WO2015120863A1

WO2015120863A1 - Microorganism lactobacillus brevis tak 124-1 ncimb42149 and its use

Info

Publication number: WO2015120863A1
Application number: PCT/EE2015/000001
Authority: WO
Inventors: Kristiina KOKK; Epp Songisepp; Merle RÄTSEP; Andres OLT; Helgi KALDMÄE; Olav KÄRT; Meelis OTS
Original assignee: Oü Tervisliku Piima Biotehnoloogiate Arenduskeskus (Bio-Competence Centre Of Healthy Dairy Products)
Priority date: 2014-02-14
Filing date: 2015-02-13
Publication date: 2015-08-20
Also published as: EA033289B1; EA201600583A1; EE201400005A; EP3105355A1; EE05756B1

Abstract

The invention provides the isolated microorganism Lactobacillus brevis TAK 124-1 NCIMB42149, which is used for securing the aerobic stability of feed and improving the fermentation of feed, for increasing the concentration of lactic acid and acetic acid, for reducing pH, hence decreasing the loss of nutrients in feed. L. brevis TAK 124-1 suppresses the function of pathogens (clostridia and enteropathogens), yeast and fungi in feed. Feed additive comprising said microorganism helps to extend shelf-life of feed.

Description

MICROORGANISM LACTOBACILLUS BREVIS TAK 124-1 NCIMB42149 AND ITS USE

TECHNICAL FIELD

The invention relates to the field of biotechnology and is useful in the production of animal feed. The invention relates to a microbiological silage additive and its use for ensuring the aerobic stability of feed and for improving fermentation quality and thereby also feed quality .

STATE OF THE 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 conservation method of 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. H. Harrison, eds . American Society of Agronomy, Madison, Wisconsin, USA. pp. 95-139) . The fermentation of silage can be divided into several phases: (1) the initial aerobic phase, (2) the main fermentation phase, (3) the^" stable storage phase, and (4) the feed-out phase, where the silo is opened and the silage is exposed to air. In order to produce well-preserved silage, the material to be ensiled must undergo correct microbial fermentation. Successful fermentation also depends on the type, quality and management of the crops, weather, 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 a number of different chemical and microbiological processes and their interactions. The majority of 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 moulds are able to 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 crop is acidified) , which in turn inhibits the activity of the spoilage microorganisms (Oude Elferink, S. J. W. H., Driehuis, F. , Gottschal, J. C, Spoelstra, S. F . 2000. Silage fermentation processes and their manipulation.- Journal FAO 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, Marlow, Bucks UK, p. 340) .

Just as important as the preservation of nutrients in the fermentation and storage phase 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 as a result of inadequate coverage of the silo.

Silages that are exposed to air spoils sooner or later due to the activity of aerobic microorganisms. The aerobic stability of silage depends on the silage crop to be ensiled, its development phase at the time of harvest, the biochemical and microbiological factors of fermentation, the physical characteristics of the silage crop, silage management, temperature, and the choice of silage additive. The aerobic stability parameter of silage is the length of time that silage is able to 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 on the basis of 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- Richtlinienfiir die Prufung von Siliermitteln auf DLG- Gutezeichen-Fahigkeit , DLG Oktober 2013), the greater the aerobic stability and the quality of the silage. In the majority of 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 are able to 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 in the course of 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 losses (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 use the residual sugars contained in silage as a source of energy; however, their first preference is lactic acid. As a result, well-preserved 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-preserved 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 butyric acid content and the number of yeasts and moulds in the silage upon the opening of the silo are important factors of the aerobic stability of silage. Negative correlation with regard to dry matter content and yeasts indicated that higher values were associated with shorter times for silage temperature to rise on exposure to air. With acetic and butyric acid, on the other hand, a greater concentration of these fermentation products were associated with silages which were relatively stable in air.

