AU680426B2 - Procedure for treatment of seed material to be germinated - Google Patents

Description

OPI DATE 15/08/94 APPLN. ID 48214/93 I AGJP DATE 13/10/94 PCT-,NU.MBER ,PCT/F.193/,00388 IIIIIi ll iiiiiiiiiii AU9348214 (51) International Patent Classification 5: (11) International Publication Number: WO 94/16053 C12 102,IM 1/4,A23 1172Al (43) International Publication Date: 21 July 1994 (21.07.94) (21) International Application Number: PCT/F193/00388 (81) Designated States: AT, AU, Btt, BG, BR, BY, CA, CH, CZ, DE, DK., ES, FI, GB, HU, JP, KP, KR, KZ, LK~ LU, LV, (22) International Filing Date: 27 September 1993 (27.09.93) MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US, VN, European patent (AT, BE, CH, DE, DK ES, FP, GB, GR, IE, IT, LU, MC, NL, PT, SE), QAPI Priority Data: patent (BF, BJ, CF, CG, CI, CM, GA, GN, NM, MR, NE, 930182 15 January 1993 (15.01.93) Fl SN, TD, TG), (71) Applicant (for all designated States except US): QY PANI. Published MOLABORATORIO BRYGGERILABORATORIUM AB With *nternational search report.

[Fl/Fl]; P1 19,.N011HlLk (FI), PL I' A'4- 03J'51 9TSft 4AlzO

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(72) Inventors; and Inventors/Applicants (for US only): HAIKARA, Aull WFlT]; Itiilahdenkatu 8 C, FIN-00210 Helsinki MAlITLA- SANDHOLM Tilna [FlFl; Ristiniementie 32 G 33, FIN- 4, LU 02320 Espoo 0 PS C) (74) Agent: PAPULA REIN LAHTELA QY; P.O. Box 981,T Salomonkatu 17 B, FIN-00101 Helsinki (Fl).

04126 (54) Title: PROCEDURE FOR TREATNvIENT OF SEED MATERIAL TO BE GERM]NATED (57) 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 mnicrobial growth.

WO 94/16053 PCT/Fi93/00388 1 PROCEDURE FOR TREATMENT OF SEED MATERIAL TO BE GER-

MINATED

FIELD OF THE INVENTION The present invention concerns a procedure for treating seed material intended to be germinated.

BACKGROUND OF THE INVENTION A germination process is in this context understood to be a process step in general which is required in order to produce a germinated product, starting from storage-dry seed. In the brewery and distillery industry, for instance, a germination process, that is the malting process, is applied in producing the important raw material of beer, or other alcoholic beverages, viz. cereal malt, such as barley malt, rye malt or any malt whatsoever. The germination process is furthermore applied in producing various commercial sprouts products, e.g. bean sprouts or any sprouts for use in human nutrition.

Germination processes are usually carried out in non-aseptic conditions. On the seeds beirg treated there occur microbes originating from the growth environment or from storage. Conditions during the germination process are mostly favourable to the microbes present on the seeds, such microbes usually multiplying during the course of the process. The microbes may exert a detrimental effect on the germinated product, or cn the end product which may ultimately be made thereof, while they may equally have beneficial influence on the product being germinated.

The main steps in brewing process are: malting, wort production, primary and secondary fermentation, and downstream processing. The purpose with malting is to produce in the kernel enzymes which in the I I I WO 94/16053 PCT/FI93/00388 2 mashing step decompose the substances of the kernel's endosperm to a form soluble in the wort. For example, the barley seed malting process is well known to comprise three steps; steeping, germination and kilning.

The cleaned and screened barley grains are steeped in water until desired moisture is achieved, e.g. on the order of 43 to 44%. Part of the steeping may be accomplished in so-called air rest. The barley is allowed to germinate in controlled conditions, and the germinated barley is kilned in a hot air current until the germination has come to an end. On termination of kilning, the rootlets are removed from the malt. Regulation of the malting steps is based on temperature, air flow and moisture/humidity control.

Malt quality is affected, on the side of malting technique and malting conditions, also by the microbial flora of the malt cereal, this flora varying significantly e.g. depending on cereal variety, weather conditions, growth site, length of growing season and storage conditions.

Barley is the cereal most often used in malting. The inherent, natural microbial flora of barley can be classified as field and storage fungi, bacteria and yeasts. The commonest field fungi of barley are: Fusarium, Alternaria, Cladosporium, Cephalosporium, Epicoccum, and Helminthosporium. The occurrence of moulds is different in different countries and different years. Wet weather conditions during the cereal's growth period, and particularly while it is being harvested, favour the growth of Fusarium mould. Fusarium contamination may be heavy indeed in rainy growth seasons. One of the commonest bacterium species on cereals is Enterobacter agglomerans. Other bacteria which should be mentioned are; Escherichia coli, and bacteria of genera Pseudomonas, Micrococcus and Bacillus, and lactic acid bacteria. The bacterial count on barley is about 10 5 to 108 CFU/g (colony forming units per g).

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WO 94/16053 PCT/F193100388 3 The moulds and bacteria in barley increase during malting, and peak concentrations are usually reached during the germination step. Fusarium moulds, in particular, and lactic acid bacteria undergo the strongest proliferation. Yeasts also increase during malting. In the kilning step the mould, yeast and bacterium concentrations go down again, as a rule. Part of the microbes present on barley have a useful effect in view of malting and of the product to be made of the malt, e.g. beer. It has been estimated that up to of some enzymes in the malt are of microbial origin. On the other hand, part of the microbes exert a detrimental influence on the barley and/or the malts.

Among the disadvantageous microbes Fusarium moulds deserve to be mentioned, which have been found to cause particularly detrimental gushing of beer more often than other moulds, the peptides produced by said moulds constituting nuclei for gas bubbles discharging from the beer bottle in the form of powerful gushing.

When more than 50% of malted kernels are contaminated with Fusarium moulds, the risk of gushing is clearly increased.

Further, gram-negative bacteria present in the barley, such as species belonging to genera Pseudomonas and Flavobacterium, and gram-positive bacteria of genus Leuconostoc have been shown to retard filtration of the mash in connection with wort production. Various microbes present in barley may also give rise to other disadvantageous effects, e.g. inhibit the germination, cause off-flavours or unfavourable changes in the analysis values of the wort and the beer.

The quality requirements of malt barley zan be specified in annually established cultivation contract and delivery terms. Moulds are a group of microbes frequently mentioned in quality specifications. Many malting plants have moreover imposed an upper limit on cettain moulds. If the proportion of Fusarium-contaminated

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WO 94/16053 PCT/FI93/00388 kernels exceeds 65% or if the corresponding proportion of Aspergillus and Penicillium moulds exceeds 50%, the barley can be classified as poor in quality or even as unfit for use in malting.

Attempts have been made to prevent gushing induced by moulds, by using barley of good quality or by blending barley, malt or beer batches. In rainy years nearly the whole barley crop may be poor in quality, in which case it may be impossible to obtain good barley.

10 Microbicidic chemicals have also been tried in order to o000 reduce the quantity of moulds, but no safe and generally approved chemical could be found.

A germinating process for producing sprouts intended to be used for nutrition offers likewise pro- 15 pitious conditions for proliferation e.g. of moulds and bacteria.. Such sprouts products will spoil rapidly.

Further still, in connection with germination increase may take place e.g. of foodstuff pathogens causing food poisoning, such as Salmonella, Yersinia and/or Listeria 20 bacteria.

SU~MA.Y OF THE INVENTION It is an aim of the present invention to ameliorate one or more of the drawbacks of the prior art.

In an aspect of the invention there is provided a *o procedure for treatment of seed material to be germinated, characterized in that to the barley kernels in a malting process or seed material going to be converted to sprouts intended to serve as nutrition, is added in connection with the germination process, a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria which has an effect inhibiting microbial growth.

It is an advantage that in one form the invention may provide a procedure by which the quantity and quality of the microbial flora can be carefully regulated during the germinating process, however, without disadvantageously affecting the quality of the germinated product or of the 'T end product potentially made of the product.

.A rA],(1 WO 94/16053 PCT/FI93100388 DETAILED DESCRIPTION OF THE INVENTION The invention is based on studies in connection of which the unexpected observation was made that lactic acid bacteria can be used towards improving the quality of a product to be germinated. The substances comprised and/or produced by the preparation of the invention, the microbicidic agents, inhibit the growth of detrimental microorganisms occurring in connection of a gerrinating process.

