METHOD FOR SELECTING A LACTOBACILLUS STRAIN FOR THE PRESERVATION OF GREEN FODDER, AND PRESERVATION OF GREEN FODDER

Abstract: The invention concerns a method for selecting a lactic acid bacterial strain for the preservation of green fodder, in which method one or more parameters describing fermentation purity and/or speed of the decomposition reaction is/are determined in a substrate simulating a forage matrix and containing an addition of a lactic acid bacterial preparation being investigated, and a lactic acid bacterial strain is selected that during a given period of time reduces the amount of ammonium nitrogen, which is measured. The invention also concerns a method for the preservation of green fodder, a lactic acid bacterial strain intended for the preservation of green fodder, and a green fodder preserved by this method.

Description: METHOD FOR SELECTING A LACTOBACILLUS STRAIN FOR THE PRESERVATION OF GREEN FODDER, AND PRESERVATION OF GREEN FODDER The present invention relates to a method for selecting a lactic acid bacterial strain for the preservation of green fodder, a method for the preservation of green fodder, use of a lactic acid bacterial strain selected by the method to preserve green fodder and to a green fodder preserved by said method. Harvesting of green fodder, such as grass fodder, as silage for feeding during the winter season has almost completely replaced the production of dry hay. An important prerequisite for large-scale use of silage is that it should be of a good quality in re- spect of preservation. The production of fodder in the form of so- called compacted ensilage without preservatives often leads to a failure of the ensiling process; as a consequence, the fodder does not meet the quality re- quirements. By using acid-based preservatives, a good fodder quality has generally been achieved even in difficult ensiling conditions. The best known acid preservation method is the AIV method, in which or- ganic and/or inorganic acids are added to the silage to reduce its pH, preferably to a level of pH 4. The acid μsed is generally formic acid in a quantity of about 4 1/t of grass (100-% acid) . However, the use of acids involves certain drawbacks, such as the corro- sive effect of acids. The use of so-called biological preservatives based on lactic acid bacteria and/or enzymes aims at enhancing the natural lactic acid fermentation of silage and thus improving its durability (e.g. patent FI 82354) . It may be possible to influence the composition of milk via biological preservation of forage. From the point of view of natural production, too, biological preservation is considered to be a better alternative than acid preservation. Consequently, the amount of initiation cultures marketed for biological preservation of green fodder has been continuously in- creasing, but the properties of commercial strains and their special features in silage are often inadequately known. A knowledge of these properties is a precondition for a controlled ensiling process, and the problem is how to select the best possible strain for the production of silage when the quality of the raw material used may vary considerably. In addition, e.g. the absence of proteolytic activity, which is one of the selection criteria for the lactic acid bacteria to be used in ensiling, is often determined on a labo- ratory substrate which does not contain the protein compounds contained in forage, so the results of the determination are not in keeping with the reality. At present there are no reliable methods in use whereby the effects of lactic acid bacteria on si- lage quality could be tested quickly and reliably in advance. The problem is that the quality of the silage is only revealed after the completion of a time- consuming ensiling process. The object of the invention is to eliminate the problems referred to above. A specific object of the invention is to disclose a method for selecting a lactic acid bacterial strain whereby the effect of the lactic acid bacterial strain on the quality properties of silage can be tested quickly and reliably in advance. A further object of the invention is to disclose the use of a reliable lactic acid bacterial strain selected by this method, which makes it possible to achieve a good lactic acid yield and minimize protein decay while preventing the activity of decay- inducing organisms during ensiling and after the silo has been opened. A further object of the invention is to disclose a method for preserving green fodder using a selected lactic acid bacterial strain and a green fodder silage preserved by this method. In the ensiling of grass forage, the critical phases of the ensiling process are concentrated in two periods. For successful preservation, the first critical phase is immediately after the silo has been closed, which is when the lactic acid bacterial strain selected as an initial culture should be able to grow fast and effectively and reduce the pH of the raw material to slow down the growth of detrimental microbes. For the fermentation process, it will be beneficial if the strain functions in a homofermentative manner, in which case the fall in the pH is primarily based on lactic acid production. By using a homofermentative lactic acid bacterial strain, it is also possible to reduce ensiling losses that may arise when homofermentative lactic acid bacteria dominate the mi- crobial population in the