WO2015120863A1 - Micro-organisme lactobacillus brevis tak 124-1 ncimb42149 et son utilisation - Google Patents

Micro-organisme lactobacillus brevis tak 124-1 ncimb42149 et son utilisation Download PDF

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WO2015120863A1
WO2015120863A1 PCT/EE2015/000001 EE2015000001W WO2015120863A1 WO 2015120863 A1 WO2015120863 A1 WO 2015120863A1 EE 2015000001 W EE2015000001 W EE 2015000001W WO 2015120863 A1 WO2015120863 A1 WO 2015120863A1
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silage
feed
tak
ncimb42149
lactobacillus brevis
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PCT/EE2015/000001
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English (en)
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Kristiina KOKK
Epp Songisepp
Merle RÄTSEP
Andres OLT
Helgi KALDMÄE
Olav KÄRT
Meelis OTS
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Oü Tervisliku Piima Biotehnoloogiate Arenduskeskus (Bio-Competence Centre Of Healthy Dairy Products)
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Priority to EA201600583A priority Critical patent/EA033289B1/ru
Priority to EP15708108.4A priority patent/EP3105355A1/fr
Publication of WO2015120863A1 publication Critical patent/WO2015120863A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
    • A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
    • A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/24—Lactobacillus brevis

Definitions

  • 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 .
  • 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.
  • 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.
  • 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.
  • silage 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.
  • 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.
  • 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) .
  • Silage may become exposed to oxygen both when the silo is opened for feeding and as a result of inadequate coverage of the silo.
  • 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.
  • silage 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.
  • Yeasts use the residual sugars contained in silage as a source of energy; however, their first preference is lactic acid.
  • 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.
  • 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.
  • Acetic and propionic acid 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 .
  • silage additives have been developed to improve the ensilage process and the nutritive value of ensiled feed.
  • 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.
  • 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
  • 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.
  • 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.
  • inoculants that produce considerable quantities of acetic acid in addition to lactic acid 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 .
  • 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) .
  • silage additives 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_1831-0
  • 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.
  • 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.
  • 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 lxlO 5 ... lxl0 6 CFU/g of the fermented feed.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • MRS broth which shows a uniformly cloudy growth after 24...48 hours of microaerobic incubation at 37 °C.
  • 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.
  • 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) .
  • strain Lactobacillus brevis TAK 124-1 NCIMB42149 exhibited no resistance to the tested antibiotics (Table 1) ⁇
  • 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 ⁇ ) . Column temperature programme: 60 °C 1 min, 20 °C/min 120 °C 10 min, detector (FID) 250 °C (Table 2) .
  • 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 ) .
  • 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.
  • 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.
  • Inhibition zone in microaerobic environment (in mm) : weak ⁇ 22.9; average 23.0...32.9; strong >33.
  • Inhibition zone in anaerobic environment weak ⁇ 11.9; average 12.0...21.9; strong >22.5
  • Clostridium spp suspension was added to the filtration-sterilised supernatant or sterile BHI broth (positive control) .
  • Antimicrobial compounds produced by Lactobacillus brevis TAK 124-1 NCIMB42149 inhibit the growth of plant-derived Clostridium spp by 23.1 per cent.
  • 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. 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) ,
  • Fig. 6 Improving the aerobic stability of timothy silage (Example 2) by means of Lactobacillus brevis strain TAK 124-1 NCIMB42149, where sub-figure
  • 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) ,
  • 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 lxlO 5 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 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 KjeltecTM 2300 analyser following the Kjeldahl method (Nx6.25). Crude fibre was determined according to the W.Henneberg and F. Stohmann method. An Agilent 7890A gas 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 KjeltecTM 2300 analyser. The pH of the silage was determined using a Hanna Instruments HI 2210 pH-meter.
  • Lactobacillus jbrevis TAK 124-1 NCIMB42149 improved the fermentation of the silage (Table 5 and Fig 1).
  • 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.
  • 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) .
  • Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation of the silage, compared to the control 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.
  • 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 IxlO 5 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
  • 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.
  • 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,
  • 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 lxlO 5 CFU per 1 g of the plant material (feed) being ensiled.
  • control silages silages, silages made with the lactic acid bacterium strain Lactobacillus brevis TAK 124-1 NCIMB42149, and silages made with a chemical silage additive
  • silages made with a chemical silage additive were prepared in five replicates. The test silages were opened after 90 days of ensiling.
  • 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.
  • the heterofermentative lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation of the silage, compared to the control silage.
  • 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 lxlO 5 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).
  • 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).
  • Example 4 of the invention the lactic acid bacterium Lactobacillus brevis TAK 124-1 NCIMB42149 improved the fermentation 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.

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Abstract

L'invention porte sur le micro-organisme isolé Lactobacillus brevis TAK 124-1 NCIMB42149, qui est utilisé pour conforter la stabilité aérobie d'un aliment pour animaux et améliorer la fermentation d'un aliment pour animaux, pour augmenter la concentration d'acide lactique et d'acide acétique, pur réduite le pH et par conséquent diminuer la perte de substances nutritives dans l'aliment pour animaux. Le L. brevis TAK 124-1 supprime la fonction d'agents pathogènes (Clostridia et les entéropathogènes), de levures et de champignons dans l'aliment pour animaux. L'additif alimentaire comprenant ledit micro-organisme contribue à étendre la durée de vie de l'aliment pour animaux.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891659A (zh) * 2022-03-02 2022-08-12 西南民族大学 一种短乳杆菌248及其应用

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