EP3688139A1

EP3688139A1 - Microorganism stainslactobacillus buchneri

Info

Publication number: EP3688139A1
Application number: EP18793358.5A
Authority: EP
Inventors: Epp Songisepp; Oksana GERULIS; Liina SADAM; Sirje KUUSIK; Merle MURUVEE; Anette NAPPA
Original assignee: BioCC OU
Current assignee: BioCC OU
Priority date: 2017-09-28
Filing date: 2018-09-28
Publication date: 2020-08-05
Also published as: JP7250783B2; JP2020534856A; CN111601879A; WO2019063056A1

Abstract

The invention provides the isolated microorganism strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 and their use as microbiological feed additives. The strains are used for ensuring aerobic stability of feed with low dry matter content (≤ 20 percentage) and improving fermentation of feed, for increasing the concentration of lactic and acetic acids in feed and for reducing pH, hence decreasing the loss of nutrients in feed. Usage of the microorganisms in ensiling suppresses the function of pathogenic microorganisms (enteropathogens) and yeasts in feed. The strains can be used for prolonging the storage life of feed made from fresh material difficult to ferment.

Description

MICROORGASNISM STRAINS LACTOBACILLUS BUCHNERI BIOCC 203 DSM 32650 AND LACTOBACILLUS BUCHNERI BIOCC 228 DSM 32651 AND THEIR. USE

TECHNICAL FIELD

The invention belongs to the field of biotechnology and is applied to feed manufacturing. The invention encompasses a microbiological silage additive and its use in feed fermentation to ensure aerobic stability, the quality of fermentation of the feed.

BACKGROUND ART

It is necessary to preserve the nutrient content of silage from the harvest and storage of the feed up to the consumption of the feed by the animal.

Silage is the material produced by the controlled fermentation of a crop of high moisture content (McDonald, P., Henderson, A. R., Heron, S.J.E. 1991 . The biochemistry of silage. 2nd ed, Chalcombe Publications, Marlow, Bucks UK, p. 340). Ensiling is a storage method of plant-based animal feed that rests on lactic acid fermentation under anaerobic conditions (Rooke, J., A. and Hatfield, G., D., 2003. Biochemistry of Ensiling. In: Silage Science and Technology, D. R. Buxton, R. E. Muck, and J. IT Harrison, eds. American Society of Agronomy, Madison, Wisconsin, USA. pp. 95-139). The fermentation of silage can be divided into four stages: (1) the anaerobic stage in the silo after the harvest, (2) the fermentation stage, (3) the stable storage stage, and (4) the silage unloading stage, where the. silo is opened, and the silage is exposed to air. To produce high-quality silage, the material to be ensiled must undergo correct microbial fermentation. Successful fermentation also depends on the type and quality of the grass plants, the techniques used in the ensiling process, climate, the development of undesirable microorganisms (e.g. Clostridium, enteropathogens. Listeria, bacilli) and fungi (yeasts and moulds), as well as the dry matter content of the material to be ensiled.

It is difficult to control the natural fermentation of feed, as the fermentation of silage is a complex combination of several different, chemical and microbiological processes and their interactions.

Most of the silage is produced with a dry matter content of 200. ,,500 g/kg. At such levels, many plant enzymes are active during the ensilage process, and numerous desirable and undesirable microorganisms, yeasts and moukis can grow in the silage under such conditions. Thus, getting the whole biological activity under control poses a remarkable challenge and can only be achieved by means of a well-managed ensilage process (Muck. R. E. 2010. Silage microbiology and its control through additives. R. Bras, Zootec. Vol, 39. July).

In a controlled ensilage process, water-soluble carbohydrates are fermented into lactic acid by lactic acid bacteria. As a result, the pH level of the material to be ensiled drops (the ensilage mass is acidified), which in turn inhibits the activity of the spoilage microorganisms (Oude Elferink, S, J. W. II. , Driehuis, P.. Gottsehal, J. C, Spoelstra, S. F. 2000. Silage fermentation processes and their manipulation. --- Journal FAG Plant Production and Protection No 161 , pp 17-30). The faster the acidity of the silage drops to pH 4, the quicker the enzymatic and microbial activity stops, the feed becomes stable and more nutrients are preserved.

It has been reported that the fermentation quality of silage can be significantly improved by means of additives containing lactic acid bacteria (McDonald, P., Henderson. A, R., Heron, S.J.E. 1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Mar!ow, Bucks UK. p. 340).

Just as important as the preservation of nutrients in the fermentation and storage stage of silage is the preservation of the nutrients in silage upon the opening of the silo. Silage may become exposed to oxygen both when the silo is opened for feeding and because of inadequate coverage of the silo.

Any silage that is exposed to air spoils sooner or later due to the activity of aerobic microorganisms. The aerobic stability of silage also depends on the silage crop to be ensiled, its growth phase at the time of harvest, the biochemical and microbiological factors of fermentation, the physical characteristics of the silage material, the organisation of silage management, temperature, and the choice of silage additive. The aerobic stability indicator of silage is the length of time that silage can resist aerobic spoilage processes, i.e. the length of time that it retains its quality upon exposure to air. The aerobic stability of silage is estimated based on the rate at which the temperature of the silage rises. The longer the temperature of the silage remains stable, i.e. the longer it does not rise above the ambient temperature by more than 3 °C (Commission Regulation (EC) No, 429/2008; DLG-Richtlinienfur die Pruiimg von Siiiemiitteln auf DLG-Gutezeichen-Fahigkeit. DLG Oktober 2013), the greater the aerobic stability and the quality of the silage. In most aerobically perishable silages, the temperature rises above the ambient temperature upon the microbial oxidation of acids and water-soluble carbohydrates into carbon dioxide and water.