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 fungi in aerobically deteriorating silades is inhibited by undissociated short-chain fatty acids (Pahlow G., Muck R.E., Driehuis F . , Oude Elferink 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 pass into microbial cells by passive diffusion, which results in the release of H⁺ ions. This reduces intracellular pH, causing the cell to perish. The extent of dissociation of an acid in silage depends on the dissociation constant of the acid (pKa) and the pH level of the silage (Zirchrom (2011) Dissociation constants of organic acids and bases. Available at: http : //www . zirchrom. com/organic . htm (accessed 3 November 2011)). Acetic and propionic acid are less dissociated than lactic acid, which explains the susceptibility of well- preserved 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 losses, 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 the feed-out phase. An effective silage additive and proper management of the silage production and feeding 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 nutritive value 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 (incl. 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 . Enzymes are often used in silage additives; however, these do not inhibit yeasts and 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 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, acids and other chemicals are corrosive on the harvesting 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 harvesting 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 on the basis of glucose fermentation: homofermenters and heterofermenters . 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 either one mole of ethanol or acetic acid. It is well 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, the majority of such bacteria inoculants 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 poorer than untreated silage, and it might even increase the heating risk of the silage.

Some silage inoculants 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, inoculants that produce considerable quantities of acetic acid in addition to lactic acid (L. brevis or L. buchneri) 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 losses. 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) .

Danner et al (Danner, H., Holzer, M. , Mayrhuber, E., Braun, R. 2003. Acetic Acid Increases Stability of Silage under Aerobic Conditions. Applied and Environmental Microbiology. Vol. 69, no. 1, pp 562-567) also report that Lactobacillus brevis, which belongs to the heterofermentative group, shows great promise with regard to improving the aerobic stability of silage.

The use of lactic acid bacteria for improving the quality of silage and providing aerobic stability has been disclosed in numerous patent applications and patents. Most commonly, Lactobacillus brevis strains are used in combination with other lactic acid bacteria.

Japanese patent application JP2006042647 (Cho Takekuni et al, National Agriculture & Bio-Oriented Research Organization, 2006) describes silage fermentation by means of Lactobacillus brevis TM2 (FERM AP-20140) . This bacterium has a low pH tolerance and high temperature resistant properties; it has an antimicrobial effect on yeasts, which cause silage to spoil. Lactobacillus brevis TM2 is used in combination with the acetic acid-producing lactic acid bacterium TM1 (FERM AP-20139) .

Japanese patent application JPH02257875 (Yano Naotatsu Et Al . , Kubota Ltd., 1990) describes silage fermentation by means of Lactobacillus brevis KB-292 (FERM P-1047) in both aerobic and anaerobic environments; this strain has particularly good fermentation abilities in the first stage of silage fermentation.

The European Register of Feed Additives includes numerous silage additives (technological additives) that contain the bacterium Lactobacillus brevis, e.g. additives containing Lactobacillus brevis DSM 21982, Lactobacillus brevis DSM 12835, Lactobacillus brevis IFA 92 (DSM 23231) (http : //ec . europa . eu/food/food/animalnutrition/feedadditive s/ comm_register_feed_additives_1831-03.pdf, downloaded 29.01.2014) . According to the scientific opinion of the European Food Safety Authority (EFSA) , the strain Lactobacillus brevis DSMZ 16680 improves the aerobic stability of silage (EFSA Journal 2014; 12(1): 3534); the same goes for the strain Lactobacillus brevis DSMZ 21982 (EFSA Journal 2012; 10 (3) :2617) .

The aim of this invention is to provide a new strain of Lactobacillus brevis for improving feed fermentation quality and prolonging the aerobic stability and storage time of silage.

DISCLOSURE OF THE INVENTION

The invention relates to the isolated microorganism strain Lactobacillus brevis TAK 124-1 NCIMB42149, as well as a feed, a feed additive and a composition comprising this strain. The feed can be a fermented feed, e.g. silage. The feed additive is, for example, a silage additive. The other components of the composition can include any necessary excipients. The afore-mentioned microorganism can be used in a lyophilised form. The microorganism Lactobacillus brevis TAK 124-1 NCIMB42149 ensures the aerobic stability of the feed.

Said microbe is 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 losses in feed.

Lactobacillus brevis TAK 124-1 NCIMB42149 promotes the lactic acid-based fermentation of feed; the resulting lactic acid accelerates the reduction in the pH level of the silage. According to the studies of its antimicrobial action, Lactobacillus brevis TAK 124-1NCIMB42149 inhibits the activity of undesirable microorganisms (pathogenic microorganisms, yeasts and moulds) in the feed. The enteropathogens in question are Listeria monocytogenes , Yersinia enterocolitica , Salmonella Enteritidis, S. enterica serovar Typhimurium, Shigella sonnei, Escherichia coli, Enterobacter sakazakii, Staphylococcus aureus etc. Clostridium spp. include Clostridium tyrobutyricum, C. butyricum, C. sporogenes etc. The invention also relates to a method for prolonging the preservation of feed, where the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149 is added to the feed during fermentation. This strain is added to the feed at a rate of lxlO⁵... lxl0⁶CFU/g of the fermented feed.