The use of lactic acid bacteria in foodstuff and animal feed industry is well known in the art. They produce in fermentative conditions such compounds which affect the composition and flavour of the products, but which also inhibit the growth of pathogenic microbes tending to spoil said products. Lactic acid bacteria have been commonly used in dairy products, meat products, vegetable fermentation and bakery products, and in fodder preservnition.

1 Addition of lactic acid bacteria or of lactic acid has been practiced in the production of a certain 'malt type, of so-called Sauermalz. This addition is made to the malt in the kilning step, prior to mashing or during mashing. The purpose with the addition is merely to cause lowering of the wort pH and, thus, to exert an influence on the course of the mashing process and on the quality of the finished beer. However, lactic acid bacteria have not heretofore been used as taught by the invention: to inhibit the growth of undesired microbes in connection with the germinating process.

The preparation of the invention can be added to the product to be germinated in any step of the germinating process.

In a particularly advantageous embodiment, lactic acid bacteria preparation, or preparation produced by a lactic acid bacteria, is added to cereal material, such as barley kernels, during the course of -L I-- WO 94/16053 PCT/FI93/00388 6 the malting process. The preparation added inhibits the growth of moulds, in particular of harmful Fusarium moulds, and of bacteria, as a result of which, for instance, the risk of beer gushing due to Fusarium mould is reduced. However, the preparation has no substantial effect on the action of the useful microbial flora as regards the quality of the malt that is obtained or of the beer therefrom produced. No harmful effects of the preparation added on malt quality have been observed either, nor has it been found to contain or to produce compounds which are harmful in view of the malt, or beer, being produced.

In a malting process, the lactic acid bacterium preparation, or the preparation produced by a lactic acid bacteria, can be added to the barley kernels before steeping, in the steeping step or in the germination step. The addition is advantageously made in the steeping or germinating step. The malting process may be carried out, in other parts, in any manner known in itself in the art. If desired, e.g. nutrients may be added to the barley to be malted, or the conditions may be regulated, e.g. lactic acid added, in order to optimize the conditions for growth of lactic acid bacteria.

It is possible in the in.vention to use any commonly available lactic acid bacterium whatsoever which possesses influence of inhibiting microbial growth. The following usable lactic acid bacterium genera may be mentioned: Lactococcus, Leuconostoc, Pediococcus and Lactobacillus. The following may be mentioned to present advantageous species: Lactococcus lactis, Leuconostoc mesenteroides, Pediococcus damnosus, Pediococcus parvulus, Pediococcus pentosaceus, Lactobacillus curvatus, and Lactobacillus plantarum, or any mixtures of these, among these the following being particularly advantageous: Lactobacillus plantarum and Pediococcus pentosaceus or mixtures thereof. Use of genetically modified lactic acid bacteria is equally

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WO 94/16053 PCT/FI93/00388 7 possible.

The lactic acid bacteria preparation may be composed of culture broth, with or without cells, or of concentrated cultcre broth (including cells). A preparation produced by lactic acid bacteria may consist of cell-free culture filtrate, of concentrated culture filtrate, of fractionated culture filtrate, or of a pure or partly purified microbicidic product.

According to a particularly advantageous embodiment, the treatment is carried out with concentrated or fractionated culture broth, which may be cell-free or may contain cells. Concentration ma- be accomplished e.g. by lyophilization or by evaporation.

The culture broth is concentrated e.g. by a factor of 2 20 Fractionation, purification of the microbicidic products, can be carried out in a manner known in itself in the art, e.g. with the aid of chromatographic methods or by ultrafiltration.

In the procedure of the invention, the microbial growth-inhibiting activity of the preparation containing lactic acid bacteria, or of preparation produced by lactic acid bacteria, to be added to the seed material corresponds e.g. to the culture broth quantity of about 10 to 10,000 ml/kg of seed material to be treated, suitably 30 to 7,000 ml/kg of seed material to be treated, e.g. 40 to 5,000 ml/kg of seed material to be treated. The preparation of the culture broth is described in the Examples section. It should be noted that in the application in hand the activity of the preparation is defined with the aid of the Culture broths employed. It is obvious to a -erson skilled in the art that preparations according to the invention possessing equivalent microbial growth-inhibiting activity can equally be produced applying other culture broths and/or procedures.

According to the invention the preparation WO 94/16053 PCT/F1I93/00388 8 contains microbicidic compounds, and/or the preparation produces microbicidic compounds during the course of the germination process. When a cell preparation is employed, cell growth can be promoted, if required, e.g. by regulating the conditions during the germination process, or by adding nutrients. The preparation may also accelerate the growth of other lactic acid bacteria present in the material to be germinated.

Since the use of lactic acid bacteria in foodstuffs is allowed and generally approved, the preparation derived from lactic acid bacteria growing is also Ssafe to use. Lactic acid bacteria belong usually to the natural microbe flora of seeds to be germinated, such Sas barley kernels. Therefore the procedure of the invention is maximally natural. It is also possible to use for lactic acid bacterium strain a strain inherently occurring on the seeds.

Thanks to the invention, it becomes in malting to reduce the detriments arising from Fusarium contaminations, such as gushing of beer.

Moreover, the procedure has unexpectedly been found to improve the filtrability characteristic in the brewing process. This has been found to be due to the fact tiat the preparation of the invention also restricts the counts of harmful species occurring in malting and retarding the filtration of the mash, e.g.

those belonging to genera Leuconostoc, Pseudomonas and Flavobacterium.

According to another advantageous embodiment, /lactic acid bacteria preparation or preparation produced by a lactic acid bacterium is added to the seed material when producing sprouts to be used for food.

Thanks to the present invention, the growth of harmful microbes can be restricted in connection with a germination process. For the first time, the invention enables biological expedients to be used in order to .prevent the growth during an industrial germination WO 94/16053 PCT/FI93/00388 9 process of detrimental bacteria occurring on seed which is to be germinated.

The procedure of the invention improves the general hygienic standard of the germination process even on the whole.

In the following the invention shall be illustrated with embodiment examples, which are merely intended to illustrate the invention, without confining it to them. The treatment of the invention is also applicable in other germination processes.

Fig. 1 shows a graph representing the microbicidic activity in a lactic acid bacteria culture filtrate, as assessed by the turbidometric method. The normal growth curve of the test organism, E.aqqglomerans E-396, and the inhibition effect exerted on the growth of the test organism by culture filtrates of the production strains L. plantarum E-76 and P. pentosaceus E-390.

In Fig. 2 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 culture broth added at different stages of the maltings.

In Fig. 3 is shown the effect on the total bacterial counts of the malting of P. pentosaceus E-390 cells added at different stages of the maltings.

In Fig. 4 are shown the total bacterial counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated culture broths, to the barley steeping water.

In Fig. 5 are shown the total bacterium counts at different stages of laboratory maltings on addition of P. pentosaceus E-390 and L. plantarum E-76 culture broths, or concentrated and fractionated culture broths, to the barley steeping water.

Fig. 6 displays the effect of lactic acid bacteria cultures, added to the malting, on mash filtrati- I on (Tepral filtration).

Figure 7 shows the effect of the addition of lactic acid bacteria on the deoxynivalenol content in the malting of barley contaminated with F.culmorum D 148 strains.

Figures 8 and 9 show the deoxynivalenol concentrations in the malting of Finnish and foreign barley batches as well as the influence of the lactic acid bacteria on the formation of deoxynivalenol (DON) in the malting of barley naturally contaminated with deoxynivalenol and the Fusarium mould.

s Figure 10 presents the mean values of the deoxynivalenol concentrations of three domestic and three foreign barley samples, i.e. the effect of the lactic acid bacteria cultures added to the first and second steeping 15 waters on the deoxynivalenol concentration in the malting of Finnish and foreign barley.

Figure 11 presents the effect of the lactic acid bacteria additions on the zearalenone content in the malting of barley contaminated with the F.culmorum D-148 20 strain.