Although the low pH level of silage inhibits the growth of undesirable microorganisms under anaerobic conditions, the low pH by itself does not suffice to prevent aerobic spoilage. The spoilage of silage in aerobic conditions mostly starts with yeasts, which can grow even at relatively low pH levels. Yeasts can grow in a broad pH range (pH 3, ,.8). The optimum pH for the growth of most yeasts is 3.5„.6.5. When silage becomes exposed to air on the opening of the silo, the acids and other compounds that have formed during fermentation are oxidised by aerobic bacteria, yeasts and moulds. The activity of yeasts results in the production of carbon dioxide, which heats up the silage - this in turn is a direct cause of dry matter loss ( McDonald, P., Henderson, A . R., Heron, S.J.E, 1991. The biochemistry of silage. 2^nd ed, Chalcombe Publications, Marlow, Bucks UK, p. 340).

Yeasts employ the residual sugar contained in silage as a source of energy; however, their first preference is lactic acid. As a result, well-fermented silages with high lactic acid content are particularly susceptible to aerobic spoilage. The activity of the yeasts causes the pH level of the silage to rise, enabling numerous other aerobic microorganisms and moulds to become active. High microbial activity in well- fermented, nutritious silage is revealed by an increase in the temperature of the silage, It has been reported (Ohyama, Y., Hara, S. and Masaki, S, (1980) Analysis of the factors affecting aerobic deterioration of grass silages. In Thomas, C. (ed.) Forage conservation in the 80s. BGS Occasional Symposium No. 11, pp. 257-261. Reading, UK: British Grassland Society) that the dry matter, acetic and propionic acid content and the number of yeasts and moulds in the silage upon the opening of the silo are important determinants of the aerobic stability of silage. Negative correlation with regard to dry matter content and yeasts indicated that a greater concentration resulted in a Faster rise in the temperature of the silage upon exposure to air. With acetic and butyric acid, on the other hand, a greater concentration of these fermentation products was associated with more stable silage.

As noted, the low pH level of silage has no direct effect on the microorganisms that cause aerobic spoilage; the acids produced during the fermentation of the silage, however, have a variable importance. The growth of yeasts is inhibited by undissociated short-chain fatty acids (Pahlow G., Muck R.E., Driehuis F., Oude Elfermk S.J..W.H. and Spoelstra S.F. (2003) Microbiology of ensiling. In: Buxton D.R., Muck R.E. and Harrison J.H, (eds.) Silage science and technology, pp. 31 -93. Madison, WI, USA: Agronomy Publication No. 42, American Society of Agronomy). Undissociated acid molecules can penetrate the cell membrane of a microbe by means of passive diffusion, which results in the release of H+ ions. This lowers the pH level inside the cell, causing the cell to perish. The dissociation rate of art acid in silage depends on the dissociation constant of the acid (pKa) and the pH level of the silage (Zirchrora (201.1) Dissociation constants oforganic acids and bases. Available at: http://www,zirchrom.com/organic.htm, (accessed 3 November 201 1)). Acetic and propionic acid are less prone to dissociation than lactic acid, which explains the susceptibility of well-fermented silage with high lactic acid content to aerobic spoilage. Acetic and propionic acid, on the other hand, effectively inhibit the growth of yeasts and moulds. Butyric acid has a similar effect. Silage with high butyric acid content has good aerobic stability; however, this indicates the activity of spoilage-causing Clostridium. Such silage exhibits extensive nutrient loss, and the high butyric acid content can cause health issues in animals. Propionic acid content in silage is rare and small; the concentration of the microorganisms producing it in silage crops is low and their competitiveness is poor.

Acetic acid content in silage is an indication of heterofermentation; since acetic acid is highly toxic to yeasts; such silages typically display great aerobic stability.

An ideal fermentation of silage reduces fermentation losses and ensures sufficient stability during the storage of the feed and unloading it from the silo for feeding. An effective silage additive and proper organisation of the production and use of silage play a. key role in the achievement of these objectives. Most silage additives have been developed to improve the ensilage process and the nutrition values of ensiled feed. .However, in addition to ensuring quick fermentation and. improving the quality of silage, silage additives are also expected to inhibit the -growth of spoilage (inch .aerobic spoilage) organisms. The main reason for using a silage additive to improve the aerobic stability of silage is to prevent the heating of the silage, the loss of nutrients and a decrease in the performance of the animals due to consumption of spoiled silage.

Silage additives often employ enzymes; however, these do not inhibit yeasts or moulds, meaning that silages prepared with enzymes have a very modest aerobic stability.

Organic acids, such as propionic, acetic and benzoic acid etc, are effective in improving the aerobic stability of silage. These are added either in large quantities, to achieve the so-called final conservation of the feed, or in smaller quantities, in the latter case, the activity of yeasts is inhibited, but total conservation is not guaranteed, and ensilage continues to depend on natural fermentation. Ammonia has also been reported to have an inhibitory effect on bacteria, yeasts and moulds. Unfortunately, organic acids and other chemicals are aggressive and damaging on the siiage equipment; strict safety requirements apply to their handling and storage.

Biological silage additives based on lactic acid bacteria are regarded as natural products; their advantages include their lack of toxicity, lack of corrosive effect on equipment, and lack of environmental risks.