DESCRIPTION OF THE STRAIN

The microbial strain Lactobacillus brevis TAK 124-1 NCIMB42149 has been isolated in Estonia from a legume-rich silage (over 75 per cent), ensiled naturally without the use of silage additives. To determine the quantitative Lactobacillus spp. composition of the silage sample obtained from the silo by means of a silage probe, serial dilution method by preparing series of suspensions with a descending degree of density in saline solution (0.9 per cent NaCl) ; these were then plated onto Rogosa agar (OXOID, U.K.), which was incubated at a temperature of 37 °C in an anaerobic environment (thermostat IG 150, Jouan, France) for 48 hours. The developed microbe colonies were described and counted and the total number of microbes was determined. Gram stained preparations were made and inspected under a microscope in order to describe the morphology of the microbes. The strain that forms the object of the invention was isolated on the basis of the colony and cell morphology characteristic of Lactobacillus spp. This was followed by a provisional and then more detailed identification as described below.

Culture-specific morphological characteristics

The characteristics were determined after growth in MRS agar and broth (OXOID, UK) . Lactobacillus brevis TAK 124-1 NCIMB42149 is a Gram-positive rod-shaped bacterium of average thickness and length, with a regular shape and no spores. Its individual cells are usually spaced apart.

Physiological and biochemical characteristics The best medium for cultivating the microbial strain Lactobacillus brevis TAK 124-1 NCIMB42149 is MRS broth, which shows a uniformly cloudy growth after 24...48 hours of microaerobic incubation at 37 °C. In a microaerobic environment (C02/02/N2 : 10/5/85) cultivated colonies, are the off-white, convex with a diameter of 1.5...2.5 mm, with undulate margin and convex.

The Lactobacillus brevis TAK 124-1 NCIMB42149 strain is an obligately heterofermentative, catalase and oxidase negative bacterium that does not hydrolyse arginine, but produces carbon dioxide upon glucose fermentation. The optimum growth temperature for the strain is 37 °C, but it also multiplies at 15 °C. To a small extent, growth can also be observed at 45 °C. The optimum pH level for growing the strain is 5.5. Lactobacillus brevis TAK 124-1 NCIMB42149 was identified on the basis of biochemical activity using the API 50CHL System (bioMerieux, France) test kit as Lactobacillus brevis (overlap with the typical strain: excellent, ID per cent - 79.0, T index 0.83). Identification on sequencing: Lactobacillus brevis (16S rRNA similarity to the typical strain: 99 per cent). Contains three plasmids of sizes 10 kb, 7 kb and 6 kb.

The strain was identified as Lactobacillus brevis by using the MALDI Biotyper (Bruker Daltonik) : score value 2.136 (secure genus identification).

The strain Lactobacillus brevis TAK 124-1 NCIMB42149 was deposited in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the UK National Collection of Industrial, Food and Marine Bacteria (NCIMB) under number NCIMB42149 on 29 May 2013. The carbohydrate fermentation profile of Lactobacillus brevis TAK 124-1 NCIMB42149 on the basis of API CHL 50 is as follows. The strain ferments: L-arabinose, ribose, D- xylose, D-glucose, D-fructose, maltose, gentiobiose, sodium 5-ketogluconate .

Antibiotic resistance

Methodology: The antibacterial susceptibility of Lactobacillus brevis TAK124-1 NCIMB42149 to antibiotics was tested by means of E-test (AB Biodisk, Solna) . Minimum inhibitory concentration was determined in accordance with the epidemiological cut-off values recommended by the European Food Safety Authority (EFSA) . Table 1. Antibacterial susceptibility of Lactobacillus brevis TAK 124-1 NCI B42149

^Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance, EFSA Journal 2012; 10(6):2740

A microbe strain is considered susceptible when its growth is inhibited at or below the cut-off concentration value of the specific antimicrobial compound (S ≤ x mg/L) .

A microbe strain is considered resistant when its growth is inhibited at a concentration above the cut-off value of the specific antimicrobial compound (R > x mg/L) .

