Methods for producing a mycobacteria-free biomass pellet or granulate preferably comprising peat material suitable for animal feed and method for detecting mycobacteria

Abstract

The present invention concerns a method for producing a Mycobacteria-free biomass pellet suitable for animal feed, the method comprising the steps of: providing biomass with variable particle size and / or length, wherein the moisture content of said biomass is more than 30 % w / w, preferably 40-60 % w / w, and subjecting said biomass to sieving and drying steps in any order, wherein in said sieving step the biomass or the heat-treated biomass is sieved in order to obtain even material preferably having a median particle length of less than 40 mm; and wherein in said drying step the biomass or the sieved biomass is heat-treated preferably at the temperature of 80-110°C, more preferably at 90-105°C, to reach a moisture content below 20 % w / w, preferably 10-15 % w / w, for the material; milling the heat-treated material in order to reduce the particle size below 1 mm; subjecting the heat-treated and milled material to a pelletizing process, preferably in a pellet press, wherein the temperature of the subjected material reaches at least 90 °C, preferably at least 100 °C, during said pelletizing process; and cooling the pellets obtained to ambient temperature, preferably with a counter flow cooler. The present invention is also directed to a method for detection of the presence of live Mycobacterium species in a sample comprising peat and / or moss, the method comprising the steps of: providing a sample comprising peat and / or moss including a sample of a processed product comprising peat and / or moss, or a sample of a processed product made of peat and / or moss; contacting said sample with liquid broth in a sterilized container, wherein said liquid broth is suitable for growing Mycobacterium species and comprises one or more antibiotics effective against other bacteria than Mycobacterium; incubating said container at a temperature range of 30 - 37 °C for at least 1 - 21 days; taking the incubated liquid broth or an aliquot thereof from said container and performing a nucleic acid amplification reaction comprising nucleic acids isolated from said incubated liquid broth or said aliquot and an oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species; wherein the presence of pathogenic Mycobacterium nucleic acid or increased presence of pathogenic Mycobacterium nucleic acid in the incubated liquid broth or said aliquot confirms the presence of pathogenic Mycobacterium in said sample.

Description

Methods for producing a Mycobacteria-free biomass pellet or granulate suitable for animal feed and for detecting Mycobacteria in said pellet or granulateFIELD

[0001] The present invention relates to animal feed products and particularly to methods for generating pelletized animal feed products. The present invention also relates to methods for detecting pathogens in animal feed products.BACKGROUND

[0002] Mycobacterium genus involves more than 100 species. Of these M. tuberculosis, M. bovis, M. microti and M. africanum are known to cause tuberculosis in humans and / or animals. Over 80 environmental mycobacteria species have been identified and nearly 50 of these species are considered as pathogenic to humans and animals.

[0003] Mycobacteria have been detected in peat bogs worldwide. Peat's high concentration of organic nutrients and low pH are optimal conditions for mycobacteria.Humic and fulvic acids have been observed to stimulate the growth of mycobacteria. The pH range of 4.5 -5.5 is an optimal pH for the growth of many Mycobacteria species which favors their growth also in acidic peat.

[0004] Matlova et al. (2005) have studied samples of commercially available peat that included processed peat for piglet feeding, natural peat and packed peat for homiculture.Matlova et al. (2005) detected potentially pathogenic mycobacteria from over 70% of the samples and all types of peat studied contained mycobacteria. M. avium subsp. hominissuis was the most frequently detected species (82.1 %). M. avium subsp. hominissuis is an environmental mycobacterium that cause opportunistic infection in pigs and hence results in economic losses.

[0005] Matlova et al., 2012, disclose that although ionisation effectively devitalized mycobacteria present in peat, this type of radiation is not currently allowed under EU legislation. Matlova et al, 2012, conclude that it will be necessary to investigate other procedures for the safe devitalization of mycobacteria in peat.

