RU2126443C1 - Method of treating germination-destined seed grain - Google Patents

Abstract

FIELD: agrotechnics. SUBSTANCE: method consists in adding antimicrobial additives as nutrient to barley seeds used in malting process or to seed grains that would be transformed into germs. Additives that suppress growth of bacteria are lactic acid bacteria preparation or preparation produced by those bacteria. EFFECT: enabled microbial protection of seed grain. 5 cl, 12 dwg, 16 tbl, 9 ex

Description

 The present invention relates to a method for processing seed intended for germination.

 In the context of the present description, the process of germination is usually understood that stage of the process in the General sense of the word, which is necessary to obtain a product of germination, when the source material is used, seeds stored in a dry state. In particular, in the brewing and distillery industries, the germination process, which is a rejuvenation process, is used to produce raw materials important for the preparation of beer or other alcoholic beverages, namely grain malt, such as barley malt, corn malt or any other malt. In addition, this germination process is used in the preparation of various commercial products, which are seedlings, for example, bean seedlings or any seedlings intended for use in human nutrition.

 Typically, germination processes are carried out under non-aseptic conditions. Microbes are found on seeds treated under these conditions, the presence of which is caused by environmental conditions during plant growth or seed storage. The conditions observed during the germination process are most favorable for the microbes present on the seeds, usually these microbes multiply plentifully during the process under consideration. These microbes can have an undesirable effect on the germination product or on the final product produced from it, and they can equally have a beneficial effect on the germinated product.

 The main stages of the brewing process are malting, wort production, primary and secondary fermentation, as well as subsequent processing. The purpose of malting is the appearance of enzymes in the grain, which, at the kneading stage, convert the endosperm of the grain into a form soluble in the resulting wort. In particular, it is well known that the process of malting barley seeds involves three stages: soaking, germination and steaming. Purified and sorted barley grains are soaked in water until they obtain the desired degree of moisture, for example in the range from 43 to 44%. Partially soaking can be carried out by the method of so-called air rest. Barley is allowed to germinate under controlled conditions, and the resulting germinated barley is steamed in a stream of hot air until germination is complete. After steaming, primary roots are removed from the malt. The regulation of the anti-aging stages is based on the control of temperature, air flow and humidity of the product or air.

 In addition to the technology and conditions of malting, the quality of the malt also depends on the microbial flora of the seeds used to obtain the malt, this flora varies significantly, for example, depending on the variety of seeds, weather conditions, place of cultivation, duration of the growing season, as well as storage conditions.

Barley is the cereal most commonly used for malting. The natural, microbial flora of barley can be subdivided into fungi that infect barley in the field, fungi that infect barley during storage, bacteria and yeast. The most famous wildflowers of barley are Fusarium, Alternaria, Cladosporium, Cephalosporium, Epicoccum and Helminthosporium. The occurrence of molds in different countries in different years is different. Wet weather during the grain growth period, especially during harvest, contributes to the growth of Fusarium mold. Fusarium-induced infection can actually be severe during rainy growing seasons. Enterobacter agglomerans is one of the best known types of bacteria found on cereals. Among other bacteria, Escherichia coli and bacteria of the genera Pseudomonas, Micrococcus and Bacillus, as well as lactic acid bacteria, should be noted. In the case of barley, the bacterial titer is approximately 10 ⁵ -10 ⁸ CFU / g (colony forming units per 1 g).

 The number of molds and bacteria in barley increases during malting, reaching the highest concentrations at the germination stage. In particular, Fusarium, as well as lactic acid bacteria, are most susceptible to reproduction. During malting, the amount of yeast also increases. At the steaming stage, the concentration of mold, yeast and bacteria, as a rule, decreases again. Some microbes found on barley have a beneficial effect in terms of rejuvenation, as well as on the product, for example beer made from the obtained malt. Judging by the estimates, up to 40-50% of some malt enzymes are of bacterial origin. On the other hand, some microbes have an undesirable effect on barley and / or malts produced.

 Among unwanted microbes, Fusarium must be noted; it was found that it more often than other molds causes undesirable foaming of beer, in which the peptides produced by these molds form the nuclei of gas bubbles released from the beer bottle in the form of intense foam. In the event that more than 50% of the grain to be rejuvenated is infected with Fusarium molds, the risk of foaming increases substantially.

 In addition, it was shown that gram-negative bacteria found in barley, such as species belonging to the genera Pseudomonas and Flavobacterium, as well as gram-positive bacteria from the genus Leuconostoc, make it difficult to filter mash to produce wort. Various microbes found on barley can also lead to other undesirable effects, including inhibiting germination, causing odors or undesirable changes in the quality indicators of the resulting wort and beer.

 Quality requirements for malting barley can be specified in the annual cultivation and supply contract. Mold fungi are a group of microbes often referred to in quality descriptions. Moreover, for many plants used for plant rejuvenation, the upper permissible limit for specific molds has been determined. If the proportion of grains infected with Fusarium exceeds 65% or the corresponding indicator for molds Aspergillus and Penicillum exceeds 50%, this kind of barley can be considered as low-quality or even unsuitable for use in malting.

 To prevent foaming caused by molds, approaches have been developed based on the use of high-quality barley or on mixing batches of barley, malt or beer. In rainy years, almost the entire barley crop may be of poor quality, in which case getting good beer may not be possible.

 The germination process used to obtain seedlings intended for use for nutrition, suggests some conditions favorable for propagation, for example, molds and bacteria. Germination products of this kind will be perishable. In addition, due to germination, an increase in the number of foodborne pathogens causing food poisoning, such as the bacteria Salmonella, Yersinia and / or Listeria, can be observed.

