JP7250783B2 - Microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 and uses thereof - Google Patents

Description

DSMZ DSM32650 DSMZ DSM32651

The present invention belongs to the field of biotechnology and is applied to feed production. The present invention encompasses a microbiological silage additive and its use in feed fermentation to increase fermentation quality and feed quality.

It is necessary to maintain the nutrient content of silage from harvested and stored forage until consumption of the forage by animals. Silage is material produced by controlled fermentation of high moisture content crops (see, for example, Non-Patent Document 1).

Silo storage is a storage method in which plant-based animal feed is subjected to lactic acid fermentation under anaerobic conditions (see, for example, Non-Patent Document 2). Silage fermentation can be divided into four stages. (1) post-harvest anaerobic stage in the silo, (2) fermentation stage, (3) stable storage stage, (4) silage unloading stage, where the silo is opened and the silage is exposed to air. exposed to In order to produce high quality silage, siled material must undergo correct microbial fermentation. The success of the fermentation also depends on the type and quality of the foliage plants, the technology used for silage storage, the climate, the development of undesirable microorganisms (clostridium, listeria, bacillus) and mold (yeasts and fungi), the silage stored. It also depends on the dry matter content of the material used.

Fermentation of silage is a complex combination of multiple different chemical and microbiological processes and their interactions, making it difficult to control spontaneous fermentation of feed.

Most silage is 200. . . Produced with a dry matter content of 500 g/kg. At such levels many plant enzymes are activated during silage storage, and a large number of desirable and undesirable microorganisms, yeasts and fungi grow in silage under such conditions. can do Therefore, controlling the activity of all microorganisms presents a formidable challenge and can only be achieved by means of well-controlled silage storage methods (3).

In controlled silo storage methods, water-soluble carbohydrates are fermented to lactic acid by lactic acid bacteria. As a result, the pH level of the material drops in silo storage (silo storage is acidic), which in turn inhibits the activity of spoilage microorganisms (4). The sooner the silage is lowered to an acidic pH of 4, the sooner enzymatic and microbiological activity ceases, the feed is stabilized, and more nutrients are preserved.

It has been reported that the quality of silage fermentation can be significantly improved by an additive containing lactic acid bacteria (Non-Patent Document 1).

Just as nutrient preservation is important during the fermentation and storage stages of silage, so is nutrient preservation in silage that has been opened from the silo. The silage can be exposed to oxygen in both cases when the silo is opened during feeding and due to inadequate covering of the silo.

Any silage exposed to air will sooner or later spoil by aerobic microorganisms. The aerobic stability of silage depends on the silage grain stored in silage, the growth phase at harvest, the biochemical and microbiological factors of fermentation, the physical characteristics of the silage material, the composition of silage management, temperature, and Depends on the choice of silage additive. An indicator of silage's aerobic stability is the length of time that silage can resist aerobic spoilage (eg, the length of time it retains its quality when exposed to air). The aerobic stability of silage is estimated based on the rate at which the temperature of the silage increases. The longer the silage is stable, for example, the longer it does not rise above 3°C in ambient temperature (European Union Commission Regulation (EC) No. 429/2008 of 25 April 2008). DLG-Richitlneienfur die Prufung von Siliermitten auf DLG-Gutezeichen-Faghigkeit, DLG Oktober 2013), aerobic stability and silage quality are excellent. In most aerobic perishable silage, the temperature rises above ambient due to microbial oxidation of acids and water-soluble carbohydrates to carbon dioxide and water.

Low pH levels in silage inhibit anaerobic undesirable microbial growth, but low pH by itself is not sufficient to prevent aerobic spoilage. Silage spoilage under aerobic conditions is often initiated by yeast, which can grow even at relatively low pH. Yeast can grow in a wide pH range (pH 3.....8). The preferred pH for most yeast growth is 3.5. . . . 6.5. When the silage is opened and the silage is exposed to air, the acids and other compounds formed during fermentation are oxidized by aerobic microorganisms, yeast and fungi. Yeast activity results in carbon dioxide production and heating of the silage, which in turn directly contributes to dry matter reduction (1).

Yeast uses the residual sugars contained in the silage as an energy source. However, their first preference is lactic acid. As a result, well-fermented silage with high lactic acid content is prone to spoilage, especially under aerobic conditions. Yeast activity raises the pH level of the silage and activates many other aerobic microorganisms and fungi. High microbial activity in well-fermented and nutritious silage revealed by increasing silage temperature

Upon opening the silage, the dry matter, acetic and propionic acid contents, and the number of yeast and fungi in the silage are important factors in determining the aerobic stability of the silage (Non-Patent Document 5). A negative relationship for dry matter and yeast indicated that very high concentrations resulted in a rapid increase in silage temperature upon exposure to air. Acetate and butyrate, on the other hand, very high concentrations of these fermentation products are associated with more stable silage.

