SK89995A3 - Procedure for treatment of seed material to be germinated - Google Patents

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

Method of seed treatment for germination

Technical field

The invention relates to a method of treating seed to be germinated.

BACKGROUND OF THE INVENTION

The germination process in the context of the present invention is generally understood to mean the technological step necessary to produce the germinated product from stored dry seeds. In the brewing and distillery industry, for example, the germination process, ie. malt formation is used in the production of an important raw material of beer or other alcoholic beverages, namely cereal malt, such as barley malt, rye malt or any other malt. The germination process continues to be used to produce various commercial key products, such as bean or other keys for human nutrition.

The germination processes are usually carried out under non-aseptic conditions. The treated seeds contain microbes originating from the place of cultivation or storage. The conditions during the germination process are generally favorable to the seeds present on the seeds, so that the microbes usually multiply during germination. The microbes can act adversely on the germinated product or the end product which can be produced therefrom, while at the same time having a beneficial effect on the germinating product.

The main stages of the brewing process are: malt production, wort production, primary and secondary fermentation and subsequent processing. The purpose of malt production is to obtain enzymes from the core part, which at the spreading stage break down the nuclear endosperm substances into a wort soluble form. For example, it is known that the process of barley malt production involves three stages: steeping, germination and kilning. The cleaned and screened barley grains are soaked in water until the desired moisture is reached, for example of the order of 43 to 44%. The soaking time may be carried out in a so-called " air climate. The barley is germinated under controlled conditions and the germinated barley is fed in a hot air stream until germination is complete. The roots are removed from the malt at the end of kilning. The regulation of the malt production stages is based on temperature, air flow and humidity control.

The quality of the malt is also influenced by the microbial flora of the malt cereal, which varies significantly depending on the type of the cereal, the weather conditions, the location of the growing season, the length of the growing season and the storage conditions.

The most commonly used cereal in malt production is barley. Its own microbial flora can be classified as field and storage fungi, bacteria and yeast. The most common barley fungi are Fusarium, Alternaria, Cladosporium, Cephalosporium, Epicoccum and Helminthosporium. The occurrence of fungi varies in different regions and different years. Moist weather conditions during the growing season of the cereal and especially during the harvesting period support the growth of Fusarium. Fusarium contamination can be truly considerable in rainy vegetation periods. One of the most common bacterial species on cereals is Enterobacter agglomerans. Other bacteria include Escherichia coli and Pseudomonas, Micrococcus and Bacillus, and lactic acid bacteria. The number of bacteria per barley is about 105 to 108 CFU / g (colony forming units per gram).

I

The number of molds and bacteria in barley increases during malt production and peak concentrations usually reach the germination stage. In particular, fungi are subject to the strongest proliferation

Fusarium and lactic acid bacteria. Yeast production also increases during malt production. When kilning, the concentration of molds, yeasts and bacteria usually decreases. Some of the microbes present on barley have a beneficial effect in terms of malting and malt products such as beer. It is estimated that up to 40 to 50% of certain enzymes in malt are of microbial origin. On the other hand, some microbes have a detrimental effect on barley and / or malts.

Among the harmful microbes, mention should be made of Fusarium, which has been found to cause particularly undesirable foaming of beer more often than other molds, the peptides produced by these molds forming cores for the accumulation and violent escape of gas bubbles from the beer bottle. If more than 50% of malt grains are contaminated with Fusarium, the risk of leakage is clearly higher.

In addition, gram-negative bacteria present in barley, such as species belonging to the genera Pseudomonas and Flavobacterium, and Gram-positive bacteria of the genus Leuconostoc have been shown to slow filtration of mash in connection with wort production. The various microbes present in barley may also produce other disadvantageous effects, for example, inhibiting germination, causing undesirable flavor or adverse changes in the wort and beer analytical values.

The quality requirements for malting barley can be specified in annual cultivation contracts and delivery times. Molds are often mentioned group of microbes in qualitative specifications. In addition, many malting companies have set an upper limit on certain molds. If the amount of grains contaminated with Fusarium exceeds 65% or if the corresponding ratio of molds to Aspergillus and Penicillium exceeds 50%, the barley may be classified as low quality or even unsuitable for use in malt production.

Attempts have been made to reduce mold-induced foaming using good-quality barley or by mixing batches of barley, malt or beer. In rainy years, almost all barley crops may have poor quality and it may be impossible to obtain a good barley. Microbicidal chemicals have also been tested to reduce the amount of fungi, but it was not possible to find a safe and generally acceptable substance.

The germination process for the production of shoots for nutritional purposes provides similarly favorable conditions for the proliferation of, for example, fungi and bacteria. Such key products are perishable quickly. In addition, pathogens in food-inducing poisoning foods such as Salmonella, Yersinia and / or Listeria may be associated with germination.

SUMMARY OF THE INVENTION

The purpose of the invention is to overcome the above-mentioned disadvantages. In particular, it is an object of the invention to find a process by which the quantity and quality of the microbial flora can be carefully controlled during germination, but without adversely affecting the quality of the germinated product or the possible end product.

As regards the features characterizing the invention, reference is made to the claims.