The goal of lowering the pH level of silage by means of lactic acid bacteria is to minimise fermentation losses. Lactic acid bacteria are divided into two groups based on glucose fermentation: homofermentative and heterofermentative species. Homofermentative lactic acid bacteria produce two moles of lactic acid from one mole of glucose, whereas heterofermentative bacteria produce one mole of lactic acid, one mole of carbon dioxide and one mole of either ethanol or acetic acid, it is wet! known that at the beginning of the fermentation process, homofermentative species dominate, but later on, as the environment becomes more acidic, heterofermentative bacteria become prevalent (Muck, R. E. 2010. Silage microbiology and its control through additives, R. Bras. Zootec. Vol. 39. July).

Silage additives based on homofermentative lactic acid bacteria improve the fermentation process of silage; however, most of such starter bacteria do little to inhibit the growth of yeasts and moulds. With the use of such a silage additive, the aerobic stability of the silage may be lower than without any silage additive, and it might even increase the heating risk of the silage.

Some silage starters contain bacteria (e.g. propionic acid bacteria) that produce propionic acid. This, unfortunately, does not improve the aerobic stability of the silage, because these microorganisms are not typically acid-tolerant, and their growth is slow. However, starters that produce large quantities of acetic acid in addition to lactic acid (L.b uchneri) do inhibit the microorganisms causing the aerobic spoilage of silage (yeasts, moulds etc.). i.e. they improve the aerobic stability of silage and prevent the spoiling of silage upon the opening of the silo or upon other kinds of exposure to air. The addition of heterofermentative lactic acid bacteria during the ensiling process lowers the pH level and reduces dry matter loss. Furthermore, some of these strains are reported to have a strong inhibitory effect on the growth of yeasts and moulds, thereby improving the aerobic stability of the silage (Jatkauskas, J,, Vrotniakiene, V„ Ohlsson, C. Lund, B. 2013. The effect of three silage inoculants on aerobic stability in grass, clover-grass, lucerne and maize silage. Agricultural and Food Science. 22: 137-144). However, different strains of the same species do not have identical properties, as due to genetic variations interspecies differences i.e. strain-specific properties occur.

The aim of this invention is to provide new strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 lor improving feed fermentation quality and prolonging the aerobic stability and storage time of silage.

DISCLOSURE OF THE INVENTION

The invention discloses the isolated microorganism strains Lactobacillus huchneri BioCC 203 DSM 32650 and Lactobacillus huchneri BioCC 228 DSM 32651, and feed, feed additive and composition comprising one or both said strains. The feed may be fermented feed, e.g. silage. The feed additive may be a silage additive. Suitable excipients can be included as other ingredients in the composition of the additive product. Said microorganisms can be used in lyophilized form.

The microorganism Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 ensure aerobic stability of the feed, including the feed difficult to ferment, e.g. feed with low (< 20 percentage) dry matter content.

The next objective of the invention is the use of the named microorganism in: accelerating the fermentation of feed, increasing the concentration of lactic acid, reducing pH, and decreasing the loss of nutrients in feed and the concentration of ammonia nitrogen and butyric acid in feed.

Said microbes (together or separately) ate used to ferment feed and improve fermentation, to increase the concentration of lactic acid and acetic acid in feed, to lower the pH level and thereby reduce nutrient loss in. feed.

Based on investigations into antimicrobial properties, Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 inhibit the growth and action of undesirable microorganisms (pathogenic microorganisms, yeasts and moulds). Said pathogens are Staphylococcus aureus, Staphylococcus saprophytics, Salmonella enterica subsp, enter ica serovar Enteritidis, Enterococcus faecalis, Escherichia coli etc.

The invention also relates to a method for prolonging the preservation of feed, where one or both of microorganisms Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus huchneri BioCC 228 DSM. 32651 are added to the feed during fermentation. In the case of use of one of the aforementioned strains is added to the feed, the rate of it is 1 x 10⁵...1 x 10⁶ CFU/g of the fermented feed.

DESCRIPTION OF THE STRAINS

The microorganism strains Lactobacillus huclmeri BioCC 203 DSM 32650 and Lactobacillus biichneri BioCC 228 DSM 32651 were isolated form ensiled naturally without the use of silage additives high quality maize (Zea mays L.) silage in Estonia. To determine the quantitative content of lactobacilli in the silage sample, a suspension (with descending concentrations) was made of solutions, using the decimal dilution method in peptone water (Sigma-Aldrich, France); and seeded on MRS (de Man Rogosa Sharpe) agar (Biolife. Italy), that was incubated at 37°C in microaerobic (10 percentage CO₂) environment (thermostat„MCO-18AIC UV" Sanyo Electronic Co, Ltd, Japan) for 48 hours. Developed colonies were described, counted and the total count of microbes was determined. To describe the morphology of microbes, preparations were made using Gram's staining method, and microscopic examinations were performed. The strains Lactobacillus huclmeri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 were isolated, based on colony and cell morphology specific to Lactobacillus spp. Provisional and detailed identification followed, this is described below.

The morphological characteristics of the cultures- of Lactobacillus buchmri BioCC 203 DSM 32650 and Lactobacillus buchmri BioCC 228 DSM 32651 were determined after growth in MRS agar and broth (Biolife, Italy).