 Microbicidal chemicals can also be used to reduce the amount of molds, but it is not possible to find safe and recognized chemicals.

 There is a known (SU copyright certificate 825616, class C 12 C 1/047, 1981) a method of processing seed germination intended for germination, in which an antimicrobial additive representing potassium permanganate is added to the seed material in connection with the germination process. At the same time, a certain amount of added chemical agent will be stored on the used seed material; this may undesirably affect the process of rejuvenation, as this agent suppresses both harmful and beneficial microflora; trace amounts of added potassium permanganate will also be preserved in the final product, which may affect the palatability of the product.

 The objective of this invention is to provide such a method of processing intended for germination of seed material, with which it would be possible to finely control the microbial flora of the seed material, inhibiting the development of undesirable microflora during seed germination and not inhibiting the effect of beneficial microflora, and obtain from these seeds the final product that does not contain foreign additives.

 This problem is solved by the fact that the proposed method of processing intended for germination of seed material, including adding to the seed material in connection with the process of germination of antimicrobial additives, characterized in that these additives added to the seed material, which is a barley grain used in the process of malting, or seed material that will be turned into seedlings intended for use as food is a preparation of lactic acid bacteria, or prep Rath produced by lactic acid bacteria which has an effect of suppressing the growth of microbes.

 It is advisable that these lactic acid bacteria belong to the genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus.

 Preferably, when the specified preparation of lactic acid bacteria or a preparation produced by lactic acid bacteria is produced from species of Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus or Lactobacillus, or Larobobactus, or a mixture of Lactobacillus, and Lactobacillus pentosaceus or a mixture thereof.

 To barley grains, such a preparation of lactic acid bacteria or a preparation produced by lactic acid bacteria, which has the effect of inhibiting the growth of Fusarium molds, can be added. In this case, it is preferable that the specified preparation of lactic acid bacteria or a preparation produced by lactic acid bacteria, was added at the stage of soaking or germination.

 The present invention is based on research, which resulted in an unexpected observation, indicating that lactic acid bacteria can be used to improve the quality of the product intended for germination. Substances containing and / or produced by the preparation of the invention, that is, microbicidal agents, inhibit the growth of undesirable microorganisms encountered during the germination process.

 The use of lactic acid bacteria in the food and feed industry is well known. Under fermentation conditions, these bacteria produce compounds that affect the composition and smell of the resulting products, but at the same time inhibit the growth of pathogenic microbes that can spoil these products. Lactic acid bacteria are commonly used in dairy products, meat products, vegetable fermentation products and baked products, as well as in the storage of fodder.

 The addition of lactic acid bacteria or lactic acid was put into practice in the production of a certain type of malt, the so-called Sauermalz. The specified additive is introduced into the malt at the stage of steaming before mashing or during mashing. The purpose of this additive is solely to reduce the pH of the resulting wort in such a way as to affect the course of the mashing process, as well as the quality of the resulting beer. However, lactic acid bacteria have not yet been used as provided by the present invention in order to inhibit the growth of unwanted microbes during the germination process.

 The preparation provided by the present invention can be added to the product intended for germination at any stage of the germination process.

 In the case of a particularly preferred embodiment, the lactic acid bacteria preparation or the preparation obtained with the aid of lactic acid bacteria is added to the grain material, such as barley grains, during the malting process. Said added preparation inhibits the growth of molds, in particular harmful Fusarium molds, as well as bacteria, which in particular reduces the risk of foaming of beer due to Fusarium mold. However, judging by the quality of the resulting malt or beer produced from it, this preparation does not significantly affect the action of beneficial microbial flora. In addition, no harmful effects of the indicated added preparation on the quality of the malt were found, nor were any substances contained in the preparation or produced by it harmful to the produced malt or beer detected.

 During the malting process, the indicated lactic acid bacteria preparation or the preparation obtained with the aid of lactic acid bacteria can be added to barley grains before soaking, at the soaking stage or during the germination stage. In the preferred case, the specified additive is carried out at the stage of soaking or germination. Other elements of the digestion process may be carried out by any method known in the art. If desired, for example, nutrients can be added to the barley to be fermented, or conditions can be regulated, including by the addition of lactic acid, in order to optimize the conditions necessary for the growth of lactic acid bacteria.

 Any widely available lactic acid bacterium having an effect in terms of inhibiting the growth of microbes can be used in the present invention. The following suitable genera of lactic acid bacteria can be mentioned: Lactococcus, Leuconostoc, Pediococcus and Lactobacillus. As preferred species, the following can be listed: Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus and Lactobacillus plantarum, or mixtures thereof, in which Lactobacusususus plantus are most preferred. It is also possible to use genetically modified lactic acid bacteria.

 The lactic acid bacteria preparation may consist of a culture fluid containing or not containing cells, or a concentrated culture fluid (containing cells). A preparation obtained with the aid of lactic acid bacteria may include a cell-free culture filtrate, a concentrated culture filtrate or a fractionated culture filtrate, or a pure or partially purified microbicidal product.

 According to a particularly preferred embodiment, said treatment is carried out using concentrated or fractionated culture fluid, which may be cell-free or may contain cells. Concentration can be achieved, for example, by lyophilization or evaporation. The specified culture fluid is concentrated, for example, by a factor of 2 - 20 - 40.

 Fractionation, including purification of microbicidal products, can be carried out using well-known methods, for example, using chromatography or ultrafiltration methods.