As mentioned, the low pH level of silage has no direct effect on microbes that cause aerobic spoilage. However, the acids produced during fermentation of silage are useful and important. Yeast growth is inhibited by non-dissociable short-chain fatty acids (Non-Patent Document 6). Non-dissociable acid molecules can penetrate the cell membrane of microorganisms by passive diffusion, releasing H ⁺ ions. This lowers the intracellular pH level and kills the cells. The diffusion rate of acid in silage depends on the dissociation coefficient (pKa) of the acid and the pH level of the silage (Non-Patent Document 7). Acetic and propionic acids tend to be less dissociated than lactic acid, which explains why good fermented silage with high lactic acid content is susceptible to spoilage under aerobic conditions. On the other hand, acetic acid and propionic acid effectively inhibit the growth of yeast and fungi. Butyric acid has a similar effect. Silage with high butyric acid content has good aerobic stability. However, this indicates the activity of Clostridium spp. causing putrefaction. Such silage exhibits significant nutrient losses and the high butyric acid content causes animal health problems. Propionic acid content in silage is rare and low. In silage grains, the concentrations of the micro-organisms that produce them are low and their competition is weak.

Acetic acid content in silage is an indicator of heterologous fermentation. Because acetic acid is highly toxic to yeast, such silage typically exhibits very high aerobic stability.

An ideal fermentation of silage would reduce fermentation losses and ensure adequate stability during storage of the feed and unloading of the feed from the silo for feeding. Effective silage additives and suitable composition of the product and use of silage play an important role in achieving these objectives. Most silage additives have been developed to improve the silage process and the nutritional value of the siled feed.

However, in addition to ensuring rapid fermentation and improving silage quality, silage additives are also expected to inhibit the growth of spoilage (including aerobic spoilage) organisms. The primary reason for using silage additives to improve the aerobic stability of silage is to prevent animal deterioration due to silage heating, nutrient loss, and consumption of spoiled silage.

Silage additives often use enzymes. However, these represent enzyme-conditioned silages with very mild aerobic stability that do not inhibit yeast or fungi. Organic acids such as propionic acid, acetic acid and benzoic acid are effective in improving the aerobic stability of silage. These are added in large or small amounts to achieve the so-called final conservation of the feed. In the latter case, yeast activity is inhibited, but ultimate conservation is not achieved, and silage storage that relies on natural fermentation continues. Ammonia has been reported to have an inhibitory effect on bacteria, yeast and fungi. Unfortunately, organic acids and other compounds are aggressive and strict safety requirements apply to their handling and storage, requiring silage equipment.

Biological silage additives based on lactic acid bacteria are considered natural products. These advantages include lack of toxicity, lack of perishability to equipment, and lack of environmental risks.

The purpose of lowering the pH level of silage due to lactic acid bacteria is to minimize fermentation losses. Lactic acid bacteria are divided into two groups based on glucose metabolism: homofermentative and heterofermentative. A homofermentative species produces 2 mol of lactic acid from 1 mol of glucose, while a heterofermentative species produces 1 mol of lactic acid, 1 mol of carbon dioxide and 1 mol of ethanol or acetic acid. Early in the fermentation process, homofermentative species predominate, but later, as the environment becomes more acidic, heterologous fermentative species become more common (3).

Silage additives based on homofermentative lactic acid bacteria improve the fermentation process of silage. However, most such starter fungi do not inhibit yeast and fungal growth. With the use of such silage additives, the aerobic stability of the silage is lower than without the silage additive and may also increase the risk of heating the silage.

Silage starters produce propionic acid-producing bacteria (eg, propionibacteria). Unfortunately, this does not improve the aerobic stability of the silage. This is because these microorganisms are typically acid tolerant and their growth is slow. However, a starter (L. buchneri) that produces a lot of acetic acid in addition to lactic acid inhibits microorganisms (yeasts, fungi, etc.) that cause aerobic spoilage of silage, i.e., they affect aerobic silage stability. and prevent spoilage of silage by opening silos or other types of exposure to air.

Addition of heterofermentative lactic acid bacteria during the silo storage process lowers the pH level and reduces dry matter loss. In addition, some of these strains have been reported to have a strong inhibitory effect on yeast and fungal growth, thereby improving the aerobic stability of silage (8). . However, different strains of the same species do not have the same properties due to differences between genetic diversity species, ie the occurrence of fungus-specific properties.