The strain Lactobacillus brevis TAK 124-1 NCIMB42149 exhibited no resistance to the tested antibiotics (Table 1) ·

FUNCTIONAL CHARACTERISTICS OF THE STRAIN Profile of short-chain fatty acids

Methodology: 24-hour old Lactobacillus brevis TAK 124-1 NCIMB42149 grown on MRS agar was suspended in saline according to the McFarland standard of 10⁹microbes/ml , 0.5 ml was inoculated in PYG medium (a 4.65 ml) and incubated under microaerobic conditions (10 per cent C0₂) in a thermostat at 37 °C for 24 and 48 hours.

The profile of short-chain fatty acids was determined with gas chromatograph HP 6890 Series GC System, using a HP- INNOWax capillary column (15 m x 0.25 mm; 0.15 μιτι) . Column temperature programme: 60 °C 1 min, 20 °C/min 120 °C 10 min, detector (FID) 250 °C (Table 2) .

Table 2. Concentration (g/1) of acetic acid, lactic acid and succinic acid in PYG medium upon the microaerobic cultivation of Lactobacillus brevis TAK 124-1 NCIMB42149 for 24 and 48 hours

Antimicrobial activity on plant-derived lactobacilli and pathogens

Streak line method was used to assess the antimicrobial activity of the lactobacillus against pathogens (Hutt P, Shchepetova J, Loivukene K, Kullisaar T, Mikelsaar M. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens . J ApplMicrobiol . 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 3) in an analogous manner to Hutt et al. (2006), and antagonistic activity (mm) was assessed based on the same.

Table 3. Antimicrobial activity of Lactobacillus brevis TAK 124-1 NCIMB42149 against plant-derived lactobacilli and pathogens on modified MRS agar, using the streak-line method (growth inhibition on the target microbe in mm) in microaerobic (10 per cent C0₂) and anaerobic (C0₂/N₂/H₂: 5/90/5 per cent) environments

Inhibition zone in microaerobic environment (in mm) : weak <26.9; average 27.0...33.9; strong >34.

Inhibition zone in anaerobic environment (in mm) : weak <14.9; average 15.0...18.9; strong >19.

A comparison of the antimicrobial activity of the disclosed strain to an heterofermentative lactic acid bacterium strain isolates (Table 4) demonstrates that Lactobacillus brevis TAK 124-1 NCIMB42149 exhibits better antimicrobial characteristics in an anaerobic environment (i.e. the typical silage environment) . Table 4. Antimicrobial activity of Lactobacillus brevis TAK 124-1 NCIMB42149 and plant-derived heterofermentative lactobacilli against pathogens on modified MRS agar, using the streak-line method (growth inhibition on the target microbe in mm) in microaerobic (10 per cent C0₂) and anaerobic (C0₂/N₂/H₂: 5/90/5 per cent) environments

Inhibition zone in microaerobic environment (in mm) : weak <22.9; average 23.0...32.9; strong >33.

Inhibition zone in anaerobic environment (in mm): weak <11.9; average 12.0...21.9; strong >22.5

Antimicrobial activity against Clostridia was evaluated as follows . Supernatant was extracted from Lactobacillus brevis TAK 124-1 NCIMB42149 after incubation in Brain Heart Infusion

(BHI) broth for 24 hours. A Clostridium spp suspension was added to the filtration-sterilised supernatant or sterile BHI broth (positive control) . The results were evaluated after 48 hours at OD620nm- Growth inhibition of Clostridium spp CD (per cent value) was calculated as follows = 100-

(OD_tX 100 / OD_c) , where

0D_t- added supernatant 0D_C- no added supernatant.

Antimicrobial compounds produced by Lactobacillus brevis TAK 124-1 NCIMB42149 inhibit the growth of plant-derived Clostridium spp by 23.1 per cent.

Its use improves the production efficiency of any silage made from forage by increasing acetic acid and lactic acid production, which in turn improves the aerobic stability and storage time of silage.

