EE05834B1 - Microorganism strain Lactobacillus buchneri BioCC 228 DSM 32651 and its use - Google Patents

Abstract

The invention provides the isolated microorganism strain Lactobacillus buchneri BioCC 228 DSM 32651 and its use as microbiological feed additive. The strain is 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 microorganism in ensiling suppresses the function of pathogenic microorganisms (enteropathogens) and yeasts in feed. The strain can be used for prolonging the storage life of feed made from fresh material difficult to ferment.

Description

TECHNICAL FIELD

The invention belongs to the field of biotechnology and finds application in the production of feed. The invention relates to a microbiological silage security additive and its use for ensuring the aerobic stability of feed, improving the quality of fermentation and thereby the quality of feed.

STATE OF THE ART

Maintaining the nutrient content of silage is necessary from the time the feed is harvested and preserved to the time the feed is consumed by the animal.

Silage is a fermented feed obtained by ensiling high-moisture plant material under controlled fermentation conditions (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 method of preserving plant animal feed based 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). Silage fermentation can be divided into four phases: 1) aerobic phase in the storage after harvesting, 2) fermentation phase, 3) stable storage phase, and 4) silage unloading phase, when the storage is open and the silo is exposed to air. Proper microbial fermentation of the silage material is important for the production of quality silage. Successful fermentation also depends on the type of grasses, their quality, the ensiling process

the technological techniques used, the weather, the development of unwanted microorganisms (e.g. clostridia, enteropathogens, listeria, bacilli) and fungi (yeast and molds), and the dry matter content of the ensiled material.

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

Most silage is produced at a dry matter content of 200-500 g/kg. At this level, many plant enzymes are active during the ensiling process and under these conditions a large number of desirable and undesirable microorganisms, yeasts and moulds can grow in the silage. Therefore, controlling all biological activity is a significant challenge and can only be achieved through a well-managed ensiling process (Muck, R. E. 2010. Silage microbiology and its control through additives. R. Bras. Zootec. Vol. 39. July).

In the controlled ensiling process, water-soluble carbohydrates are fermented into lactic acid by lactic acid bacteria. As a result, the pH of the ensiled material drops (the ensiled mass becomes acidic), which in turn suppresses the vital activity of 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 faster the enzymatic and microbial activity ceases, the feed becomes stable and more nutrients are preserved.

It has been previously documented that the quality of silage fermentation can be significantly improved by 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 preserving nutrients during the fermentation and storage phases of silage, it is also important to preserve nutrients in the silage when the silo is opened. Silage can be exposed to oxygen both when the silo is opened for feeding purposes and as a result of errors in the covering of the silo.

All silos, when exposed to air, sooner or later deteriorate as a result of the active life activity of aerobic microorganisms. In addition, the aerobic stability of the silo is affected by the silage crop being ensiled and its growth phase at the time of harvesting, the biochemical and microbiological factors of fermentation, the physical and organizational factors of the silage material, the temperature and the choice of silage safety additive. The aerobic stability of the silo is considered to be the time for which the silo can resist aerobic deterioration processes, i.e. how long the silo remains of good quality when exposed to air. The aerobic stability of the silo is assessed by the rate of increase in the silo temperature. The longer the silo temperature remains stable, i.e. the silo temperature does not exceed the ambient temperature by more than 3°C (Commission Regulation (EC) No 429/2008; DLG-Richtlinien für die Prüfung von Siliermitteln auf DLG-Gütezeichen-Fähigkeit October 2013), the more aerobically stable and better the silo is. In most aerobically decomposing silos, the temperature rises above ambient temperature due to microbial oxidation of acids and water-soluble carbohydrates to carbon dioxide and water.

Although the low pH of silage under anaerobic conditions suppresses the growth of unwanted microorganisms, the low pH is not

sufficient in itself to prevent aerobic spoilage. Silage spoilage under aerobic conditions is mostly initiated by yeasts, which can grow at quite low pH. Yeasts are able to grow in a wide pH range (pH 3-8). The optimal pH for the growth of most yeasts is 3.5-6.5. When the silo is exposed to air when the storage is opened, the acids and other compounds produced during fermentation are oxidized by aerobic bacteria, yeasts and molds. The life activity of the yeasts produces carbon dioxide and this causes the silo to heat up, which in turn is a direct cause of dry matter losses (McDonald, P., Henderson, A. R., Heron, S.J.E.