McDonald, P.; , Henderson, A.; R. , Heron, S.; J. E. 1991. The biochemistry of silage. 2nd ed. Chalcombe Publications, Marlow, Bucks UK, p. 340 Rooke, J.; , A. and Hatfield, G.; , D. , 2003. Biochemistry of Engineering. In: Silage science and Technology. D. R. Buxton, R.; E. Muck, andJ. H. Harrison, eds. American Society of Agronomy, Madison, Wisconsin, USA. pp. 95-139 Muck, R.; E. 2010. Silage microbiology and its control through additives. R. Bras. Zootec. vol. 39. July Oude Elferink, S.; J. W. H. , Driehuis, F.; , Gottshal, J, C.; , Spoelstra, S.; F. 2000. Silage fermentation processes and their manipulation. -Journal FAO Plant Production No. 161, pp 17-30 Ohyama, Y.; , Hara, S.; and Masaki, S.; (1980) Analysis of the factors affecting aerobic determination of gas silages. In Thomas, C.I. (ed.) Forage conservation in the 80s. BGS Occasional Symposium No. 11, pp257-261. reading, UK: British Grassland Society) Pahlow, G.; , MuckR. E. , Driehuis F.; , Oude Elferink S.; J. W. H. and Spoolstra S.; F. (2003) Microbiology of engineering. In: BuxtonD. R. MuckR. E. and HarrisonJ. H. (eds.) Silage science and technology, pp31-93). Madison, Wis., USA: Agronomy Publication No. 42, American Society of Agronomy Zirchrom (2011) Dissociation constant of organic acids and bases. Available at: http://www.zirchrom. com/organic. htm (accessed 3 November 2011) Jatkauskas, J.; , Vrotniakiene, V.; , Ohlsson, C.; , Lund, B.; 2013. The effect of three silage inculants on aerobic stability in glass, clover-grass, lucerne and maize silage. Agricultural and Food Science. 22:137-144 Hutt P. Shcheptova J.; Loivukene K. Kullisaar T. Mikelsaar M. Antagonistic Activity of probiotic lactobacilli and biofidobacteria against entro- and uropahogens. J Appl. Microbiol. 2006;100(6):1324-32)

It is an object of the present invention to provide novel strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 that improve the quality of feed fermentation and increase the aerobic stability and shelf life of silage. be.

The present invention discloses isolated microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651, and feeds, feed additives and compositions comprising one or both of said strains. . The feed may be fermented feed, such as silage. The feed additive can be a silage additive. Suitable excipients may be included as other ingredients in the composition of the additive. Said microorganisms can be used in lyophilized form.

The microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 are suitable for use in feeds, including feeds that are difficult to ferment, such as feeds with low (≦20%) dry matter content. Ensures stability under temper.

The next object of the present invention is to accelerate the fermentation of the feed, increase the concentration of lactic acid, lower the pH and the loss of nutrients in the feed and the concentration of ammonia nitrogen and butyric acid in the feed. is to reduce

Said microorganisms (together or separately) are used to ferment the feed and to improve the fermentation, to increase the concentration of lactic and acetic acid in the feed, to lower the pH, thereby reducing the Decrease loss of nutrients.

Based on investigations of antimicrobial properties, the microbial strains Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchnei BioCC 228 DSM 32651 inhibit the growth and action of undesirable microorganisms (pathogenic microorganisms, yeasts and fungi). The enteropathogenic bacteria include Staphylococcus aureus, Staphylococcus saprophyticus, Salmonella enterica subsp. Escherichia coli), and the like.

The present invention also relates to a method of prolonging the storage of feed by adding one or both of the microorganism strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 to the feed during fermentation.

When using one of the above strains, it is added to the feed at a rate of 1×10 ⁵ to 1×10 ⁶ CFU/g of fermented feed.

(Description of the strain)
The microbial strains Lactobacillus buhneri BioCC 203 DSM32650 and Lactobacillus buhneri BioCC 228 DSM32651 were obtained from naturally siled high quality corn (Zea mays L.) silage in Estonia (without the use of silage additives). Isolated. To determine the quantitative content of lactobacilli in silage samples, suspensions of the solution (with decreasing concentrations) were made using the small dilution method in peptone water (Sigma-Aldrich, France). , MRS (de Man Rogosa Sharp) agar (Biolife Italy), which was incubated in a microaerobic environment (10% CO ₂ ) at 37° C. for 48 hours (thermostat “MCO-18AIC UV” Sanyo Electric Co., Ltd.). ,Japan). Colonies that arose were described, counted, and total microbial counts were determined. To delineate microbial morphology, specimens were prepared using Gram staining and examined microscopically. Based on the colony and cell morphology characteristic of Lactobacillus spp, the microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 were isolated. This was followed by preliminary and detailed identifications, which are described below.

The morphological characteristics of cultures of the microbial strains Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 were determined after growth on MRS agar and broth (Biolife Italy).

Lactobacillus buchneri BioCC 203 DSM32650 is a solitary, short-chain, Gram-positive, bacillus-like, non-motile, non-spore-forming bacterium. It exists in elongated cells during culture in MRS medium.

Lactobacillus buchneri BioCC 228 DSM32651 is a solitary, short-chain, Gram-positive, bacillus-like, non-motile, non-spore-forming bacterium. It exists in long and elongated cells during culture in MRS medium.