Lactobacillus brevis TAK 124-1 NCIMB42149 has no harmful effect on animal health, human health or the environment, as the species L. brevis is listed by EFSA among taxonomical units with QPS status (Qualified Presumption of Safety) (EFSA. Introduction of a Qualified Presumption of Safety (QPS) approach for assessment of selected microorganisms referred to EFSA. *EFSA J *2007; 587, 1-16). LIST OF FIGURES

Fig. 1 - Example 1

Improving the quality of ryegrass silage fermentation by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where a - ethanol, g/kg in dry matter; b - acetic acid, g/kg in dry matter; c - propionic acid, g/kg in dry matter; d - butyric acid, g/kg in dry matter; e - lactic acid, g/kg in dry matter; f - pH; g - ammonia nitrogen in total nitrogen, per cent;

Fig. 2 - Example 2

Improving the quality of timothy silage fermentation by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where a - ethanol, g/kg in dry matter; b - acetic acid, g/kg in dry matter; c - propionic acid, g/kg in dry matter; d - butyric acid, g/kg in dry matter; e - lactic acid, g/kg in dry matter; f - pH; g - ammonia nitrogen in total nitrogen, per cent;

Fig. 3 - Example 3

Improving the quality of red clover silage fermentation by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where a - ethanol, g/kg in dry matter; b - acetic acid, g/kg in dry matter; c - propionic acid, g/kg in dry matter; d - butyric acid, g/kg in dry matter; e - lactic acid, g/kg in dry matter; f - pH; g - ammonia nitrogen in total nitrogen, per cent; Fig. 4 - Example 4

Fig. 5 - Improving the aerobic stability of ryegrass silage (Example 1) by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where sub-figure A - ambient temperature and control silage temperature (5 replicates) ,

B - ambient temperature and the temperature of silage made with Lactobacillus brevis strain TAK 15 124-1 NCI B42149 (5 replicates) ,

C - ambient temperature and the temperature of silage made with a chemical silage additive (5 replicates) ;

Fig. 6 - Improving the aerobic stability of timothy silage (Example 2) by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where sub-figure

A - ambient temperature and control silage temperature (5 replicates ) ,

B - ambient temperature and the temperature of silage made with Lactobacillus brevis strain TAK 15 124-1 NCIMB42149 (5 replicates ) ,

C - ambient temperature and the temperature of silage made with a chemical silage additive (5 replicates);

Fig. 7 - Improving the aerobic stability of red clover silage (Example 3) by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where sub-figure

A - ambient temperature and control silage temperature (5 replicates) ,

Fig. 8 - Improving the aerobic stability of ryegrass silage (Example 4) by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where sub-figure A - ambient temperature and control silage temperature (5 replicates) ,

B - ambient temperature and the temperature of silage made with Lactobacillus brevis . strain TAK 15 124-1 NCIMB42149 (5 replicates ) ,

C - ambient temperature and the temperature of silage made with a chemical silage additive (5 replicates).

DESCRIPTION OF THE EMBODIMENTS

The following examples describe the improvement of the fermentation quality of silage produced from various crops, ensuring the aerobic stability of silage, inhibiting pathogenic microorganisms, yeasts and moulds, and prolonging the storage time of silage by means of the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149.

Two embodiments (Examples 1 and 4) relate to the fermentation of ryegrass silage with varying water-soluble carbohydrates content (1.52 and 2.85 per cent, respectively) . Example 2 relates to the fermentation of timothy, and Example 3 relates to the fermentation of red clover .

Example 1. Improving the fermentation quality of ryegrass silage and ensuring the aerobic stability of the silage by means of the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

The experiment was conducted with hybrid ryegrass, where the water-soluble carbohydrates content in the crop to be ensiled was 1.52 per cent. The crop was mowed and then wilted for 24 hours. The wilted crop was harvested, chopped and test silages were prepared. The control silage was made without any silage additive. The second test variation was inoculated with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149. The third silage was made with a formic acid-based chemical silage additive of the following composition: 42.5 per cent of formic acid, 30.3 per cent of ammonium formate, 10 per cent of propionic acid, 1.2 per cent benzoic acid, 1 per cent of ethyl benzoate, 15 per cent of water. The chemical silage additive was used as a so-called positive control and added to the ensiled crop in a ratio of 3 1/t. The lyophilised lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 was added to the ensiled material in the form of an aqueous solution with a concentration of lxlO⁵ CFU per 1 g of the plant material (feed) being ensiled. All test variations (control silages, silages made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149, and silages made with a chemical silage additive) were prepared in five replicates. The test silages were opened after 90 days of ensiling.