The invention is based on studies in which it has unexpectedly been found that lactic acid bacteria can be used to improve the quality of the germinated product. The substances contained and / or produced by the preparation according to the invention, i. microbicidal agents, inhibit the growth of harmful microorganisms that occurs in connection with the germination process.

The use of lactic acid bacteria in the food and feed industry is known. Under fermentation conditions, these bacteria produce compounds that affect the composition and flavor of the product, but which also inhibit the growth of pathogenic microbes leading to the deterioration of these products. Lactic acid bacteria are commonly used in dairy products, meat products, plant fermentation products and bakery products and in the preservation of feed.

The addition of lactic acid bacteria or lactic acid is practiced in the production of a certain type of malt, the so-called Sauermalz. This addition to malt takes place at the kiln stage, before or during spreading. Its purpose is only to reduce the pH of the wort and, as a consequence, to influence the course of the spreading process and the quality of the finished beer. However, lactic acid bacteria have not been used so far in the manner proposed by the invention, i. to inhibit the growth of unwanted microbes in connection with the germination process.

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

In a particularly preferred embodiment, the lactic acid bacteria preparation or the lactic acid bacteria preparation is added to the cereal material, such as barley grains, during the malt production process. The addition of the formulation inhibits the growth of fungi, particularly harmful Fusarium fungi, and bacteria, thereby reducing, for example, the risk of foaming of beer due to Fusarium fungi. However, the preparation does not have a significant effect on the action of a useful microbial flora in terms of the quality of the malt obtained or the beer produced therefrom. No harmful effects of the added composition on malt quality were observed or found to contain or produce compounds harmful to malt or beer produced.

In the malt production process, the lactic acid bacteria preparation or the lactic acid bacteria preparation may be added to the barley grains prior to steeping, during steeping or during germination. Preferably, the addition is carried out in a steeping or germination step. In other parts, the malt production process may be carried out in any manner known per se. If desired, nutrient additives can be added to the malted barley to be processed, or conditions such as lactic acid added can be controlled to optimize the conditions for the growth of lactic acid bacteria.

Any commercially available lactic acid bacterium capable of inhibiting microbial growth can be used in the present invention. Useful types of lactic acid bacteria include Lactococcus, Leuconostoc, Pediococcus and Lactobacillus. Preferred species are Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus and Lactobacillus plantarum, or any mixtures thereof, with Lactobacillus plantarum or Pediococososus pentosaceus being particularly preferred. The use of genetically modified lactic acid bacteria is also possible.

The lactic acid bacterial preparation can be a culture medium, with or without cells, or a concentrated culture medium (including cells). The preparation produced by the lactic acid bacteria may consist of a cell-free culture filtrate or a fractionated culture filtrate or a pure or partially purified microbicidal product.

According to a particularly preferred embodiment, the treatment of the ear is rotated with a concentrated or fractionated culture medium, which may be cell-free or cell-containing. The concentration can be carried out, for example, by lyophilization or evaporation. The culture medium is concentrated, for example, fak

Om torom 2-20-40.

Fractionation, i. The purification of the microbicidal products can be carried out in a manner known per se, for example by means of chromatographic methods or by ultrafiltration.

In the process according to the invention, the activity in inhibiting microbial growth in a preparation comprising lactic acid bacteria or in a preparation produced by lactic acid bacteria added to the seeds, for example an amount of culture medium of about 10 to 10,000, preferably 30 to 7000, for example 40 to 5000 ml / kg treated seeds. The preparation of the culture medium is described in the Examples. It should be noted that in this application the efficacy of the formulation is defined by the culture media used. However, one of ordinary skill in the art will appreciate that compositions of the invention having equivalent inhibitory activity on microbial growth can also be prepared using other culture media and / or procedures.

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

Since the use of lactic acid bacteria in foods is permitted and generally approved, the use of a lactic acid bacteria-derived preparation is also safe. Lactic acid bacteria usually belong to the natural microbial flora of seeds subjected to germination, such as barley grains. Therefore, the process according to the invention is maximally natural. It is also possible to use a strain originally found on seeds as a lactic acid bacteria strain.

Thanks to the invention, damage caused by Fusarium contamination, such as beer foaming, can be reduced in malt production.

In addition, it has unexpectedly been shown that the process improves filterability characteristics in the brewing process. This has been found to be due to the fact that the composition of the invention also limits the number of harmful species occurring in the production of malt and retarding mash filtration, for example belonging to the genera Leuconostoc, Pseudomonas and Flavobacterium.

According to a further preferred embodiment, the lactic acid bacterial preparation or the preparation produced by lactic acid bacteria is added to the seeds in the production of sprouted shoots for food use.

The invention makes it possible to limit the growth of harmful microbes in connection with the germination process. In particular, the invention makes it possible to use biological means to prevent the growth of harmful bacteria on seeds during an industrial germination process.

In general, the process of the invention improves the general hygiene standard of the germination process.

BRIEF DESCRIPTION OF THE DRAWINGS

Hereinafter, the invention is described in more detail by way of examples, which are intended to illustrate the invention without, however, limiting it thereto. The treatment according to the invention is also applicable in other germination processes.