Lactobacillus buchneri BioCC 203 DSM 32650 is a Gram-positive regular rod-shaped non-motile non-spore forming bacterium, occurring singly and in short chains. Elongated cells occur during cultivation in MRS broth,

Lactobacillus buchneri BioCC 228 DSM 32651 is a Gram-positive regular rod-shaped non-motile non-spore forming bacterium, occurring singly and in short chains. Long and slender cells occur during cultivation in MRS broth,

PHYSIOLOGICAL-BIOCHEMICAL CHARACTERISTICS

MRS broth (for 48-72 hours) is suitable for cultivating the microbial strain Lactobacillus buchneri BioCC 203 DSM 32650 microaerobically or anaerobically, after which a homogenous turbid growth occurs. After 48 hours of cultivation at 37 degrees in a microaerobic (10 percentage CO₂) or -anaerobic (CC₂/Na₂/H₂: 5/90/5 percentage) environments the colonies are grayish white, 1.5-2 millimeters, flat, shiny, translucent, with rough texture and ismbonate.

The microbial strain Lactobacillus buchneri BioCC 203 DSM 32650 is obligately heterofemientative. catalase and oxidase negative, hydrolyzes arginine and produces carbon dioxide during the fermentation of glucose.

The optimal growth temperature of the strain Lactobacillus buchneri BioCC 203 DSM

32650 is 37 degrees; the strain also replicates at 15 degrees. To a small extent, growth can also be observed at 45 degrees. The optimum pH range for growing the strain is 5.7- 6.2.

MRS broth (for 48-72 hours) is suitable for cultivating the microbial strain of Lactobacillus buchneri BioCC 228 DSM 32651 microaerobically; after which a homogenous turbid growth occurs. After 48 hours of cultivation at 37 degrees in a microaerobic ( 10 percentage CO₂) or anaerobic (CO₂/N₂/H₂: 5/90/5 percentage) environments the colonies are grayish white, 1.5-2 millimeters, flat, shiny, translucent, with rough texture and umbonate.

The microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 is obligate!}' heterofermentative, catalase and oxidase negative, hydrolyzes. arginine and produces carbon dioxide during the fermentation of glucose.

The optimal growth temperature of the strain Lactobacillus buchneri BioCC 228 DSM

32651 is 37 degrees; the strain also replicates at 15 degrees. To a small extent, growth can also be observed at 45 degrees. The optimum pH range for growing the strain is 5.7- 6.2.

The microbial strain Lactobacillus buchneri BioCC 203 DSM 32650 was identified as Lactobacillus buchner using MALDI Biotyper (Bruker Daltonik).

The microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 was identified as Lactobacillus buchneri using MALDI Biotyper (Bruker Daltonik).

Lactobacillus buchneri strain BioCC 203 was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorgajtiisms for the Purposes of Patent Procedure in Deutsche Sammlung von Mikroorganismen und Zellkuifuren GmbH under number DSM 32650 on 25 September 2017.

The address of DSMZ: Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

Lactobacillus buchneri- strain BioCC 228 was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH under number DSM 32651 on 25 September 2017.

The address of DSMZ; Inhoffenstr. 7B, D-38124 Braunschweig, Germany.

Resistance to anitibotics

Methods: Antibiotic susceptibility of the Lactobacillus buchneri strains BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 were analysed according to ISO10932:2010 standard with VetMIC Lact-I and VetMIC Lact-2 plates (SVA National Veterinary institute, Uppsala, Sweden) in anaerobic (CO₂/N₂/H₂: 5/90/5 percentage) conditions at + 37 degrees for 48 hours. The minimum inhibitory co.neentrati.ons (MIC) were compared with MIC cut-off values reported by EFSA, Table 1. Minimum Inhibitory Concentration (MiC)-values (mg/L)of antibiotics for Lactobacillus buhneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651.

human and veterinary importance. EFSA Journal 2012. 10(6), 2740,

For the assessment of bacteria used as feed additives, strains can be categorised as susceptible or resistant, to antimicrobials;

Susceptible (S): a bacterial strain is defined as susceptible when, it is inhibited at a concentration of a specific antimicrobial equal or lower than the established cut-off value (S≤ x mg/L). Resistant (R): a bacterial strain is defined as resistant when it is not. inhibited at a concentration of a specific antimicrobial higher than the established cut-off value (R > x mg/L). Results of antibiotic susceptibility of strains LactohacUlus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 are presented in Table 1. The minimum inhibitor)' concentrations for Lactobacillus buchneri strains BioCC^' 203 DSM 32650 and BioCC 228 DSM 32651 did not exceed the M IC cut-off values proposed for obligate hetero fermentative Lactobacillus proposed by EFSA.

FUNCTIONAL PROPERTIES OF S TRAINS

Growth in the presence of various sugars

The -purpose of the. experiment was to investigate the ability of the strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 to grow in the presence of various sugars and acidify the culture meedium.