The aerobic stability of the silages was tested according to the method described by Honig (Honig, H., 1990: Evaluation of the aerobic stability. In: Proceedings of the Eurobac 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 °C. Changes in temperature over time were measured in the course of 9 days (216 hours). Ambient temperature and test silage temperatures were recorded once an hour, using Comet Temperature Data Logger S0141 devices. Silage samples were analysed using generally accepted methods (AOAC. 2005. Official methods of analysis of AOAC International, 18th ed. Association of Official Analytical Chemists International, Gaithersburg, MD, USA) . In order to determine dry matter content, the silage sample was dried to constant weight in a thermostat at 130 °C. To establish crude ash content, the silage sample was ashed in a muffle furnace at 550 °C for six hours. Protein content was determined using a Kjeltec™ 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 chromatograph was used for determining the acids, ethanol and 2.3-butanediol content of the silage. The proportion of ammonia nitrogen in total nitrogen was established with a Kjeltec™ 2300 analyser. The pH of the silage was determined using a Hanna Instruments HI 2210 pH-meter.

The heterofermentative lactic acid bacterium Lactobacillus jbrevis TAK 124-1 NCIMB42149 improved the fermentation of the silage (Table 5 and Fig 1). The addition of Lactobacillus brevis TAK 124-1 NCIMB42149 more than doubled the lactic acid content of the silage (p<0.01) and increased the acetic acid content more than 2.4-fold (p<0.01), compared to the control silage. Lactobacillus brevis TAK 124-1 NCIMB42149 also resulted in a statistically significant improvement (p<0.01) in the following fermentation indicators: the pH level of the silage, the proportion of ammonia nitrogen in total nitrogen, the content of 2.3-butanediol , and a lower nutrient losses in fermentation. Of the microorganisms responsible for the spoiling of silage, Lactobacillus brevis TAK 124-1 NCIMB42149 inhibited the population of yeasts (p<0.05) and Clostridium spp . (p<0.01), compared to control silage (Table 6) .

Table 5. Chemical composition, nutritive value and fermentation quality parameters of ryegrass silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149 (Example 1)

Table 6. Microbiological parameters of the fermentation guality of ryegrass silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149 (Example 1)

below the limit of analytical determination **- cannot be calculated

In the aerobic stability test, the untreated control silage heated up at 32 hours (Table 5 and Fig 5) , while the silage made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 remained aerobically stable for 99 hours (p<0.01). The result also showed a statistically significant advantage (p<0.05) over the corresponding parameter ^' of the silage made with a chemical silage additive (59 hours).

In this example, the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation of the silage, compared to the control silage. By producing more lactic and acetic acid and inhibiting the growth of the microorganisms (yeasts and Clostridium spp.) that cause the spoiling of the silage, Lactobacillus brevis TAK 124-1 NCIMB42149 provides well-preserved and stable silage both upon storage in a silo and upon the opening of the silo for feeding by preventing the silage from heating. In conclusion, the use of Lactobacillus brevis TAK 124-1 NCIMB42149 increased the lactic acid and acetic acid concentration in silage, lowered the pH level of silage, reduced nutrient (dry matter) losses in the feed, inhibited the activity of pathogenic microorganisms and yeasts, ensured a better aerobic stability of the silage, and prolonged the storage time of the silage.

Example 2. Improving the fermentation quality of timothy silage and ensuring the aerobic stability of the silage by means of the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

The experiment was conducted with timothy {Phleum pratense L. ) , where the water-soluble carbohydrates content in the crop to be ensiled was 1.10 per cent. The crop was mowed and then wilted for 24 hours. The wilted crop was harvested, chopped and test silages were prepared. The control silage was made without any silage additive. The second test variation was inoculated with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149. The third silage was made with a formic acid-based chemical silage additive of the following composition: 42.5 per cent of formic acid, 30.3 per cent of ammonium formate, 10 per cent of propionic acid, 1.2 per cent benzoic acid, 1 per cent of ethyl benzoate, 15 per cent of water. The chemical silage additive was used as a so-called positive control and added to the ensiled material in a ratio of 3 1/t. The lyophilised lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 was added to the ensiled material in the form of an aqueous solution with a concentration of IxlO⁵ CFU per 1 g of ensiled plant material (feed) . All test variations (control silages, silages made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149, and silages made with a chemical silage additive) were prepared in five replicates. The test silages were opened after 90 days of ensiling . The aerobic stability of the test silages was tested and the silage samples were analysed using the methods

described in Example 1.