silage. The strain also has to be competitive, and it preferably prevents the growth of detrimental microbes in the forage matrix. There may be large variations in the natural flora of lactic acid bacteria in the raw material in respect of both quality and quantity, and the strain has to be able to rapidly gain a dominating position in relation to the competing background growth. This position should also be maintained during the first few weeks, after which the microbiological activity in the silage is stabilized. A second critical phase is at the time after the opening of the silo, which is when the environmental conditions gradually become aerobic, enabling the onset of aerobic attack. The quality of silage is generally estimated on the basis of certain chemical quality parameters describing fermentation purity and/or the intensity of decomposition reactions. The official quality classification is based on the amounts of lactic, acetic and butyric acids contained in the silage and the proportion of ammonium nitrogen in the total amount of nitrogen and on acidity. For silage stability, a good microbiological silage quality is also beneficial. A poor microbiological quality is indicated by increased quantities of clostridia and decay- inducing yeasts and molds . The method of the invention is characterized by what is presented in claim 1. The method for the preservation of green fodder, the use of a selected lactic acid bacterial strain and the silage according to the invention are characterized by what is presented in claims 7, 12 and 14. The invention is based on research work in which the effect of different lactic acid bacterial strains on the preservation of green forage was investigated and in which it was established that it is beneficial if the selected strain reduces the amount of ammonium nitrogen in a grass extract substrate at the beginning of the ensiling process. The method of the invention for selecting a lactic acid bacterial strain aims at ensuring good conditions for the activity of the lactic acid bacterial strain in biological preservation of green for- age. The method comprises the determination of one or more parameters describing fermentation purity and/or speed of the decomposition reaction in a substrate simulating a forage matrix and containing an addition of a lactic acid bacterial preparation being investi- gated. 'Lactic acid bacterial preparation' refers to an antimicrobial preparation containing bacteria and/or produced by bacteria. In the method, the effect of the bacterial strain on the amount of ammonium nitrogen in the substrate simulating a forage matrix is determined. In regard of silage quality, the smaller the quantity of proteins and peptides decaying into ammonia in the silage, the better. The change in the quantity of ammonium nitrogen is determined as for a given period of time, usually 24 hours. Lactic acid bacterial strains that reduce the amount of ammonium nitrogen during that period have proved to be effective and reliable strains. The quantity of ammonium nitrogen can be determined by a spectrophotometric method, an ion- selective method using e.g. an ion-selective electrode, by a chromatographic method, e.g. via liquid chromatography, or by some other method applicable for the determination of ammonium nitrogen. In an embodiment of the method, the applicability of a lactic acid bacterial strain can be determined on the basis of a fall in the pH value of the substrate simulating a forage matrix. The strain has to be able to reduce the pH of the substrate quickly and effectively. Lactic acid bacterial strains that reduce the pH < 4 within a certain period of time, preferably 14 hours, have proved to be effective and reliable strains. As a substrate simulating a forage matrix, a grass extract substrate prepared from vegetable material is used. The grass extract substrate is prepared e.g. by dissolving grass, such as meadow fescue, timothy, clover or equivalent, chopped in water, by fil- tering, autoclaving or sterile-filtering and concentrating the solution, diluting the concentrate as appropriate and finally adding some glucose, e.g. about 1 %, into it. This method can be used to test any lactic acid bacterial strains. The tests were performed using lactic acid bacterial preparations isolated by the VTT (State Technical Research Centre) which have proved to be able to produce several antimicrobial compounds of a small molecular size. Examples of the strains used are Lactobacillus plantarum (VTT E-78076, VTT E- 79098) , Pediococcus parvulus (VTT E-88315) , Pediococ- cus pentosaceus (VTT E-90390) and/or mixtures of these . It is possible to verify whether the method works by checking how the bacterial strain selected by the method performs in an actual forage matrix in a mini-silo. Good conditions for the activity of the lactic acid bacterial strain are ensured via an estimation of the central quality parameters for the final product, determination of the competitiveness of the strain and measurement of the ensiling loss on the basis of gas formation. The competitiveness of the strain can be determined e.g. by the PCR technique or an equivalent technique. Gas formation can be measured e.g. by a semi-quantitative method using an injector syringe provided with a measurement scale or by an equivalent technique. (The requirements regarding si- lage quality are presented in decree no. 181/94, § 