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

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r i v^ t A" 'v Example 1: THE MICROBICIDIC EFFECT OF VARIOUS LACTIC ACID BACTERIA STRAINS ON MICROBES OCCURRING IN MALTING In the experiment, the microbicidic effect on microbes occurring in malting exerted by preparations produced by various lactic acid 'bacteria strains were studied. The sterile-filtrated culture broth was used for preparations.

1. Production strains: The following lactic acid bacteria strains were used for production strains: Os 0 4 *Sa Lactobacillus lactis 15 ssp. lactis sso. diacitilactis Leuconostoc mesenteroides sspo. mesenteroides szLp. mesenteroides ssp. mesenteroides Pediococcus damnosus Pediococcus uarvulus Pediococcus pentosaceus Pediococcus pentosaceus 25 Pediococcus Dentosaceus Pediococcus oent1.osaceus VTT-E-9 04 14 VTT-E-90423 (E-'414) (E-423) 0600 0 00 S 0 VTT-E-90389 (E-389) VTT-E-90415 (E-415) VTT-E--90466 (E-466) VTT-E-76065 VTT-E-88315 (E-315) VTT-E-70-067 (E-67) VTT-E-76068 (E-68) VTT-E-88317 (E-317) VTT-E-90390 (E-390) (Z'SM 7389) VTT-E-90391 (E-391) VTT-E-78076 (E-76) (DSM 7388) VTT-E--79098 (E-98) Lactobacillus curvatus Lactobacillus olantarum Lactobacillus olantarun The strains were obtained from the VTT, Collection of Industrial Microorganins (Biotechnical Laboratory, Finland). Lactobacil lus plantarui (1-76) was deposited with DSM (Deutsche Sammiung von MikoogaisenU-4Zlkutrn under accession number 7388 on January 1993. The strain E-76 (DSM 7388) had been isolated from beer \~fi

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11 Sby known techniques used for liquid products, and analyzed/identified using well known analysis methods. Pediococcus pentosaceus (E-390) was deposited with DSM (Deutsche Sammlung von Mikroorganismen und Zellkulturen) under accession number 7389 on 15 January 1993. The strain E-390 (DSM 7389) had been isolated from homogenized samples of split barley kernels and identified/analyzed using techniques well known in the art [see the article of Haikara, A. and Home, Mash Filtration difficulties caused by split barley kernels: a Microbiological problem, in the publication of EBC Congress 1991 (Quality Control)]. The deposits are under the provisions of the Budapest Treaty.

2. Test strains: Various harmful microbial species occurring at malting were used for test strains, as well as lactic acid bacteria strains which served as production strains, among others.

Harmful moulds were represented in the tests 20 by ?u sarrum moui £[Gibb e a a- r.r cLS.r r i s.um avenaceum) VTT-D-80141 (D-141) and VTT-D-80147 (D- 147), and Fusa. lum culmorum VTT-D-80148 (D-148) and VTT-D-80149 (D-149), Collection of Industrial Microorganisms, Biotechnical Laboratory of VTT], and one As- 25 pergillus species.

Harmful gram-negative bacteria were represented by two strains from genus Enterobacter and by one species each from genus Flavobacterium and genus Pseudomonas. The lactic acid bacteria consisted of those strains which were employed as production strains, plus the strain Lactococcus sp. E-416.

3. Cultivation of production strains, and preparation of sterile filtered culture broth: The lactic acid bacteria were cultivated in MRS broth (MRS BROTH, Oxoid). The Pediococcus strains E-67 and E-68 were aerobically cultivated at 0 C, all other production strains anaerobically at *099 9 9* 9t 9.

9 9 9 9 *0* 0 S3 *r 99..

9 .9.

9~ .99. 9*99 4~ r 49r WO 94/116053 PCT/FI93/00388 12 0 C, cultivation period varying from 2 to 5 days. The cells were then centrifuged and the supernatant was sterile-filtered.

4. Cultivation of test strains: Spore suspensions were produced from the Fusarium moulds by cultivating the mould strain in CMC (carboxymethyl cellulose) solution at 25 0 C, 5 to 6 days as a shaking culture, dispersing the spore formations in TWEEN solution, filtering the suspension and recovering the filtrate.

The spore suspension of Aspergillus mould was produced directly on PD agar (25°C, 3 days) (Potato dextrose, Difco).

Gram-negative bacteria were aerobically cultivated in NB broth (Nutrient broth, Difco) for 1 day, the Enterobacter strain at 30 0 C, and the Flavobacterium and Pseudomonas strains at 25 0

C.

Lactic acid bacteria were cultivated as described under 3. above.

5. Examination of the microbicidic effect of the lactic acid bacteria: The microbicidic activity in the culture broth was assessed by the disk method, or turbidometrically.

5.1. Disk method for assessment of microbicidic activity: Sterile-filtered culture broth, or a dilution thereof, was pipetted onto a filter paper disk (12.7 mm dia.), 100 pl. The disks were placed on Plate Count agar dishes into which 0.3 ml of test organism dilution, 10 2 had been pour plated. The specimens were cultivated for 24 hrs at 30 0 C, whereupon the diameter in mm of the inhibition zone that had formed was measured.

5.2. Turbidometric method for assessment of microbicidic activity: An automatic turbidometer (Bioscreen, Labsys- WO 94/16053 PCT/FI93/00388 13 tems) was employed in the procedure.

The sample contained 10% by vol. of the test organism and 10% by vol. of the sterile-filtered culture broth of the production strain, calculated on the sample volume, and growth medium. For controls, the sterile filtrate preparation was replaced with distilled water of which the pH had been adjusted with lactic acid to identical level with the sterile-filtered preparation.

The growth medium used was, in the case of each test strain, the same medium as in the test strain cultivation.

The growth conditions for Fusarium and Aspergillus moulds were: 5 days, 25 0 C, powerful shaking.

Those for gram-negative bacteria were: Enterobacter strain, 300C, all others 25 0 C, 2 days, and shaking. The growth conditions for lactic acid bacteria were: 3 days, 30°C, and siaking.

The apparatus determined from the samples the absorbance at visible light wavelength 420-580 nm.

After cultivation, the growth curve of each sample could be made up and the area subtended by the curve calculated.

The microbicidic effect of a production strain on the growth of the test strain was expressed by an inhibition percentage, obtained by comparison of the growth area sizes found with the control and with the sterile-filtered preparation of the production strain, respectively.

6. Examination of the fungicidic effect of certain lactic acid bacteria: The fungicidic effect of six lactic acid bacteria strains, E-76, E-98, E-315, E-317, E-414 and E-415, separately on all Fusarium moulds employed as test strains was studied. In the test, a visual examination was made of turbidity formation in mould cultures to which sterile-filtered preparation from each WO 94/16053 PCT/FI93/00388 14 lactic acid bacteria cultivation had been added at various dilutions, stated in Table 3.

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

Cultivation took place as a test tube culture in CMC broth, at 250C, during 5 days. The results were visually read.

7. Results: Tables 1 and 2, and Fig. 1, display the microbicidic activities determined for the different lactic acid bacteria strains by the disk method and by the turbidometric method, respectively.

Table 3 displays the visually determined fungicidic activities.

TABLE 1. Microbicidic activities of lactic acid bacteria culture broths, by the disk method.

II i II Illactic acid Cell count, Culture inhibition Ii Ibacterium jCFU/ml ibroth, pH Izone dia., mmll II I II SE-76 3.2x10 8 3.70 1 19 S E-98 1.2x0 8 3.72 i 17 ii I E-315 1.6x10 8 3.90 I 16 E-317 l 1.0x10 3.86 i 16 SE-390 1.9x10 8 3.92 I J i I Cell count of test organism E-396 4.0x10 7 CFU/ml; on dish 1.

2 x10 CFU/ml.

TABLE 2. Microbisidic effect of lactic acid bacteria on the growth of various microbes.

Production strain Cell Pedicccusconc.

Lactococcus LDeuconosto Pedicoccus Lactobacillus con.