(physiological-biochemical properties)
MRS cultures (48-72 hours) are suitable for culturing the microorganism strain Lactobacillus buchneri BioCC 203 DSM32650 under microaerobic or anaerobic conditions, after which homogeneous turbid growth occurs. break out. After culturing at 37° C. for 48 hours in a microaerobic environment (10% CO ₂ ) or an anaerobic environment (CO ₂ /N ₂ /H ₂ : 5/90/5%), the colonies are greyish white, 1.5-2 mm, flat, glossy, transparent, rough, with projections.

The microbial strain Lactobacillus buchneri BioCC 203 DSM32650 is obligately heterofermentative, catalase- and oxidase-negative, hydrolyzes arginine and produces carbon dioxide during glucose fermentation.

The optimal growth temperature for the microbial strain Lactobacillus buchneri BioCC 203 DSM32650 strain is 37°C and the strain also replicates at 15°C. It was confirmed that replication was possible to some extent even at 45°C. The optimum pH for fungal growth is 5.7-6.2.

MRS cultures (48-72 hours) are suitable for culturing the microorganism strain Lactobacillus buchneri BioCC 2228 DSM32651 under microaerobic conditions, followed by homogenous turbid growth. After culturing at 37° C. for 48 hours in a microaerobic environment (10% CO ₂ ) or an anaerobic environment (CO ₂ /N ₂ /H ₂ : 5/90/5%), the colonies are greyish white, 1.5-2 mm, flat, glossy, transparent, rough, with projections.

The microbial strain Lactobacillus buchneri BioCC 228 DSM32651 is obligately heterofermentative, catalase- and oxidase-negative, hydrolyzes arginine and produces carbon dioxide during glucose fermentation.

The optimal growth temperature for the microbial strain Lactobacillus buchneri BioCC 228 DSM32651 strain is 37°C and the strain also replicates at 15°C. It was confirmed that replication was possible to some extent even at 45°C. The optimum pH for fungal growth is 5.7-6.2.

Microbial strain Lactobacillus buchneri BioCC 203 DSM32650 was identified as Lactobacillus buchner based on biochemical activity using a MALDI Biotyper (Bruker Daltonik).

The microbial strain Lactobacillus buchneri BioCC 228 DSM32651 was identified as Lactobacillus buchneri based on biochemical activity using a MALDI Biotyper (Bruker Daltonik).

The microorganism strain Lactobacillus buchneri BioCC 203 has been deposited on September 25, 2017 under the number DSM 32650 with Mikrooganismen and Deutshe Sammlung of Zellkulturen GmbH according to the "Deposit of Microorganisms for the Purpose of Patent Procedure" under the Budapest Treaty.
DSMZ address: Inhoffenstr. 7B, D-38124 Braunschweig, Germany

The microorganism strain Lactobacillus buchneri BioCC 228 has been deposited on September 25, 2017 under the number DSM 32651 with Mikrooganismen and Deutshe Sammlung of Zellkulturen GmbH according to the "Deposit of Microorganisms for the Purposes of Patent Procedure" under the Budapest Treaty.
DSMZ address: Inhoffenstr. 7B, D-38124 Braunschweig, Germany

(antibiotic resistance)
Method: The susceptibility of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 to antibiotics was determined under anaerobic conditions (CO2/ _N2 / _H2 : 5/90/5%) at ₊ 37°C. Analyzed according to ISO 10932:2010 standard on VetMIC Lact-1, VetMIC Lact-2 plates (SVA National Veterinary Institute, Uppsala, Sweden) for 48 hours. Minimum inhibitory concentrations (MICs) were compared to MIC cutoff values reported by EFSA.

For evaluating bacteria for use as feed additives, strains can be classified as susceptible or antimicrobial.
Susceptibility (S): A bacterial strain is defined as susceptible if its growth is inhibited at concentrations that are either equal to or below the cut-off value (S≤xmg/L) for a particular antimicrobial. be.

Resistance (R): A bacterial strain is defined as resistant if its growth is not inhibited at concentrations above the cut-off value (R>xmg/L) for a particular antimicrobial.

Table 1 shows the antibiotic sensitivity results of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 strains. Lactobacillus buchneri BioCC 203 DSM32650 and BioCC 228 DSM32651 strains do not exceed the MIC cut-off value proposed for obligately heterofermentative lactobacilli proposed by the EFST.

Functional characteristics of strains (growth in the presence of various sugars)
The purpose of this experiment was to investigate the ability of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 strains to grow in the presence of various sugars and to acidify the medium.

Methods: Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 cultured on MRS agar were prepared according to McFarland Tobidity Standard No. 5 (1.5×10 ⁹ microorganisms/ml) in peptone water and added to modified MRS broth (glucose, fructose, trehalose, xylose or maltose, or glucose, fructose, trehalose (ratio, 1:1:1). ) were inoculated at a final concentration of 1.5×10 ⁶ microbes/ml, also at a final concentration of 20 g/L.

The suspension was incubated in a thermostat microaerobically (10% _CO2 ) or anaerobic (CO2/ _{N2 / H2} : 5/90/5%) at 25°C for 24, 48 and 72 hours. incubated. Under the anaerobic environment, the medium was previously reduced for 24 hours. A possible number of both strains was registered. Yield, generation number (n), and growth rate (V) were calculated as follows.

