TECHNICAL FIELD

The object of the invention is a new strain of Lactobacillus buchneri A, deposited in the Collection of Industrial Microorganisms in the Institute of Agricultural and Food Biotechnology in Warsaw, under the number KP 2047 p, a composition and a multi- component preparation for starch-rich plant preservation, their use and a method for plant preservation.

BACKGROUND ART

Currently, there is a high demand for enrichment of starter cultures for preparations for forage ensiling with new, not genetically modified strains of lactic acid bacteria isolated from the natural environment. The involvement of these strains in forage ensiling processes contributes to the improvement in quality, aerobic stability and nutritive value of silage, as well as being safe for animals and natural environment.

Patent PL209677 discloses the strain Lactobacillus buchneri KPP 907 p, which is characterized by the ability to synthesize 1 ,2-propanediol, without showing, however, any ability to metabolize it and utilize it in bacterial growth.

In modern methods of roughage ensiling, additives of bacterial or bacterial-enzymatic preparations are used in order to improve silage quality. Preparations of this type are also disclosed in numerous patents, e.g. EP0369198 and EP0563133, as well as PL180272, PL 180 329, PL 190232, PL 208392, PL 209677, PL 210287, PL 212635, WO 2008073848 Al, WO 9729644 Al .

US patent no. 6403084 discloses inoculants for forage ensiling, which comprise mixed cultures of homofermentative and heterofermentative lactic acid bacteria of the Lactobacillus genus and the Enterococcus faecium species. Some of the strains are genetically modified by chemical or radiation mutagenesis, conjugation, transduction or transformation, and thus should not be used in countries where use of GMO in agriculture is prohibited, such as Poland. Such strains are also not allowed in organic farming. DISCLOSURE OF THE INVENTION

The aim of the invention was to isolate, from ensiled corn grains, a new strain of the Lactobacillus buchneri species that, without genetic modifications, will be characterized by a desired efficacy of 1 ,2-propanediol synthesis and the ability to metabolize it to propionic acid, both compounds improving the durability and aerobic stability of starch-rich fodder, as well as the design of a multi-component preparation for ensiling process stimulation and improvement of starch-rich silage durability. The new strain of Lactobacillus buchneri species, due to its properties, will preferably be used for preparation of silage, as well as starch-rich plant silage for biogas production.

The Lactobacillus buchneri A strain according to the invention was isolated from ensiled corn grains, which were stored for three years in anaerobic conditions, by multiple selection of colonies growing on solid MRS media, containing 1 ,2-propanediol as carbon source.

The first object of the invention is a new bacterial strain, which was defined as Lactobacillus buchneri A and deposited in the Collection of Industrial Microorganisms in the Institute of Agricultural and Food Biotechnology in Warsaw, under the number KKP 2047 p.

The second object of the invention is a composition comprising a bacterial strain of Lactobacillus buchneri A, deposited in the Collection of Industrial Microorganisms in the Institute of Agricultural and Food Biotechnology in Warsaw, under the number KKP 2047 p.

The invention also relates to a multi-component preparation for starch-rich plant preservation, comprising a bacterial starter culture of the Lactobacillus genus, the starter culture consisting of a mixture of strains: Lactobacillus buchneri A KKP 2047 p, Lactobacillus plantarum K KKP 593 p, Lactobacillus plantarum S KKP 2021 p, Lactobacillus fermentum N KKP 2020 p, Lactobacillus reuteri M KKP 2048 p, wherein the preparation preferably also contains enzymes, vitamins, carriers and emulsifiers.