[0006] Consequently, there is a need in the present field for alternative methods for production of peat-based feeds and other biomass-based animal feeds, the safety of which have been improved.SUMMARY

[0007] In the present disclosure, we provide a solution to at least part of the problems discussed above. The present inventors have shown that by a novel pelletizing process disclosed herein, it is possible to prepare a feed pellet which does not contain Mycobacteria although the biomass used as starting material naturally contains these bacteria. The present invention also provides an improved method for detecting the presence or absence of Mycobacteria in the pellets produced.

[0008] The invention is defined by the features of the independent claims. Some specific embodiments are defined in the dependent claims.

[0009] According to a first aspect of the present invention, there is provided a method for producing a Mycobacteria-free biomass pellet suitable for animal feed, the method comprising the steps of: a) providing biomass with variable particle size and / or length, wherein the moisture content of said biomass is more than 30 % w / w, preferably 40-60 % w / w, and subjecting said biomass to sieving and heating steps in any order, wherein in said sieving step the biomass or the heat-treated biomass is sieved in order to obtain even material preferably having a median particle length of less than 40 mm; and wherein in said heating step the biomass or the sieved biomass is heat-treated preferably at the temperature of 80-110°C, more preferably at 90-105°C, to reach a moisture content below 20 % w / w, preferably 10-15 % w / w, for the material; b) milling the heat-treated material obtained from step a) in order to reduce the particle size below 1 mm; c) subjecting the heat-treated and milled material obtained in step b) to a pelletizing process, preferably in a pellet press, wherein the temperature of the subjected material reaches at least 90 °C, preferably at least 100 °C, during said pelletizing process; and d) cooling the pellets obtained from step c) to ambient temperature, preferably with a counter flow cooler.

[0010] According to a second aspect of the present invention, there is provided a pellet suitable for animal feed comprising at least 60 % w / w of peat, preferably at least 80 % w / w ofpeat having moisture content of less than 20 % w / w, preferably 5-15 % w / w, wherein said pellet is produced by the method of the present disclosure and is devoid of Mycobacterium.

[0011] According to a third aspect of the present invention, there is provided method for detection of the presence of live pathogenic Mycobacterium species in a sample comprising peat and / or moss, the method comprising the steps of: a) providing a sample comprising peat and / or moss including a sample of a processed product comprising peat and / or moss, or a sample of a processed product made of peat and / or moss; b) contacting said sample with liquid broth in a sterilized container, wherein said liquid broth is suitable for growing Mycobacterium species and comprises one or more antibiotics effective against other bacteria than Mycobacterium,' c) incubating said container at a temperature range of 30 - 37 °C, preferably for less than 28 days, more preferably 1-21 days; d) taking the incubated liquid broth or an aliquot thereof from said container and performing a nucleic acid amplification reaction comprising nucleic acids isolated from said incubated liquid broth or said aliquot and an oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species; wherein the presence of pathogenic Mycobacterium nucleic acid or increased presence of pathogenic Mycobacterium nucleic acid in the incubated liquid broth or said aliquot confirms the presence of pathogenic Mycobacterium in said sample.BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIGURE 1. Example of animal feed peat pellet manufacturing process. This figure illustrates a schematic flow chart of one example process for producing feed pellets from peat as a starting material.

[0013] FIGURE 2 illustrates a schematic flow chart of a process for detecting pathogenic Mycobacteria from a biological sample.

[0014] FIGURE 3 shows results from PCR analyses from peat raw material and peat pellets produced by the Mycobacteria detection method of the present disclosure. Peat pellets were shown to be devoid of Mycobacteria although the raw material contained the bacteria. MA, Mycobacterium avium subsp. avium,' MP, Mycobacterium avium subsp. paratuberculosis.EMBODIMENTS

[0015] Biomass gathered from bogs such as peat or moss can be a good source of protein, fiber and energy but it is known that feeding such natural biomass to animals may expose them to pathogenic microbes including Mycobacteria. In the present invention, we provide a pelletizing method, which provides biomass pellets treated so that pathogenic microbes are killed during the process.

