example Soak germination malting 4th 3.2 x1ο ² 1.4x1ο ⁴ 9,5x102 5th 1,3x10 ' 7,5x106 2,6x106 6th 4,8x10 ' 5.0 x ^{10 5} 4.0 x ^{10 4} 7th 7,3x102 2,4x10 * 2,6x10 ' 8th 1.2x10 ' 1,4x10 ' 2,3x106

The lactic acid bacteria content of barley is 7.5 x 10 ² colony forming units / g.

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HU 220 583 Bl

Table 10

Number of lactic acid bacteria. Colony forming units / ml during malting with addition of P. pentosaceus E-390 and L. plantarum E-76 to barley soaking water. The numbering is as in Example 5

Example Soak germination malting 9th 8.0 x 10 ² 4.2 x1ο ⁶ δ, ΟχΙΟ ³ 10th 8.0 x 10 ² 5.0x1ο ⁶ 5.6x1ο ⁴ 11th 2,7x10 ' 2.1 χ 10 ⁷ 8.5 χ ΙΟ ⁵ 12th 2.2 x ^{10 3} 3.4 χ 10 ⁵ 2.5 χΙΟ ³ 13th 1.2 x ^{10 3} 1.4χ10 ⁷ 2,3 χ ΙΟ ³ 14th 4.1 χ 10 ⁷ 3.5 χ ΙΟ ⁷ 1.1 χ ΙΟ ⁴ 15th 4.8 x ^{10 3} 4.8 χ ΙΟ ⁶ 3,9x105 16th 3.5 x 10 ² 2,1 χ ΙΟ ⁵ 1.7x1ο ⁴

The lactic acid bacteria content of the fetus is 7.5 χ 10 ² colony forming units / g.

Table 77

Effect of P. pentosaceus E-390 and L. plantarum E-76 on malt quality in barley soaking water. The numbering is as given in Example 5.

Analysis Sample First Second Third 4th 5th 6th 7th 8th Moisture, % 3.6 3.6 3.5 3.7 3.7 3.6 3.9 3.9 Extraktumtartalom, fine % Ups. 81 80.8 80.6 80 81.2 81 80.7 81.2 Extraktumtartalom, rough % Ups. 76.8 77 76.1 75.2 77 76.4 74.8 77.1 Extraktumkülönbség, % 4.2 3.8 4.5 4.9 4.3 4.7 5.9 4.1 Szacharifikáció, minute <10 <10 <10 <10 <10 <10 <10 <10 Cefretisztaság clean clean clean clean clean clean clean clean Filterability, fine ground, ml / min 320/45 320/44 315/48 315/40 320/47 315/66 320/49 315/52 Filterability, rough ground, ml / min 260/86 265/64 270/59 255/73 260/68 260/76 255/90 275/120 mash 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 aminonitrogen, mg / 1 154 156 155 139 158 150 130 154 modifications, % 74 72 73 73 72 75 77 76 homogeneity, % 61 65 63 58 67 66 62 68 α-amylase, E / g 178 182 148 128 179 156 144 174 β-glucanase e / kg 434 420 360 360 422 412 361 425

HU 220 583 Bl

Table 12

Effect of P. pentosaceus E-390 and L. plantarum E-76 on malt quality in barley soaking water. The numbering is as given in Example 5.

Analysis Sample 9th 10th 11th 12th 13th 14th 15th 16th Moisture, % 3.5 3.6 3.6 3.5 3.8 3.5 3.5 3.6 Extraktumtartalom, fine % Ups. 80.5 80.5 81.2 80.5 80.3 81 81.1 80.4 Extraktumtartalom, rough % Ups. 78.4 77.9 77.4 73.3 73.5 77.6 76.6 75.1 Extraktumkülönbség, % 2.1 2.6 3.7 7.2 6.8 3.4 4.5 5.4 Szacharifikáció, minute <10 <10 <10 <10 <10 <10 <10 <10 Cefretisztaság clean clean clean clean clean clean clean clean Filterability, fine ground, ml / min 320/40 315/47 320/44 315/42 320/55 315/49 330/40 320/40 Filterability, rough ground, ml / min 290/120 270/76 260/51 265/120 245/120 285/68 305/120 265/12 mash pH 6.04 6.05 5.99 5.97 5.94 5.95 5.98 5.99 Viscosity, cP L54 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 aminonitrogen, mg / 1 149 145 149 128 117 149 139 120 modifications, % 85 85 86 82 74 81 75 71 homogeneity, % 63 66 76 57 57 68 51 47 alpha-amylase, Sky 161 172 161 123 72 148 125 101 β-glucanase e / kg 365 375 365 282 282 371 342 308

The results show that the treatments of the present invention particularly reduce the amount of Fusarium mold and total bacteria in the various malting steps. It is further noted that besides broths, concentrated and fractionated broths have a particularly beneficial effect on, for example, Fusarium molds.