Methods: The 24h old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 cultivated on MRS agar were suspended in peptone water according to the McFariand Turbidity Standard No 5 (1.5.x 10⁹ microbes/ml), seeded at final density of 1.5 x 10⁶ microbes/mi into modified MRS broth containing 20 g / L either of glucose, fructose, trehalose, xylose or maltose or mix of glucose, fructose, trehalose (in ratio 1 : 1 :1 ), with a final concentration also of 20 g / L. Suspenisons were incubated in a thermostat microaerobically (10 percent CO₂) and anaerobic-ally (CO₂ / N₂ / H₂: 5/90/5 percent) at 25 degrees for 24, 48 and 72 hours, in the case of anaerobic environment, the medium was beforehand reduced guring 24 hours. The viable counts of both strains were registered, the yield, generation number in) and growth rate (V) were calculated as follows:

Yield - log Ni-Iog No where N_l is the cell concentration at any given time; No is the initial cell concentration

n = log N_l-log No / log 2, where N_l is the cell concentration at any given time; No is the initial cell concentration

V - log Ni-Iog No / 0.301 x t. w N_t is the cell concentration at any given time; No is the initial cell concentration and t is the specified period of time in hous. The grovsth of the strain of Lactobacillus buchneri BioCC 203 DSM 32650 was one generation faster than that of the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 2) during the first 24 hours of microaerobic cultivation In a culture medium containing glucose, fructose, xylose or a mixture, of glucose, fructose and trehalose. Table 2. The effect of different sugars on the growth dynamics of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during microaerobic (10 percent CO₂) cultivation at 25 degrees for 24, 48 and 72 hours.

N - number of n generations; V - growth rate; G - MRS broth with glucose; F - MRS broth with fructose; T - MRS broth with trehalose; M - MRS broth with a mixture of glucose - fructose - trehalose; X - MRS broth with xylose; Ma - MRS broth with maltose

During the first 24 hours of anaerobic cultivation, the growth of Lactobacillus buchneri BioCC 203 DSM 32650 was on average two generations faster in medium containing fructose or a mixture of glucose, fructose and trehalose; within 48 hours for three generations faster in glucose containing medium and about 1.5 generations faster in fructose and xylose containing medium compared to the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 3). Lactobacillus buchneri BioCC 228 DSM 32651 was slower growing, being able to outrun Lactobacillus buchneri BioCC 203 DSM 32650 after 48 hours of culturing in medium containing fructose or xylose and in a medium containing a mixture of glucose, fructose and trehalose.

Table 3. The effect of different sugars on the growth dynamics of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during anaerobic (CO₂/N₂/H₂: 5/90/5 percent) cultivation at 25 degrees for 24, 48 and

72 hours.

N - number of n generations; V - growth rate; G - MRS broth with glucose; F - MRS broth with fructose; T - MRS broth with trehalose; M - MRS broth with a mixture of glucose - fructose - trehalose: X - MRS broth with xylose; Ma - MRS broth with maltose

Example 1. Organic acid and alcohol profile

The purpose of the experiment was to determine the profile of organic acids and alcohols of strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 durina microaerobic and anaerobic cultivation. Methods: The 24h old cultures of Lactobacillus buchmri BioCC 203 and Lactobacillus buchmri BioCC 228 cultivated on MRS agar (Biolife. Italy) were suspended in peptone water according to the McFarland Turbidity Standard No 5 (1.5x 10⁹ microbes/ml), seeded into MRS broth (Biolife. Italy) at final density of 1.5 x 10* microbes/ml and incubated microaerobieaily ( 10 percentage CO₂) and anaerobic-ally (CO₂/N₂/H₂: 5/90/5 percentage) in thermostat at 25 degrees for 24. 48 and 72 hours.

Gas chromaiographycaily was organic acid and alcohol profile determined by a gas chromatograph Agilent 6890A capillary column CP-Wax 52 CB (30 m x 0.25 mm 0.25 μm). Column temperature programm 75 degrees 1 min hold. 10 degrees /min to 1 15 degrees 3 min hold. 20 degrees /min to 190 degrees 5 min hold. Detector (FID) 280 degrees.

Liquid chromatographycally organic acids were determined on a Shimadzu Prominence HPLC System. The samples were separated on Aminex HPX-87H ion-exclusion column (300 mm x 7.8 mm). The temperature of column was thermostated at 60degrees. flow rate was 0.6 ml/min and. organic acids were detected with PDA detector at 210 nm, Time of the analysis was 26 min.

In the. profile of organic acids and alcohols, the strain-specific characteristics of the strains Lactobacillus buchmri BioCC 203 DSM 32650 and BioCC 228 DSM 32651 were apparent (Table 4). Lactobacillus buchmri BioCC 203 DSM 32650 was a significantly stronger producer of ethanot, acetic acid and lactic acid during cultivation m both niicroerobic and anaerobic environment. Lactobacillus buchmri BioCC 228 DSM 32651 was capable of producing pyruvate in anaerobic environment (Table 4).

During the first 24 hours of microaerobic and anaerobic cultivation, the strain Lactobacillus buchmri BioCC- 203 DSM 32650 utilised approximately 99.5 percent and 97.8 percent respectively of the citrate initially present in the culture medium.

During anaerobic cultivation, Lactobacillus buchmri BioCC 228 DSM 32651 consumed 4,8 percent of the citrate initially present, in the culture medium. Unlike Lactobacillus huchnen BioCC 203 DSM 32650, the strain Lactobacillus buchneri BioCC 228 DSM 32651 was able to produce 5.9 percent citrate during 72 hours of microaerobic cultivation. Table 4. Organic acid and alcohol profile (mg7m) in MRS broth of the mieroaerobic (10 percentage CO₂) and anaerobic (CO₂/N₂/H₂: 5/90/5 percentage) cultivation of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228

DSM 32651 for 24 h, 48 h and 72 h at 25 degrees

Example 2. Organic acid and alcohol profile in maize plant supernatant

The purpose of the experiment was to determine the profile of organic acids and alcohols of strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during plant material fermentation.