1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Marlow, Bucks UK, p. 340).

Yeasts use the residual sugar in the silo as an energy source, but they primarily prefer lactic acid. Therefore, well-fermented silos with a high lactic acid content are particularly susceptible to aerobic spoilage. As a result of the activity of yeasts, the pH of the silo begins to rise, which creates an opportunity for several other aerobic microorganisms and molds to become active. Active microbial activity in well-fermented, nutrient-rich silos is manifested by an increase in the temperature of the silo.

It has been found (Ohyama, Y., Нага, 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 important factors affecting the aerobic stability of silage are the dry matter, acetic acid and propionic acid content of the silage and the number of yeast and mold fungi at the time of opening the silo. The negative relationship between dry matter and yeast showed that higher concentrations caused a faster increase in the temperature of the silage when exposed to air. On the other hand, acetic acid and butyric acid showed that their

A higher concentration of fermentation products was associated with a more stable silage.

As mentioned, the low pH of silage does not have a direct effect on aerobic spoilage microorganisms, but the acids produced during silage fermentation are of different importance. Yeast growth 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 are able to pass through the microbial cell membrane by passive diffusion, resulting in the release of H+ ions. This lowers the intracellular pH and, as a result, the cell dies. The extent to which an acid dissociates in silage depends on the acid dissociation constant (pKa) and the pH 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 acids dissociate less than lactic acid, which explains the susceptibility of well-fermented lactic acid silage to aerobic spoilage. In contrast, acetic and propionic acids are effective inhibitors of yeast and molds. Butyric acid has a similar effect. Butyric acid silage is aerobically stable, but this indicates the activity of spoilage clostridia. Such silage has high nutrient losses and high butyric acid content can cause health problems in animals. Propionic acid is rarely present in silage and in small quantities, and the concentration of microorganisms producing it in silage cultures is low and their competitiveness is low.

The acetic acid content in the silage indicates heterofermentative fermentation, and since acetic acid is highly toxic to yeasts, such silages are usually very aerobically stable.

Ideal silage fermentation reduces fermentation losses and ensures sufficient stability during storage and unloading of the feed for feeding. Effective silage additives, proper management of silage preparation and feeding play a key role in achieving these goals. Most silage additives are developed to improve the ensiling process and the nutritional value of the ensiled feed. However, in addition to rapid silage fermentation and quality improvement, silage additives are also expected to suppress the growth of spoilage organisms (including aerobic spoilage). The main reasons for using silage additives to improve the aerobic stability of silage are to prevent heating of the silage, nutrient losses and reduced animal performance that may be caused by feeding spoiled silage.

Enzymes are often used in silage additives, but they do not inhibit yeasts and molds, which is why silages made with enzymes have very modest aerobic stability.

Organic acids, such as propionic, acetic and benzoic acids, are effective in improving the aerobic stability of silage. They are added either in large quantities to achieve the so-called final preservation of the feed, or in smaller quantities. In the latter case, the activity of yeasts is suppressed, but complete preservation is not ensured and ensiling continues to depend on natural fermentation. Ammonia has also been found to have an inhibitory effect on aerobic bacteria and yeasts and molds. Unfortunately, organic acids and other chemicals have a corrosive effect and damage silage equipment, and there are strict safety requirements for handling and storing them.

Biological silage additives based on lactic acid bacteria are considered natural products and have the advantage of being non-toxic, non-corrosive to equipment and non-hazardous to the environment.

The aim of lowering the pH of silage using lactic acid bacteria is to minimise fermentation losses. Lactic acid bacteria are divided into two groups based on glucose fermentation: homofermentative and heterofermentative. Homofermentative lactic acid bacteria produce two moles of lactic acid from one mole of glucose, while heterofermentative bacteria produce one mole of lactic acid, one mole of carbon dioxide and one mole of either ethanol or acetic acid. It is known that homofermentative species dominate at the beginning of the fermentation process, but later, as the environment becomes more acidic, heterofermentative bacteria gain the upper hand (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, but most of these bacterial starter cultures have little inhibition of yeast and mold growth. It may happen that when using such a silage additive, the aerobic stability of the silage is lower than in silage without the additive and may even increase the risk of silage heating.