From the analysis of the malt, it can be concluded that the addition of the broth of E-76 and E-390 strains improves the filterability of the mash made from the malt and also reduces the β-glucan content of the malt compared to the control malt.

Example 6

Effect of formulations produced by the lactic acid bacteria added during malting Figure 6 is a graph showing the effect of the lactic acid bacterial strains of the present invention on the filterability of the malt produced with malt treated with this (120 ml of barley). The strains mentioned in Example 1 were used in this assay: E-390, E-416, E-98, E-317, E390, E-76 and E-315. The assay was performed using the Tepral Screening Procedure (BIOS. 19, 1988, Grandclerc, J. et al., "Simplification of the Methode of the Filtration with a Tepral Description," page 88-92).

It is clear from the results that the process of the invention improves the filterability of the mash.

The following examples illustrate the effect of lactic acid bacteria on the concentration of mycotoxins, deoxivalenol and zearalenone produced by Fusarium molds in barley malting process.

Example 7

Effect of lactic acid bacteria on deoxivalenol (DON) concentration in malting barley infected with F. culmorum Dl 48

1. Applied strains

The lactic acid bacterial strains used in the assay were Lactobacillus plantarum (E-76) and Pediococcus pentosaceus (E-390).

The bacteria were grown under anaerobic conditions in MRS medium at 30 ° C for 34 days. Between studies, the bacterial strains were maintained in culture medium at 4 ° C under anaerobic conditions.

HU 220 583 Bl

2. Barley

The barley version used is Kymppi barley harvested in 1994, to which 2 ml of Fusarium culture solution was added per 100 g. The Fusarium strain was cultured in shaken CMC solution for about 7 days to create spores. F. culmorum VTT D-80148 has a spore density of about 10 ⁶ spores / ml. The contaminated barley was incubated at 25 ° C for 3 days. The barley was stirred daily and dried overnight at 30 ° C to recover its original moisture content.

3. Malting procedure

The 1000g barley soaking program was as follows: washing (1 hour), first soaking (7 hours), first aeration pause (16 hours), second soaking (8 hours), second aeration pause (15 hours), and dipping. The soaking temperature is 12 ° C and the target moisture content of the barley is 46% by the end of the soaking. In order to ensure breathing and metabolism of barley, aeration pauses are taken between the soaking stages and barley samples are placed in aeration space during the breaks. The soaking vessels are also provided with aeration. After the second aeration, the barley samples are measured to determine their moisture content. If the moisture content is below 46%, the barley samples are submerged in water for a short time. During soaking, the ratio of barley to water is 1: 1.5. The barley samples are then transferred to the germination box.

The barley samples were germinated in aeration germinated box at 14 ° C for 6 days. The barley samples are wetted and loosened daily to adjust the moisture content to the desired 46%. The green malt thus obtained is dried using a 21 hour temperature program as in Example 3, paragraph 3. Finally, the germs are mechanically removed from the malt.

As a control, a malting process without addition of lactic acid bacteria was used.

4. Lactic acid bacterial preparation

Table 13 shows the pH, cell number and microbicidal activity of the lactic acid bacterial cultures added to the malting process (as determined by the method described in Example 1, paragraph 5).

Table 13

Tejsavbaktc- preferably sodium strain PH of the culture solution cell Number (PMY / ml) Inhibition zone (mm) E-76 3.72 O8 + 5,1E 17 E-390 3.77 l, 7E + 09 15

5. Dosing of lactic acid bacteria preparations

The lactic acid bacterial preparation is added at the beginning of the soaking steps 1 and 2. The ratio of the lactic acid bacterial culture to water during the malting process was maintained at 8%.