Yield = logN ₁ -logN ₀ , where N ₁ is the cell concentration at a given time and N ₀ is the initial cell concentration.
n=log N ₁ -log _{N 0} /log 2, where N ₁ is the cell concentration at a given time and N ₀ is the initial cell concentration.
V=logN ₁ -logN ₀ /0.301×t, w, N ₁ is the cell concentration at a given time, N ₀ is the initial cell concentration, t is the time at a particular epoch (h).

The growth of Lactobacillus buchneri strain BioCC 203 DSM32650 during the first 24 hours in microbial culture in a culture medium containing glucose, fructose, xylose or a mixture of glucose, fructose and trehalose was higher than that of Lactobacillus buchneri strain BioCC 228 DSM32651. One generation ahead of growth (Table 2).

Compared to Lactobacillus buchneri strain BioCC 228 DSM32651, during the first 24 hours of anaerobic culture, Lactobacillus buchneri strain BioCC 203 DSM32650 showed an average of 2 Generations early, within 48 hours of anaerobic culture, 3 generations earlier in media containing glucose and approximately 1.5 generations earlier in media containing fructose and xylose (Table 3).

Lactobacillus buchneri strain BioCC 228 DSM32651 grows slowly and outperforms Lactobacillus buchneri strain BioCC 203 DSM32650 after 48 hours of culture in media containing fructose or xylose and media containing a mixture of glucose, fructose and trehalose. can do

Example 1 Profile of Organic Acids and Alcohols The purpose of this experiment was to examine the organic acids and is to determine the alcohol profile.

Method: Lactobacillus buchneri BioCC 203 and Lactobacillus buchneri BioCC 228 cultures after 24 hours on MRS agar (Biolife Italy) were plated on a McFarland Tobidity standard no. 5 (1.5×10 ⁹ microorganisms/ml) in peptone water, inoculated in MRS culture medium (Biolife Italy) at a final concentration of 1.5×10 ⁶ microorganisms/ml, and incubated under microaerobic conditions ( 10% CO ₂ ) and anaerobic (CO ₂ /N ₂ /H ₂ : 5/90/5%) in a thermostat at 25° C. for 24, 48 and 72 hours.

Organic acid and alcohol profiles were determined by gas chromatography on a Gas Chromatograph 6890A capillary column CP-Wax 52CB (30 m x 0.25 mm 0.25 µm). Column temperature program 75 degrees 1 minute hold. 10 degrees/minute to 115 degrees and hold for 3 minutes. 20 degrees/minute to 190 degrees and hold for 5 minutes. Detector (FID) 280 degrees.

Organic acids were determined by liquid chromatography on a Shimadzu Prominence HPLC system. Samples were separated on an Aminex HPX-87H ion exchange column (300 mm x 7.8 mm). The column temperature was maintained at 60 degrees, the flow rate was 0.6 ml/min and organic acids were detected by a PDA detector at 210 nm. Analysis time was 26 minutes.

In the organic acid and alcohol profiles, strain-specific features of Lactobacillus buchneri BioCC 203 DSM32650 and BioCC 228 DSM32651 strains were revealed (Table 4). Lactobacillus buchneri BioCC 203 DSM32650 produced significantly more ethanol, acetic acid and lactic acid during cultivation under both microaerobic and anaerobic conditions. Lactobacillus buchneri BioCC 228 DSM32651 was able to produce pyruvate in an anaerobic environment (Table 4).

During the first 24 hours of microaerobic and anaerobic culture, Lactobacillus buchneri BioCC 203 DSM32650 strain uses approximately 99.5% and 97.8% of the citric acid originally present in the culture medium, respectively. bottom.

During anaerobic cultivation, the Lactobacillus buchneri BioCC 228 DSM32651 strain consumed approximately 4.8% of the citric acid originally present in the culture medium. Unlike Lactobacillus buchneri strain BioCC 203 DSM32650, Lactobacillus buchneri strain BioCC 228 DSM32651 produced 5.9% citric acid during 72 hours of microaerobic culture.

Example 2 Profile of Organic Acids and Alcohols in Supernatants of Corn Plants and to determine the alcohol profile.

Method: 226 g of corn (Zea mays L.) plants at the vegetable growth stage (V6-V8) were crushed, watered, homogenized in a laboratory blender Bagmixer 400 (Interscience, France) for 6 minutes, filtered and centrifuged. Separation (10 minutes at 5000 rpm at ambient temperature) and sterilization at 121 degrees for 5 minutes.

After 24 hours on MRS agar (Biolife Italy) cultures of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 were plated on McFarland Tobidity standard no. 5 (1.5×10 ⁹ microbes/ml) in peptone water and inoculated into the supernatant of corn plants at a final concentration of 1.5×10 ⁶ microbes/ml, under microaerobic conditions. (10% CO ₂ ) and anaerobic (CO ₂ /N ₂ /H ₂ : 5/90/5%) in a thermostat at 25° C. for 24, 48 and 72 hours.