Some silage starter cultures contain bacteria (e.g. propionic acid bacteria) that produce propionic acid. Unfortunately, the aerobic stability of the silage is not improved, as these microorganisms are generally not acid-tolerant and grow slowly. However, some starter cultures that produce large amounts of acetic acid in addition to lactic acid (L. buchneri) inhibit microorganisms (yeasts and

mold, etc.), i.e. they improve the aerobic stability of the silo and prevent the silo from spoiling when the storage is opened or when exposed to air.

The addition of heterofermentative lactic acid bacteria to silage lowers pH and reduces dry matter losses. In addition, some of these strains have been observed to have a strong inhibitory effect on the growth of yeasts and molds, thereby increasing the aerobic stability of 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 there are intraspecific differences, or strain-specific properties, due to genetic variations.

The aim of the present invention is to provide a new strain of Lactobacillus buchneri

Lactobacillus buchneri BioCC 228 DSM 32651 for improving the quality of feed fermentation, aerobic stability and shelf life of silage.

ESSENCE OF THE INVENTION

The invention relates to an isolated microorganism strain Lactobacillus buchneri BioCC 228 DSM 32651, a feed containing the aforementioned microorganism, a feed additive and a composition. The feed may be a fermented feed, e.g. silage. The feed additive is, for example, a silage additive. Other components of the composition may be necessary excipients. The said microorganism can be used in lyophilized form.

The microorganism Lactobacillus buchneri BioCC 228 DSM 32651 ensures aerobic stability of the feed.

This microbial strain is used to ferment feed and improve fermentation, increase the concentration of lactic acid and acetic acid in feed, lower pH, and thereby reduce nutrient losses in feed.

Based on antimicrobial studies, Lactobacillus buchneri BioCC 228 DSM 32651 suppresses the activity of undesirable microorganisms (pathogenic microorganisms, yeasts and molds). These enteropathogens include Staphylococcus aureus, Staphylococcus saprophyticus, Salmonella enterica subsp. enterica serovar Enteritidis, Enterococcus faecalis, Escherichia coli, etc.

The invention also relates to a method for extending the shelf life of feed, in which the microorganism Lactobacillus buchneri BioCC 228 DSM 32651 is added to the feed during fermentation. When using the said strain, its content is 1x105-1x106 cfu/g of fermentable feed.

STRAIN DESCRIPTION

The microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 was isolated naturally, i.e. from high-quality corn silage (Zea mays L.) fermented without additives in Estonia. To determine the quantitative lactobacillary composition of the silage sample, a suspension of decreasing density was made from the material in peptone water (Sigma-Aldrich, France) using the dilution series method and inoculations were performed on MRS (de Man Rogosa Sharpe) agar (Biolife, Italy), which was incubated at 37 degrees in a microaerobic environment (10 percent CO2) (thermostat "МСО-18А1С UV" Sanyo Electronic Co, Ltd, Japan) for 48 hours. The outgrown microbial colonies were described, counted and the total number of microbes was determined. To describe the morphology of the microbes, Gram-stained preparations were made and microscopically examined. The subject of the invention

The strain Lactobacillus buchneri BioCC 228 DSM 32651 was isolated based on the characteristic nest and cell morphology of Lactobacillus spp. A provisional and then more precise identification followed, which is described below.

The culture morphological characteristics of the strain Lactobacillus buchneri BioCC 228 DSM 32651 were determined after cultivation in MRS agar and broth medium (Biolife, Italy).

Lactobacillus buchneri BioCC 228 DSM 32651 is a non-spore-forming, non-motile Gram-positive rod bacterium with a regular shape. Its individual cells are usually arranged singly or in short chains. Elongated slender cells may be present when cultured in MRS broth.

Physiological-biochemical characteristics

The most suitable medium for culturing the microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 is MRS broth, which shows uniform turbid growth after 48 hours of microaerobic or anaerobic incubation at 37 degrees Celsius. In a microaerobic (10 percent CO2) or anaerobic (CO2/N2/H2: 5/90/5 percent) environment, 48-hour-old colonies are grayish-white, 1.5-2 millimeters, flat, shiny, translucent, with a rough texture and a raised center.

The optimal growth temperature of the microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 is 37 degrees, it also multiplies at 15 degrees, and grows slightly at 45 degrees. The optimal pH range for growing the strain is 5.7-6.2.

The microbial strain Lactobacillus buchneri BioCC 228 DSM 32651 is obligately heterofermentative, catalase and oxidase negative, hydrolyzes arginine and produces carbon dioxide during glucose fermentation.