 In accordance with the method provided by the present invention, intended to add to the seeds activity that inhibits the growth of microbes and is part of the preparation containing lactic acid bacteria or obtained using lactic acid bacteria, corresponds, for example, the specified culture fluid in an amount of from about 10 to 10,000 ml / kg of treated seeds, in an acceptable case, from 30 to 7000 ml / kg of treated seeds, including from 40 to 5000 ml / kg of processed seeds. The preparation of this culture fluid is described in the Examples section. It should be noted that the direct parameters for the use of the activity of the specified drug are determined depending on the purpose of application of the aforementioned culture fluid. It is obvious to a person skilled in the art that preparations provided by the present invention having equivalent activity in terms of inhibiting the growth of microbes can be equally obtained using other culture liquids and / or methods.

 According to the present invention, said preparation includes microbicidal compounds and / or produces microbicidal compounds during the germination process. If a cell preparation is used, cell growth can be induced if necessary, for example, by regulating the conditions during the germination process or using nutritional supplements. The indicated preparation can also enhance the growth of other lactic acid bacteria found on seeds intended for germination.

 Since the use of lactic acid bacteria in the food industry is permitted and widespread, this preparation obtained from growing lactic acid bacteria is also safe to use. Lactic acid bacteria usually belong to the natural microbial flora of germinated seeds, such as barley grains. Thus, the method provided by the present invention is as natural as possible. In addition, a strain naturally occurring on the corresponding seeds can be used as a strain of lactic acid bacteria.

 Thanks to the present invention, during fermentation, it becomes possible to reduce undesirable effects resulting from infection of Fusarium, such as foaming of beer.

 In addition, it was unexpectedly discovered that the proposed method improves the filterability in the beer production process. It was found that this effect is due to the fact that the drug provided by the present invention also limits the number of harmful species that occur during fermentation and interfere with the filtration of the resulting mass, including those belonging to the genera Leuconostoc, Pseudomonas and Flavobacterium.

 According to another preferred embodiment, the lactic acid bacteria preparation or the preparation obtained from lactic acid bacteria is added to the seeds if the resulting seedlings are used for food.

 Thanks to the present invention, it is possible to limit the growth of harmful microbes during the germination process. This is the first time that the invention involves the use of biological agents to prevent the growth of unwanted bacteria found on germinated seeds during the industrial germination process.

 The method provided by the present invention improves the overall hygiene standard of said whole germination process.

 Hereinafter the present invention will be illustrated by examples of embodiment, the sole purpose of which is to illustrate the present invention, not reducing it to the examples given. The processing provided by the present invention is also applicable to other germination processes.

 Figure 1 presents a graph showing the microbicidal activity contained in the filtrate culture of lactic acid bacteria and determined using the turbidometric method. The normal growth curve of the test organism E. agglomerans E-396, as well as the inhibitory effect on the growth of the aforementioned test organism by filtrates of the culture of producing strains L. plantarum E-76 and P. pentosaceus E-390.

 Figure 2 shows the effect of the culture fluid P. pentosaceus E-390, added at different stages of malting, on the total titer of bacteria during malignancy.

 In FIG. Figure 3 shows the effect of P. pentosaceus E-390 cells added at different stages of malignancy on the complete bacterium titer during malignancy.

 In FIG. Figure 4 shows the complete titer of bacteria during various stages of laboratory malignancy when added to the water used to soak barley, P. pentosaceus E-390 and L. plantarum E-76 culture liquids, or concentrated culture liquids.

 In FIG. Figure 5 shows the complete titer of bacteria during various stages of laboratory malting when added to the water used to soak barley, P. pentosaceus E-390 and L. plantarum E-76 culture liquids, or concentrated and fractionated culture liquids.

 Figure 6 shows the effect of cultures of lactic acid bacteria added at the stage of malting, on the filtration of the resulting mass (Terpal-filtering).

 In FIG. Figure 7 shows the effect of the addition of lactic acid bacteria on the deoxynivalenol content during the malting of barley infected with strain F. culmorum D 148.

 In FIG. Figures 8 and 9 show the concentration of deoxynivalenol during the deposition of portions of Finnish and foreign barley, as well as the effect of the lactic acid bacteria on the formation of deoxynivalenol (DON) during deposition of barley naturally infected with dexinivalenol and Fusarium mold.

 Figure 10 shows the average concentration of deoxynivalenol of three home and three foreign barley samples.

 11 shows the effect of the addition of lactic acid bacteria on the content of zearalenone during the malting of barley infected with the strain F.culmorum D 148.

 12 shows the effect of the addition of lactic acid bacteria on the content of zearalenone during the malting of barley infected with the F.graminearium VTT strain D-95470 (D-148).

 Example 1. The microbicidal effect exerted by various strains of lactic acid bacteria on microbes encountered during malting.

 In the experiment, the microbicidal effect exerted by drugs produced by different strains of lactic acid bacteria on microbes encountered during malting was studied. As the specified drug used culture fluid obtained by sterile filtration.

 1. Producing strains.

The following strains of lactic acid bacteria were used as producing 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 pentosacues - VTT-E-88317 (E-317)
Pediococcus pentosaceus - VTT-E-90390 (E-390) - (DSM 7389)
Lactobacillus curvatus - VTT-E-90391 (E-391)
Lactobacillus plantarum - VTT-E-78076 (E-76) - (DSM 7388)
Lactobacillus plantarum - VTT-E-79098 (E-98)
The above strains were obtained from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland). Strain Lactobacillus plantarum (E-76) was deposited in DSM (Deutsche Sammlung von Mikroorganismen und Zeilkulturen) under the number 7388. Strain E-76 (DSM 7388) was isolated from beer using a technique known for liquid products, and analyzed / identified with using well-known analysis methods. The strain Pediococcus pentosaceus (E-390) was deposited in DSM (Deutsche Sammlung von Mikroorganismen und Zeilkulturen) under the number 7389. The strain E-390 (DSM 7389) was isolated from homogenized samples of cracked barley grains and analyzed / is well known / identified 1]. Depositories comply with the terms of the Budapest Treaty.