Test strain E-414 E-423 E-389 E-466 E-65 E-315 E-67 E-68 E-317JE-390 E-391 E-76 E-98 CFU/ml Moulds Aspergillus niger D-5 -28 -36 -39 -38 -16 -33 -21 -20 -24 -28 -44 -39 -41 2,0x10 4 Fusarium avenaceum D-1411) -36 -36 -49 -55 -33 -59 -42 -44 -52 -47 -42 -79 -74 6,0x10 3 "it D-147 1 -12 -20 -36 -66 -8 -63 -27 -43 -50 -53 -37 -70 -67 2,0x10 4 culmorum D-148 1 -5 -17 -29 -26 -9 -25 -10 -16 -20 -24 -21 -35 -30 2,5x10 3 SD-149 1 3 -6 -20 -30 -10 -26 -18 -9 -20 -23 -9 -39 -32 6,0x10 2 Gram-negative bacteria Enterobacter agglonerans E-396 -89 -92 -94 ND -96 -92 -93 -96 -91 -93 -93 -93 -93 6,0x10 4 Flavobacterium sp. E-399 2 -98 -99 -99 -98 -99 -97 -98 -98 -98 -98 -99 -96 -97 2 6x10 6 Pseudcmonas fluorescens E-397 2 -98 -98 -97 -98 -97 -97 -98 -98 -97 -97 -97 -98 -97 1,0x0I- Lactic acid bacteria Lactococcus lac( 5is. 4 sLt lactis E-414 -37 -33 -24 -47 -46 -57 -39 -48 -53 -44 -41 -56 -56 5,0x10 4 ssp.diacitilactis E-423 -15 -29 -35 -28 -43 -52 -39 -87 -37 -33 -34 -43 -42 4,0x10 4 Lactococcus sp. E-416 -15 -23 -16 -61 -37 -41 -31 -35 -36 -41 -31 -57 -58 2,0x10 4 Lauconostoc mesenteroides 4 ss. mesenteroides -389 2 -19 -30 -26 -50 -39 -45 -11 -38 -47 -41 -38 -57 -58 4 ,0x10 Pedicoccus parvulus E-315 6 -6 -7 -7 -7 -8 -6 -23 -8 -7 -7 -12 -12 1 0x10 pentosaceus E-68 10 19 21 15 4 20 10 8 9 3 17 18 5 6,0x10 4 SE-390 0 -3 -1 -71 -6 -5 -3 -6 -5 -8 -5 -7 -9 2,0x10 Lactbacillus plantarum E-76 -2 -6 -5 -12 -9 -14 -12 -10 -16 -14 -10 -22 -22 2,0x105 o E-98 -1 -3 -3 -8 -9 -6 -10 -6 -4 -6 -41 -8 -11 1, 0x10 0 \o '0 ul ND: not determined; negat. number: inhibits growth; posit. number: stimulates growth strong inhibition; 35-55% fairly strong inhibition; 15-34% fairly weak inhibition; 15% weak or no inhibition 1)inducing beer gushing; 2 )retards mash filtration i i: i i IPII~-~ WO 94116053 WO 9416053PCT/I93 100388 TABLE 3. Effect of lactic acid bacteria culture broth Fusarium fungi causing gushing of beer.

distinct growth; no growth D-141 Lactic acid CULTUREBROTH Dilution bacterium strain 1:6 2:6 3:6 4:6 5:6 6:6 E-76 E-98 1+ E-317 1+ E-414 Control IPH control D-147 Lactic acid CULTUREBROTH Dilution strain 1:6 2:6 3:6 14:6 5:6 6:6 E- 6+ E-315 E-317 E~-414 E-415 Control H control I D-148, Lactic acid CULTUREBROTH Dilutionbacterium strain -1:6 12:6 3: 6 -4:6 5:6 6:6 E-76 E-98 E-315 E-317 J+ E-414 E-415 Control H control SUBSTITUTE

SHEE~T

WO 94/16053 PCT/F193/00388 17 D-149 Lactic acid CULTURE BROTH Dilution bacterium strain 1:6 2:6 3:6 4:6 5:6 6:6 E-76 E-98 E-315 E-317 E-414 E-415+ Control pH control The results reveal that the lactic acid bacteria strains mentioned inhibit the growth of harmful Fusarium moulds and other detrimental microbes occurring in the malting process, exerting substantially no influence on useful microbes. The results demonstrate the usability of lactic acid bacteria in the procedure of the present invention.

Example 2: THE MICROCIDIC EFFECT OF CERTAIN LACTIC ACID BACTERIA STRAINS ON FOODSTUFF PATHOGENS AND ON MICROBES DETRIMENTAL TO FOODSTUFFS In the experiment a study was made of the microcidal effect on foodstuff pathogens and on micro- 1-s detrimental to foodstuffs of preparations produced the aid of Pediococcus pentosaceus VTT-E-90390 (E- 390) and Lactobacillus plantarum VTT-E-78076 (E-76) strains. As test organisms, strains belonging to genera Bacillus, Yersinia, Listeria, Pseudomonas, Salmonella and Staphylococcus were employed.

As a preparation, a sterile culture broth of lactic acid bacteria, prepared according to Example 1, was used. All the other test strains were cultivated for 16 to 18 hrs in Iso-Sensi&est broth (Oxoid) except the Listeria strain, which was grown in tryptose/phos- SUBSTITUTE SHEET WO 94/16053 PCT/FI93/00388 18 phate broth. Cultivating temperature was 30 0 C. except for the Salmonella, Listeria and Staphylococcus strains, 37°C.

The microbicidic activity was determined by the turbidometric method, described in Example 1. Experimental conditions, as regards growth substrate and temperature, were as described in the foregoing. The incubation time was 24 hrs, except for Bacillus and Yersinia strains, 48 hrs.

The results are shown in Table 4, which reveals that addition of the lactic acid bacteria preparation of the invention causes inhibition of the growth of foodstuff pathogens and microbes detrimental to foodstuffs.

TABLE 4. Growth inhibition caused by P.

E-390 and L. plantarum E-76.

pentosaceus Production strains E-390 E-76 Test organisms Reduction of growth area, Bacillus cereus 93 ATCC91339 Yersinia enterolitica 85 54 ELI351 Listeria monocytogenes 41 49 KTL4126) Pseudomonas fluorescens 59 97 ELI97 Pseudomonas fragi 84 93 ATCC4973 Salmonella infantis 30 98 Staphylococcus aureus 73 86 EL1200 Pathogens occurring in foodstuffs ATCC: American Type Culture Collection ELI: VTT, Food Research Laboratory KTL: National Public Hea'th Institute SUBSTITUTE SHEET -I I WO 94/16053 PCT/FI93/00388 19 Example 3: THE EFFECT OF LACTIC ACID BACTERIA PREPARA- TIONS AND OF PREPARATIONS PRODUCED BY LACTIC ACID BAC- TERIA ON THE MICROFLORA OF MALTING AND ON MALT QUALITY 1. Strain employed: The lactic acid bacteria strain Pediococcus pentosaceus VTT-E-90390 (E-390) was used in the experiment.

MRS broth was used for growth medium of inoculum. The bacteria were anaerobically cultivated for 2 days in 10 ml MRS broth, temperature 30 0 C. The inoculation volume was 1% of the growth solution volume.

2. Barley: Kymppi barley of the harvest year 1990 was used, in which the proportion of Fusarium mould-contaminated kernels was 3. Malting process: 1000 g barley batches were rinsed in a water bath at 12 0 C for 1 hr. The rinsing water was replaced with the first steeping water, and this was replaced with the second steeping water after 5 hrs. Air rest was commenced 16 hrs thereafter. The purpose with air rest was to eliminate the water on the surface of the kernels.

Its duration was 8 hrs. During the steepings, the barley attained 44% moisture. The barley was aerated throughout the steeping process.

Steeping was succeeded by germination. The barley was germinated in a germination boxes for 6 days, at 14 0 C. In order to maintain the moisture on the 44% level, the barley batches were moistened and turned every day. The green malt thus obtained was dried in a 21-hour temperature programme. The temperature was 50 0 C for hrs. During the next 4.5 hrs it was raised to 60 0 C, where it was held 4 hrs. The temperature was further raised uniformly during 5 hrs, up to 85 0 C and held there the remaining 3 hrs. The ultimate moisture content of the malt became about Finally, the rootlets were mechanically removed.

I g WO 94/16053 PCT/F193/00388 A malting run without any additions whatsoever served as control.

4. Lactic acid bacteria preparation and preparation produced by lactic acid bacteria: Lactic acid bacteria cells isolated from the culture broth and culture broths containing microbicidal compounds were employed for preparations, in combination as well as separately. Culture broth including cells was added 120 ml per kg of barley, or the cells were separated from 120 ml of culture broth. This separation was done by centrifuging the culture broth, and the cells were suspended in water. In the cases in which culture broth was added, the culture broth was used as such. The cell counts of the preparations added were on the order of about 108 to 109 CFU/ml.