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

Lactobacillus buchneri strain BioCC 228 was deposited for patent examination in the culture collection Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH on September 25, 2017 under the number DSM 32651, in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms.

Resistance to antibiotics

Methodology: The antibiotic susceptibility of Lactobacillus buchneri strain BioCC 228 DSM 32651 was tested under anaerobic (CO2/N2/H2: 5/90/5 percent) conditions at 37 degrees for 48 hours according to ISO10932: 2010 using VetMIC Lact-1 and VetMIC Lact-2 plates (SVA National Veterinary Institute, Uppsala, Sweden). The Minimum Inhibitory Concentrations (MICs) of the Lactobacillus buchneri strains were compared with the MIC breakpoints provided by the European Food Safety Authority (EFSA).

Table 1. Minimum inhibitory concentration (MIC) values (mg/L) of antibiotics by Lactobacillus bucneri BioCC 203 DSM 32650 and Lactobacillus bucneri BioCC 228 DSM 32651.

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

For the evaluation of microbes used as feed additives, strains are classified as susceptible or resistant to antimicrobial agents:

Susceptible (S): A bacterial strain is defined as susceptible if it is inhibited by a concentration of a specific antimicrobial agent equal to or less than the established breakpoint (S ≤ x mg/L).

Resistant (R): A bacterial strain is defined as resistant if it is not inhibited by a concentration of a specific antimicrobial agent higher than the established breakpoint (R> x mg/L).

The antibiotic susceptibility results of Lactobacillus buchneri strains BioCC 203 DSM 32650 and BioCC 228 DSM 32651 are presented in Table 1. The minimum inhibitory concentrations of Lactobacillus buchneri strains BioCC 203 DSM 32650 and BioCC 228 DSM 32651 did not exceed the MIC breakpoints for heterofermentative lactobacilli as reported by EFSA.

FUNCTIONAL PROPERTIES OF STRAINS

The aim of the experiment was to investigate the ability of the strain Lactobacillus buchneri BioCC 228 DSM 32651 to grow in the presence of various sugars.

Methodology: 24-hour-old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 grown on MRS (Biolife, Italy) agar

were suspended in peptone water according to McFarland density standard no. 5 (1.5 x 109 microbes/ml), inoculated at a final density of 5 x 105 microbes/ml into modified MRS broth containing 20 g/L of either glucose, fructose, trehalose, xylose, maltose or a mixture of glucose, fructose and trehalose in a ratio of 1:1:1, with a final concentration of sugars of 20g/L. The suspensions were incubated in a thermostat microaerobic (10 percent CO2) and anaerobically (CO2/N2/H2: 5/90/5 percent) at 25 degrees for 24, 48 and 72 hours. In the case of an anaerobic environment, the medium was reduced for 24 hours before the start of the experiment. During the experiment, the germinal number of the strains was determined, the yield, the number of generations (n) and the growth rate (V) were calculated as follows:

Yield = log N1- log N0 where N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0

n= log N1-log N0 / log 2, where N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0

V= log N1-log N0 / 0.301 x t, where worm N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0, and t represents a specific time.

The strain Lactobacillus buchneri BioCC 203 DSM 32650 grew one generation faster during the first 24 hours of microaerobic cultivation in a medium containing glucose, fructose, xylose, and a mixture of glucose, fructose, and trehalose compared to the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 2).

Table 2. Effect of different sugars on the growth dynamics of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 in microaerobic (10 percent C02) cultivation at 25 degrees for 24, 48 and 72 hours.

n-number of 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, Lactobacillus buchneri BioCC 203 DSM 32650 grew on average two generations faster in a medium containing a mixture of fructose and glucose, fructose and trehalose, and within 48 hours, it grew three generations faster in a medium containing glucose and ca. 1.5 generations faster in a medium containing fructose and xylose compared to the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 3).

Lactobacillus buchneri BioCC 228 DSM 32651 had slower growth, being able to outgrow the strain Lactobacillus buchneri BioCC 203 DSM 32650 after 48 hours of cultivation in a medium containing a mixture of fructose and xylose and glucose, fructose and trehalose.

Table 3. Effect of different sugars on Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651

growth dynamics and anaerobic (CO2/N2/H2: 5/90/5 percent) cultivation at 25 degrees for 24, 48 and 72 hours.

n-number of generations; V-growth rate; G-MRS broth with glucose; FHMRS 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. Profile of organic acids and alcohols

The aim of the experiment was to determine the organic acid and alcohol profile of the strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 when cultivated in microaerobic and anaerobic environments.