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In FIG. 1 is a graph showing the microbicidal activity in a lactic acid bacteria culture filtrate as determined by the turbidometric method. It represents the normal growth curve of the test organism, E. agglomerans E-396, and the inhibitory effect on test organism growth induced by the filtrates of the culture strains of the production strains L. plantarum E-76 and P. pentosaceus E-390.

In FIG. 2 shows the effect on the total number of bacteria in malt production with the addition of P. pentosaceus E-390 culture medium added at various stages of malt production.

In FIG. 3 shows the effect on the total number of bacteria in malt production with the addition of P. pentosaceus E-390 cells added at various stages of malt production.

In FIG. 4 shows total bacterial counts at various stages of laboratory malt production with the addition of culture media or concentrated culture media of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water.

In FIG. 5 shows total bacterial counts at various stages of laboratory malt production with the addition of culture media or concentrated and fractionated culture media of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water.

Fig. 6 shows the effect of lactic acid bacteria cultures added in malt production on mash filtration (Tepral filtration).

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Microbicidal effect of different strains of lactic acid bacteria on malt production microbes

In the experiment, the microbicidal effect on microbes occurring in malt production induced by preparations produced by various strains of lactic acid bacteria was studied. Sterile filtered culture medium was used for the preparations.

1. Production strains:

The following strains of lactic acid bacteria were used as 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 (E390) 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 a collection of industrial microorganisms VTT (Biotechnical Laboratory, Finland). Lactobacillus plantarum (E-76) was deposited with DSM (Deutsche Sammlung von Microorganismen und Zellkulturen) under number 7388. The strain E-76 (DSM 7388) was isolated from beer by known methods used for liquid products and was analyzed / identified by known analytical methods. Pediococcus pentosaceus (E-390) was deposited with DSM (Deutsche Sammlung von Microorganismen und Zellkulturen) under number 7389. The strain E-390 (DSM 7389) was isolated from homogenised samples of split barley grains and identified / analyzed by known methods, see Haikar article, A. and Horne, S., Mash Filtration difficulties caused by split barley kernels: A Microbiological Problem, in EBC 1991 publication (Quality Control). The deposit complies with the provisions of the Budapest Treaty.

2. Test strains:

Various harmful microbial species occurring in malt production, as well as lactic acid bacteria strains, used as production strains were used as test strains.

The harmful fungi were represented in the tests by Fusarium fungi [Gibberella avenacea (formerly Fusarium avenaceum) VTT-D-80141 (D-141) and VTT-D-80147 (D-147) and Fusarium culmorum VT.TD-80148 (D-148) and VTT-D-80149 (D-149), the Collection of Industrial Microorganisms, Biotechnical Laboratory of VTT], and one Aspergillus species.

The harmful gram-negative bacteria were represented by two strains of the genus Enterobacter and one species of the genera Flavobacterium and Pseudomonas. Lactic acid bacteria were made up of those strains used as production strains and, moreover, Lactococcus sp. E-416th

3. Cultivation of production strains and preparation of sterile culture filtrate from culture medium:

Lactic acid bacteria were cultured in MRS broth (MRS BROTH, Oxoid). Pediococcus strains were cultured aerobically at 25 ° C and all other production strains anaerobically at 30 ° C; the culture time ranged from 2 to 5 days. The cells were then centrifuged and the supernatant was sterile filtered.

»4. Cultivation of test strains:

Spore suspensions were made from Fusarium fungi by culturing the strain in CMC (carboxymethylcellulose) solution at 25 ° C by shaking for 5 to 6 days, dispersing the formed spores in TWEEN solution, filtering the suspension, and collecting the filtrate.

Aspergillus spore suspension was obtained directly on PD agar (25 ° C, 3 days; potato dextrose, Difco).

Gram-negative bacteria were cultured aerobically in NB medium (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 cultured as described in paragraph 3.

5. Investigation of the microbicidal effect of lactic acid bacteria:

The microbicidal activity of the culture medium was evaluated by disk method or by turbidometry.

1.5 Disc method of microbicidal activity evaluation:

I

Pipette the sterile filtered culture medium or its diluted solution at 100 μΐ onto a filter paper disc (12.7 mm diameter). The discs are placed on Plate Count agar plates into which 0.3 ml of a 10-2 dilution of test organism is poured. The samples were cultured for 24 h at 30 ° C, and the diameter of the inhibition zone formed in mm was measured.

5.2. Turbidometric method for the evaluation of microbicidal activity:

*

An automatic turbidometer (Bioscreen, Labsystems) was used in the procedure.

The sample contained 10% by volume of test organism and 10% by volume of sterile filtered culture medium of the production strain, calculated on sample volume, and growth medium. In the controls, the sterile filtrate preparation was replaced with distilled water, the pH of which was adjusted to the same value as the sterile filtered preparation with lactic acid.

The growth medium used was the same for each test strain as for the cultivation of the test strain.

Growth conditions for Fusarium and Aspergillus: 5 days, 25 ° C, vigorous shaking; for Gram-negative bacteria - Enterobacter strain 30 ° C, all others 25 ° C and shaking; for lactic acid bacteria 3 days, 30 ° C and shaking.