Additions of lactic acid bacteria preparation: The additions of lactic acid bacteria preparation were made either to the barley, to the beginning of steeping I, to the beginning of steepings I and II, or to the beginning of germination.

6. Analyses performed: Samples were drawn from each malting step.

6.1 Moulds were assessed as follows. The percentage of kernels contaminated with Fusarium moulds was determined by means of CZAPEK IPRODION DICLORAL agar (CZID agar), which is selective to Fusarium moulds, and a moist filter paper (EBC-Analytica Microbiologica, Part II, 1987). Fusarium moulds were identified by their typical colony and spore morphology and by the red colour.

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

6.2 Lactic acid bacteria were assessed on MRS agar in the case of cultures added as well as malting I- I WO 94/16053 PCT/FL93/00388 21 samples.

6.3 The total bacterial coumts were assessed on Plate Count agar (Difco).

6.4 Chemical characteristics of the malt were determined employing methods known from the context of malting (EBC-Analytica, 1987, 4th Ed.).

7. Results: In Table 5 are given the counts of Fusarium moulds as well as lactic acid bacteria in the different malting steps on addition of E-390 culture broth.

In Figs 2 and 3 are presented the total bacterial counts in different malting steps for E-390 culture broths and for E-390 cells.

Table 6 presents the results of malt analysis on addition of E-390 culture broth or E-390 cells.

WO 94116053 WO 9416053PCT/F193/00388 22 TABLE 5. Effect of P. pentosaceus E-390 culture broth a.,dded at different salting steps, on Fusarius and lactic acid bacteria counts during salting.

Fusarium moilds (percentage of con- ___taminated_ Addlition step jBarley Steeping iGermi- Malt .Control 55 72 196 120 ml CB to 3 6 77 3 barl.ey 12 0 ml CB*) to 55 62 98 14 1st. st. water 120 ml CB*) tol1. 55 42 90 9 2. st. water_____ 120 ml CB~ to .55 791 3 germination Lactic acid bacteria (cell density, CFU/g) Control 6,0x10 1 I 3,4x10 2 2,2x10 4 3,0x10 3 120 ml CB*) to 1,2x10 1,lxlO 2,2x10 1,8x10 D~arley______ 120 ml CB*) to 1st 6,OxlO' 3,1lX10 7 2,8x10 7 1,4xl10 7 st. water 120 ml CB~ to 1. 6, 01xl10 7,4x1 0 7 8,3xl0 7 5,3x10 1 7 2. st. water 120 ml CB") to 6,0x10 1 1,5x10' 2,7xI10 8 2,6x10 8 6 germination *CB; culture broth SUBSTITUTE-

SHEET

I I_ TABLE 6.Effect of culture broth or cells of P. pentosaceus E-390 added at various steps of salting on salt quality.

0 tA Analysis 8glucans Moisture 1 I I Extract Coarse grind Extract differ.

%d.m.

:la- Filt- Sac- is- I I city ration char-icos- Modi- omo- -amyfica- gene- lase ft- r\n 4- 1- B-glu- Rootcanase lets actifine grind ml/h ific.

time min.

~rvii vity Il/a vity Il/kn Sample mq/1% %d.m. aa/l rr/k Control 604 4,1 180,1 77,8 2,3 clear 300 <10 1,59 163 82 69 202 365 120 ml CB' to 496 4,3 80,3 77,3 3,0 clear 305 <10 1,51 164 80 57 207 355 37 barley 120 ml CB" to 482 4,2 80,,5 77,5 2,9 clear 310/56 <10 1,50 171 76 51 194 316 1st st. water min 120 ml CB"' to 482 4,3 80,0 76,9 3,1 clear 305 <10 1,49 168 80 64 186 360 31 1+2 st. water 120 ml CB' to 955 4,2 79,0 74,9 4,1 opal. 310 <10 1,68 129 69 59 159 386 21 germination Cells (108 CFU/g) 606 4,3 80,3 77,4 2,9 clear 315/61 <10 1,54 167 79 59 223 359 38 to barley min Cells (108 CFU/g) 79 59 187 376 39 to 1st st. water Cells (108 CFU/g) 81 67 178 353 36 to 1+2 st. water Cells (108 CFU/g) 804 4,3 79,5 76,6 2,9 opal. 290 <10 1,63 148 i81 69 179 392 to germination 1 not analysed 'CB; culture broth r I I WO 94/16053 PCT/F193/00388 24 The results here obtained reveal that treatments according to the invention reduce in particular the Fusarium mould quantity and the total bacterial count in different steps of malting.

Addition of preparation had no detrimental effect on malt quality. Quite the opposite is true: treatment conforming to the invention improved the filtration of the mash derived from the wort and lowered the B-glucan content of the malt.

Example 4: PRODUCTION OF PREPARATIONS PRODUCED BY LACTIC ACID BACTERIA 1. Preparation of concentrated culture broths: The production strains, Pediococcus pentosaceus VTT-E-90390 (E-390) and Lactobacillus plantarum VTT-E- 78076 (E-76) were cultivated in a fermentor in a 15 1 volume. Inoculum volume was 6% to culture was made on MRS medium, at 30 0 C, 2 days in microaerophilic conditions. The culture broths were concentrated tenfold and twenty-fold by lyophilizing and evaporating procedure.

The microbicidic activity of the concentrates was ascertained by the disk method.

2. Preparation of a purified solution containing microbicidic compounds: The lactic acid bacteria culture broth was purified by gel chromatographic fractionation according to molecule size. The fractions found by disk method and by turbidometry to be active were collected and rerun through the gel column.

Example 5: THE EFFECT OF LACTIC ACID BACTERIA PREPARA- TIONS AND OF PREPARATIONS PRODUCED BY LACTIC ACID BAC- TERIA ON THE MICROFLORA OF MALTING AND ON MALT QUALITY 1. Strains employed and preparations produced from the strains: The following bacterial strains were used: Lactobacillus plantarum VTT-E-78076 (E-76) WO 94/16053 PCT/FI93/00388 2 Pediococcus pentosaceus VTT-E-90390 (E-390) The strains were obtained from VTT, Collection of Industrial Microorganisms (Biotechnical Laboratory, Finland).

The preparations were made as described in Example 1, item Example 3, item and Example 4, items 1. and 2.

2. Barley: The barley employed was Kymppi barley of the harvest year 1991.

3. Malting process: Malting was carried out as described in Example 3, however with duration of the germination step, 8 days.

The final moisture content of the malt came to be less than 4. Malti: g: Two laboratory maltings were made. MElting without any additions served as control.

4.1 First malting: Culture broths without cells, concentrated tenfold, and untreated culture broth including cells were used for preparations in the laboratory malting. Preparation was added at the beginning of steeping I or at the beginning of steepings I and II. Malting trials 1 to 8 were carried out as follows.

Test No. 1: Control, Kymppi barley 1991; Test No. 2: At beginning of steepings I and II, 120 ml E-76 culture broth with cells is added: Test No. 3: At beginning of steeping I, 120 ml tenfold concentrated E-76 culture filtrate is added; Test No. 4: At beginning of steepings I and II, 120 ml tenfold concentrated E-76 culture filtrate is added; Test No. 5: At beginning of steepings I and II, 120 ml E-390 culture broth is added; Test No, 6: At beginning of steeping I, 120 ml tenfold concentrated E-390 culture filtrate is added; Test No. 7: At beginning of steepings I and II, 120 ml WO 94/16053 PCT/F9300388 26 tenfold concentrated E-390 culture filtrate is added; Test No. 8: At beginning of steepings I and II, 60 ml E- 76 culture broth with cells and 60 ml E-390 culture broth with cells are added.

4.2. Second malting: In the laboratory malting, twenty-fold concentrated culture filtrates without cells, purified microbicidic fractions without cells and untreated culture broths with cells were used for preparations. The steeping water pH was controlled. Preparation was added at the beginning of steepings I and II. Malting trials 9 to 16 were carried out as follows.