Methodology: 24-hour-old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 grown on MRS (Biolife, Italy) agar were suspended in peptone water according to McFarland density standard no. 5 (1.5 x 109 microbes/ml), inoculated into MRS (Biolife, Italy) broth at a final density of 1.5 x 106 microbes/ml and incubated microaerobically in a thermostat (10 percent CO2)

and anaerobically (CO2/N2/H2: 5/90/5 percent) at 25 degrees for 24, 48 and 72 hours.

The profile of organic acids and alcohols was determined by gas chromatography using an Agilent 6890A gas chromatograph, using a CP-Wax 52 CB capillary column (30 m x 0.25 mm, 0.25 pm). Column temperature program: 75 degrees for 1 minute, 10 degrees/min at 115 degrees held for 3 minutes, 20 degrees/min at 190 degrees held for 5 minutes, detector (FID) 280 degrees.

Organic acids were determined by liquid chromatography using a Shimadzu Prominence BPLC System, an ion separation column was used. Liquid chromatograph Aminex HPX-87H (300 mm x 7.8 mm). Column temperature was 60 degrees, flow rate was 0.6 ml/min and a PDA detector was used to detect organic acids at a wavelength of 210 nm. Analysis time was 26 min.

The profile of organic acids and alcohols clearly showed strain-specific properties of strains Lactobacillus buchneri BioCC 203 DSM 32650 and BioCC 228 DSM 32651 (Table 3). Lactobacillus buchneri BioCC 203 DSM 32650 is a significantly stronger producer of ethanol, acetic acid and lactic acid when cultivated in both microaerobic and anaerobic environments. Lactobacillus buchneri BioCC 228 DSM 32651 is capable of producing pyruvate in anaerobic environments (Table 4).

When cultivated in a microaerobic environment, the strain Lactobacillus buchneri BioCC 203 DSM 32650 consumed approximately 99.5 percent of the citrate present in the growth medium during the first 24 hours of cultivation, and in an anaerobic environment, 97.8 percent of the citrate present in the growth medium.

When cultivated in an anaerobic environment, Lactobacillus buchneri BioCC 228 DSM 32651 had consumed 4.8 percent of the citrate present in the growth medium. Unlike the strain Lactobacillus buchneri BioCC 203 DSM 32650, Lactobacillus buchneri BioCC 228 DSM 32651 had consumed 4.8 percent of the citrate present in the growth medium in a microaerobic environment.

When cultivated in the medium, it is capable of producing 5.9 percent citrate within 72 hours.

Table 4. Profile of organic acids and alcohols (mg/m) in MRS broth of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during microaerobic (10 percent CO2) and anaerobic (CO2/N2/H2: 5/90/5 percent) cultivation for 24, 48 and 72 hours at 25 degrees.

Example 2. Organic acid and alcohol profile in corn supernatant

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

Methodology: 226 g of maize plants (Zea mays L.) in the vegetative growth phase (V6-V8) were chopped, homogenized with water in a laboratory blender Bagmixer 400 (Interscience, France) for 6 minutes, filtered, centrifuged at room temperature at 5000 rpm for 10 minutes and sterilized at 121 degrees for 5 minutes.

24-hour-old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 grown on MRS agar (Biolife, Italy) were suspended

in peptone water according to McFarland density standard No. 5 (1.5 x 109 microbes/ml), the corn plant supernatant was seeded at a final density of 1.5 x 106 microbes/ml and incubated microaerobically (10 percent CO2) in a thermostat at 25 degrees for 24, 48 and 72 hours.

The profile of organic acids and alcohols was determined by gas chromatography using an Agilent 6890A gas chromatograph, using a CP-Wax 52 CB capillary column (30 m x 0.25 mm, 0.25 pm). Column temperature program: 75 degrees for 1 minute, 10 degrees/min at 115 degrees held for 3 minutes, 20 degrees/min at 190 degrees held for 5 minutes, detector (FID) 280 degrees.

Organic acids were determined by liquid chromatography using a Shimadzu Prominence HPLC System, using an ion separation column with a Liquid Chromatograph Aminex HPX-87H (300 mm x 7.8 mm). The column temperature was 60 degrees, the flow rate was 0.6 ml/min, and a PDA detector at a wavelength of 210 nm was used to detect organic acids. The analysis time was 26 min.