Methods: 226 g of maize {Zee mays L.) plants in vegetative growth stage (V6-V8) were chopped, homogenized in laboratory blender Bagmixer 400 (interscience. France) for 6 minutes with water.' filtered, centrifuged (5000 rpm 10 minutes at ambient temperature) and sterilized at 121 degrees for 5 minutes

The 24b old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 cultivated on MRS agar (Biolife. Italy) were suspended in peptone water according to the McFarland Turbidity Standard No 5 (i.5x 10⁹ microbes/ml), seeded into maize plant supernatant at. final density of 1 .5 x 10° microbes/ml and incubated microaerobieally ( 10 percentage CO₂) and anaerobically (CO₂/N₂/H₂: 5/90/5 percentage) in thermostat at 25 degrees for 24 h 48 h and 72 h. Gas chromatographycally was organic acid and alcohol profile determined by a gas chromatograph Agilent 6890A capillary column CP-Wax 52 CB (30 m x 0.25 mm. 0.25 μm ). Column temperature programm 75 degrees 1 mm hold. 10 degrees /rain to 1 15 degrees 3 min hold. 20 degrees /min to 190 degrees 5 min hold. Detector (FID) 280 degrees.

Liquid chromatographvcally organic acids were determined on a Shimadzu Prominence HPLC System. The samples were separated on Aminex HPX-87H ion-exclusion column (300mm x 7.8mm). The temperature of column was thermostated at 60 degrees, flow rate was 0.6 ml/min and organic acids were detected with PDA detector at 210 nm. Time of the analysis was 26 min.

Table 5. Organic acid and alcohol profile (mg/m) in maize plant supernatant of the microaerobie (10 percentage CO₂) cultivation of Lactobacillus huchneri BioCC 203 DSM 32650 and Lactobacillus huchneri BioCC 228 DSM 32651 for 24 h. 48 h and 72 h at. 25 degrees

in the fermentation test of the plant material, Lactobacillus huchneri BioCC 203 DSM 32650 proved to be stronger producer of ethanol and lactic acid compared to Lactobacillus huchneri. BioCC 228 DSM 32651 (Table 5).

Example 3, Antimicrobial activity towards pathogens

The purpose of the experiment was to test the antimicrobial activity of Lactobacillus huchneri BioCC 203 DSM 32650 and Lactobacillus huchneri BioCC 228 DSM 32651 to entero bathogens during microaerobie and anaerobic cultivation at 25 degrees.

To evaluate the antimicrobial properties of Lactobacillus huchneri BioCC 203 DSM 32650 and Lactobacillus huchneri BioCC 228 DSM 32651 towards pathogens, streak- line procedure was used (Hutt P. Shchepeiova J. Loivukene K. Kullisaar T. Mikelsaar M. Antagonistic activity of probiotic laetohaciili and bifidobacteria against entero- and uropathogens. J Appl. Microbiol. 2006; 100(6); 1324-32)). To determine the growth inhibition on the target microbes, the growth-free zone, was measured in millimetres. Arithmetic mean and standard error were calculated on the basis of the results of the sample (Table 6) in an analogous manner to Hurt et al. (2006), and antagonistic activity (ram) was assessed based on the same.

Table 6. The antimicrobial activity of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 against pathogens using the streak- line method on modified MRS agar medium in microaerobic (10 percentage CO₂) and anaerobic (CO₂/N₂/.H₂: 5/90/5 percentage) environments (target microbe growth inhibition zone in mm)

17.1 1; strong >17.12. Inhibition zone in anaerobic environment (mm-s): weak < 10.67; average 1.0.66-14.94; strong >14,95. in a microaerobic environment, both strains expressed equally strong antimicrobial activity (Table 6). In an anaerobic environment. Lactobacillus buchneri BioCC 203 DSM 32650 has a slightly higher inhibitory effect on tested pathogenic microbes.

Example 4, Antifungal activity

The aim of the experiment was to evaluate the effect of Lactobacillus buchner BioCC 203. DSM 32650 and Lactobacillus buchner BioCC 228 DSM 32651 supernatant against yeasts of maize silage origin using agar well-di ffusion method. A 48-hour lactobacillus culture suspension was prepared in peptone water according to McParland Turbidity Standard No. 5 ( 1.5 x 10⁹ microbes / ml), inoculated into MRS (Biolife. Italy) broth with a final volume of 1 ,5 x 10⁶ microbes / ml, incubated micro aerobicaily (10 percentage CO₂) and anaerobic (CO₂ / N₂ / H2: 5/90/5 ercentage) at 25 degrees for 48 and 72 hours, Microbial cells were removed by centrifugation (4500 rpm. 10 min). The supernatant was sterilized by filtration and concentrated by freeze-drying. The freeze-dried supernatant was resuspended to a 10-fold concentration of 10 mM acetic acid. Six strains of wild yeasts (Candida spp) isolated from maize silage were plated in a uniform layer at ihe PCA (Plate Count Agar; Liofilchem srl. Italy) medium. Wells with a diameter of 6-mm were cut in the agar aseptically and 100 μl of samples of supernatant was added to the wells. After incubation at 25 degrees, the antifungal effect was recorded as follows: no suppression; + weak suppression, growth, of the yeast disturbed; ++ strong suppression, growth of the yeast suppressed with detectable clear zones; +++ very strong suppression, growth of the yeast suppressed with large clear zones.