The device detected absorbance at 420 to 580 nm wavelengths of visible light. After cultivation, it was possible to construct the growth curve of each sample and calculate the area below that curve.

The microbicidal effect of the production strain on the test strain was expressed by the percentage of inhibition obtained from the comparison of the growth area sizes found in the control and control groups, respectively. a sterile filtered preparation of the production strain.

6. Examination of the fungicidal effect of certain lactic acid bacteria:

The fungicidal effect of the six lactic acid bacteria strains E-76, E-98, E-3J.5, E-317, E-414 and E-415, in particular on all Fusarium fungi used as test strains, was studied. In the assay, a visual evaluation of haze formation was performed in fungal cultures to which a sterile filtered preparation from culturing each lactic acid bacterium at various dilutions was added, as shown in Table 3.

In the controls, sterilized Milli-Q water and Milli-Q water adjusted to pH 3.6 with lactic acid were used instead of culture medium.

Cultivation was carried out in tubes in CMC medium for 5 days at 25 ° C. Results could be read visually.

7. Results:

In Tables 1 and 2 and FIG. 1 shows the microbicidal activity determined for various lactic acid bacteria, respectively. turbidometric method.

Table 3 summarizes the visually determined fungicidal activity.

Table 1. Microbicidal activities of lactic acid bacteria culture media by the disk method

lactic acid bacteria CFU / ml cell count culture medium, pH diameter of inhibition zone, mm E-76 3,2.10 ⁸ 3.70 19 E-98 1,2.10 ⁸ 3.72 17 E-315 1.6.10 ⁸ 3.90 16 E-317 1,0.10 ^a 3.86 16 E-390 1,9.10 ⁸ 3.92 15

cell number of test organism E-396: 4.0.10 ⁷ CFU / ml; on dish: 1.2.10 ⁵ CFU / ml

ND: not specified; negative number: inhibits growth; positive number: stimulates growth;

> 55% strong inhibition; 35-55% fairly potent inhibition; 15-34% fairly weak inhibition; 15% little or no inhibition induces beer foaming; ² ^ slows the filtration of the mash

Table 3. Effect of lactic acid bacteria on Fusarium molds causing beer foaming + distinct growth; - no growth

D-141 strain of bacteria milk fermentation dilution of the culture medium 1: 6 2: 6 3; about 4: 6 6: 6 E-76 + + - - - E-98 + + - _ _ E-315 + + + _ _ E-317 + + + _ _ E-414 + + + + - - E-415 + + + _ _ inspection + pH control +

D-147 lactic acid bacteria strain dilution of the culture medium 1: 6 2: 6 3: 6 4: 6 6: 6 E-76 + + - - - - E-98 + + - _ _ _ E-315 + + + _ _ E-317 + + + _ _ E-414 + + + + - - E-415 + + + _ _ _ inspection + pH control +

D-148 lactic acid bacteria strain dilution of the culture medium 1: 6 2: 6 3: 6 4: 6 6: 6 E-76 + _ - - - E-98 + + _ _ _ _ E-315 + + _ _ _ _ E-317 + + - E-414 + + + - - - E-415 + + _ _ _ _ inspection -¼ 1 pH control +

D-149 lactic acid bacteria strain dilution of the culture medium 1: 6 2: 6 3: 6 4: 6 6: 6 E-76 + + _ _ _ _ E-98 + + - E-315 + + ' + - - - E-317 + + + - - - E-414 + + + - - - E-415 + + + - - - inspection + pH control +

The results of the tests show that said lactic acid bacteria strains inhibit the growth of harmful Fusarium fungi and other undesirable microbes occurring in the malt production process, while substantially not affecting useful microbes. The results obtained demonstrate the utility of lactic acid bacteria in the process of the invention.

Example 2

Microbicidal effect of certain strains of lactic acid bacteria on food pathogens and on food harmful microbes

In the experiment, the microbicidal effect of the preparations obtained with the strains Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) on food pathogens and on food harmful microbes was conducted. The strains belonging to the genera Bacillus, Yersinia, Listeria, Pseudomonas, Salmonella and Staphylococcus were selected as test organisms.

Sterile lactic acid bacteria culture medium prepared as described in Example 1 was used. All other strains tested were cultured for 16-18 hours in Iso-Sensitest (Oxoid) except Listeria strain grown in tryptose / phosphate. The culture temperature was 30 ° C except for the Salmonella, Listeria and Staphylococcus strains where it was 37 ° C.

The microbicidal activity was determined by the turbidometric method described in Example 1. The experimental conditions for the nutrient substrate and temperature were the same as described above. The incubation time was 24 h except for the Bacillus and Yersinia strains when it was 48 h.

The results are summarized in Table 4, which shows that the addition of the lactic acid bacterium preparation of the invention inhibits the growth of food pathogens and microbes harmful to food.