 2. Tester strains.

 Among the test strains, among others, various types of harmful microbes that were found during malignancy, as well as strains of lactic acid bacteria that served as producing strains, were used.

 Harmful molds were presented in the tests by Fusarium mold [Gibberella avenacea (formerly Fusarium avenaceum) VTT-D-80141 (D-141) and VTT-D-80147 (D-147), as well as Fusarium culmorum VTT-D-80148 ( D-148) and VTT-D-80149 (D-149), Collection of Industrial Microorganisms, Biotechnical Laboratory of VTT] and one species of Aspergillus.

 The harmful gram-negative bacteria were represented by two strains from the genus Enterobacter, as well as one species from the genus Flavobacterium and one from the genus Pseudomonas. Strains of lactic acid bacteria included strains used as producing strains, as well as the strain Lactococcus sp. E-416.

 3. The cultivation of producing strains and obtaining a sterile filtered culture fluid.

Lactic acid bacteria were cultured in MRS medium (MRS BROTH, Oxoid). Strains Pediococcus E-65, E-67 and E-68 were cultured under aerobic conditions at a temperature of 25 ^o C, all other producing strains under anaerobic conditions at a temperature of 30 ^o C, the cultivation period was from 2 to 5 days. Subsequently, the cells were besieged by centrifugation, and the resulting supernatant was sterile filtered.

 4. The cultivation of tester strains.

Fusarium mold spore suspension was obtained by culturing a strain of this fungus in a CMC (carboxymethyl cellulose) solution at a temperature of 25 ^° C for 5-6 days as an intensively mixed culture, suspending the resulting spore particles in a TWEEN solution, filtering the resulting suspension and selecting the filtrate.

Aspergillus mold spore suspension was obtained directly on PD-arape (Potato Dextrose, Difco) at a temperature of 25 ^° C. for 3 days.

Gram-negative bacteria were cultured under aerobic conditions 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 in accordance with paragraph 3 (see above).

 5. Determination of the microbicidal effect of lactic acid bacteria.

 The microbicidal activity of the obtained culture fluid was evaluated using the disk method, or turbidometrically.

 5.1. Disc method for determining microbicidal activity.

Sterile filtered culture fluid or its dilution was instilled in 100 μl onto filter paper discs (12.7 mm diameter). These discs were placed on agar plates for counting plaques, seeded with liquid culture of the tester organism (0.3 ml dilution 10 ^-2 ). The obtained samples were cultured for 24 h at a temperature of 30 ^o C, after which the diameter (in mm) of the formed suppression zone was measured.

 5.2. Turbidometric method for determining microbiological activity.

 An automatic turbidometer (Bioscreen / Labsystems) was used in this method.

 The test sample consisted of 10% by volume of the culture of the test organism, 10% by volume of sterile filtered culture fluid (calculated in relation to the volume of the sample) and growth medium. In the case of controls, the sterile filtered preparation was replaced with distilled water, the pH of which was adjusted with lactic acid to a value corresponding to the sterile filtered preparation.

 In the case of each test strain, the growth medium was the medium used to cultivate the corresponding test strain.

The cultivation of molds Fusarium and Aspergillus was carried out for 5 days at a temperature of 25 ^o C with vigorous stirring. The appropriate conditions for gram-negative bacteria were: in the case of the Enterobacter strain, 30 ^° C, in the remaining cases 25 ^° C, with stirring for 2 days. The cultivation of lactic acid bacteria was carried out for 3 days at a temperature of 30 ^o C and stirring.

 Using the aforementioned device, the ability of the studied samples to absorb visible light with a wavelength of 420-580 nm was determined. After cultivation, a growth curve was built for each sample and the area under the resulting curve was calculated.

 The microbicidal effect of the producing strain affecting the growth of the test strain was expressed as the percentage of inhibition determined by comparing the area under the growth curves in the control and using a sterile filtered preparation of the producing strain, respectively.

 6. Determination of the fungicidal effect of specific lactic acid bacteria.

 We studied the fungicidal effect produced by six strains of lactic acid bacteria E-76, E-98, E-315, E-317, E-414 and E-415, separately for all Fusarium molds used as tester strains. In this experiment, a visual assessment of the turbidity of mold cultures was carried out, to which various dilutions of a sterile-filtered preparation obtained by culturing each of the strains of lactic acid bacteria were added (Table 3).

 In control studies, Milli-Q water or Milli-Q water adjusted to pH 3.6 with lactic acid was used instead of the culture fluid.

Cultivation was carried out in test tubes with CMC medium at a temperature of 25 ^o C for 5 days. The results were taken into account visually.

 7. Results.

 In the table. 1 and 2, as well as in FIG. 1 shows the microbicidal activity detected for different strains of lactic acid bacteria by the disk method or using the turbidometric method, respectively.

 In the table. 3 shows visually detectable fungicidal activities.

 The results obtained indicate that the aforementioned strains of lactic acid bacteria inhibit the growth of harmful Fusaruim mold and other unwanted microbes that occur during the fermentation process, while having virtually no effect on beneficial microbes. The results obtained show the possibility of using lactic acid bacteria in the method provided by the present invention.