Antimicrobial compounds produced by Lactobacillus bitchmri BioCC 228 DSM 32651 inhibit yeast growth of plant origin more strongly than those of the strain Lactobacillus buchneri BioCC 203 DSM 32650, Lactobacillus biiehneri BioCC 228 DSM 32651 produced yeast growth inhibitory compounds already during 48 hours of cultivation, creating a wide clear growth inhibition zone on the agar medium around the well, as the supernatant of the strain BioCC 203 DSM 32650 only disturbed the growth of yeasts. Example 5. Growth dynamics during the fermentation of plant material

The aim of the experiment was to evaluate the eflect of Lac to bacillus buchner BioCC 203 DSM 32650 and Lactobacillus buchner BioCC 228 DSM 32651 growth dynamics during the fermentation of plant material

Methods. 226 g of maize (Zea mays L,) plants in vegetative growth stage (V6-V8) was chopped, homogenised with laboratory blender Bagmixer 400 (Interscience, France) for 6 minutes, filtered, centrifuged at room temperature for 5000 rpm for 10 minutes, and sterilized at 121 degrees for 5 minutes

A 48-hour lactobaciilus culture suspension was prepared in peptone water according to McFarland Turbidity Standard No. 5 (1 ,5 x 10⁹ microbes / ml), inoculated into MRS (Biolife. Italy) broth with a final volume of 1.5 x 10⁶ microbes / ml, incubated micro aerobic-ally (10 percentage C0₂) and anaerobic (C02 / N₂ / H₂ : 5/90/5 ercentage) at 25 degrees for 24, 48 and 72 hours.

The viable counts of both strains were registered, the yield, generation number (n) and growth rate (V) were calculated as follows:

Yield - log N_l-log No where N_l is the cell concentration at any given time; No is the initial cell concentration

n log Ni-iog No / log 2, where N_l is the cell concentration at any given time; No is the initial cell concentration

V - log N_l-log No / 0.301 x t, w N_l is the cell concentration at any given time; N<, is the initial cell concentration and t is the specified period of time in hous.

Table 7. The growth dynamics of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during microaerobic (10 percent C⁽¾) cultivation at 25 degrees for 24, 48 and 72 hours.

n - generation number; V - growth rate

During the first 24 hours of microaerobic cultivation, the strain Lactobacillus buchneri BioCC 203 DSM 32650 was four generations faster and 2.4 times faster in 48 hours than the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 7).

Example 6. Investigation of ihe effect of the silage additives L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 on fresh material medium to easy to ferment

Aim of investigation was determination of the effect of the silage additive L.b uchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 on aerobic stability and fermentation quality of maize (Zea mays, maize variety 'Cathy') silage (dry matter content > 30 percentage).

The silage trial was conducted, in 1 ,5 1 laboratory scale-silos with freshly chopped maize at dough stage of maturity. The following investigations were conducted: determination of the pH-Values and fermentation quality at day 90,

Two tests for aerobic stability were carried out The first test was done after a storage period of 49 days with twice air stress (24 hours; at day 28 and day 42).

The test for aerobic stability was carried out in a temperature-conirolled room at approx.

20 degree. Temperatures were recorded every four hours with a PS-ES Datalogging system.

The chemical composition of the fresh material is presented in Table 8, Table 8. The chemical composition of the fresh material

The application of L huchneri BioCC 203 DSM 32650 and L huchneri BioCC 228 DSM 32651 led to significant increase of acetic acid and 1 ,2-propanediol in comparison with imtreated (Table 9).

Table 9. Chemical composition, nutritional values and fermentation quality indicators of maize silage from maize variety 'Cathy using microorganisms L huchneri after a storage period of 90 days.

n.d.- not deteced

The aerobic stability test done after a storage period of 49 days showed a significant increase of the aerobic stabilities for the L.b uchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 treated silages of nearly 2 to 2.5 days in comparison with the untreated control (control :3.9 days vs. L.b uchneri BioCC 203 DSM 32650: 6.3 days and L.b uchneri BioCC 228 DSM 32651 : 5.8 days).

Extending the storage time up to 90 days resulted in an increased aerobic stabilitiy for both either the untreated control with 7,9 days, 10.6 days for the L. buchneri BioCC 203 DSM 32650 treated and 1 1.4 days for the L. buchneri BioCC 228 DSM 32651 silages. This difference of less than three days for L. buchneri BioCC 203 DSM 32650 and more than three days for L. buchneri BioCC 228 DSM 32651 was found to be statistically significant.

Example 7. Investigation of the effect of the silage additives £. buchneri BioCC 203 DSM 32650 and Lbuchneri BioCC 228 DSM 32651 on fresh ntaterial difficult to ferment

The aim of the experiment was to evaluate the fermentation quality and aerobic stability of silage made from freshly chopped whole plant maize (Zea mays, maize variety 'Darker ) with low dry matter content (< 20 percentage) using the microorganism strains L, buchneri BioCC 203 DSM 32650 and Lbuchneri BioCC 228 DSM 32651. Strains L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 were added to the ensiled material in the form of an aqueous solution with a concentration ot 1 x 10⁵ CFU per 1 g of the plant material (feed) being ensiled. A3! test variations (control silages, silages made with the lactic acid bacteria strains L.b uchneri BioCC 203 DSM 32650 and Lhmhnen BioCC 228 DSM 32651, and control silages made without silage additive were prepared in five replicates. All test silages were opened after 90 days of ensiling.

The chemical composition of the fresh material is presented in Table 10.