Table 4. Growth inhibition induced by P. pentosaceus E-390 and L. plantarum E-76

Production strains E-390 E-76 Tested organisms reduction of growth area,% Bacillus cereus ATCC91339 93 80 Yersinia enterolitica ELI351 *) 85 54 Listeria monocytogenes KTL4126 *) 41 49 Pseudomonas fluorescens ELI97 59 97 Pseudomonas fragi ATCC4973 84 93 Salmonella infantis ELI5 *) 90 98 Staphylococcus aureus ELI200 *) 73 86

*) Food Pathogens ATCC: American Type Culture Collection ELI: VTT, Food Research Laboratory

KTL: National Public Health Institute

Example 3

Effect of lactic acid preparations and preparations produced by lactic acid bacteria on microflora in malt production and malt quality

1. Strain used:

The lactic acid bacteria strain Pediococcus pentosaceus VTT-E-90390 (E-390) was used in the experiment.

MRS nutrient medium was used as inoculum growth medium. The bacteria were cultured anaerobically for 2 days in 10 ml of MRS medium at 30 ° C. The injection volume was 1% of the volume of the culture solution.

2. Barley:

Kymppi barley from the 1990 crop was used in which the proportion of grains affected by Fusarium was 55%.

3. Malt production process:

The 1000 g barley batches were washed with a water bath at 12 ° C for 1 hour. The wash water was replaced with the first soaking water and replaced with the second soaking water after 5 hours. Then after 16 h a rest period in the air was started. The purpose of this period was to remove water from the grain surface. It took 8 hours. During steeping the barley reached a humidity of 44%. During the steeping process, the barley was aerated.

and

The steeping was followed by germination. Barley germination was carried out for 6 days at 14 ° C. In order to maintain the moisture at 44%, the barley batches were moistened every day and flooded. The green malt thus obtained was dried according to the 21h temperature program. The temperature was 50 ° C for 4.5 h. During a further 4.5 it was raised to 60 ° C where it was maintained for 4 h. Thereafter, the temperature was raised uniformly up to 85 ° C over a period of 5 h and maintained at that value for 3 h. The final moisture content in the malt was about 4%. Finally, the roots were mechanically removed.

The malt production process without any additives served as a control.

4. Preparation of lactic acid bacteria and preparation produced by lactic acid bacteria

Lactic acid bacteria cells isolated from culture media and culture media containing microbicidal compounds, in combination or separately, were used for the preparations. Per kg of barley, 20 ml of culture medium, including cells, was added or cells were separated from 120 ml of culture medium. This separation was performed by centrifugation of the culture medium and the cells were suspended in water. In cases where a culture medium was added, the culture medium was used as such. The number of cells in the formulations added was of the order of about 10 8 to 10 9 CFU / ml.

5. Addition of lactic acid bacteria:

The lactic acid preparation was added to either the barley, at the beginning of steeping I, at the beginning of steeping I and II, or at the beginning of germination.

6. Analyzes carried out:

Samples were taken from each stage of malt production.

6.1. Molds were evaluated as follows: The percentage of grains contaminated with Fusarium molds was determined by

J agar CZAPEK IPRODION DICLORAL (CZID agar), which is selective for Fusarium fungi, and wet filter paper (EBC-Analytica Microbiologica, Part II, 1987). Fusarium fungi have been identified by their typical colony and spore morphology and by the red color.

Aspergillus and Penicillium were evaluated using selective malt salt agar (EBC-Analytica Microbiologica, Part II, 1987). Other common molds were evaluated on a moistened filter paper.

2.6 Lactic acid bacteria were evaluated on MRS agar for both added cultures and malt samples.

3.6 Total bacterial counts were determined on Plate * Count agar (Difco).

4.6 The chemical characteristics of malt were determined by analytical methods known in the field of malt production (EBC-Analytica, 1987, 4th ed.).

7. Results:,

Table 5 shows the numbers of Fusarium molds and lactic acid bacteria at various stages of malt production with the addition of E-390 culture medium.

Figures 2 and 3 show the total numbers of bacteria at various stages of malt production for E-390 culture media and for E-390 cells.

Table 6 shows the results of malt analysis when E-390 culture medium or E-390 cells were added.

Table 5. Effect of P. pentosaceus E-390 culture medium added at different stages of malt production, on Fusarium counts and lactic acid bacteria during malt production

Fusarium molds (number of contaminated grains) degree of addition barley maceration germination malt inspection 55 72 96 25 120 ml KM *) to barley 3 6 77 3 120 ml KM *) to water for 1st steeping 55 62 98 14 120 ml KM *) to water for 1st and 2nd soaking 55 42 90 9 120 ml KM *) to germinate 55 78 91 3 Lactic acid bacteria (cell density, CFU / g) inspection 6,0x1ο ¹ 3,4Χ1Ο ² 2,2χ10 ⁴ 3,0-10 ³ 120 ml KM *) to barley 1,2X10 ⁸ ΙΧΙΟ, ΙΧΙΟ ⁸ 2,2Χ1Ο ⁸ 1,8Χ1Ο ⁸ 120 ml KM *) to water for 1st steeping ΙΟ, ΟχΙΟ ¹ 3,1-10 ⁷ 2,8Χ1Ο ⁷ 1,4Χ1Ο ⁷ 120 ml KM *) to water for 1st and 2nd soaking 6,0x1ο ¹ 7,4χ10 ⁷ 8,3χ10 ⁷ 5,3χ10 ⁷ 120 ml KM *) to germinate β, ΟχΙΟ ¹ 1,5χ10 ⁸ 2,7χ10 ⁸ 2,6Χ1Ο ⁸