The heterofermentative lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 produced more acetic acid in the silage (Table 7 and Fig 2), compared to the corresponding parameters of the control silage fermented by means of the natural population of lactic acid bacteria (p<0.05) and the chemical additive treated silage (p<0.01). The microbiological parameters of the test silage are presentedin Table 8.

The greater acetic acid content of the silage improved the aerobic stability of the feed on the opening of the silo, inhibiting the activity of aerobic microorganisms and thereby preventing the silage from heating.

Table 7. Chemical composition, nutritive value and fermentation quality parameters of timothy silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

Item Control TAK 124-1 Chemical additive

Mean SD Mean SD Mean SD

Dry 323.5 25.5 298.9 8.7 250.3 9.2 matter,

g/kg

In dry

matter,

g/kg

Table 8. Microbiological parameters of the fermentation quality of timothy silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

• **- cannot be calculated

The temperature of the silage made with the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 remained stable throughout the test period (>216 hours) in the aerobic stability test (Table 7 and Fig 6) . The control silage and the silage made with a chemical silage additive heated up at 164 hours (p<0.01) and 126 hours (p<0.01), respectively.

Example 2 shows that the use of Lactobacillus brevis TAK 124-1 NCIMB42149 increased the acetic acid concentration in silage and ensured the aerobic stability of the silage, prolonging the storage time of the silage.

Example 3. Improving the fermentation quality of red clover silage and ensuring the aerobic stability of the silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149 The experiment was conducted with tetraploid red clover {Trifolium pratense L. ) , where the water-soluble carbohydrates content in the crop to be ensiled was 0.62 per cent. The crop was mowed, chopped and test silages were prepared. The control silage was made without any silage additive. The second test variation was inoculated with the lactic acid bacterium strain TAK 124-1 NCIMB42149. The third silage was made with a formic acid-based chemical silage additive of the following composition: 42.5 per cent of formic acid,

30.3 per cent of ammonium formate, 10 per cent of propionic acid, 1.2 per cent benzoic acid, 1 per cent of ethyl benzoate, 15 per cent of water. The chemical silage additive was used as a so-called positive control and added to the ensiled crop in a ratio of 3 1/t. The lyophilised lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 was added to the ensiled crop in the form of an aqueous solution with a concentration of lxlO⁵ CFU per 1 g of the plant material (feed) being ensiled. All test variations (control silages, silages made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149, and silages made with a chemical silage additive) were prepared in five replicates. The test silages were opened after 90 days of ensiling.

The aerobic stability of the test silages was tested and the silage samples were analysed using the methods described in Example 1.

The lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation parameters of the red clover silage (Table 9 and Fig 3) . The addition of Lactobacillus brevis TAK 124-1 NCIMB42149 more than doubled the lactic acid content (p<0.01) and thereby lowered the pH level of the silage, compared to the control silage. This served to decrease the content of undesirable propionic acid (p<0.01), 2.3- butanediol (p<0.01) and ammonia nitrogen (p<0.01) as well as dry matter losses (p<0.01). The microbiological parameters are presented in Table 10.

Table 9. Chemical composition, nutritive value and fermentation quality parameters of red clover silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

Table 10. Microbiological parameters of the fermentation quality of red clover silage using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

*- below the limit of analytical determination 10² **- cannot be calculated

Silage made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 remained aerobically stable for 197 hours (p<0.01) in the aerobic stability test, while the untreated control silage heated up at hour 113 (Table 9 and Fig 7) .

Thus, in the above Example, the heterofermentative lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation of the silage, compared to the control silage.

The use of Lactobacillus brevis TAK 124-1 NCIMB42149 increased the lactic acid concentration in silage, lowered the pH level of silage, reduced nutrient (dry matter) losses in the feed, ensured a better aerobic stability of the silage onthe opening of the silo, and prolonged the storage time of the silage.