Table 5. Profile of organic acids and alcohols (mg/m) in the supernatant of corn plants during microaerobic cultivation of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 for 24, 48 and 72 hours at 25 degrees.

In a plant material fermentation experiment, Lactobacillus buchneri BioCC 203 DSM 32650 proved to be a stronger producer of ethanol and also lactic acid compared to Lactobacillus buchneri BioCC 228 DSM 32651 (Table 5).

Example 3. Antimicrobial activity against pathogens.

The aim of the experiment was to test the antimicrobial activity of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 against enteropathogens when cultivated in microaerobic and anaerobic environments at 25 degrees.

The line culture method was used to evaluate the antimicrobial properties of the lactobacillus strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 against pathogens (Hütt P, Shchepetova J, Loivukene K, Kullisaar T, Mikelsaar M. Antagonistic activity of probiotic lactobacilli and bifidobacteria against entero- and uropathogens. J Appl Microbiol. 2006; 100(6):1324-32).

To determine the inhibition of target microbes, the pathogen growth-free zone was measured in millimeters. Analogously to Hütt et al. (2006), the arithmetic mean and standard error were calculated based on the results of the sample used and the antagonistic activity of the strains was assessed based on this.

Table 6. Antimicrobial activity of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 against pathogens on modified MRS agar medium by the streak plate method (target microbe growth inhibition in millimeters) in microaerobic (10 percent CO2) and anaerobic (CO2/N2/H2: 5/90/5 percent) environments.

Zone of inhibition in microaerobic environment (mm-s): weak 17.12. Zone of inhibition in anaerobic environment (mm-s): weak 14.95.

In a microaerobic environment, both strains had equally strong antimicrobial activity (Table 6). In an anaerobic environment, Lactobacillus buchneri BioCC 203 DSM 32650 had somewhat stronger activity against the tested pathogens.

Example 4. Antifungal activity.

The aim of the experiment was to evaluate the effect of the supernatant of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 against yeasts isolated from corn silage using the hole-diffusion method on agar medium.

A 48-hour-old lactobacillus culture was suspended in peptone water according to McFarland density standard no. 5 (1.5 x 109 microbes/ml), inoculated into MRS broth (Biolife, Italy) at a final density of 1.5 x 106 microbes/ml and incubated microaerobic (10 percent CO2) and anaerobically (CO2/N2/H2: 5/90/5 percent) at 25 degrees for 48 and 72 hours. Microbial cells were removed

by centrifugation (4500rpm, 10 min). The supernatant was sterilized by filtration and concentrated by lyophilization. The lyophilizate was resuspended in 10-fold 10 mM acetic acid. Six wild yeast strains isolated from corn silage were plated as a single layer on PCA medium (Plate Count Agar; Liofilchem srl, Italy). 6-millimeter holes were cut sterilely in the medium, into which 100 μΐ of sterile supernatant was added. After incubation at 25 degrees, the width of the growth inhibition zone was evaluated as follows: - -no effect, + weak effect, yeast growth disturbed, ++ yeast growth inhibited, clear inhibition zone visible, +++ yeast growth strongly inhibited, wide clear inhibition zone visible.

Antimicrobial compounds produced by Lactobacillus buchneri BioCC 228 DSM 32651 inhibit the growth of plant-derived yeasts more strongly compared to the strain Lactobacillus buchneri BioCC 203 DSM 32650. Lactobacillus buchneri BioCC 228 DSM 32651 produced compounds that strongly inhibited yeast growth within 48 hours, creating a wide, clear zone of inhibition on the agar medium around the well containing the supernatant, while the supernatant of strain BioCC 203 only interfered with the growth of yeasts.

Example 5. Growth dynamics during fermentation of plant material

The aim of the experiment was to investigate the growth dynamics of the strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 during the fermentation of plant material.

Methodology: 226 g of maize plants (Zea mays L.) in the vegetative growth phase (V6-V8) were chopped, homogenized with water in a laboratory blender Bagmixer 400 (Interscience, France) for 6 minutes, filtered, centrifuged at room temperature at 5000

rpm for 10 minutes and sterilized at 121 degrees for 5 minutes.