[0016] Feed pelletizing is, as conventionally practiced, a thermoplastic molding operation in which finely divided particles of a feed material are pressed through a die and formed into compact, easily handled pellets (see, e.g., US20120301598). A pellet mill, also known as a pellet press, is a type of mill or machine press used to create pellets from powdered starting material. Pellet mills are generally used to produce animal feed, wood pellets, and fuel pellets. There are two common types of large-scale pellet mills: flat die mills and ring die mills. In a flat die mill, the powder is introduced to the top of the die and as the die rotates a roller presses the powder through the holes in the die. A cutter on the other side of the die cuts the exposed pellet free from the die. In the ring die, there are radial slot throughout the die. Powder is fed into the inside of the die and spreaders evenly distribute the powder. Two rollers then compress the powder through the die holes. Cutters are used to cut the pellets free from the outside of the die. In a pellet press, heat and pressure produced in the compaction process of the pellet have an effect on the conversion of loose materials into dense, solid pellets. Pressure compacts the material, while heat can aid in binding and densification.

[0017] Before the pelletizing step, the biomass material is delivered to the plant, unloaded, preferably cleaned (e.g., by magnetic separator to remove any unsuitable foreign matter), and conveyed for processing. According to an embodiment, the biomass may comprise at least about 60%, preferably at least 80%, peat, moss, wood, bark, or lignocellulose with variable particle size and / or length. In an embodiment, the moisture content of said biomass is more than 30 % w / w, preferably 40-60 % w / w. According to an exemplary embodiment, biomass in the form of mostly peat is delivered to the plant, stored in piles and managed with a conveyor inside the facility (see Figure 1). In an embodiment, the biomass delivered to the plant is first subjected to sieving and drying steps in any order, wherein in said sieving step the biomass or the heat-treated biomass is sieved in order to obtain even material preferably having a median particle length of less than 40 mm; and wherein in said drying step the biomass or the sieved biomass is heat-treated preferably at the temperature of 80-110°C, more preferably at90-105°C, to reach a moisture content below 20 % w / w, preferably 10-15 % w / w, for the material. In an embodiment, the drying step is performed with a disc dryer, preferably a steam- heated thermal rotary disc dryer providing heat indirectly through the metallic disc structures filled with hot steam. In a preferred embodiment, the biomass stays in the dryer for about 15 minutes.

[0018] In a subsequent milling step, the sieved and heat-treated material is milled in order to reduce the particle size below 1 mm. In a preferred embodiment, the milling step is performed in a hammer mill.

[0019] In a preferred embodiment, the milling step comprises two consecutive milling sub-steps, preferably a first sub-step of pre-milling the material to the median particle size of 1-2 mm and a second sub-step of fine milling the pre-milled material to the particle size of 100- 400 pm.

[0020] Subsequently, after the milling step, the milled biomass material can be processed in a conditioning chamber which receives steam and subjects the milled biomass to elevated temperatures and moisture for a set period of time. In some embodiments, the duration of conditioning may be anywhere from around 1 second to about 10 minutes but, in some particular embodiments, approximately a 2-5 seconds conditioning time can be sufficient. The temperature of the milled biomass may be raised to between about 60 - 65 °C or even up to 100 °C, dependent upon process conditions. Preferably, this conditioning step improves the final product's Pellet Durability Index (PDI), i.e. hardness of the pellet, providing durability so that the pellets can withstand repeated transfers in feed handling systems.

[0021] The milled and optionally steam conditioned biomass is then supplied to a pelletizer (i.e. a pellet mill die chamber) where the biomass is pressed / extruded through a die with suitable hole diameter and length to generate the feed pellets. Afterwards, the hot newly- formed pellets are cooled to ambient temperature (e.g., 15 - 25 °C), typically with forced air through vertical cooling systems.

[0022] In a preferred embodiment, the hole diameter in the die is 2-8 mm.