In the multi-component preparation the weight ratio of individual strains of Lactobacillus buchneri A KKP 2047 p, Lactobacillus plantarum K KKP 593 p, Lactobacillus plantarum S KKP 2021 p, Lactobacillus fermentum N KKP 2020 p, Lactobacillus reuteri M KKP 2048 p in the mixture is from 1.8 to 2.5: from 0.8 to 1.2: from 0.6 to 1 : from 0.8 to 1.2: from 1 to 1.2, most preferably 2:1 : 1 : 1 : 1, wherein the preparation contains from 0.5% to 1% w/w of enzymes with main activities: endo-l,4-p-D-glucanase EC 3.2.1.4, beta-glucanase EC 3.2.1.6, xylanase EC 3.2.1.8 and vitamin B[ ₂ in quantity from 0.05% to 0.1% w/w, as well as carriers in quantity from 88% to 90% w/w. Preferably, carriers in the preparation are soluble starch, sucrose, glucose and lactose, and preferably lecithin is used as an emulsifier.

The invention also relates to the use of the new bacterial strain of Lactobacillus buchneri A KKP 2047 p, the composition containing Lactobacillus buchneri A KP 2047 p, or the multi-component preparation according to the invention for the stimulation of ensiling process, preservation and/or improvement of starch-rich plant durability.

Preferably, the plant is corn.

The invention also relates to a method of silage production or starch-rich plant preservation, which comprises a step of adding the new bacterial strain of Lactobacillus buchneri A KKP 2047 p, the composition containing Lactobacillus buchneri A KKP 2047 p, or the multi-component preparation according to the invention, to a starch-rich plant material. Preferably, the addition step is performed by spraying after dissolving the preparation in water. A particularly preferable starch-rich plant material are corn grains.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be easier to understand with regard to the description below, constituting however only an example of an embodiment, with reference to figures, where:

Figure 1 shows a comparison of 1 ,2-propanediol synthesis dynamics in MRS medium during 28 days of culture of L. buchneri A KPP 2047 p and L. buchneri KKP 907 p strains.

Figure 2 shows a comparison of 1 ,2-propanediol content in MRS medium on the 28th day of culture of L. buchneri A KPP 2047 p and L. buchneri KKP 907 p strains.

Figure 3 shows a dynamics of changes in the 1 ,2-propandiol and propionic acid content in culture of the L. buchneri A KPP 2047 p strain during 28 days of culture in MRS medium.

Figure 4 shows a dynamics of 1 ,2-propanediol use and propionic acid synthesis by the L. buchneri A KPP 2047 p strain during 28 days of culture in medium with glucose and 1,2- propanediol (in molar ratio 1 :2).

DESCRIPTION OF EMBODIMENTS

The new strain of Lactobacillus buchneri A according to the invention, was isolated from ensiled corn grains, which were stored for three years in anaerobic conditions, by multiple selection of colonies growing on solid MRS media, containing 1 ,2-propanediol as carbon source. The new isolated strain was identified as Lactobacillus buchneri A and deposited in the Collection of Industrial Microorganisms in the Institute of Agricultural and Food Biotechnology, under the number KKP 2047 p.

The strain of Lactobacillus buchneri A KKP 2047 p is characterized by the following morphological features:

- bacterial colonies growing in anaerobic conditions on agar- solidified MRS medium, specific for the Lactobacillus genus (De Man J.D., Rogosa M., Sharpe M.E.: A medium for the cultivation of Lactobacilli, J. Appl. Bact., 23, 130-135, 1960) have an oval shape, are white, with a size of about 0.5 mm.

- bacterial cells do not produce spores, are gram-positive, have a form of short rods, occur singly or together in chains, which is characteristic for the Lactobacillus buchneri species. Optimal temperature of the strain's growth is 35°C. On the basis of the obtained results of lactic acid fermentation of 49 carbohydrates contained in the API test and computer data analysis, it was established that the new bacterial strain isolated from ensiled corn grains ferments the following carbohydrates: glucose, fructose, galactose, D-xylose, L-arabinose, ribose, maltose, melibiose, sucrose, melicitose and raffinose.

Genetic identification was performed by sequencing of a 16S rRNA gene fragment and comparison of obtained results with the ones published in genetic databases for genetic identification of this species.

The new bacterial strain was identified as Lactobacillus buchneri with 99% compatibility with the test strain species.