6. Analysis performed

The concentration of deoxyvalenol in each barley sample is determined by gas chromatography. A mass spectrometer is used to detect and quantify deoxivalenol.

7. Results

Figure 7 shows the effect of administration of lactic acid bacteria on the deoxivalenol content of malting barley contaminated with F. culmorum strain D 148.

The results obtained show that L. plantarum strain E-76 has a deoxyivalenol content of malt which is 50% lower than that of the reference maltate and that of P. pentosaceus E-390 strain has a 25% lower level of deoxivalenol at his reference malt.

Example 8

Effect of lactic acid bacteria on malting of barley containing natural deoxyvalenol impurity and Fusarium molds

1. Applied strains

Lactobacillus plantarum (E-76) and Lactobacillus acidophilus VTT E-87276 (E-276) strains of lactic acid bacteria were used in the assay.

The Lactobacillus plantarum strain was grown as described in Example 7 and the Lactobacillus acidophilus strain was cultured at 37 ° C.

2. Barley

The barley varieties used are low-quality Kustaa, Loviisa and Jo 1599 from the 1993 harvest.

3. Malting procedure

The malting process was carried out as described in Example 7, except that the barley portions were 100 g and the barley: water ratio during steeping was 1: 4.0.

The control is a malting process without the addition of lactic acid bacteria.

4. Lactic acid bacteria

Table 14 shows the pH, cell number, and microbicidal activity of the lactic acid bacterial cultures added to the malting process (as determined by the method described in Example 1, paragraph 5).

Table 14

malted áipaváltozat Tejsavbaktériiim strain PH of the culture solution cell Number (PMY / ml) Inhibition zone (mm) Kustaa E-76 3.68 2.6E + 09 23 E-276 3.96 l, 6E + 08 21 Loviisa E-76 3.68 2,6E + 09 23 E-276 3.96 l, 6E + 08 21 Its 1599 E-76 3.68 2,6E + 09 23 E-276 3.96 l, 6E + 08 21

HU 220 583 Β1

B. Foreign barley

1. Applied strains

A.l. above.

2. Barley

The barley used is as shown in Figs. low quality foreign barley.

The soaking was carried out as described in Example 7 except that barley portions of 20 g were used and the barley: water ratio was set to 1: 17.0.

The barley is germinated for 6 days in plastic bags placed in a plastic box on a metal frame, while reaching a target moisture content of 46%. The bags are rotated daily. To maintain moisture, pour some water into the bottom of the germination box. The green malt was dried in a 50 ° C incubator for 17 hours, then the temperature was raised to 85 ° C and the barley dried for an additional 4 hours. Germs are removed mechanically.

4. Lactic Acid Bacterial Preparation Table 15 shows the pH, cell number and microbicidal activity of the lactic acid bacterial cultures added to the malting process (as determined by the method described in Example 1, paragraph 5).

Table 15

Malted barley version lactic acid bacteria tribe PH of the culture solution cell Number (PMY / ml) Inhibition zone (mm) Foreign No. 1 E-76 3.82 9,5E + 08 23 E-276 4.00 3.5E + 08 20 Foreign No. 2 E-76 3.82 9,5E + 08 23 E-276 4.00 O8 + 3,5E 20 Foreign No. 2 E-76 3.70 7.5E + 09 23 E-276 4.01 4,4E + 08 20

5. Dosing of lactic acid bacteria preparations

The lactic acid bacterial preparation is added at the beginning of the soaking steps 1 and 2. The ratio of the lactic acid bacterial culture to water during the malting process was maintained at 8%. 30

6. Analysis performed

The deoxivalenol concentration of each barley sample was determined by ELISA test (Ridascreen).

7. Our results

Table 16 and Figures 8 and 9 show the effect of deoxivalenol concentrations on malting of Finnish and foreign barley batches, as well as the effect of deoxivalenol (DON) on malting barley containing natural deoxivalenol impurity and Fusarium molds. in.

Figure 10 shows the mean values of deoxivalenol concentrations of three domestic and three foreign barley samples, i.e., the effect of lactic acid cultures of lactic acid cultures added to first and second soaking water on deoxivalenol concentrations obtained by malting Finnish and foreign barley, as determined by ELISA. happened. Our results are presented as mean values.