[0023] As described in this disclosure, the present pellet producing process results in reduced presence or even absence of Mycobacteria in the final pellet product. Without wishing to be bound by a theory, it is the inventors’ opinion that the pelletizing process of the present invention with at least two separate heat treatments, the first during the drying step and thesecond during the compression / extrusion step in the pelletizer, subject the material to such conditions that Mycobacteria is killed in the process. It is also notable that when the dried and milled biomass moves in a conveyor towards the pelletizer, it takes several minutes. During this time, the biomass may cool down. Cooling can have the effect that microbes die more easily in the following pelletizing step as the varying temperature conditions during the process expose the bacteria to constant stress. Further, it is suggested that the friction created in the flow of the material can also have an effect on the survival of bacteria during the process as the compression friction in the compaction / extrusion increases the temperature of the biomass material and promotes the death of bacteria.

[0024] In a preferred embodiment, the temperature of the compressed materials reach at least 90 °C, preferably at least 100 °C, during the pelletizing process. In a more preferred embodiment, the temperature of the material under compression during the pelletizing process is between 90 - 120 °C, most preferably 90 - 100 °C.

[0025] In a preferred embodiment, the pellets obtained have a diameter in the range of 2- 8 mm.

[0026] In another embodiment, the pellets obtained have moisture content of less than 20 % w / w, preferably 5-15 % w / w.

[0027] In another embodiment, the length of the pellet obtained in step d) is in the range of 10 - 50 mm.

[0028] In another specific embodiment, the present method comprises a further step of crushing or granulating the pellets in order to produce granulates or powder.

[0029] In another embodiment, the pellets obtained are devoid of Mycobacterium. In order to confirm the absence of Mycobacteria in the final pellet product, the present inventors have developed a special method for the quick detection of Mycobacteria in the pellets produced by the present method.

[0030] Accordingly, the present invention is also directed to a pellet suitable for animal feed comprising at least 60 % w / w of peat, preferably at least 80, 90 or 95 % w / w of peat having moisture content of less than 20 % w / w, preferably 5-15 % w / w, wherein said pellet is produced by the pelletizing method defined in the present disclosure and is devoid of Mycobacterium.

[0031] As it is well-known in the art that pathogenic Mycobacteria are very slowly growing bacteria, detection of these bacteria from environmental samples by culturing methods is hampered by the presence of other environmental faster growing bacteria as well as other non-pathogenic Mycobacteria species. Culturing methods also require long incubation time (i.e. several weeks, usually 6-8 weeks) before the detection or non-detection can be confirmed. Quicker PCR-based methods on the other hand cannot distinguish between living Mycobacteria and the dead ones.

[0032] To monitor efficiently the safety of the feed pellets produced by the method according to the present disclosure particularly in relation to the presence of living pathogenic Mycobacteria, the detection method results must be provided in a shorter time span than what can be achieved by traditional culturing methods. In the present invention, at least part of the problems of the prior art has been overcome by providing a detection method which combines culturing and nucleic acid amplification-based methods in a novel manner.

[0033] In particular, the present invention is further directed to a method for detection of the presence of live pathogenic Mycobacterium species in a sample comprising peat and / or moss, the method comprising the steps of: a) providing a sample comprising peat and / or moss including a sample of a processed product comprising peat and / or moss, or a sample of a processed product made of peat and / or moss; b) contacting said sample with liquid broth in a sterilized container, wherein said liquid broth is suitable for growing Mycobacterium species and comprises one or more antibiotics effective against other bacteria than Mycobacterium,' c) incubating said container at a temperature range of 30 - 37 °C, preferably for less than 28 days, more preferably 1 - 21 days; d) taking the incubated liquid broth or an aliquot thereof from said container and performing a nucleic acid amplification reaction comprising nucleic acids isolated from said incubated liquid broth or said aliquot and an oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species; wherein the presence of pathogenic Mycobacterium nucleic acid or increased presence of pathogenic Mycobacterium nucleic acid in the incubated liquid broth or said aliquot confirms the presence of pathogenic Mycobacterium in said sample.