KM *); culture medium

Table 6. Effect of culture medium or P. pentosaceus E-390 cells added at various stages of malt production on malt quality

analysis β-Glu mok- ' ex- thick difference Cana bone tract milling extract. sample mg / 1 % % dry. % dry. % dry. inspection 604 4.1 80.1 77.8 2.3 120 ml KM *) to barley 496 4.3 80.3 77.3 3.0 120 ml KM *) to water for 1st steeping 482 4.2 80.5 77.5 2.9 120 ml KM *) to water for 1st and 2nd soaking 482 4.3 80.0 76.9 3.1 120 ml KM *) to germinate 955 4.2 79.0 74.9 4.1 cells (10 ⁸ CFU / g) to barley 606 4.3 80.3 77.4 2.9 cells (10 ⁸ CFU / g) k - - - - - water for the 1st steeping cells (10 ⁸ CFU / g) - - - - - de for 1st and 2nd soaking cells (10 ⁸ CFU / g) 804 4.3 79.5 76.6 2.9 sprouting

Table 6. Effect of culture medium or P. pentosaceus E-390 cells added at various stages of malt production on malt quality

- continuation of the table

analysis sample clarity filter. fine. grinding ml / h sachari fiction time min viscosity cP FAN mg / 1 inspection limpid 300 <10 1.59 163 120 ml KM *) to barley limpid 305 <10 1.51 164 120 ml KM *) to water limpid 310/56 <10 1.50 171 for 1st steeping min 120 ml KM *) to water for 1st and 2nd soaking limpid 305 <10 1.49 168 120 ml KM *) to germinate opal. 305 <10 1.68 129 cells (10 ⁸ CFU / g) limpid 315/61 <10 1.54 167 k j barley min cell (10 ⁸ CFU / g) to water for first dipping of the cell (10 ⁸ CFU / g) to water  " - - - - - de for 1st and 2nd soaking cells (10 ⁸ CFU / g) to germinate opal. 290 <10 1.63 148

Table 6. Effect of culture medium or P. pentosaceus E-390 cells added at various stages of malt production on malt quality

- continuation of the table

analysis modi- homo- aktivi- aktivi- korien- f iká- Genita ta a-a- ta β-gl Patterns cia mylázy glucanase rivers sample % % U / g U / kg g / kg inspection 82 69 202 365 35 120 ml KM *) to barley 80 57 207 355 37 120 ml KM *) to water for 1st steeping 76 51 194 316 30 120 ml KM *) to water for 1st and 2nd soaking 80 64 186 360 31 120 ml KM *) to germinate 69 59 159 386 21 cells (10 ⁸ CFU / g) to barley 79 59 223 359 38 cell (10 ^CF CFU / g) to water for 1st steeping of the cell (10 ⁸ CFU / g) to water 79 59 187 376 39 81 67 178 353 36 de for 1st and 2nd soaking cells (10 ⁸ CFU / g) 81 69 179 392 35 sprouting

- not analyzed

KM *) culture medium

The results obtained show that the treatment according to the invention reduces, in particular, the number of Fusarium molds and the total number of bacteria at various stages of malt production.

The addition of the preparation does not adversely affect the quality of the malt. The opposite is true: the treatment according to the invention improved the filtration of the wort obtained from the wort and reduced the β-glucan content in the malt.

Example 4

Manufacture of preparations produced by lactic acid bacteria

1. Preparation of concentrated culture media:

The production strains Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) were cultured in a 15 L fermenter. The inoculum volume was 6-7%, cultivated for 2 days. at 30 ° C in MR S medium under microaerophilic conditions. The culture media were concentrated ten and twenty times by lyophilization and evaporation. The microbicidal activity of the concentrates was determined by the disc method.

2. Preparation of a purified solution containing microbicidal compounds:

The lactic acid bacteria culture medium was purified by size exclusion gel chromatography. Fractions detected by the disk method and turbidometry as active were collected and returned to the gel column.

Example 5

Effect of lactic acid preparations and preparations produced by lactic acid bacteria on microflora in malt production and malt quality

1. Used strains and preparations made from the following strains:

The following bacterial strains were used: Lactobacillus plantarum VTT-E-78076 (E-76) of Pediococcus pentosaceus VTT-E-90390 (E-390).

The strains were obtained from a collection of industrial microorganisms VTT (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:

Kymppi barley from the 1991 crop was used.

3. Malt production process:

Malt production was carried out as in Example 3, but with a germination degree of 8 days. The final moisture content in the malt decreased below 5%.

4. Malt production:

Two laboratory malt production was carried out. The process without any additives served as a control.

4.1. First production:

Cell-free culture media, concentrated ten times, and untreated culture media, including cells, were used for laboratory malt formulations. The preparation was added to barley or at the beginning of steeping I and II. The following 8 experiments were performed:

and

Test no. 1: control, Kymppi barley 1991.

Test no. 2: 120 ml of E-76 cell culture medium is added at the beginning of steeping Ia II.

Test no. 3: at the beginning of steeping I, 120 ml of a 10-fold concentrated E-76 culture filtrate are added.