Organic acid and alcohol profiles were determined by gas chromatography on a Gas Chromatograph 6890A capillary column CP-Wax 52CB (30 mx 0.25 mm 0.25 µm). Column temperature program 75 degrees 1 minute retention. 10 degrees/minute to 115 degrees and hold for 3 minutes. 20 degrees/minute to 190 degrees and hold for 5 minutes. Detector (FID) 280 degrees.

Organic acids were determined by liquid chromatography on a Shimadzu Prominence HPLC system. Samples were separated on an Aminex HPX-87H ion exchange column (300 mm x 7.8 mm). The column temperature was maintained at 60 degrees, the flow rate was 0.6 ml/min, and the organic acids were detected by a PDA detector at 210 nm. Analysis time was 26 minutes.

In plant material fermentation tests, Lactobacillus buchneri BioCC 203 DSM 32650 demonstrated stronger production of ethanol and lactic acid compared to Lactobacillus buchneri BioCC 228 DSM 32651 (Table 5).

Example 3 Antimicrobial activity against pathogenic bacteria The purpose of this experiment was to examine the gut of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 strains in microaerobic and anaerobic culture at 25°C. It is to test the antimicrobial activity against pathogenic bacteria.

The streak-line technique was used to evaluate the antimicrobial activity of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 strains against pathogens (Non-Patent Document 9).

The growth-free zone was measured in millimeters to determine growth inhibition on the target microorganism. Calculated means and standard errors were calculated based on the results of the samples (Table 6) by a method similar to Hutt et al. (2006), and on the same basis the opposing activities (mm) were evaluated.

Under microaerobic conditions, both strains exhibited similar strength of antimicrobial activity (Table 6). Under anaerobic conditions, Lactobacillus buchneri BioCC 203 DSM32650 has a slightly higher inhibitory effect against the enteropathogens tested.

Example 4 Antifungal Activity The purpose of this experiment was to measure corn silage from the supernatant of Lactobacillus buchneri BioCC 203 DSM 32650 and Lactobacillus buchneri BioCC 228 DSM 32651 strains using the agar well-diffusion method. To evaluate the effect on the yeast of origin.

For 48 hours, the Lactobacillus culture suspension was added to McFarland Tobidity Standard No. 5 (1.5×10 ⁹ organisms/ml) in peptone water and plated in MRS agar (Biolife Italy) cultures to a final concentration of 1.5×10 ⁶ organisms/ml; Incubation was carried out at 25° C. for 48 and 72 hours under microaerobic (10% CO ₂ ) and anaerobic (CO ₂ /N ₂ /H ₂ : 5/90/5%) conditions. Microbial cells were removed by centrifugation (4500 rpm, 10 min). The supernatant was sterilized by filtration and concentrated by lyophilization. The lyophilized supernatant was resuspended 10-fold with 10 mM acetic acid. Six strains of wild-type yeast (Candida sp.) isolated from corn silage were uniformly plated on PCA (Plate Count Ager, Liofilchem srl, Italy) medium. A 6 mm diameter well was sterilely agar cut and 100 μl of supernatant sample was added to the well. After incubation at 25°C, the antifungal action was recorded as follows.

No inhibition; + weak inhibition, inhibits yeast growth; ++ strong inhibition, inhibition of yeast growth with detectable clearing zone; +++ very strong inhibition, inhibition of yeast growth with wide clearing zone

The antimicrobial compound produced by Lactobacillus buchneri BioCC 228 DSM32651 inhibits the growth of plant-derived yeast more strongly than that produced by Lactobacillus buchneri BioCC 203 DSM32650. Lactobacillus buchneri BioCC 228 DSM32651 already produces yeast growth inhibitory compounds during 48 hours of culture, creating broad clear zones of growth inhibition on the agar medium around the wells, whereas the supernatant of BioCC 203 DSM32650 strain only interferes with yeast growth.

Example 5 Growth Dynamics During Fermentation of Plant Material The purpose of this experiment was to evaluate the effect of Lactobacillus buchneri BioCC 203 DSM32650 and Lactobacillus buchneri BioCC 228 DSM32651 strains during fermentation of plant material. be.

Method: 226 g of corn (Zea mays L.) plants at vegetable growth stage (V6-V8) were crushed, watered, homogenized in a laboratory blender Bagmixer 400 (Interscience, France) for 6 minutes, filtered, Centrifuge at 5000 rpm for 10 minutes at room temperature and sterilize at 121 degrees for 5 minutes.

McFarland Tobidity Standard No. 5 (1.5×10 ⁹ microbes/ml) of Lactobacillus in peptone water was prepared and subjected to MRS (Biolife Italy) at a final concentration of 1.5×10 ⁶ microbes/ml. The cells were inoculated in culture medium and incubated microaerobically (10% CO ₂ ) and anaerobicly (CO ₂ /N ₂ /H ₂ : 5/90/5%) at 25° C. for 48 and 72 hours.