4 item 1, by the Ministry of Agriculture and Forestry.) Lactic acid bacterial strains selected by the method in question perform well in the ensiling process. Usable lactic acid bacterial preparations include lactic acid bacterial strains isolated at the State Technical Research Centre (VTT) , which have been found to produce several anti-microbial compounds of a small molecular size. Small-molecule anti-microbial compounds complement the pH effects of lactic acid bacte- rial strains towards preventing the growth of decay- inducing organisms. A strain that proved to be beneficial in the preservation of green forage is Lactoba- cillus plantarum (VTT E- 78076) . The Lactobacillus piantarum (VTT E-78076) strain has been described e.g. in FI patent 94875. In addition, it has been found that this strain can withstand formic acid when its concentration in the substrate is below 1 % by volume. In the method of the invention for the preservation of green fodder, the selected lactic acid bacterial preparation is added into the silage in an amount of about 10 5 - 10 7 CFU/g of grass, preferably 10 6 CFU/g of grass. The lactic acid bacterial preparation may be added together with one or more preserva- tives and possibly with one or more lactic acid bacterial preparations . The preservative used may consist of e.g. enzymes capable of breaking up vegetable fiber and having e.g. cellulase activity, or formic acid, benzoic acid and/or sorbic acid and their derivatives and/or other known preservatives. In an embodiment of the preservation method, formic acid together with a lactic acid bacterial preparation is added to the forage. The amount of for- mic acid used is e.g. 1/2.5 - 1/20 of the amount used in normal conservation using an acid, i.e. about 1.6 - 0.2 1/t of grass, preferably about 1/10, i.e. about 0.4 1/t of grass. The lactic acid bacterial preparation is preferably added to the grass on the field in conjunction with chopping, because otherwise the natural microbes in the grass growth may turn the scales in a competition situation and cause the fermentation processes to take an undesirable course. The invention also concerns the use of a lactic acid bacterial strain selected by the selection method of the invention for the preservation of green forage. The strain used in the invention is preferably Lactobacillus plantarum (VTT E-78076) . Further comprised in the sphere of the invention is a silage preserved by the preservation method of the invention. The invention makes it possible to easily and quickly select a reliable initiation culture for the preservation of green forage. By applying this new method to select the lactic acid bacterial strain to be used, it will be possible to improve the reliability of the ensiling process so as to ensure ample consumption of silage and a good hygienic milk quality. In the following, the invention will be described in detail in the following examples. Example 1. This experiment was carried out to study the effects of four different lactic acid bacterial preparations on the amount of NH4 nitrogen in a grass sub- strate and on the reduction in its pH . The strains used were Lactobacillus plantarum VTT E-78076 (=E76) and VTT E-78098 (=E98) and Pediococcus parvulus VTT E- 88315 (=E315) and Pediococcus pentosaceus VTT E-90390 (=E390) . The grass substrate used was a timothy meadow fescue and clover substrate which was prepared by dissolving 1 kg of chopped grass in a water bath of 5 1 of water at 50 °C for 2 hours, whereupon the liquor was filtered, autoclaved at 120 °C for 15 min and concentrated until a 20 % solution was obtained. For the test, the liquor was additionally diluted with water to obtain a concentration of about 10 %, and glucose in an amount of 1 % was added into it . The quantity of NH4 nitrogen was determined using an ion-selective electrode. The effect of the initiation culture on the amount of NH 4 nitrogen in the grass substrate and on the reduction of its pH is presented in Table 1. Table 1 Table 1 shows that the L. piantarum and P . pentosaceus E390 strains reduced the amount of NH 4 nitrogen in a timothy - meadow fescue substrate within 24 hours and that all four strains reduced the sub- strate pH to a value below 4 within 14 hours. The reduction in the pH of the clover liquor was smaller because clover has a larger buffer capacity. Example 2. These tests were carried out to study the conditions for the activity of a selected lactic acid bacterial strain in a forage matrix on a mini-silo scale by estimating the quality parameters of the silage, determining the competitiveness of the strain and determining the ensiling loss on the basis of gas formation. The strain used was the Lactobacillus plantarurn VTT E-78076 (=E76) strain, which was also used in example 1. Chopped grass was stowed into 120-ml mini-silos, which were filled with 80 g of grass. The level of dosage of lactic acid bacteria was 10 6 CFU/g of grass. By way of control, compacted ensilage treatment without preservatives and treatment with formic acid (FA) 4 1/t of grass were used. The experiment lasted 84 days and nights. A. Silage quality The quality of the silage in different lots ensiled was determined after 84 days following the ensiling. The silage quality parameters are presented in Table 2. From the results given in Table 2, it can be seen that the selected lactic acid bacterial strain clearly improved the ensiling