Test no. 4: 120 ml of a 10-fold concentrated E-76 culture filtrate are added at the beginning of soaking I and II.

Test no. 5: 120 ml of E-390 culture medium is added at the beginning of steeping Ia.

Test no. 6: at the beginning of soaking I, 120 ml of a 10-fold concentrated E-390 culture filtrate are added.

Test no. 7: at the beginning of steeping I and II, 120 ml of a 10-fold concentrated E-390 culture filtrate are added.

2.4 Second production:

Twenty times thickened cell-free culture filtrates and untreated cell culture media were used for laboratory malt formulations. The pH of the steeping water was checked. The formulations were added at the beginning of steeping I and II. Experiments 9-16 were conducted:

Test no. 9: control, Kymppi barley 1991.

Test no. 10: na early soaking I and II the was added 120 ml water with pH 3, 8th Test no. 11: on early soaking I and II the was added 120 ml culture E i-76 cells. Test no. 12: on early soaking I and II the was added 120 ml fractionated concentrate] E-76, pH 3 8th Test no. 13: on early soaking I and II the was added 120 ml

Twenty times concentrated E-76 culture filtrate.

Test no.

14: at the beginning of soaking

I and II are added 120 ml of E-390 cell culture medium.

Test no.

15: at the beginning of soaking

I and II are added 120 ml of fractionated concentrate E-390, pH 3.8.

Test no.

16: at the beginning of steeping I and II, 120 ml of a twenty-fold concentrated E-390 culture filtrate are added.

5. Analyzes carried out:

Samples were taken from each stage of malt production.

The numbers of fungi, lactic acid bacteria and total bacteria and the physico-chemical quality characteristics of the malt were determined as described in Example 3.

6. Results:

Fig. Figures 4 and 5 show the total number of bacteria in each stage of malt production using cell culture media of lactic acid bacteria with cells or concentrated or fractionated culture filtrates in steeping water.

Tables 7, 8, 9 and 10 show the concentrations of Fusarium and other mildew and lactic acid bacteria at each malt production step in both processes.

Tables 11 and 12 show the results of the malt analysis from both malt production processes.

- 31 Table 7. Mycoflora in malt production with the addition of preparations p. pentosaceus E-390 and L. planta rum E-76 to barley steeping water (results expressed as percentage of contaminated grains). Sample Numbering in Example 5.

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FP; filter paper

Table 8. Lactic acid counts (CFU / ml) in malt production by addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water. Sample Numbering in Example 5.

example maceration germination malt 1 5,5.10 ³ 3,3.10 ⁶ 2,5.10 ² 2 7,3.10 ⁶ 1,2.10 ⁷ 5,5.10 ⁵ 3 1,4.10 ³ 4,4.10 ³ 1,1.10 ³ 4 3,2.10 ² 1,4.10 ⁴ 9,5.10 ² 5 1.3.10 ⁷ 7,5.10 ⁶ 2,6.10 ⁶ 6 4,8.10 ³ 5,0.10 ⁵ 4,0.10 ⁴ 7 7,3.10 ² 2,4.10 ⁴ 2,6.10 ³ 8 1,2.10 ⁷ 1.4.10 ⁷ 2,3.10 ⁶

Lactic acid bacteria content on barley: 7.5.10 ² CFU / g.

I

- 33 Table 9. Mycoflora in malt production with addition of P · pentosaceus E-390 and L. plantarum to barley steep water (results expressed as percentage of contaminated grains). Sample Numbering in Example 5.

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FP; filter paper

Table 10. Lactic acid counts (CFU / ml) in malt production with the addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water. The sample numbering is in Example 5.

example maceration germination malt 9 8,0.10 ² 4,2.10 ⁶ 8,0.10 ³ 10 8,0.10 ² 5,0.10 ⁶ 5,6.10 ⁴ 11 2.7.10 ⁷ 2,1.10 ⁷ 8,5.10 ⁵ 12 2,2.10 ³ 3,4.10 ⁵ 2,5.10 ³ 13 1,2.10 ³ 1.4.10 ⁷ 2,3.10 ³ 14 4,1.10 ⁷ 3,5.10 ⁷ 1,1.10 ⁴ 15 4,8.10 ³ 4.8.10 ⁶ 3,9.10 ⁵ 16 3,5.10 ² 2,1.10 ⁵ 1,7.10 ⁴

Lactic acid bacteria content on barley: 7.5.10 ² CFU / g.

Table 11. Effect of addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water on malt quality. Sample Numbering in Example 5.

analysis sample 1 2 3 4 humidity% 3.6 3.6 3.5 3.7 fine extract% dry matter 81 80.8 80.6 80 Coarse extract% dry matter 76.8 77 76.1 75.2 extract difference% 4.2 3.8 4.5 4.9 sugar min <10 <10 <10 <10 clear wort clear clear clear clear fine filtration 320/45 320/44 315/48 315/40 fraction ml / min coarse filtration 260/86 265/64 270/59 255/73 fraction ml / min pH of wort 5.93 5.88 5.86 5.86 viscosity cP 1.69 1.57 1.59 1.57 β-glucans mg / l 784 659 705 736 free amino nitrogen mg / l 154 156 155 139 modification% 74 72 73 73 homogeneity% 61 65 63 58 α-amylase U / g 178 182 148 128 β-glucanase U / kg 434 420 360 360

Table 11. Effect of addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water on malt quality. Sample Numbering in Example 5.