A possible number of both strains was registered. Yield, generation number (n), and growth rate (V) were calculated as follows.
Yield = logN ₁ -logN ₀ , where N ₁ is the cell concentration at a given time and N ₀ is the initial cell concentration.
n=log N ₁ -log _{N 0} /log 2, where N ₁ is the cell concentration at a given time and N ₀ is the initial cell concentration.
V=logN ₁ -logN ₀ /0.301×t, w, N ₁ is the cell concentration at a given time, N ₀ is the initial cell concentration, t is the time at a particular epoch (h).

Lactobacillus buchneri strain BioCC 203 DSM32650 was 4 generations earlier than Lactobacillus buchneri BioCC 228 DSM32651 strain during the first 24 hours of culture of the organism and 2.4 times faster at 48 hours (Table 7). .

(Example 6)
silage additive, L.I. Buhnelli BioCC 203 DSM32650 and L. Exploring the effect of Buchneri BioCC 228 DSM32651 on readily fermentable fresh material

The purpose of this search was to investigate the aerobic stability and fermentation quality of corn (Zea mays, corn sp. "Cathy") silage (dry matter content ≥ 30%) with silage additives Lactobacillus buchneri BioCC 203 DSM32650 and , to determine the effect of Lactobacillus buchneri BioCC 228 DSM32651 strain.

Silage trials were conducted in 1.5 l lab-scale-silos with freshly ground corn at the dough stage of maturity.

The following explorations were conducted. Two tests were carried out for the pH value at 90 days and the fermentation quality aerobic stability. A first test was performed after a storage period of 49 days with two air stresses (days 28 and 42: 24 hours). Testing for aerobic stability was conducted in a temperature-controlled room at approximately 20°C. Temperatures were recorded every 4 hours with a PS-ES Data logging system.
The chemical composition of the fresh material is shown in Table 8.

L. Buhnelli BioCC 203 DSM32650 and L. Application of Buchneri BioCC 228 DSM32651 resulted in a significant increase in acetic acid and 1,2-propanediol compared to untreated (Table 9).

An aerobic stability test performed after a storage period of 49 days showed that the aerobic stability of L. Buhnelli BioCC 203 DSM32650 and L. Silage treated with L. buhneri BioCC 228 DSM 32651 showed a significant improvement of approximately 2-2.5 days (control: 3.9 days vs. L. buhneri BioCC 203 DSM 32650: 6.3 days, L. buhneri BioCC 203 DSM 32650: 6.3 days, L. Buchneri BioCC 228 DSM32651: 5.8 days).

Extending the storage period to 90 days was 7.9 days for both untreated controls and 7.9 days for L. Buchneri BioCC 203 DSM 32650 treatment was 10.6 days; This resulted in an increased aerobic stability of 11.4 days for Buchneri BioCC 228 DSM32651 silage. L. Buchneri BioCC 203 DSM32650 difference less than 3 days, L. A difference of more than 3 days to Buchneri BioCC 228 DSM32651 was found to be statistically significant.

(Example 7)
silage additive, L.I. Buhnelli BioCC 203 DSM32650 and L. Exploring the effect of Buchneri BioCC 228 DSM32651 on difficult-to-ferment fresh material

The purpose of this experiment was to develop freshly ground, low dry matter content (≤20%) whole corn plants (Zea mays To evaluate the fermentation quality and aerobic stability of silage made from corn species 'Dorka').

L. Buhnelli BioCC 203 DSM32650 and L. Buhneri BioCC 228 DSM32651 strain was added in the form of an aqueous solution to the siled material at a concentration of 1×10 ⁵ CFU/g of plant material (feed) to be siled. Five replicates of all test types (control silage, silage made with lactic silage bacterial strains L. buchneri BioCC 203 DSM 32650 and silage made with L. buchneri BioCC 228 DSM 32651, control silage made without silage additives were prepared. All test silages were opened after 90 days of silage storage.

The chemical composition of the fresh material is shown in Table 10.
Ｈｏｎｉｇ （Ｈｏｎｉｇ， Ｈ．，１９９０：Ｅｖａｌｕａｔｉｏｎ ｏｆ ｔｈｅ ａｅｒｏｂｉｃ ｓｔａｂｉｌｉｔｙ． Ｉｎ： Ｐｒｏｃｅｅｄｉｎｇｓ ｏｆ ｔｈｅ Ｅｕｒｏｂａｃ Ｃｏｎｆｅｒｅｎｃｅ， Ｓｗｅｄｉｓｈ Ｕｎｉｖｅｒｓｉｔｙ ｏｆ Ａｇｒｉｃｕｌｔｕｒｅ Ｓｃｉｅｎｃｅｓ， Ｕｐｐｓａｌａ／Ｓｗｅｄｅｎ， Ｓｐｅｃｉａｌ Ｉｓｓｕｅ）に記載の方法に従って、９０日の保存後、サイレージのAerobic stability was tested. Silage is considered aerobically unstable when the temperature measured at the geometric center exceeds 3 degrees above ambient temperature. Long-term changes in temperature were measured over 9 days (217 hours). Ambient and test silage, Comet temperature data Logger S0141 equipment was used to record hourly intervals. Silage samples were analyzed using well-established methods (AOAC, 2005. Official methods of analysis of AOAC International, ^18th ed. Association of Official Analytical Chemists International, Gaithersburg, USA).