Test No. 9: Control, Kymppi barley 1991; Test No. 10: At beginning of steepings I and II, 120 ml water is added, pH 3.8; Test No. 11: At beginning of steepings I and II, 120 ml E-76 culture broth with cells is added; Test No. 12; At beginning of steepings I and II, 120 ml E-76 fractionated concentrate is added, pH 3.8; Test No. 13: At beginning of steepings I and II, 120 ml twenty-fold concentrated E-76 culture filtrate is added; Test No. 14: At beginning of steepings I and II, 120 ml E-390 culture broth with cells is added; Test No. 15: At beginning of steepings I and II, 120 ml E-390 fractionated concentrate is added, pH 3.8; Test No. 16: At beginning of steepings I and II, 120 ml twenty-fold concentrated E-390 culture filtrate is added.

Analyses performed: Samples were drawn from each malting step.

Moulds, lactic acid bacteria, total bacterial counts and physical and chemical quality characteristics of the malt were determined as described in Example 3.

6. Results: Figures 4 and 5 show the total bacterial counts at different steps in the malting process on application WO 94/16053 PCT/FI93/00388 27 of lactic acid bacteria culture broths with cells or of concentrated or fractionated culture filtrates in the steeping waters.

In Tables 7, 8, 9 and 10 are presented the concentrations of Fusarium moulds and other moulds, and of lactic acid bacteria in different malting steps, in both malting runs.

Tables 11 and 12 present the results of malt analysis from both malting runs.

8_1_ TABLE 7. Myco flora at malting on addition of P. pentosaceus E-390 and L- plantarum E-76 preparations to the barley steeping water (results given as contaminated kernels). Numbering of samples in Example Sample Bar- Steeping _Germination Malt Mould genus ley 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 Fusarium FP 35 80 64 60 22 76 76 48 68 72 88 88 94 96 80 90 94 26 22 2 3 25 17 7 24 CZID 36 74 72 46 38 84 54 32 7 184 82 80 82 84 78 80 96 36 22 10 2 24 20 8 24 Alternaria 26 6 18 22 20 18 22 12 24 18 10 8 2 10 2 2 10 18 8 17 5 18 5 11 16 Cephalosporium 37 22 38 34 36 42 12 28 24 72 20 42 80 42 32 36 124 2 3 5 4 7 2 6 2 Cladosporium 17 4 20 6 56 34 10 44 20 6 6 30 52 14 1 0 36 4 2 0 4 1 1 1 0 Rhitzopus 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 16 0 48 13 0 110 Mucor 0 2 4 0 2 8 14 0 4 0 10 28 64 24 44 34 14 65 79 78 88 68 83 96 73 Stemphylium 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 6 0 1 Epicoccum 2 0 0 0 0 0 0 0 0 00 0 2 0 0 0 2 1 1 0 0 3 0 1 3 Helminthosporium 10 26 46 10 10 36 16 14 30 48 64 60 42 50 54 38 58 28 26 14 3 11 23 8 Acremoniella 0 0 00 0 0 0 0 0 1000 0 O 0 0 0 0 0 0 0 0 0 Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Aspergillus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 Penicillium 3 0 2 2 2 0 0 2 2 4 2 2 2 120 0 0 00 01 0 Other fungi 1 8 00 000 0 0 0 0 0 0 0 02 0 0 0 00 0 000 FP; filter paper 0 0 o

OO

i rr- -u i j WO 94116053 WO 94/6053 CT/IF93 /00388 29 TABLE 8. Lactic acid bacteria counts. (CFU/ml) at malting on addition of P. pentosaceus E-390 and L. plantarum E-76 preparations to the barley steeping water. Numbering of samples in Example Example Steeping Germination Malt 1 5,5x10 3 3,3xl10 2,5xl10 2 2 7,3xl10 5 1,2xl10 7 5,5x10 3 1,4xl& 4,4xl10 3 1 ,1x10 3 4 3,2xl10 2 1,4xl10 4 9,5x10 2 1,3xl10 7 7 5 xl 1 0 6 2,6x10 6 64,8x10 3 5,0x10 5 4,0x10 4 7 7,3x10 2 2,4x10 4 2,6xl10 3 18 l,2x10 7 1,4xl10 7 2,3xl10 6 Lactic acid bacteria CFU/g.

content of the barley: 7,5x1 0 2 SUBSTITUTE SHEET TABLE 9. Myco flora at malting on addition of P. pentosaceus E-390 and L- plantarum E-76 preparations to the barley steeping water (results given as contaminated kernels). Numbering of samples in Example Sample Bar- Steeping Germination Malt Mould genus ley 9 10 11 12 13 14 15 16 9 10 11 12 13 14 15 16 9 10 I1 12 13 14 15 16 Fusarium FP* 35 78 84 32 30 0 44 36 28 98 86 94 98 59 96 94 72 23 28 37 14 1 16 65 13 CZID 36 78 78 30 14 0 26 28 22 98 98 92 66 12 92 92 54 38 31 36 7 1 24 44 11 Alternaria 26 6 2 34 10 10 24 20 14 8 2 14 24 0 0 14 6 17 16 25 13 2 19 27 3 Cephalosporium 37 18 18 34 24 0 36 44 44 52 18 56 52 4 36 36 58 5 3 6 11 1 14 13 Cladosporium 17 6 8 14 16 70 18 24 42 14 6 2 16 54 2 6 20 1 0 0 1 7 1 0 1 Rhitzopus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 13 20 0 0 0 0 11 0 Mucor 0 0 2 2 2 4 2 0 2 36 14 52 40 76 22 56 66 80 78 78 91 98 93 84 Stemphylium 0 0 0 0 0 2 00 0 0 0 0 0 2 2 0 2 0 0 4 4 1 0 3 2 0 Epicoccum 2 0 0 2 00 0 0 0 0 0 0 0 0 0 2 0 0 1 0 0 0 0 0 Helminthosporium 10 32 28 44 18 10 26 28 26 66 50 66 82 6 78 66 58 46 58 46 52 7 76 55 17 Acremoniella 0 00 0 0 0 0 0 0 44 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Trichothecium 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 Aspergillus 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Penicillium 3 2 2 0 2 0 0 2 0 0 0 10 16 30 4 10 01 0 0 0 0 0 Other fungi 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 FP; filter paper i i Ilr err i -s~-Pls~ WO 94/16053 WO 9416053PCT/I93/00388 31 TABLE 10. Lactic acid bacteria counts. (CFU/ml) at malting on addition of P. pentosaceus E-390 and L.

plantarum E-76 preparations to the barley steeping water.

Numbering of samples in Example Example Steeping Germination Malt 9 8,0X10 2 4,2xl10 6 8,0X10 3 8,0x10 2 5,0xi0 6 5,6x10 4 11 2,7x10 7 2,1xl10 7 8,5xl10 12 2,2x10 3 3,4xl10 5 2,5xl10 3 13 1,2xl10 3 1,4x10 7 2,3x10 3 144,1x10 7 3,5x10 7 1 1x10 4 154,8x10 3 4,8x10 3,9x10 673,5xl10 2 2,1x10 5 1,7x10 4 Lactic acid bacteria CFU/g.