- continuation of the table

analysis sample 5 6 7 8 humidity% 3.7 3.6 3.9 3.9 fine extract% dry matter 81.2 81 80.7 81.2 Coarse extract% dry matter 77 76.4 74.8 77.1 extract difference% 4.3 4.7 5.9 4.1 sugar min <10 <10 <10 <10 wort clarity clear clear clear clear fine filtration 320/47 315/66 320/49 315/52 fraction ml / min coarse filtration 260/68 260/76 255/90 275/120 fraction ml / min pH of wort 5.86 5.86 5.97 5.99 viscosity cP 1.55 1.58 1.59 1.58 β-glucans mg / l 610 696 822 657 free amino nitrogen mg / l 158 150 130 154 modification% 72 75 77 76 homogeneity% 67 66 62 68 α-amylase U / g 179 156 144 174 β-glucanase U / kg 442 412 361 425

Table 12. Effect of addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water on malt quality. Sample Numbering in Example 5.

analysis sample 9 10 11 12 humidity% 3.5 3.6 3.6 3.5 fine extract% dry matter 80.5 80.5 81.2 80.5 Coarse extract% dry matter 78.4 77.9 77.4 73.3 extract difference% 2.1 2.6 3.7 7.2 sugar min <10 <10 <10 <10 wort clarity clear clear clear clear fine filtration 320/40 315/47 320/44 315/42 fraction ml / min coarse filtration 290/120 270/76 260/51 265/120 fraction ml / min pH of wort 6.04 6.05 5.99 5.97 viscosity cP 1.54 1.65 1.51 1.53 β-glucans mg / l 442 522 397 553 free amino nitrogen mg / l 149 145 149 128 modification% 85 85 86 82 homogeneity% 63 66 76 57 α-amylase U / g 161 172 161 123 β-glucanase U / kg 365 375 365 282

Table 12. Effect of addition of P. pentosaceus E-390 and L. plantarum E-76 to barley steeping water on malt quality. Sample Numbering in Example 5.

- continuation of the table

analysis sample 13 14 15 16 humidity% 3.8 3.5 3.5 3.6 fine extract% dry matter 80.3 81 81.1 80,4 Coarse extract% dry matter 73.5 77.6 76.6 75.1 extract difference% 6.8 3.4 4.5 5.4 sugar min <10 <10 <10 <10 wort clarity clear clear clear clear fine filtration 320/55 315/49 330/40 320/40 fraction ml / min coarse filtration 245/120 285/68 305/120 265/12 fraction ml / min pH of wort 5.94 5.95 5.98 5.99 viscosity cP 1.5 1.49 1.49 1.52 β-glucans mg / l 612 381 463 576 free amino nitrogen mg / l 117 149 139 120 modification% 74 81st 75 71 homogeneity% 57 68 51 47 α-amylase U / g 72 148 125 101 β-glucanase U / kg 282 371 342 308

The results obtained now show that the treatments according to the invention reduce, in particular, the amount of Fusarium and the total number of bacteria at various stages of malt production. Furthermore, it is clear that, in addition to culture media, concentrated and fractionated culture filtrates, in particular, have a beneficial effect with respect to Fusarium molds, for example.

From the malt analyzes, it can be concluded that the addition of the culture medium of strains E-76 and E-390 resulted in improved filterability of the wort obtained / from malt; β-glucan content is also lower in individual malts than in control sla39

Example 6

Effect of lactic acid filtration products produced by lactic acid bacteria on malt production

In FIG. 6 is a diagram showing the effect of the treatment of the invention with formulations derived from lactic acid bacteria strains (120 ml culture medium per kg barley) on mash filtration obtained using the malt so treated.

The strains listed 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 filter method (BIOS 19, 1988, Grandclerc, J. et al., Simplification of the Methylation and Brassin Filtration of the Description of the Method, pp. 88-92).

The results show that the treatment according to the invention improves the filtration of the mash.

Claims (5)

PATENT CLAIMS 1. A method of treating seed for germination, characterized in that a preparation of lactic acid bacteria or a product produced by the process of germination is added to the barley grains in the malt production process or to be converted into nutritional shoots. lactic acid bacteria having an inhibitory effect on microbial growth. Method according to claim 1, characterized in that the lactic acid bacteria belong to the genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus. 3. The method of claim 1 or 2, characterized in that the preparation of lactic acid bacteria, or a preparation produced by lactic acid bacteria has been derived from species Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus _{(pentosaceus,} Lactobacillus curvatus or Lactobacillus plantarum or mixtures thereof, preferably from the species Lactoba * cillus plantarum or Pediococcus pentosaceus, or mixtures thereof. Method according to any one of claims 1 to 3, characterized in that a lactic acid bacterial preparation or a product produced by lactic acid bacteria having an inhibitory effect on Fusarium fungal growth is added to the barley grains. The method of claim 4, wherein the lactic acid bacteria preparation or the lactic acid bacteria preparation is added in a steeping or germination step.