The results of genetic analysis of a 16 S rRNA fragment from the strain Lactobacillus buchneri A KKP 2047 p defined in this invention, are shown in SEQ ID NO:l . The strain according to the invention, in comparison to the known strain Lactobacillus buchneri KPP 907 p, is characterized by more than three times higher efficiency of 1 ,2-propanediol synthesis, as shown on figures 1 and 2.

Unexpectedly, the new strain Lactobacillus buchneri A KKP 2047 p also has the ability to use 1,2-propanediol for bacterial growth, and therefore to metabolize it, which is not an obvious feature for the Lactobacillus buchneri species.

During culture of the bacteria of the Lactobacillus buchneri A KKP 2047 p strain according to the invention, in MRS medium with glucose as a carbon source, the content of lactic acid and acetic acid was increasing until the 3 ^rd day, and then fell, while the content of 1 ,2-propanediol and propionic acid was increasing until the 21 ^st day of culture, whereupon the content of 1 ,2-propanediol showed a downward trend, while the content of propionic acid continued to increase, reaching the value of 26 mg/100 ml of medium on the 28 ^l day of culture (Fig. 3).

In bacterial culture for 28 days, temperature of 35°C, in medium containing glucose and 1 ,2-propanediol (in molar ratio 1 :2), 57% of 1 ,2-propanediol content was used for the production of biomass and organic acids, including propionic acid, wherein the effectiveness of propionic acid synthesis was higher than during culture of Lactobacillus buchneri A KKP 2047 p in medium with glucose, without the addition of 1,2-propanediol (Fig. 4).

Another object of the invention is a multi-component preparation for starch-rich plant preservation, comprising a bacterial starter culture of the Lactobacillus genus, the starter culture consisting of a mixture of strains: Lactobacillus buchneri A KKP 2047 p, Lactobacillus plantarum K KKP 593 p, Lactobacillus plantarum S KKP 2021 p, Lactobacillus fermentum N KKP 2020 p, Lactobacillus reuteri M KKP 2048 p, wherein the preparation preferably also contains enzymes, vitamins, carriers and emulsifiers.

The preferable preparation according to the invention contains a bacterial starter culture of the Lactobacillus genus, enzymes, vitamins, as well as carriers and an emulsifier, and is characterized by the fact that the starter culture comprises the strains: Lactobacillus buchneri A KKP 2047 p, Lactobacillus plantarum K KKP 593 p, Lactobacillus plantarum S KKP 2021 p, Lactobacillus fermentum N KKP 2020 p, Lactobacillus reuteri M KKP 2048 p, wherein the weight ratio of individual strains in the mixture is from 1.8 to 2.5: from 0.8 to 1.2 : from 0.6 to 1.0 : from 0.8 to 1.2 : from 1.0 to 1.2, most preferably 2: 1 :1 : 1 : 1.

In addition, the multi-component preparation prepared according to the invention contains from 0.5% to 1.0% w/w of enzymes with main activities: endo-l,4-P-D-glucanase EC 3.2.1.4 (cellulase), beta-glucanase EC 3.2.1.6 (endo-l,3(4)-P-glucanase), xylanase EC 3.2.1.8 (endo-l,4-p-xylanase) and vitamin B _i2 in quantity from 0.05% to 0.1 % w/w, as well as carriers and an emulsifier in quantity from 88% to 90% w/w.

Preferable carriers in the preparation are soluble starch, sucrose, glucose and lactose, and preferably lecithin is used as an emulsifier.

All strains used in the multi-component preparation were isolated from plant environment, have GRAS status and are deposited in the Collection of Industrial Microorganisms in the Institute of Agricultural and Food Biotechnology.

Unexpectedly, it has been found that a synergistic activity of bacterial strains contained in the multi-component preparation with enzymes, which is particularly advantageous in the presence of a coenzyme for the propionic acid synthesis reaction - vitamin Bi ₂ , results in multidirectional activity of the preparation, that: inhibits mold and pathogenic bacteria growth, reduces the level of contamination by aflatoxin Bl and ochratoxin A and increases the durability of starch-rich feed silage, e.g. corn grains and whole corn plants, without loss of quality or energy value of silage, for at least three years, and extends their aerobic stability after opening the silos to 16 days.