result as compared with compacted ensilage. In all fodder groups, the amount of entero- bacteria and molds was below detection level . Table 2 meadow fescue. B. Competitiveness of the lactic acid bacterial strain The competitiveness of the selected strain was determined by the PCR technique after 1, 5, 21 and 84 days following the beginning of the ensiling process. Table 3 shows the amount of lactic acid bacteria (log CFU/g) . The amount of formic acid (FA) is given in 1/t (of 100-% acid) . Table 3. Table 4 presents the amounts of L. piantarum found at different points of time (as a percentage of the LAB strains checked) . Table 4. ""Five strains were isolated and checked by an API 50 CHL test. 2 Twenty-five strains were isolated and checked by an API 50 CHL test. Table 5 presents the proportion of L. iantarum E76 genotype in the LAB strains. Table 5. Five strains isolated 2 Twenty-five strains isolated C. Measurement of ensiling loss on the basis of gas formation Table 6 presents the gas formation describing the ensiling loss, as observed during the ensiling period. Table 6. *Red clover used as raw material. In other tests, timothy - meadow fescue. Based on a measurement of gas formation, the ensiling loss was lowest in the case of silage pre- served using the Lactobacillus plantarum VTT E-78076 preparation. The effect of the strain on the amount of ammonium nitrogen in the grass extract substrate as determined in laboratory anticipates the intensity of protein decay that will occur in the silage. Example 3. This test was carried out to study the conditions for the activity of lactic acid bacterial preparations in a timothy - meadow fescue forage matrix. The test arrangements were the same as in example 2. Table 7 shows the quality parameters for different silages. Table 7. It can be seen from Table 7 that the amount of ammonium nitrogen, which describes protein decay, was largest in silages preserved using the E315 and E390 strains. The amount of ammonium nitrogen was smallest in silages preserved using the E76 and E98 strains, which in the test described in example 1 reduced the amount of ammonium nitrogen. Example 4 This test was carried out to study the effect on silage quality of the addition of a combination of a lactic acid bacterial preparation and an amount of formic acid somewhat below the traditional dosage level. Timothy - meadow fescue was stowed into mini- silos as in Example 2. The lactic acid bacterial preparation used was the Lactobacillus plantarum VTT E-78076 preparation, and formic acid was used in concentrations of 0, 0.2, 0.4, 0.8 and 1.6 1/t of grass. Chemical and microbiological analyses were performed after 84 days of ensiling in conjunction with the opening of the silo. Moreover, gas formation in the silage was measured during ensiling. The chemical composition of the silage is presented in Table 8. Table 8 + lactic acid bacteria added - no lactic acid bacteria added It can be seen from the results that the addition of lactic acid bacteria and formic acid reduced gas formation as compared with compacted ensilage. The best result was obtained by using a combination of lactic acid bacteria and formic acid in an amount of 0.4 1/t of grass. Several chemical quality parameters also indicated that this silage was the best in respect of quality. The silage had the highest sugar content and the lowest acetic acid and ammonium nitro- gen content. In microbiological analyses, no enterobacte- ria were found in these silages, and the quantities of clostridia were under 50 CFU/g, except in the silage which had been treated with the least amount of formic acid (0.2 1/t) . In respect of molds, the silages were likewise of a blameless quality. Example 5 This test was carried out to study the effect of combined use of a selected lactic acid bacterial strain and formic acid on the quality of silage in pilot silos. The test silages were prepared from timothy - meadow fescue grass that had been mowed using a field chopper, slightly pre-dried and harvested using a precision chopper. The lactic acid bacterial prepa- ration used was the Lactobacillus plantarum VTT E- 78076 preparation (10 6 CFU/g of grass, solution 5 1/t of grass) and the controls were untreated compacted ensilage and silage treated with formic acid. The silages were prepared in pilot silos (300-400 kg of si- lage/silo) . The lactic acid bacterial preparation was applied in the precision chopper. The chemical composition of these silages is presented in Table 9. Table 9 Although the raw material had been pre-dried, its dry matter content remained rather low. The sugar content was also small. The lactic acid bacteria had a favorable effect on several quality parameters. Silage with the selected lactic acid bacterial strain added to it had a lower pH and contained less propionic acid, ethanol and ammonium nitrogen. Moreover, adding the lactic acid bacterial preparation directly on the field after the grass has been cut has a beneficial effect on the quality of the silage because, if there is a delay between the chopping of the grass and the application of the preservation bacteria, the natural microbial flora of the grass mass may become dominant, which would hamper the ensiling process. The invention is not restricted to the examples of its embodiments described above; instead, many variations are possible within the scope of the inventive idea defined in the claims.