[0034] In a preferred embodiment, said sample has been pretreated by one or more of the following steps selected from the group consisting of:i) pre-cultivating the sample in order to germinate spores of environmental bacteria, preferably by contacting said sample with PBS buffer and incubating at a temperature range of 30 - 37 °C overnight; ii) filtering the sample in order to reduce the amount of peat fibres in the sample; and iii) decontaminating the sample from other bacteria than Mycobacterium.

[0035] In another preferred embodiment, said decontaminating in step iii) is performed by contacting the sample with a disinfectant, preferably NALC-NaOH.

[0036] In another preferred embodiment, the incubation in step c) lasts 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, or 21 days, more preferably 3-21, 4-21, or 5-21 days, even more preferably 5-10 or 5-15 days. In an embodiment, the incubation in step c) lasts at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 ,17, 18, 19, 20, or 21 days but less than 28 days.

[0037] In another preferred embodiment, said liquid broth suitable for growing Mycobacterium species comprises the ingredients of Middlebrook 7H9 broth (Atlas, Ronald M.; James W. Snyder, 2006, Handbook of media for clinical microbiology. 2ndedition, CRC Press. ISBN 978-0-8493-3795-6).

[0038] In another preferred embodiment, said liquid broth suitable for growing Mycobacterium species comprises ingredients selected from the group consisting of: ammonium sulfate, L-glutamic acid, sodium citrate, pyridoxine, biotin, disodium phosphate, monopotassium phosphate, ferric ammonium citrate, magnesium sulfate, calcium chloride, zinc sulfate, and copper sulfate.

[0039] In another preferred embodiment, said liquid broth suitable for growing Mycobacterium species is supplemented with ingredients selected from the group consisting of: oleic acid, albumin, casein peptone, catalase, Mycobactin J, and dextrose.

[0040] In another preferred embodiment, one or more of said antibiotics is / are selected from the group consisting of: vancomycin, polymyxin B, trimethoprim, azlocillin, and nalidixic acid.

[0041] In another preferred embodiment, said liquid broth suitable for growing Mycobacterium species further comprises a fungicide, preferably cycloheximide or amphotericin B.

[0042] In another preferred embodiment, the detection method comprises further steps of taking a sample of the biomass provided in step a) or an aliquot of the liquid broth from said container before step c) and performing a nucleic acid amplification reaction comprising said aliquot or nucleic acids isolated from said sample or aliquot and oligonucleotide primers specific to at least one pathogenic Mycobacterium species in order to detect the presence and / or amount of pathogenic Mycobacterium nucleic acid in the sample before performing step c).

[0043] In another preferred embodiment, the amount of pathogenic Mycobacterium nucleic acid detected in the sample before performing step c) is used in step d) for the detection of increased presence of pathogenic Mycobacterium nucleic acid in said liquid broth during the incubation step c) in an aliquot taken after the onset of step d).

[0044] In another preferred embodiment, step d) is performed at least two times at different time points after the onset of step d).

[0045] In another preferred embodiment, the amplification results of pathogenic Mycobacterium nucleic acid from any aliquot from any time point during step c) is used in step d) for the detection of increased presence of pathogenic Mycobacterium nucleic acid in said liquid broth in a subsequent time point during the incubation step c).

[0046] In another preferred embodiment, said nucleic acid amplification reaction of step d) further comprises one or both of the following oligonucleotide primer pairs:- i) an oligonucleotide primer pair detecting bacterial growth in the sample; and- ii) an oligonucleotide primer pair detecting the presence of pathogenic species of Mycobacterium genus.

[0047] In another preferred embodiment, said oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species in step d) is an oligonucleotide primer pair specific to a strain or strains of Mycobacterium avium complex (MAC), preferably to Mycobacterium avium subspecies avium, or Mycobacterium avium subspecies paratuberculosis.

[0048] In another preferred embodiment, said sample of a processed product comprising peat in step a) is a sample of the pellet produced by the pelletizing method as described in the present disclosure.