 Example 2. The microbicidal effect exerted by certain strains of lactic acid bacteria on food pathogens, as well as on microbes that are undesirable for food products.

 In the present experiment, we studied the microbicidal effect exerted by preparations obtained with the use of the Pediococcus pentosaceum VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) strains with respect to food pathogens as well as microbes undesirable for food. The strains belonging to the genera Bacillus, Yersinia, Listeria were used as tester organisms. Pseudomonas, Salmonella, and Staphylococcus.

A sterile culture medium of lactic acid bacteria obtained in accordance with Example 1 was used as a preparation. All other test strains were cultured for 16-18 hours in Iso-Sensitest (Oxoid) medium, with the exception of the Listeria strain, which was grown in tryptose phosphate medium. The cultivation temperature was equal to 30 ^o C, with the exception of strains of Salmonella, Listeria and Staphylococcus, in which case it was equal to 37 ^o C.

 Microbicidal activity was determined using the turbidometric method described in example 1. The conditions of the experiment in terms of growth substrate and temperature correspond to the above. The incubation time was 24 hours, with the exception of strains of Bacillus and Yersinia, for which it was 48 hours

 The results obtained are presented in table. 4 indicate that the addition of a lactic acid bacteria preparation of the present invention causes a suppression of the growth of foodborne pathogens as well as microbes undesirable for foodstuffs.

 Example 3. The effect of preparations of lactic acid bacteria, as well as preparations obtained with the help of lactic acid bacteria, on the microflora of malting and the quality of the malt.

 1. Used strains.

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

The growth medium for the inoculum was MRS medium. These bacteria were cultured for 2 days in 10 ml of MRS medium at a temperature of 30 ^o C. The volume of the inoculum was equal to 1% of the volume of the growth solution.

 2. Barley.

 We used Kymppi barley cultivar in 1990, in which the proportion of grains infected with Fusaruim mold was 55%.

 3. The process of malting.

Portions of barley weighing 1000 g were rinsed in a water bath at a temperature of 12 ^o C for 1 h. The water used for rinsing was replaced with the first water for soaking, which after 5 hours was replaced with a second water for soaking. 16 hours after this, the stage of air rest was carried out. The purpose of the air rest stage was to remove water from the surface of the grains. The duration of this stage was 8 hours. During the soaking, the barley moisture reached 44%. During the soaking step, barley was aerated.

Following the soaking, germination was carried out. Barley was germinated in germination boxes for 6 days at a temperature of 14 ^o C. In order to maintain humidity at 44%, these portions of barley were moistened and ted daily. Thus obtained green malt was dried according to a 21-hour temperature program. Over a period of 4.5 hours, the temperature was 50 ^° C. Over the next 4.5 hours, it was raised to 60 ^° C. and then maintained at that level for 4 hours. Over the next 5 hours, the temperature was uniformly raised to 85 ^° C. and kept at this level for the remaining 3 hours. The final moisture content in the malt became equal to 4%. At the end of this process, the primary roots were mechanically separated.

 A posture performed in the absence of any additives was considered as a control.

 4. The preparation of lactic acid bacteria and the preparation obtained with the help of lactic acid bacteria.

To obtain these preparations, lactic acid bacteria cells isolated from the corresponding culture fluids were used, as well as culture fluids containing substances toxic to microbes, both in combination with each other and separately. The culture fluid containing the cells was added in an amount of 120 ml per 1 kg of barley, or cells isolated from 120 ml of culture fluid were added. The specified selection was carried out by centrifugation of the corresponding culture fluid, after which the selected cells were suspended in water. In those cases where culture fluid was added, said culture fluid was used as such. Cell titres of added preparations ranged from about 10 ⁸ to 10 ⁹ CFU / ml.

 5. Additives of lactic acid bacteria preparations.

 Lactic acid bacteria preparations were added either to barley, either at the beginning of the first stage of soaking, or at the beginning of the first and second stages of soaking, or at the beginning of germination.

 6. Ongoing analyzes.

 Samples were taken at each stage of malting.

 6.1. Accounting molds was carried out as follows. The percentage of grains infected with Fusarium mold was determined using CZAPEK IPRODION DICLORAL agar (CZIP agar), which is selective for Fusarium molds, as well as wet filter paper [2]. Fusarium molds were identified based on the typical morphology of their colonies and spores, as well as on the basis of red color.

 The counting of Aspergillus and Penicillum molds was carried out using selective malt salt agar [2]. Accounting for the other most common molds was carried out on wet filter paper.

 6.2. Lactic acid bacteria were counted on MRS agar both in the case of added cultures and in the analysis of malization samples.

 6.3. All bacterial titers were counted on agar for plaque counting (Difco).

 6.4. Chemical indicators of malt were determined using methods known in relation to malting [3].

 7. Results.

 In the table. Figure 5 shows the titers of Fusarium molds and lactic acid bacteria at various stages of malting with the addition of E-390 culture fluid.

 In FIG. Figures 2 and 3 show the complete bacterial titers at various stages of malting in the case of adding culture fluid of strain E-390 and E-390 cells.

 In the table. Figure 6 shows the results of the analysis of malt when adding culture fluid of strain E-390 or E-390 cells.

 These results show that the treatment according to the invention reduces, in particular, the amount of Fusarium mold and the total bacterial titer at various stages of malting.

 The addition of the drug does not have an undesirable effect on the quality of the malt. The opposite is true: the processing according to the invention improves the filtration of paste obtained from the wort and reduces the content of β-glucan in the malt.

 Example 4. Obtaining drugs produced using lactic acid bacteria.