The invention is illustrated by non-limiting examples of embodiments.

EXAMPLES

Example 1

Characterization of the Lactobacillus buchneri A KKP 2047 p strain.

Table 1 shows the results of lactic acid [L+D], acetic acid, 1 ,2-propanediol and propionic acid synthesis by the new strain Lactobacillus buchneri A KKP 2047 p (MRS medium (by Man, Rogosa and Sharpe, 1960), carbon source: glucose), culture temperature 35°C, duration of culture 28 days, individual samples for study were collected on 1, 2, 3, 10, 21 and 28 day of culture.

Table 1. The dynamics of synthesis of organic acids, 1 ,2-propanediol and growth for Lactobacillus buchneri A KKP 2047 p in MRS medium (by Man, Rogosa and Sharpe, 1960). Duration of culture 28 days, temperature 35°C, the results shown are average of three experiments.

During culture of the bacteria of the Lactobacillus buchneri A KKP 2047 p strain, in

MRS medium with glucose as a carbon source, the content of lactic acid and acetic acid was increasing until the 3 ^r day, and then fell, while the content of 1,2-propanediol and propionic acid was increasing until the 21 ^st day of culture, whereupon the content of 1,2-propanediol showed a downward trend, while the content of propionic acid continued to increase, reaching the value of 26 mg/100 ml of medium on the 28 ^th day of culture (Table 1 and Fig. 3).

Table 2 shows the results of lactic acid [L+D], acetic acid and propionic acid content, as well as the percentage utilization of 1,2-propanediol added to the medium, by the bacteria of Lactobacillus buchneri A KKP 2047 p strain, cultured in MRS medium, where portion of glucose was replaced with 1 ,2-propanediol (the molar ratio of glucose to 1,2-propanediol was 1 :2). The bacteria were cultured in temperature of 35°C, up to 28 days.

Table 2. The influence of 1,2-propanediol on bacterial biomass growth and metabolite synthesis by Lactobacillus buchneri A KKP 2047 p. Duration of culture 28 days, temperature of 35°C, the results shown are average of three experiments.

In bacterial culture conditions for 28 days, in medium containing glucose and 1,2- propanediol (in molar ratio 1 :2), 57% of 1,2-propanediol content was used for the production of biomass and organic acids, including propionic acid, wherein the effectiveness of propionic acid synthesis was higher than during culture of Lactobacillus buchneri A KKP 2047 p in medium with glucose, without the addition of 1 ,2-propanediol (Table 2 and Fig. 4). Example 2

Conditions of biomass propagation of Lactobacillus buchneri A KKP 2047 p strain and preparation of dry formulation.

After multiplication of the inoculum (test tube stage and flask stage in three steps) bacterial culture was carried out in a medium of the following composition (in g/1 of water): sucrose - 20, yeast extract - 10, corn steep liquor - 5, diammonium phosphate - 0.2, magnesium sulphate - 0.08, ammonium sulphate - 0.35, manganese sulphate - 0.01 ; in fermenters with a capacity of 150 1 at 35°C, pH controlled with aqueous ammonia to the value 5.8.

Centrifuge with a spin ratio above 13000 x g was used for biomass separation.

After culture in conditions given above, 1000 g of bacterial biomass was obtained with a 30% DM (dry mass) content.

Bacterial biomass obtained after centrifugation, after mixing with carriers such as potato soluble starch, lactose, glucose and sucrose, was treated with fluidization drying in temperature not exceeding 40°C. Dry granular bacterial preparation with dry mass content of around 94% contained 2 x 10 ⁹ bacterial colony forming units in 1 g.

Example 3

Production of multi-component bacterial preparation

The multi-component bacterial preparation according to the invention was obtained in such a way that for every individual strain the bacterial preparation was produced separately in granular form, by methods described in Example 2.