Example 4. Improving the fermentation quality of ryegrass silage and ensuring aerobic stability using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

The experiment was conducted with hybrid ryegrass, where the water-soluble carbohydrates content in the crop to be ensiled was 2.85 per cent. The crop was mowed and then o wilted for 3 hours. The wilted crop was harvested, chopped and test silages were prepared. The control silage was made without any silage additive. The second test variation was inoculated with the lactic acid bacterium strain TAK 124-1 NCIMB42149. The third silage was made with a formic acid- based chemical silage additive of the following composition: 42.5 per cent of formic acid, 30.3 per cent of ammonium formate, 10 per cent of propionic acid, 1.2 per cent benzoic acid, 1 per cent of ethyl benzoate, 15 per cent of water. The chemical silage additive was used as a so-called positive control and added to the ensiled material in a ratio of 3 1/t. The lyophilised lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149 was added to the ensiled crop in the form of an aqueous solution with a concentration of lxlO⁵ CFU per 1 g of the plant material (feed) being ensiled. All test variations (control silages, silages made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149, and silages made with a chemical silage additive) were prepared in five replicates. The test silages were opened after 90 days of ensiling. The aerobic stability of the test silages was tested and the silage samples were analysed using the methods described in Example 1.

The lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 produced 3 times more acetic acid in the silage (Table 11 and Fig 4), compared to the corresponding parameter of the control silage fermented by means of the natural population of lactic acid bacteria (p<0.01). The TAK 124-1 NCI B42149 silage also contained more lactic acid (p<0.01) than the untreated control silage (87.9 and 48.8 g per 1 kg in dry matter, respectively) . The greater lactic acid content ensured the lower pH level of the TAK 124-1 NCI B42149 silage (p<0.01). Unlike the control silage, the silage made with the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 exhibited no yeast content (p<0.01) (Table 12). The inhibition of yeasts and the higher acetic acid content in the silage improved the aerobic stability of the feed on the opening of the silo. The temperature of the silage made with the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCI B42149 remained stable throughout the test period (216 hours) in the aerobic stability test (Table 11 and Fig 8). The control silage and the silage treated with chemical additive heated at hour 26 and hour 43, respectively. The use of Lactobacillus brevis TAK 124-1 NCIMB42149 resulted in a statistically significant improvement (p<0.01) in the aerobic stability of the silage, compared to the control silage and the silage made with a chemical silage additive. Table 11. Chemical composition, nutritive value and fermentation quality parameters of the 2nd ryegrass silage experiment (Example 4) using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

Table 12. Microbiological parameters of the fermentation quality of the 2nd ryegrass silage experiment (Example 4) using the microorganism Lactobacillus brevis TAK 124-1 NCIMB42149

**- cannot be calculated

In Example 4 of the invention, the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation of the silage. By inhibiting the yeasts that cause the spoiling of the silage, Lactobacillus brevis TAK 124-1 NCIMB42149 provides well-preserved and stable silage during storage in a silo and prevents the heating of the silage upon the opening of the silo. In Example 4, the use of Lactobacillus brevis TAK 124-1 NCIMB42149 increased the lactic acid and acetic acid concentration in silage, lowered the pH level of silage, inhibited the activity of pathogenic microorganisms and yeasts, ensured the aerobic stability of the silage, and prolonged the storage time of the silage.

Claims

1. An isolated microorganism strain Lactobacillus brevis TAK 124-1 NCIMB42149.

2. The microorganism strain of claim 1 in a lyophilised form.

3. A feed comprising the microorganism strain of any of claims 1 and 2.

4. The feed according to claim 3, in the form of fermented feed, e.g. silage.

5. A composition comprising the microorganism strain of any of claims 1 and 2.

6. The use of the microorganism strain of any of claims 1 and 2 as a feed additive.

7. The use of the microorganism strain of any of claims 1 and 2 for ensuring the aerobic stability of silage.

8. The use of the microorganism strain of any of claims 1 and 2 for the fermentation of feed.

9. The use of the microorganism strain of any of claims 1 and 2 for improving the fermentation of feed, increasing the lactic acid and acetic acid concentration in feed, lowering the pH level and thereby reducing nutrient loss in feed .

10. The use of the microorganism strain of any of claims 1 and 2 for inhibiting the activity of pathogenic microbes and the growth of yeasts during the fermentation of feed by adding the microorganism strain of any of claims 1 and 2 to the feed being fermented.

11. The use of claim 10, wherein the pathogenic microbes are Clostridium spp. and enteropathogens.

12. A method for prolonging the preservation of feed, wherein the microorganism strain of any of claims 1 and 2 is added to the feed during fermentation.