 1. Obtaining concentrated culture fluid.

The producing strains of Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E-78076 (E-76) were cultured in a fermenter in a volume of 15 l. The inoculum volume was from 6 to 7%, cultivation was carried out in MRS medium at a temperature of 30 ^o C for 2 days under microaerophilic conditions. The resulting culture liquids were concentrated tenfold and twentyfold by lyophilization and evaporation. The microbicidal activity of the obtained concentrates was evaluated using the disc method.

 2. Obtaining a purified solution containing microbicidal compounds.

 The lactic acid bacteria culture fluid was purified by gel chromatography fractionation according to the size of the molecules. The fractions that showed activity in the study by the disk method or by using the turbidometric method were combined and run again through a gel column.

 Example 5. The effect of lactic acid bacteria preparations and preparations obtained with the help of lactic acid bacteria on the microflora of malting and on the quality of malt.

1. Used strains and preparations obtained using these strains:
The following bacterial strains were used: Lactobacillus plantarum VTT-E-78076 (E-76) and Pediococcus pentosaceus VTT-E90390 (E-390).

 These strains were obtained from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland).

 The preparation of preparations was carried out as described in example 1, paragraph 3; Example 3, paragraph 1; Example 4, paragraphs 1-2.

 2. Barley.

 We used Kymppi barley from the 1991 crop.

 3. The process of malting.

 The malting was carried out as described in example 3, except that the duration of the germination stage was 8 days. The final moisture content in the resulting malt became less than 5%.

 4. Posture.

 The malting was carried out in laboratory conditions. A posture performed in the absence of any additives was considered as a control.

 4.1. The first option is malting.

 Cell-free culture fluids ten times concentrated, as well as untreated culture fluids containing cells, were used as drugs used in laboratory malting. These preparations were added at the beginning of the soaking stage I or at the beginning of the soaking stages I and II. Studies of malposition 1-8 were carried out as follows.

Test N 1: Control, Kymppi 1991 Barley;
Test N 2: At the beginning of the soaking stages I and II add 120 ml containing cell culture fluid of strain E-76;
Test N 3: At the beginning of the soaking stage I add 120 ml of concentrated 10 times the filtrate of the culture fluid of strain E-76;
Test N 4: At the beginning of the soaking stages I and II add 120 ml of concentrated 10 times the filtrate of the culture fluid of strain E-76;
Test N 5: At the beginning of the soaking stages I and II add 120 ml of culture fluid of strain E-390;
Test N 6: At the beginning of the soaking stage I add 120 ml of concentrated 10 times the filtrate of the culture fluid of strain E-390;
Test N 7: At the beginning of the soaking stages I and II add 120 ml of concentrated 10 times the filtrate of the culture fluid of strain E-390;
Test N 8: At the beginning of the soaking stages I and II add 60 ml of cell culture fluid of strain E-76 and 60 ml of cell culture fluid of strain E-390 are added.

 4.2. The second option is malting.

 Preparations used in laboratory malting were cell-free 20-fold concentrated culture filtrates, their cell-free purified fractions containing microbicidal activity, as well as untreated, cell-containing culture fluids. The pH of the water for soaking was monitored. The drugs were added at the beginning of the soaking stages I and II. Studies of complications 9-16 were carried out as follows.

Test No. 9: Control, Kymppi 1991 Barley;
Test N 10: At the beginning of the soaking stages I and II add 120 ml of water, pH 3.8;
Test N 11: At the beginning of the soaking stages I and II add 120 ml containing cell culture fluid of strain E-76;
Test N 12: At the beginning of the soaking stages I and II add 120 ml of fractionated concentrate of strain E-76, pH 3.8;
Test N 13: At the beginning of the soaking stages I and II add 120 ml of concentrated 20 times the filtrate of the culture fluid of strain E-76;
Test N 14: At the beginning of the soaking stages I and II add 120 ml containing cell culture fluid of strain E-390;
Test N 15: At the beginning of the soaking stages I and II add 120 ml of fractionated concentrate of strain E-390, pH 3.8;
Test N 16: At the beginning of the soaking stages I and II add 120 ml of concentrated 20 times the filtrate of the culture fluid of strain E-390.

 5. Ongoing analyzes.

 Sampling was carried out at each stage of malting.

 Titers of molds and lactic acid bacteria, full bacterial titers, as well as physical and chemical quality indicators of the resulting malt were determined as described in example 3.

 6. Results.

 In FIG. Figures 4 and 5 show the complete bacterial titers at different stages of the malting process when lactic acid bacteria with cells or concentrated or fractionated filtrates of culture liquids are used for soaking culture liquids in water.

 In the table. Figures 7, 8, 9, and 10 show the concentrations of Fusarium molds and other molds, as well as lactic acid bacteria at different stages of malposition for both variants of malignancy.

 In the table. 11 and 12 present the results of a study of malt for both malting options.

 Our results indicate that the treatments provided by the present invention reduce, in particular, the content of bacterial Fusarium fungi, as well as the complete bacterial titer at various stages of malting. In addition, it was noted that not only culture fluids, but, in particular, concentrated and fractionated culture filtrates have a favorable effect on Fusarium molds.

 Based on the analyzes of malt, we can conclude that the addition of culture fluid of strains E-76 and E-390 contribute to the improvement of the filterability of the wort obtained from the corresponding malt; also the content of β-glucan in the indicated malt is lower than in the control.

 Example 6. The effect of drugs obtained using lactic acid bacteria and used as additives in the process of malting, on the filtration of the resulting mass.