To determine the dry matter content, silage samples were dried to constant weight in a thermostat at 130°C. To determine crude ash content, silage samples were calcined at 550 degrees for 6 hours in a muffle furnace. Protein content was measured using a Kjeltec™ 2300 analyzer followed by the Kjeldahl method (Nx 6.25). Crude fibers are available from W.W. Henneberg and F. Determined according to the Stohamm method. An Agilent 7890A gas chromatograph was used to determine the acid and ethanol content in the silage. The ratio of ammonia nitrogen in total nitrogen was determined with a Kjeltec™ 2300 analyzer. Silage acidity was determined using a Hanna Instrument HI2210 pH meter.

The dry matter content of all silages remained ≦18% (Table 11). However, silage from corn variety 'Dorka' and L. Buhnelli BioCC 203 DSM32650 or L. Silage treated with Buchneri BioCC 228 DSM 32651 showed good fermentation characteristics (Table 11). Lactic acid predominated in all silages. L. Silage treated with Buchneri BioCC 203 DSM 32650 is L. Silage made with Buchneri BioCC 228 DSM32651 and had higher acetic acid and 1,2-propanediol concentrations compared to the control. Ethanol content was low in all silages (ranging from 4.1 to 8.6 g/kg).

Fermentation quality of siled material and microbial index of corn silage are shown in Table 11. The amount of fungi in the siled material was relatively high, with chostridia and yeast below the level of detection. L. Buhnelli BioCC 203 DSM32650 or L. Silage samples treated with the Buchneri BioCC 228 DSM32651 strain contained very high concentrations of lactic acid bacteria (>8.0 log ₁₀ CFU/g silage) and the added strains outnumbered the endogenous lactic acid bacteria. The amount of lactic acid bacteria in untreated control silage was 4.56 log ₁₀ CFU/g silage.

In the aerobic stability test, 4 out of 5 replicates of untreated silage were elevated in temperature. The average aerobic stability of control silage amounted to 149 hours (ie 6.2 days). L. Buhnelli BioCC 203 DSM32650 and L. Silage treated with Buchneri BioCC 228 DSM 32651 was aerobically stable until the end of the test (ie 217 hours (ie 9.04 days)). Thus, L. Buhnelli BioCC 203 DSM32650 and L. Treatment of fresh material with Buchneri BioCC 228 DSM32651 strain increased the aerobic stability of the silage by 2.84 days.

In conclusion, L. Buhnelli BioCC 203 DSM32650 and L. Buchneri BioCC 228 DSM32651 surprisingly showed very strong growth in silage made from difficult to ferment fresh material. L. Buhnelli BioCC 203 DSM32650 and L. The use of Buchneri BioCC 228 DSM32651 increases the lactic and acetic acid content of silage made from low dry matter (≦20%) silage material and inhibits microbial and yeast activity, thereby reducing silage from heating. silage, thereby ensuring improved aerobic stability of the silage upon opening the silo and thus extending the shelf life of the silage.

Claims (14)

Isolated microbial strain Lactobacillus buchneri BioCC 203 DSM32650. Isolated microbial strain Lactobacillus buchneri BioCC 228 DSM32651. 3. The microbial strain of claim 1 or 2 in lyophilized form. A composition comprising one or more of the microbial strains of any one of claims 1-3. A feed comprising the microbial strain according to any one of claims 1-3. The feed according to claim 5, which is a fermented feed . Use of the microorganism according to any one of claims 1-3 as a feed additive. Use of the microorganism according to any one of claims 1 to 3 to support the aerobic stability of silage. 9. Use according to claim 8, wherein the silage is made from feed with a low dry matter content (≤20%). Use of the microorganism according to any one of claims 1-3 in feed fermentation. A composition comprising one or more microorganisms according to any one of claims 1 to 3,
Accelerates the fermentation of feed,
increasing the concentration of lactic acid and acetic acid in the feed,
by lowering the pH and thereby
A composition that reduces loss of nutrients in feed. of claims 1 to 3 for suppressing the action of pathogenic microorganisms and inhibiting the growth of yeast by adding the microorganism according to any one of claims 1 to 3 to the feed to be fermented. Use of the microorganism according to any one of the clauses. 13. Use according to claim 12, wherein the pathogenic microorganism is an enteropathogen. A method for prolonging the storage of feed, wherein a microorganism according to any one of claims 1 to 3 is added to the feed to be fermented.