24-hour-old cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 grown on MRS agar (Biolife, Italy) were suspended in peptone water according to McFarland density standard no. 5 (1.5 x 109 microbes/ml), inoculated into corn plant supernatant at a final density of 1.5 x 106 microbes/ml and incubated microaerobically (10 percent CO2) in a thermostat at 25 degrees for 24, 48 and 72 hours.

During the experiment, the germinal number of the strains was determined and the yield, number of generations (n) and growth rate (V) were calculated as follows:

Yield = log N1 - log N0 where N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0

n= log N1-log N0 / log 2, where N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0

V= log N1-log N0 / 0.301 x t, where N1 is the number of bacteria at some point in time; N0 is the number of bacteria at time 0, and t represents a specific time.

Table 7. Growth dynamics of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 in microaerobic (10 percent CO2) cultivation at 25 degrees for 24, 48 and 72 hours.

n-number of generations; V-growth rate;

When cultivated in corn supernatant in a microaerobic environment, the strain Lactobacillus buchneri BioCC 203 DSM 32650 grew four generations faster in the first 24 hours and 2.4 generations faster in 48 hours compared to the strain Lactobacillus buchneri BioCC 228 DSM 32651 (Table 7).

Example 6. Investigation of the effect of silage additives L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 on easily ensiled plant material.

The aim of the experiment was to evaluate the effect of the silage additives L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 on the fermentation quality and aerobic stability of silage (dry matter content ≥ 30 percent) made from maize (Zea mays, maize variety "Cathy").

The experiment was conducted with green waxy corn chopped in 1.5 liter laboratory silo containers.

The following studies were carried out: Determination of pH level and fermentation quality at 90-1 day. Two aerobic stability tests were carried out. The first test was performed after a 49-day storage period with two aerobic stresses (24 hours apart, on day 28 and day 42). The aerobic stability test was carried out in a temperature-controlled room at approximately 20 degrees. The temperatures were recorded every 4 hours using the PS-ES Datalogging system.

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

Table 8. Chemical composition of the starting material

* DM percentage + (8x (WSC/BC))

The addition of L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 caused a significant increase in acetic acid and 1,2-propanediol compared to the control silage (Table 9).

Table 9. Chemical composition, nutritional value and fermentation quality indicators of silages prepared from the "Cathy" corn variety using the microorganisms L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 after 90 days of storage

n.d.- not determined

In an aerobic stability test conducted after a 49-day storage period, silages prepared with L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 were significantly more stable (by approximately 2 to 2.5 days) compared to the control silage, control: 3.9 days vs. L. buchneri BioCC 203 DSM 32650: 6.3 days and L. buchneri BioCC 228 DSM 32651: 5.8 days.

When the storage time was extended to 90 days, aerobic stability increased by 7.9 days in the control silage, 10.6 days in the silage prepared with L. buchneri BioCC 203 DSM 32650, and 11.4 days in the silage prepared with L. buchneri BioCC 228 DSM 32651. The 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 statistically significant.

Example 7. Investigation of the effect of silage additives L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 on difficult-to-ensilage material.

The aim of the experiment was to evaluate the microorganism isolated from whole-harvested low dry matter (≤ 20 percent) maize (Zea mays, maize variety 'Dorka') using the strains L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651

fermentation quality and aerobic stability of the prepared silage.

Strains L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 were added to the ensiled material as an aqueous solution at a concentration of 1x105 cfu/g of ensiled plant material (feed). All experimental variants (control silages, silages prepared with lactic acid bacteria strains L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 and control silages prepared without silage preservative) were prepared in five replicates. The experimental silos were opened after 90 days of ensiling. The experimental silos were opened after 90 days of ensiling. The chemical composition of the starting material is presented in Table 10.

The aerobic stability test of the silage was carried out after 90 days of storage using 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). The silage was considered aerobically unstable if the temperature measured at its geometric center exceeded the ambient temperature by 3 degrees. Temperature changes over time were measured for 9 days (216 hours). The room (ambient temperature) and test silo temperatures were recorded every hour using Comet Temperature Data Logger S0141.

Silage samples were analyzed according to generally accepted methodologies (AOAC.

2005. Official methods of analysis of AOAC International, 18th ed. Association of Official Analytical Chemists International, Gaithersburg, MD, USA).

To determine the dry matter content, the silage sample was dried in a thermostat at 130 degrees to a constant weight. To determine the crude ash content, the silage sample was burned in a muffle furnace for six hours at a temperature of 550 degrees. The protein content was determined using the Kjeltec™ 2300 analyzer using the Kjeldhal method (Nx6.25). Crude fiber was determined using the W. Henneberg and F. Stohmann methodology.