 Presented in FIG. Figure 6 shows the effect of treatment with preparations obtained with the help of strains of lactic acid bacteria (120 ml of culture liquid per 1 kg of barley) provided for by filtration of the mass produced from appropriately treated malt.

 The strains listed in Example 1 are used in this experiment: E-390, E-416, E-98, E-317, E-390, E-76, and E-315. The analysis was performed using the Terpal filtration method [4].

 From the obtained results it is seen that the processing provided by the present invention improves the filtration of the mass produced.

 The following examples show the effect of the lactic acid bacterium on the concentration of mycotoxins, deoxynivalenol and zearalenone produced by Fusarium mold when barley is malignant.

 Example 7. The effect of lactic acid bacteria on the concentration of deoxynivalenol (DON) during the posture of barley infected with mold F.culmorum D148.

 1. Used strains.

 Lactic acid bacteria strains Lactobacillus plantarum (E-76) and Pediococcus pentsaceus (E-390) were used in this test.

These bacteria were cultured under anaerobic conditions in MRS nutrient solution at 30 ^o C for 3-4 days. Between tests, bacterial strains were stored in their nutrient solution under anaerobic conditions at 4 ^o C.

 2. Barley.

The 1994 Kymppi barley variety was used, to which 2 ml of Fusarium culture solution per 100 g of barley was added. The Fusarium strain was grown on a rocking chair in a CMC solution for about 7 days before spore formation. The spore density of the F.culmorum VTT D-80148 (D-148) culture solution was about 10 ⁶ spores / ml of the solution. Infected barley was incubated at 25 ^° C. for 3 days. The barley was stirred daily and dried overnight at 30 ^° C. to its initial moisture content.

 3. The process of malting.

The soaking program for 1000 g serving of barley was as follows:
washing for 1 h
first soak for 7 hours
the first break for airing 16 hours,
second soaking for 8 hours,
second break for airing 15 hours,
immersion.

The soaking temperature was 12 ^° C. and the desired barley moisture was 46% at the end of the soaking. In order to verify the respiration and metabolism of barley, between the stages of soaking were taken breaks for ventilation and samples of barley during the breaks were transferred to the aerated area. Aeration of the vessels for soaking was also provided. After a second break for ventilation, barley samples were weighed to determine the moisture content. If the humidity was below 46%, barley samples were immersed in water for a short period of time. The ratio of barley and water when soaked was 1: 1.5. After this, barley samples were placed in germination boxes.

The barley samples were germinated in aerated germination boxes for 6 days at 14 ^° C. To bring the humidity to the desired level of 46%, the barley samples were moistened and agitated daily. The green malt thus obtained was dried using a 21-hour temperature program, as in step 3 of Example 3. Finally, seedlings were mechanically removed from the malt.

 The malting performed without the addition of lactic acid bacteria was used as a control test.

 4. The preparation of lactic acid bacteria.

 In the table. 13 shows the pH, cell titer and microbicidal activity (disk method, example 1, paragraph 5) of a lactic acid bacteria culture added during malting.

 5. Addition of a lactic acid bacteria preparation.

 The lactic acid bacteria preparation was added at the beginning of the first and second soaking stages. The ratio of the culture of lactic acid bacteria to water during malting is maintained at 8%.

 6. The performed analyzes.

 The concentration of deoxynivalenol in each barley sample was determined by gas chromatography. A mass spectrometer was used to recognize and quantify deoxynivalenol.

 7. Results.

 In FIG. Figure 7 shows the effect of the addition of lactic acid bacteria on the deoxynivalenol content during the malting of barley infected with the strain F.culmorum D 148.

 From the results it is seen that under the influence of the strain L. plantarum E-76, malt turns out to be 50% less deoxynivalenol in comparison with untreated malt, and under the influence of the strain P. pentosaceus E-390 in the malt it is 25% less than deoxynivalenol compared to raw malt.

 Example 8. The effect of lactic acid bacteria on the formation of deoxynivalenol (DON) during the deposition of barley, naturally contaminated with deoxynivalenol and Fusarium mold.

 1. Used strains.

 Lactic acid bacteria strains Lactobacillus plantarum (E-76) and Lactobacillus acidophilus VTT E-87276 (E-276) were used in this test.

The strain of Lactobacillus plantarum was cultured as in example 7. The cultivation temperature for the strain of Lactobacillus acidophilus was 37 ^o C.

 2. Barley.

 We used low-quality barley varieties Kustaa, Loviisa and Jo 1599 from the 1993 crop.

3. The process of malting
The malting was carried out in the same way as in example 7, with the exception of portions of barley (100 g) and the ratio of barley: water when soaked (1: 4.0).

 The malting performed without the addition of lactic acid bacteria was used as a control test.

 4. The preparation of lactic acid bacteria.

 In the table. 14 shows the pH, cell titer and microbicidal activity (disk method, example 1, paragraph 5.1) of a lactic acid bacteria culture added during malting.

 B. Foreign barley.

 1. Used strains.

 Conforms to clause A.1.

 2. Barley.

 Used barley consisted of grades 1-3 of foreign barley of low quality.

 The malting was carried out in the same way as in example 7, with the exception of portions of barley (20 g) and the ratio of barley: water when soaked (1: 17.0).

Barley was germinated for 6 days in plastic bags placed in a plastic box in a metal frame to the desired moisture content of 46%. Packets were rotated daily. To maintain moisture, a certain amount of water was poured onto the bottom of the box for germination. Green malt was dried in an incubator at 50 ^° C for 17 hours, after which the temperature was raised to 85 ^° C, and barley was dried for another 4 hours. Sprouts were removed mechanically.