Claims: CLAIMS 1. Method for selecting a lactic acid bacterial strain for the preservation of green fodder, said method comprising the determination of one or more pa- rameters describing fermentation purity and/or speed of the decomposition reaction in a substrate simulating a forage matrix and containing an addition of a lactic acid bacterial preparation being investigated, charac t e ri zed in that, in the method, the change in the amount of ammonium nitrogen in the grass extract substrate is measured during a given period of time and a lactic acid bacterial strain that reduces the amount of ammonium nitrogen is selected. 2. Method as defined in claim 1, chara c - t e r i z e d in that the change in the amount of ammonium nitrogen is measured during 24 hours. 3. Method as defined in claim 1 or 2, characteri zed in that, in the method, the pH of a sample is measured and a lactic acid bacterial strain that reduces the pH of the substrate to a value < 4 within a given length of time is selected. 4. Method as defined in claim 3, charac t e ri zed in that the pH is measured during 14 hours . 5. Method as defined in any one of claims 1 - 4, charac t e ri z e d in that good conditions for the activity of the lactic acid bacterial strain are ensured in the sample matrix using a mini-silo scale by estimating the quality parameters of the green fod- der, determining the competitiveness of the lactic acid bacterial strain and/or measuring the gas formation. 6. Method as defined in any one of claims 1 - 5, c harac t eri z e d in that the lactic acid bac- terial strain selected is Lactobacillus plantarum (VTT E-78076, VTT E-79098) , Pediococcus parvulus (VTT E- 88315) , Pediococcus pentosaceus (VTT E-90390) and/or a mixture of these, preferably Lactobacillus plantarum (VTT E-78076) and/or a mixture of these. 7. Method for the preservation of green fodder, charac t er i zed in that a lactic acid bac- terial preparation selected by a method according to any one of claims 1 - 6 is added to the green fodder. 8. Method as defined in claim 7, charac t e ri z ed in that the amount of lactic acid bacterial preparation added is about 10 s - 10 7 CFU/g of grass, preferably 10 6 CFU/g of grass. 9. Method as defined in claim 7 or 8, charac teri zed in that the bacterial preparation is added to the fodder together with one or more preservatives . 10. Method as defined in claim 9, char ac t eri zed in that the preservative is formic acid or a salt of it. 11. Method as defined in claim 9 or 10, charac t e ri zed in that formic acid or a salt of it is used in an amount of about 1.6 - 0.2 1/t of grass, preferably about 0.4 1/t of grass. 12. Use of a lactic acid bacterial strain selected by a method according to any one of claims 1 - 6 for the preservation of green fodder. 13. Use as defined in claim 12, charac t e ri zed in that the lactic acid bacterial strain selected is Lactobacillus plantarum (VTT E-78076, VTT E-79098) , Pediococcus parvulus (VTT E-88315) , Pediococcus pentosaceus (VTT E- 90390) and/or a mixture of these, preferably Lactobacillus plantarum (VTT E- 78076) and/or a mixture of these. 14. Green fodder preserved by a method according to any one of claims 7 - 11.

Inventors: HAIKARA AULI, HUHTANEN PEKKA, JAAKKOLA SEIJA, JALAVA TAINA, SKYTTAE EIJA, VON WRIGHT ATTE, HAIKARA, AULI, HUHTANEN, PEKKA, JAAKKOLA, SEIJA, JALAVA, TAINA, SKYTTAE, EIJA, VON WRIGHT, ATTE
Applicants: HAIKARA AULI, HUHTANEN PEKKA, JAAKKOLA SEIJA, JALAVA TAINA, MAATALOUDEN TUTKIMUSKESKUS, SKYTTAE EIJA, VALTION TEKNILLINEN, VON WRIGHT ATTE, HAIKARA, AULI, HUHTANEN, PEKKA, JAAKKOLA, SEIJA, JALAVA, TAINA, SKYTTAE, EIJA, VALTION TEKNILLINEN TUTKIMUSKESKUS, VON WRIGHT, ATTE
Application year: 2000
Main classification: A23K3
IPC codes: A23K3