The acid and ethanol content of the silage was determined using an Agilent 7890A gas chromatograph. The ammonia nitrogen content of the total nitrogen was determined using a Kjeltec™ 2300 analyzer. The acidity of the silage was determined using a Hanna Instruments HI 2210 pH meter.

Table 10. Chemical composition of the starting material

All silages had a dry matter content of ≤18 % (Table 11). However, silages prepared from maize cultivar 'Dorka' and L. buchneri strain BioCC 203 DSM 32650 or L. buchneri strain BioCC 228 DSM 32651 had good fermentation parameters (Table 11). Lactic acid was the dominant acid in all silages. Silages prepared with L. buchneri strain BioCC 203 DSM 32650 had higher acetic acid and 1,2-propanediol concentrations compared to silage prepared with L. buchneri strain BioCC 228 DSM 32651 and the control silage. Ethanol content in all silages was low (4.1 to 8.6 g/kg).

Table 11. Chemical composition, nutritional value and bioavailability of silages prepared from the maize variety 'Dorka' using the microorganisms L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651

fermentation quality indicators after 90 days of storage Microbiological indicators of fermentation quality of silage and corn silage are presented in Table 12. The amounts of molds were relatively high in the silage, the levels of clostridia and yeasts were below the smear limit. Silage samples prepared with the strain L. buchneri BioCC 203 DSM 32650 or L. buchnerl BioCC 228 DSM 32651 contained very high amounts of lactic acid bacteria (>8.0 log10 pmu / g silage) and the added strains dominated the endogenous lactic acid bacteria in the silage samples. The amount of lactic acid bacteria in the control silage was 4.56 log10 pmu / g silage.

Table 12. Microbiological indicators of fermentation quality in the starting material and in silage samples prepared from the corn variety Dorka using L. buchneri BioCC 203 DSM 32650 or L. buchneri BioCC 228 DSM 32651

*- below the limit of determination

**- not calculable

In the aerobic stability test of the silage, four out of five control silos became hot. The average aerobic stability was 149 hours (i.e. 6.2 days). Silages prepared with strains L. buchneri BioCC 203 DSM 32650 and L. buchneri BioCC 228 DSM 32651 were aerobically stable until the end of the test, i.e. 217 h (i.e.

9.04 days). Thus, the aerobic stability of silage prepared from the starting material with strains L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 was extended by 2.84 days.

In conclusion. The strains L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 proved to be very vigorous starter cultures in silage made from difficult-to-ensilage material. The use of L. buchneri BioCC 203 DSM 32650 and L.buchneri BioCC 228 DSM 32651 increased the lactic acid content in silage with a low dry matter content (≤ 20 percent), inactivated microbial and yeast activity, protected the silo from heating and thereby improved the aerobic stability of the silo after opening, extending the storage life of the silo.

Claims (13)

1. Isolated microorganism strain Lactobacillus buchneri BioCC 228 DSM 32651. 2. The microorganism according to claim 1 in lyophilized form. 3. A composition comprising the microorganism according to claim 1 or 2. 4. Feed containing any strain of microorganism according to claims 1-2. 5. Feed according to claim 4, which is fermented feed, e.g. silage. 6. Use of any microorganism according to claim 1 - 2 as a feed additive. 7. Use of any microorganism according to claim 1 - 2 to ensure aerobic stability of silage. 8. Use of the microorganism according to claim 7, wherein the silage is made from feed with a low dry matter content (≤ 20 percent). 9. Use of a microorganism according to any of claims 1-2 for fermenting feed. 10. A composition containing one or more microorganisms according to any of claims 1-2 for use in improving feed fermentation, increasing the concentration of lactic acid and acetic acid in the feed, lowering the pH and thereby reducing nutrient losses in the feed. 11. Use of a microorganism according to any one of claims 1-2 in the fermentation of feed, for suppressing the action of pathogenic microbes and inhibiting the growth of yeasts, which consists in adding a microorganism according to any one of claims 1-2 to the feed to be fermented. 12. Use of the microorganism according to claim 11, wherein the pathogenic microbes are enteropathogens. 13. A method for extending the shelf life of feed, wherein a microorganism according to any of claims 1-2 is added to the feed before fermentation.