 4. The preparation of lactic acid bacteria.

 In the table. 15 shows the pH, cell titer and microbicidal activity (disk method, example 1, paragraph 5.1) of the lactic acid bacteria culture added during malting.

 5. Addition of a lactic acid bacteria preparation.

 The lactic acid bacteria preparation was added at the beginning of the first and second soaking stages. The ratio of the culture of lactic acid bacteria to water during malting was maintained at 8%.

 6. The performed analyzes.

 The concentration of deoxynivalenol in each barley sample was determined using the Elisa test (Ridascreen).

 7. Results.

 In the table. 16 and in FIG. Figures 8 and 9 show the concentration of deoxynivalenol during the deposition of portions of Finnish and foreign barley, as well as the effect of the lactic acid bacteria on the formation of deoxynivalenol (DON) during the deposition of barley naturally contaminated with deoxynivalenol and Fusarium mold.

 In FIG. 10 shows the average concentration of deoxynivalenol of three home and three foreign barley samples, i.e. the effect of lactic acid bacteria cultures added to water for the first and second soaking on the concentration of deoxynivalenol (DON) during the deposition of Finnish and foreign barley, determined using the Elisa test (Ridascreen) . The results are given as average values.

 It can be seen from the results obtained when a culture solution of L. plantarum E-76 was added, especially when malting foreign barley samples, the concentration of deoxynivalenol is higher in untreated malt than in the presence of a lactic acid bacterium. Under the influence of the strain L.plantarum, the concentration of deoxynivalenol (average for three samples) is 68% lower than in untreated malt, and under the influence of the strain Lactobacillus acidophilus (E-276), the concentration of deoxynivalenol is 75% lower than in untreated malt. Portions of Finnish barley have low concentrations of deoxynivalenol, which can be further reduced by malting.

 Example 9. The effect of lactic acid bacteria on the content of zearalenone (ZEN) in the malt during the malting of barley infected with strains of Fusarium culmorum D-148 and F.graminearum D-470.

 A: 1. A lactic acid bacteria strain Lactobacillus plantarum (E-76) was used.

 This strain, barley (infection time 7 days), the malting process, the preparation of lactic acid bacteria and the additives of the preparation of lactic acid bacteria used in this example were the same as in paragraphs 1-5 of example 7.

 6. The performed analyzes.

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

 7. Results.

 11 shows the effect of the addition of lactic acid bacteria on the content of zearalenone during the malting of barley infected with the strain F.culmorum D 148.

 From the results obtained, it is seen that under the influence of the strain L. plantarum E-76, malt turns out to be 46% less zearalenone compared to untreated malt.

 B: 1. Used strains.

 Lactic acid bacteria strains Lactobacillus plantarum (E-76), Lactobacillus acidophilus VTT E-87276 (E-276) and Pediococcus pentsaceus (E-390) were used in this test.

 Cultivation was carried out as described in example 7.

 2. Barley.

 A Kymppi barley variety of the 1994 crop was used, which was infected with F.graminearum VTT D95470 (D-470) culture solution, as described in paragraph 2 of Example 7.

 The malting process, the lactic acid bacteria preparation, and the lactic acid bacteria preparation additives used in this example were the same as in paragraphs 3-5 of Example 7.

 Additionally, the culture of the lactic acid bacteria L. acidophilus E-276 added during malting had a pH of 4.05, a cell titer of 5.8E + 08 PMY / ml and an inhibition zone of 14 m (disk method, example 1, paragraph 5.1).

 6. The performed analyzes.

 The content of zearalenone in each barley sample was determined using the Elisa test (Ridascreen).

 7. Results.

 In FIG. 12 shows the effect of the addition of lactic acid bacteria on the content of zearalenone during the deposition of barley infected with the F.graminearium VTT strain D-95470 (D-148).

 From the results obtained, it is seen that under the influence of a lactic acid bacterium, malt turns out to be 37-55% less than zearalenone compared to untreated malt.

List of references
1. Haikara A. and Home S., Mash filtration difficulties caused by split barley kernels: a Microbiological problem. Proc. EEC Congress 1951 (Quality Control).

 2. EEC-Analytica Microbiologia, Part II, 1987.

 3. EEC-Analytica, 1987, 4th Ed.

 4. Grandclerc J. et al., Simplification de la methode de filtration du brassin tepral description de la methode. 1988 BIOS 19: 88-92.

Claims (5)

 1. A method of processing intended for germination of seed material, comprising adding to the seed material in connection with the process of germination of antimicrobial additives, characterized in that the additives are added to the seed material, which is barley grains used in the process of malting, or seed material, which will be turned into seedlings intended for use as food, are a preparation of lactic acid bacteria or a drug produced by lactic acid bacteria mi, which has the effect of inhibiting the growth of microbes.  2. The method according to claim 1, characterized in that said lactic acid bacteria belong to the genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus.  3. The method according to claim 1 or 2, characterized in that the specified preparation of lactic acid bacteria or a preparation produced by lactic acid bacteria, is produced from species of Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococuscus pentosaceus, Lactobacusus plant Pentusaceusus, Lactobacusum curtus curculus from a mixture thereof, preferably from Lactobacillus plantarum and Lactobacillus pentosacens species, or from a mixture thereof.  4. The method according to claim 1, or 2, or 3, characterized in that a preparation of lactic acid bacteria or a preparation produced by lactic acid bacteria, which has the effect of inhibiting the growth of Fusarium molds, is added to barley grains.  5. The method according to claim 4, characterized in that the specified preparation of lactic acid bacteria or a drug produced by lactic acid bacteria is added at the stage of soaking or germination.

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