The aerobic stability of the silages was tested after a storage period of 90 days according to the method described by Honig (Honig, H., 1990; Evaluation of the aerobic stability. In: Proceedings of the Eurobae Conference, Swedish University of Agricultural Sciences, Uppsala/Sweden, Special Issue). Silage was considered aerobically unstable if the temperature measured at its geometric centre exceeded the ambient temperature by 3 degrees. Changes in temperature over time were measured for 9 days (217 hours). Ambient temperatures and test silage temperatures were recorded once an hour, using Comet Temperature Data Logger SO 141. devices.

Silage samples were analysed using well-established methods (AOAC. 2005. Official methods of analysis of AOAC International, 18th ed. Association of Official Analytical Chemists International, Gaithersburg, MD, USA).

To determine dry matter content, the silage sample was dried to constant weight in a thermostat at 130 degrees. To establish crude ash content, the silage sample was incinerated in a muffle furnace at 550 degrees for six hours. Protein content was determined using a KjeltecTM 2300 analyser following the Kjeldahl method (Nx6.25). Crude fibre was determined according to the W.Henneberg and F. Stohmann method. An Agilent 7890A gas chroraatograph was used for determining the acid and ethanol content of the silage. The proportion of ammonia nitrogen in total nitrogen was established with a Kjeltec™ 2300 analyser. The acidity of the silage was determined using a Hanna instruments Hi 2210 pH-meter. Table 10. The chemical composi tion of the fresh material

The -dry matter content of all silages remained <18 percentage (Table 1 1). However, silages from the maize variety 'Dorka' and treated with the L.b uchneri strain BioCC 203 DSM 32650 or L. buchneri strain BioCC 228 DSM 32651 had good fermentation characteristics (Table 1 1). Lactic acid was the dominant acid in aii silages. The silage treated with the L. buchneri strain BioCC 203 DSM 32650 had higher concentration of acetic acid and 1 ,2-propanediol compared to the silage made with the L.b uchneri strain BioCC 228 DSM 32651 and control.

The eihanol content was low in all silages (range from 4.1 to 8.6 g / kg).

Table 1 1 , Chemical composition, nutritional values and fermentation quality indicators of maize silage from maize variety 'Dorka' using microorganisms /,, huchneri BioCC 203 DSM 32650 and L huchneri BioCC 228 DSM 32651 after a storage period of 90 days,

Microbiological indicators of the fermentation quality of ensiled material and maize silage are presented in Table 1 1. In the ensiled material the quantities of moulds were relatively high, Clostridia and yeasts were below detection limit. Silage samples teated with the strains L.b uchneri BioCC 203 DSM 32650 or L. buchneri BioCC 228 DSM 32651 contained very high quantities of lactic acid bacteria (>8.0 log₁₀ CFU / g silage) and the added strains dominated over endogenous lactobiota. The amount of lactic acid bacteria counts in untreated control silage was 4.56 log₁₀ CFU / g silage.

Table 1 1. Microbiological indicators of the fermentation quality of fresh material and maize silage from maize variety 'Dorka" treated with microorganisms L buchneri BioCC 203 DSM 32650 or L.b uchneri BioCC 228 DSM 32651

- can't be calculated

in the aerobic stability test, four out of five replicates of the untreated silage heated up, The average aerobic stability of the control silage turned out to be 149 hours (u.e. 6.2 days). Silages treated with strains L.b uchneri BioCC 203 DSM 32650 and L.b uchneri BioCC 228 DSM 32651 were aerobically stable till the end of the test. i.e. until 217 hours (i.e. 9.04 days). Thus, treatment of the fresh material with strains L.b uchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 increased the aerobic stability of silages for 2,84 days. in conclusion: strains L.b uchmri BioCC 203 DSM 32650 and L buchmri BioCC 228 DSM 32651 expressed surprisingly very vigorous growth in silages made of fresh material difficult to ferment. The use of L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 increased the lactic acid and acetic acid content of silage made from low dry matter (≤ 20 percentage) ensiled material, inhibited the activity of microorganisms and yeasts, thereby preventing the silage from heating, which ensured the improvement of aerobic stability of the silage on the opening of the silo and thus prolonged the storage time of the silage.

Claims

1. Isolated microorganism strain Lactobacillus huckneri BioCC 203 DSM 32650.

2. Isolated microorganism strain Lactobacillus buchmri BioCC 228 DSM 3265 \ .

3. The microorganism strain of claim 1 or claim 2 in !yophilised form,

4. A composition comprising one or more microorganisms according to any of claims 1 to 3.

5. Feed containing the microorganism strain according to any of claims 1 to 3.

6. Feed according to claim 5, which is a fermented feed, e.g. silage.

7. Use of a microorganism according to any of claims 1 to 3 as a feed additive.

8. Use of a microorganism according to any of claims 1 to 3 to support the aerobic stability of silage.

9. The use according to claim 8, wherein silage is made from feed of low dry matter content (≤ 20 percentage).

10. Use of a microorganism according to any of claims 1 to 3 for the fermentation of a feed.

1 1. A composition comprising one or more microorganisms according to any of claims 1 to 3 to accelerate the fermentation of the feed, to increase the concentration of lactic acid and acetic acid in the feed, to reduce the pH, and hence decrease the loss of nutrients in feed.

12, Use of a microorganism according to any of claims 1 to 3 to suppress the effect of pathogenic microbes and to inhibit yeast growth, by adding the microorganism, strain according to any of claims 1 to 3 to the feed to be fermented,

13. The use according to claim 12, wherein pathogenic microbes are enteropathogens.

14. Method for prolonging the preservati on of feed, where the microorganism according to any of claims 1 to 3 is added to feed before fermentation.