content of the barley: 7,5x10 2 SUBSTITUTE

SHEET

TABLE 11. Effect of P. pentosaceus E-390 and L. plantarum E-76 preparations added to the barley steeping water on malt quality. Numbering of samples in Example Sample Analysis 1 2 3 4 5 6 7 8 Moisture 3,6 3,6 3,5 3,7 3,7 3,6 3,9 3,9 Extract content, fine dry m. 81 80,8 80,6 80 81,2 81 80,7 81,2 Extract content, coarse dry m. 76,8 77 76,1 75,2 77 i 6,4 74,8 77,1 Extract difference 4,2 3,8 4,5 4,9 4,3 4,7 5,9 4,1 Saccharification min. <1 <1<0 <10 <10 <10 <10 <10 Wort clarity clear clear clear clear clear clear clear clear Filtration, fine grind ml/min 320/45 320/44 315/48 315/40 320/47 315/66 320/49 315/52 Filtration, coarse grind ml/min 260/86 265/64 270/59 255/73 260/68 260/76 255/90 275/120 Wort pH 5,93 5,88 5,86 5,86 5,86 5,86 5,97 5,99 Viscosity cP 1,69 1,57 1,59 1,57 1,55 1,58 1,59 1,58 B-glucans mg/l 784 659 705 736 610 696 822 657 Free amino nitrogen mg/l 154 156 155 139 158 150 130 154 Modification 74 72 73 73 72 75 77 76 Homogeneity 61 65 63 58 67 66 62 68 a-amylase U/g 178 182 148 128 179 156 144 174 B-glucanase U/kg 434 420 360 1360 442 412 361 425 i- TABLE 12. Effect of P. pentosaceus E-390 and L. plantarum E-76 preparations added to the barley steeping water on malt quality. Numbering of samples in Example Sample Analysis 9 10 11 12 13 14 15 16 Analysis Moisture 3,5 3,6 3,6 3,5 3,8 3,5 3,5 3,6 Extract content, fine dry m. 80,5 80,5 81,2 80,5 80,3 81 81,1 80,4 Extract content, coarse dry m. 78,4 77,9 77,4 73,3 73,5 77,6 76,6 75,1 Extract difference 2,1 2,6 3,7 7,2 6,8 3,4 4,5 5,4 Saccharification min. <10 <10 <10 <10 <10 <10 <10 Wort clarity clear clear clear clear clear clear clear clear Filtration, fine grind ml/min 320/40 315/47 320/44 315/42 320/55 315/49 330/40 320/40 Filtration, coarse grind ml/min 290/120 270/76 260/51 265/120 245/120 285/68 305/120 265/12 Wort pH 6,04 6,05 5,99 5,97 5,94 5,95 5,98 5,99 Viscosity cP 1,54 1,65 1,51 1,53 1,5 1,49 1,49 1,52 B-glucans mg/l 442 522 397 553 612 381 463 576 Free amino nitrogen mg/l 149 145 149 128 117 149 139 120 Modification 85 85 86 82 74 81 75 71 Homogeneity 63 66 76 57 57 68 51 47 a-amylase U/g 161 172 161 123 72 148 125 101 B-glucanase U/kg 365 375 365 282 282 371 342 308 WO 94/1605' PCT/F93/00388 34 The results now obtained reveal that treatments according to the invention reduce, in particular, the Fusarium mould quantity and the total bacterial count in the different malting steps. It is further noted that, in addition to culture broths, concentrated and fractionated culture filtrates in particular have a favourable effect e.g. as regards Fusarium moulds.

From the malt analyses the inference can be drawn that culture broth additions of strains E-76 and E-390 had an effect of improving the filtrability of the wort made from the malt; the B-glucan contents are also lower in the respective malts than in the control malt.

0 S S* 15 EXAMPLE 6: THE EFFECT ON MASH FILTRATION OF PREPARATIONS PRODUCED BY LACTIC ACID BACTERIA, ADDED TO THE MALTING In Fig. 6 a diagram is reproduced, showing the effect of treatment according to the invention with 20 preparations derived from lactic acid bacteria strains (120 ml culture broth per kg of barley) on the filtration of the mash produced using malt thus treated.

The strains mentioned in Example 1 were used in this experiment: E-390, E-416, E-98, E-317, E-390, E-76,, and E-315. The test was carried out using the Tepral filtration method (BIOS. 19, 1988, Grandclerc, J. et al.,, "Simplification de la methode de filtration du brassin tepral description de la m6thode", p. 88-92).

It is evident from the results that treatment according to the invention improves the filtration of the mash.

00o0 oo** 0 0 06 0O 0n o *o o* 06 c* S o 0« C 65 S6 00

B

S

6 In the following examples, the effect of lactic acid bacteria on the concentrations of mycotoxins, deoxynivalenol and zearalenone produced by Fusarium moulds in a barley malting process are studied.

EXAMPLE 7: EFFECT OF LACTIC ACID BACTERIA ON DEOXYNIVALENOL CONCENTRATION (DON) IN THE MALTING OF BARLEY CONTAMINATED WITH THE F.culmorum D 148 MOULD 1. Strains used In the test, the Lactobacillus plantarum (E- 76) and Pediococcus pentsaceus (E-390) strains of lactic acid bacteria were used.

The bacteria were anaerobically cultivated in MRS nutrient solution at 30 °C for 3-4 days. Between the tests, the bacterial strains were kept in their nutrient solution in anaerobic conditions at 4 °C.

2. Barley: The barley variety used was "Kymppi" barley of the harvest year 1994, to which was added 2 ml of 20 Fusarium culture solution/100 g of barley. The Fusarium strain was grown in shaken CMC solution for about 7 days to produce spores. The spore density of the F.culmorum VTT D-80148 (D 148) culture solution was about 106 spores/ml of solution. The contaminated bar- 25 ley was incubated at 25 °C for 3 days. The barley was stirred daily and the barley was dried overnight at °C to its original moisture.

3. Malting process The steeping program for 1000 g barley batches was as follows: washing (1 l st steeping (7 h), i st aeration break (16 2 n d steeping (8 2 nd aeration break (15 h) and dipping. The steeping temperature 12 °C and the target moisture of the barley was 46 at the end of the steeping. To ensure the breathing and metabolism of the barley, aeration breaks were provided between the steeping stages and the barley samples were moved to an aerated space during the bre- Lp- Y- I aks. The steeping vessels were also provided with aeration. After the second aeration break, the barley samples were weighed to determine their moisture content. When the moisture was below 1 6 the barley samples were dipped in water for a short time. The ratio of barley to water during steeping was 1:1.5. After this, the barley samples were moved to a germination box.

The barley samples were germinated in an aerated germination box for 6 days at 14 oC. To adjust the moisture to the 46 target level, the barley samples were moistened and fluffed up daily. The green malt thus obtained was dried using a 21 h temperature programme, as in item 3 of Example 3. Finally, the rootlets were mechanically removed from the malt.

A malting run without the addition of lactic acid bacteria was used as a control test.

4. Lactic acid bacteria preparation Table 13 presents the pH, cell counts and microbicidal activity (disc method, Example 1, item of the lactic acid bacteria cultures added to the malting process.

Table 13 *a C

CC

C C

C

CCC C

CC..

C

C C

CC

CC..

Strain of lactic acid bacteria E-76 E-390 pH of culture solution 3.72 3.77 Cell count (PMY/ml) Inhibition zone (mm) 17 5. lE+08 1.7E+09 i Additions of lactic acid bacteria preparation: Lactic acid bacteria preparation was added at the beginning of the first and second steeping stages.

The ratio of lactic acid bacteria culture to water was '3P maintained at 8 during the malting process.

(f i r. g (BP I~ ISI ~r 4PrC~ rr I 6. Analyses performed: The deoxynivalenol concentration of each barley sample was determined via gas chromatography. A mass spectrometer was used for the recognition and quantization of deoxynivalenol.

7. Results: Fig. 7 shows the effect of the addition of lactic acid bacteria on the deoxynivalenol content in the malting of barley contaminated with F.culmorum D 148 strains.

It can be seen from the results obtained that the L.plantarum E-76 strain had the effect that the malt showed 50 less deoxynivalenol than the reference malt, and that the P.pentosaceus E-390 strain had the effect that the malt showed 25 less deoxynivalenol than the reference malt.

EXAMPLE 8: EFFECT OF LACTIC ACID BACTERIA ON THE FORMATION OF DEOXYNIVALENOL (DON) IN THE MALTING 20 OF BARLEY NATURALLY CONTAMINATED WITH DEOXYNIVALENOL AND FUSARIUM MOULDS.

1. Strains used: 0 In the test, the Lactobacillus plantarum (E- 76) and Lactobacillus acidophilus VTT E-87276 (E-276) 25 strains of lactic acid bacteria were used.

The Lactobacillus plantarum strain was cultivated as in Example 7; the culture temperature for the Lactobacillus acidophilus strain was 37 OC.

2. Barley: The barley varieties used were low-quality Kustaa, Loviisa and Jo 1599 of the harvest year 1993.

3. Malting process: The malting process was carried out as in Example 7, except for the size of the barley batches (100 g) and the barley-to-water ratio during sceeping s." a^'rc IP -Ps41~ A malting run without the addition of lactic acid bacteria :as used as a control test.

4. Lactik- acid bacteria preparation: Table 14 presents the pH, cell counts and microbicidal activity (disc method, Example 1, item 5.1) of the lactic acid bacteria cultures added to the malting process.