[0049] It is to be understood that the embodiments of the invention disclosed are not limited to particular structures, process steps, or materials disclosed herein, but are extended toequivalents thereof as would be recognized by those ordinarily skilled in the relevant arts. It should also be understood that terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting.

[0050] Reference throughout this specification to “one embodiment”, “preferred embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment”, “in a preferred embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment.

[0051] As used herein, a plurality of items, structural elements, compositional elements, and / or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary. In addition, various embodiments and example of the present invention may be referred to herein along with alternatives for the various components thereof. It is understood that such embodiments, examples, and alternatives are not to be construed as de facto equivalents of one another, but are to be considered as separate and autonomous representations of the present invention.

[0052] Furthermore, the described features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided, such as examples of lengths, widths, shapes, etc., to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well- known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

[0053] While the forgoing examples are illustrative of the principles of the present invention in one or more particular applications, it will be apparent to those of ordinary skill in the art that numerous modifications in form, usage and details of implementation can be made without the exercise of inventive faculty, and without departing from the principles and concepts of the invention. Accordingly, it is not intended that the invention be limited, exceptas by the claims set forth below.

[0054] The verbs “to comprise” and “to include” are used in this document as open limitations that neither exclude nor require the existence of also un-recited features. The features recited in depending claims are mutually freely combinable unless otherwise explicitly stated. Furthermore, it is to be understood that the use of "a" or "an", i.e. a singular form, throughout this document does not exclude a plurality.EXPERIMENTAL SECTIONEXAMPLEMycobacteria strains used as culture controls.As culture standards, well-defined strains of both Mycobacterium avium subsp. avium and Mycobacterium avium subsp. paratuberculosis are used. These laboratory strains were obtained from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures: DSM 44156 Mycobacterium avium subsp. avium and DSM 44133 Mycobacterium avium subsp. paratuberculosis.The Mycobacterium avium subsp. avium strain is maintained on the 7H10 agar slants at +4°C or for longer storage liquid glycerol stocks are prepared on Middlebrook 7H9 growth medium and stored at -80°C.The Mycobacterium avium subsp. paratuberculosis requires supplementation with Mycobactin J in the medium (2ug / L), otherwise maintenance is the same.Peat sampling25 g of peat sample from peat raw material and from the peat pellets of the present disclosure were selected and pre-incubated with 200 ml of 0.0068 M PBS buffer, pH 6.8 prior to grinding the samples.Decontamination30 ml of each ground sample was filtered and transferred for a decontamination in a tube with 10 ml of NALC-NaOH solution for 15 min. 30 ml of 0.0068 M PBS buffer, pH 6.8 was added and the tube was centrifuged (30 min / 3000 g / 4 °C). After centrifugation, the supernatant was removed and 250 pl of 0.0068 M PBS buffer, pH 6.8 was added to the tube.CulturingThe decontaminated sample was added to a tube of culture media MGIT® (i.e. modified Middlebrook 7H9 broth) with supplements as recommended by the manufacturer (Becton, Dickinson and Company, NJ, USA) and incubated at 37 °C with separate culture controls as described above. The incubation was continued for 2 weeks.DNA extractionAfter the culturing step, 1 ml of culture media was moved from the incubated tube to an Eppendorf-tube and centrifuged at 11 000 g for 2 min. The supernatant was discarded and the pellet was eluted. DNA from the pellet was extracted with microbial DNA extraction kit: MN 740235.250 (MACHEREY-NAGEL GmbH & Co. KG, Duren, Germany). The extracted DNA was eluted to 100 pl of elution buffer.PCR reactionsPCR reaction were performed with BIORAD SsoFast EvaGreen on BIORAD CFX 96 thermocycler. PCR conditions were optimized according to the PCR supermix instructions and according to the melting temperatures of each oligo pair separately. The PCR products’ sizes and specificity were verified in agarose gels.PCR with 4 different oligo pairs was used for verification:1) Bacterial load (16S RNA)• BSF8: 5’-AGAGTTTGATCCTGGCTCAG-3’ (SEQ ID NO:1)• BSR1541 : 5’-AAGGAGGTGATCCAGCCGCA-3’ (SEQ ID NO:2)2) Pan-mycobacteria presence (16S RNA)• KY18-F: 5’-CACATGCAAGTCGAACGGAAAGG-3’ (SEQ ID NOG)• KY75-R: 5'-GCCCGTATCGCCCGCACGCTCACA-3' (SEQ ID NO:4)3) Mycobacteria avium subsp. avium (IS 1245). P41-F: 5’-GGTGAGCGGATCACTCAAG-3’ (SEQ ID N0:5)• P40-R: 5'-GAATCCGCAGTTCCAGGTC-3’ (SEQ ID N0:6)4) Mycobacterium avium subsp. paratuberculosis (IS900)• IS900-F 5'-CCGCTAATTGAGAGATGCGATTGG-3' (SEQ ID N0:7) • IS900-R 5'-AATCAACTCCAGCAGCAGCGCGGCCTCG-3' (SEQ ID NO: 8)PCR setupIn reaction:10 pl SsoFast supermix1.6 pl Primer mix (5mM for each primer)1 pl sample7.4 H2O20 plPCR protocol:Verification of the PCR products in agarose gel The PCR product(s) size and number (in case of possible unspecific reactions) were verified in 1.5% TAE agarose gel stained with EtBr and visualized on ChemiDoc XRS+ System (BIO-RAD). The results are shown in Table 1 and Figure 3. The size of the PCR product is 175 bp for Mycobacterium avium sp. avium with P41 / P40 oligos or 229 bp for Mycobacterium avium sp. paratuberculosis with IS900 oligos.Table 1.7H9: Middlebrook 7H9 broth; PANTA: Panta ™ antibiotic mix; C / V : cycloheximide / vancomycinCITATION LISTNon-patent literatureMatlova L, Dvorska L, Ayele WY, Bartos M, Amemori T, Pavlik I. Distribution of Mycobacterium avium complex isolates in tissue samples of pigs fed peat naturally contaminated with mycobacteria as a supplement. J Clin Microbiol. 2005 Mar;43(3): 1261-8.Matlova L, Kaevska M, Moravkova M, Beran V, Shitaye JE, Pavlik I. Mycobacteria in peat used as supplement for pigs: failure of different decontamination methods to eliminate the risk. Veterinami Medicina, 57, 2012 (4):212-217.Patent literature US20120301598