The obtained preparation for each bacterial strain was characterized by the dry mass content of 93-94%.

Afterwards, the dried preparations of the strains: Lactobacillus buchneri A 2047 p, Lactobacillus plantarum K KKP 593 p, Lactobacillus plantarum S KKP 2021, Lactobacillus fermentum N KKP 2020 p, Lactobacillus reuteri M KKP 2048 p were mixed in weight ratio 2: 1 :1 : 1 :1 and supplemented with 1% w/w of enzymatic preparation with main activities: endo-l,4-P-D-glucanase EC 3.2.1.4, beta-glucanase EC 3.2.1.6, xylanase EC 3.2.1.8 and vitamin B ₁₂ in quantity 0.1% w/w. The number of bacterial colony forming units (cfu) was around 2 x 10 ⁹ in 1 g of dry preparation. Example 4

The multi-component bacterial preparation obtained in example 3 was used for corn grain silage production. The preparation was used in spray form, after dissolving in drinking water, in a dose of 5 g per 1 ton of corn grain silage.

Silage produced with and without the addition of the preparation was stored in silos for 3 years, then after silo opening its content of: lactic, acetic and butyric acids and 1 ,2- propanediol, as well as its metabolites: 1-propanol and propionic acid was determined. The silage quality was evaluated according to Flieg-Zimmer scale (Zimmer E., Wirtschaftseig. Futter, 12, p. 299, 1966) and digestibility of dry organic matter, content of starch and protein, and aerobic stability of silage were compared. The obtained results are shown in Tables 3 and 4.

Table 3. Organic acids, 1 ,2-propanediol and 1-propanol content in corn grain silage, with or without the addition of the preparation, after 3 -year storage period. The results shown are average of three experiments in production scale.

Under the influence of the multi-component preparation the corn grain silage was characterized by very high quality, high content of: lactic acid [1.73%] and propionic acid [54.8 mg/100 g]. However, the silage without the addition of the preparation was characterized by poor quality and after three years of storage was not suitable for animal feed.

Table 4. Influence of the multi-component preparation on starch and total protein content, digestibility of dry organic mass and aerobic stability of corn grain silage. The results shown are average of three experiments in production scale. Dry organic Total

Humidity Starch

mass Aerobic ent protein

Silage: cont ility in

[%] digestibility stab

content

[%]

[%] days

[%]

without the

40.0 50.4 81.4 5.27 4

preparation with the

36.8 54.5 85.4 6.66 16

preparation

Under the influence of the multi-component preparation digestibility of dry organic mass (DM) of silage increased by 4%, while starch and protein loss was limited by 7.5 and 21.0% respectively, in relation to silage without the preparation. High lactic acid and acetic acid content in silage with the preparation has ensured high quality of corn grain silage, even after 3 years of storage. The addition of the preparation has improved silage aerobic stability. Silage with the addition of the preparation in quantity of 5.0 g/t showed aerobic stability for 16 days after opening the silo, which is a sufficient period for its use as animal feed.

In parallel microbiological purity of silage produced with or without the preparation was assessed and contamination with the Salmonella sp., Escherichia coli bacteria and molds was determined. The results on the influence of the preparation on the improvement in the purity of silage is shown in Table 5.

Table 5. Influence of the preparation on the improvement in corn grain silage purity. The results shown are average of three experiments in production scale.

In corn grain silage with the addition of the multi-component preparation, pathogenic bacteria of the Salmonella sp. genus and the Escherichia coli species were eliminated in 100%. The preparation also caused a reduction in the number of molds by around 3 log cfu/g DM of silage and a reduction in aflatoxin Bi and ochratoxin A by around 75 % in comparison to silage without the preparation.

Animal feed contaminated with molds, aflatoxin B _ls ochratoxin A and pathogenic microorganisms, after the lactic acid fermentation process involving the multi-component preparation may safely be used in feeding of livestock.