Table 14_ Barley Strain of pH of cul- Cell count Inhibition variety lactic ture solu- (PMY/ml) zone (mm) malted acid bac- tion teria Kustaa E-76 3.68 2.6E+09 23 E-276 3.96 1.6E+08 21 Loviisa E-76 3.68 2.6E+09 23 E-276 3.96 1.6E+08 21 Jo 1599 E-76 3.68 2.6E+09 23 E-276 3.96 1.6E+08 21 B. FOREIGN BARLEY g* sea 1. Strains vsed: Corresponding to item A.I.

15 2. Barley: The barley used consisted of low-quality fo- "reign barley varieties 1 3.

The steeping was performed as in Example 7, *~except for the size of the barley batches (20 g) and the barley-to-water ratio (1:17.0).

The barley was germinated for 6 days in plastic bags placed in a plastic box on a metal frame to the target moisture 46 The bags were turned daily.

To preserve the moisture, some water was poured onto the bottom of the germination box. The green malt was dried in a in incubator at 50 'C for 17 hours, whereupon the temperature was raised to 85 0 C and the barley 39 wa.s dried for a further 4 hours. The rootlets were removu~d mechanica.Ily.

Lactic acid bacteria preparation: Table 15 presents the pH, cell counts and microbicidal activity (disc method, Example 1, item 5.1) of the lactic acid bacteria cultures added to the malting process.

'0 Barley Strain pH of cu.- Cell count Inhibition variety of lac- ture solu- (PMY/ml) zone (mm) malted tic acid tion bacteria Forei~zi E-76 3.82 9.5E+08 23 no. 1 E-276 4.00 3,5E+08 Foreign E-76 3.82 9.5E+08 23 no. 2 E-276 4.00 3.5E+08 Foreign E-76 3.70 7.5E+09 23 no. J E-276 4.01 4.4E+08 Additions o C lactic acid bacteria preparation: 5 Lactic acid bacte;Aa preparation was added at th,' beginpning the first and second. steeping stages.

The, ratio of lacti C acid bacteria culture to water wz',* maIntaIn-ed at 8 during trie malting process.

6. Analyses performed: 0 The deoxynivalenol concentration of each barley sample was det~ermined via a r ELISA test (Ridascreen).

7. Results:

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0 9* ~B~B~LIWPr-6~ I Table 16 and Fig. 8 and 9 show the deoxynivalenol concentrations in the malting of Finnish and foreign barley batches as well as the influence of the lactic acid bacteria on the formation of deoxynivalenol (DON) in the malting of barley naturally contaminated with deoxynivalenol and the Fusarium mould.

Fig. 10 presents the mean values of the deoxynivalenol concentrations of three domestic and three foreign barley samples, i.e. the effect of the lactic acid bacteria cultures added to the first and second steeping waters on the deoxynivalenol concentration in the malting of Finnish and foreign barley, determined via the ELISA test (Ridascreen). The results are given as average values.

Table _1 Sample

DON

(ua/ka)

~L~E~YL;I

20 fee.

00*00* s~o 25 :04 0 40.

030 *0 0000 01 3 25 000~; 0000 0 "330 Barley Kustaa -93 Loviise -93 Jo 1599 -93 Kymppi -94 Foreign no. 1 Foreign no. 2 Foreign no. 3 Malt Kustaa -93 reference Kustaa -93 E-76 Kustaa -93 E-276 Loviisa -93 reference Loviisa -93 E-76 Loviisa -93 E-276 Kymppi -94 Foreign no. 1 reference Foreign no. 1 E-76 Foreign no. 1 E-276 34 115 72 56 296 212 673 29 23 66 53 109 262 73 56 i 11IHIP r ll~ LI~~ ~B)~i~oi~i~a~s~arrarrrrrr~-l 41 Foreign no. 2 reference 92 Foreign no. 2 E-76 87 Foreign no. 2 E-276 59 Foreign no. 3 reference 263 Foreign no. 3 E-76 Foreign no. 3 E-276 38 E-76 Lactobacillus plantarum E-276 Lactobacillus acidophilus It could be seen from the results obtained that when L.plantarum (E-76) culture solution was added, especially in the malting of foreign barley samples, their deoxynivalenol concentration is higher in the reference malt than when lactic acid bacteria are present. The L.plantarum strain had the effect that the deoxynivalenol concentration (average for three samples) was 68 lower than in the reference, and that the Lactobacillus acidophilus (E-276) strain had 20 the effect that the deoxynivalenol concentration was 75 lower than in the reference. The Finnish barley batches had low deoxynivalenol concentrations, and these may have been further decreased during the malting.

EXAMPLE 9: EFFECT OF LACTIC ACID BACTERIA ON THE ZEARALENONE CONTENT (ZEN) OF MALT IN THE MALTING •OF BARLEY CONTAMINATED WITH THE FUSARIUM CULMORUM D- 148 AND F.GRAMINEARUM D-470 STRAINS.

A:l. The strain used is the Lactobacillus plantarum (E-76) strain of lactic acid bacteria.

The strains, barley (contamination time 7 days), malting process, lactic acid bacteria preparation and additions of lactic acid preparation used in this example were as in items 1 5 of Example 7.

6. Analyses performed: "I r k 42 The zearalenone content was determined from each barley sample via liquid chromatography.

7. Results: Fig. 11 presents the effect of the lactic acid bacteria additions on the zearalenone content in the malting of barley contaminated with the F. culmorum D-148 strain.

It can be seen from the results that the L plantarum E-76 strain had the effect of reducing the zearalenone content of the malt by 46 as compared with the reference malt.

B:l. Strains used: In the test, the Lactobacillus plantarum (E- 76), Lactobacillus acidophilus (E-276) and Pediococcus pentosaceus (E-390) strains of lactic acid bacteria were used.

The cultivation was performed as in Example 7.

20 2. Barley: The barley used was Kymppi barley of the harvest year 1994, which was contaminated with F. graminearum VTT D-95470 (D-470) culture solution as described under item 2 in Example 7.

25 The malting process, lactic acid bacteria preparation and additions of lactic acid bacteria preparation were as in items 3 5 of Example 7.

S0 In addition, the L. acidophilus E-276 lactic i" acid bacteria culture added to the malting process had a pH value of 4.05, a cell count of 5.8E+08 PMY/ml and an inhibition zone of 14 m (disc method, Example 1, item 5.1).

6. Analyses performed: The zearalenone content was determined from each barley sample via an ELISA test (Ridascreen).

7. Results: q II I 43 Fig. 12 presents the effect of the lactic acid bacteria additions on the zearalenone content inl the malting of barley contaminated with the F. graminearum. VTT D-95470 (D-148) strain.

It can be seen from the results that the lactic acid bacteria had the effect of reducing the zearalenone content of the malt by 37 -55 as compared with the reference malt.

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S@ 0 0,S 4*00.

i

Claims (6)

1. The procedure for treatment of seed material to be germinated, characterized in that to the barley kernels in a malting process or seed material going to be converted to sprouts intended to serve as nutrition, is added in connection with the germination process, a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria which has an effect inhibiting microbial growth.

2. Procedure according to claim 1, characterized in that the lactic acid bacterium belongs to genus Lactococcus, Leuconostoc, Pediococcus or Lactobacillus.

3. Procedure according to claim 1 or 2, characterized in that the lactic acid bacteria preparation or the 15 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 from a mixture of these, advantageously from species Lactobacillus plantarum or Pediococcus pentosaceus or from a mixture of these.

4. Procedure according to any one of claims 1 to 3, characterized in that to barley kernels is added a lactic acid bacteria preparation or a preparation produced by 25 lactic acid bacteria having an effect inhibiting the growth of Fusarium moulds.

5. Procedure according to claim 4, characterized in that the lactic acid bacteria preparation or the preparation produced by lactic acid bacteria is added in the steeping or 30 germination step.

6. A procedure for the treatment of seed material to be germinated, comprising adding to the seed material in connection with a germination process, a lactic acid bacteria preparation or a preparation produced by lactic acid bacteria which has an effect inhibiting microbial growth, substantially as hereinbefore described with reference to any one of Examples 1 to 6. Dated this 30th day of April 1997 OY PANIMOLABORATORIO-BRYGGERILABORATORIUM AB By their Patent Attorneys p-f GRIFFITH HACK r