Table 16

Sample DON (pg / kg) Barley Gust -93 34 Lovisa -93 115 Its 1599 -93 72 Dozen -94 56 Foreign No. 1 296

Sample DON (pgAcg) Foreign No. 2 212 Foreign No. 3 673 Malt Gust -93 reference 29 Gusts -93 + E-76 55 Gusts -93 + E-276 23 Loviisa -93 reference 66 Lovisa -93 + E-76 53 Loviisa -93 + E-276 109 Dozen -94 Foreign Reference # 1 262 Foreign No. 1 + E-76 73 Foreign No. 1 + E-276 56 Foreign Reference 2 92 Foreign No. 2 + E-76 87 Foreign No. 2 + E-276 59 Foreign Reference 3 263 Foreign No. 3 + E-76 40 Foreign No. 3 + E-276 38 E-76 = Lactobacillus plantarum E-276 = Lactobacillus acidophilus

The results obtained show that the concentration of deoxivalenol in the reference maltate is higher than that of lactic acid bacteria when L. plantarum (E-76) is added, especially when malting foreign barley samples18.

EN 220 583 Bl malt when malting. The L. plantarum strain had a 68% lower deoxivalenol concentration (as an average of three samples) than the reference sample and the Lactobacillus acidophilus (E-276) strain had a 75% lower deoxivalenol concentration than the reference sample. Finnish barley lots have low deoxivalenol concentrations and this can be further reduced by malting.

Example 9

Effect of lactic acid bacteria on malting of barley contaminated with strains of Fusarium culmorum D148 and F. graminearum D-470 on zearalenone (Zen) content

A: l. Lactic acid bacterial strain used: Lactobacillus plantarum (E-76) strain.

The strains, barley (contamination time 7 days), malting procedure, preparation of the lactic acid bacteria and addition of the lactic acid formulations used are as described in Example 7, 1-5. of this Regulation.

6. Tests performed

The zearalenone content of each barley sample was determined by liquid chromatography.

7. Our results

All. Fig. 4A shows the effect of the addition of lactic acid bacteria on the zearalenone content of malting barley contaminated with F. culmorum strain D-148.

The results show that L. plantarum strain E76 reduces the zearalenone content of malt by 46% compared to the reference malt.

B: 1. Applied strains

Lactic acid bacterial strains used in the assay: Lactobacillus plantarum (E-76), Lactobacillus acidophilus (E-276) and Pediococcus pentosaceus (E-390).

The cultivation was carried out as described in Example 7.

2. Barley

The barley used was Kymppi barley harvested in 1994 and contaminated with F. graminearum VTT D-95470 (D-470) as described in Example 7 (2).

The malting process, the lactic acid bacterial formulations and the lactic acid bacterial formulations are administered as described in Examples 7 to 3-5. are identical.

Furthermore, the L. acidophilus E-276 lactic acid bacterial culture added to the malting process had a pH of 4.05, a cell number of 5.8E + 08 CFU / ml, and an inhibition zone of 14 mm (

Example 1 5.1. above).

6. Tests performed

The zearalenone content of each barley sample was determined by ELISA test (Ridascreen).

7. Results

Figure 12 shows the effect of the addition of lactic acid bacteria on the zearalenone content of barley contaminated with F. graminearum strain VTT D-95470 (D-148).

The results show that lactic acid bacteria reduce the zearalenone content of malt by 37-55% compared to the reference maltate.

Claims (5)

PATENT CLAIMS CLAIMS 1. A method of treating seed material for germination, comprising the step of producing a microorganism inhibitory lactic acid bacterial composition or a lactic acid bacterial composition for the barley seeds to be used in the malting process or for seed germination for nutritional purposes. 2. The method of claim 1, wherein the lactic acid bacterium is a bacterium of the genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus. The process according to claim 1 or 2, characterized in that Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus or Lactobacillus plantarum species or a mixture thereof, preferably Lactobococcus or , or a lactic acid bacterial composition derived from a mixture thereof, or a composition produced by these lactic acid bacteria. 4. A process according to any one of claims 1 to 3, characterized in that the barley seeds are supplemented with a lactic acid bacterial composition having an inhibitory effect on Fusarium mold or a product produced by a lactic acid bacterium. The method of claim 4, wherein the lactic acid bacterial composition or the lactic acid bacterial composition is added to the soaking or germination step.