Claims

CLAIMS1. A method for producing a Mycobacteria-free biomass pellet suitable for animal feed, the method comprising the steps of: a) providing biomass with variable particle size and / or length, wherein the moisture content of said biomass is more than 30 % w / w, preferably 40-60 % w / w, and subjecting said biomass to sieving and drying steps in any order, wherein in said sieving step the biomass or the heat-treated biomass is sieved in order to obtain even material preferably having a median particle length of less than 40 mm; and wherein in said drying step the biomass or the sieved biomass is heat-treated preferably at the temperature of 80-110°C, more preferably at 90-105°C, to reach a moisture content below 20 % w / w, preferably 10-15 % w / w, for the material; b) milling the heat-treated material obtained from step a) in order to reduce the particle size below 1 mm; c) subjecting the heat-treated and milled material obtained in step b) to a pelletizing process, preferably in a pellet press, wherein the temperature of the subjected material reaches at least 90 °C, preferably at least 100 °C, during said pelletizing process; and d) cooling the pellets obtained from step c) to ambient temperature, preferably with a counter flow cooler.

2. The method according to claim 1, wherein said biomass comprises peat, moss, wood, bark, lignocellulose, or a combination thereof.

3. The method according to claim 1 or 2, wherein said sieved biomass comprises sieved peat material.

4. The method according to claim 3, wherein said sieved peat material comprises at least 60 % w / w of peat, preferably 80 % w / w of peat.

5. The method according to any one of claims 1-4, wherein the heat-treating in step a) is performed with a disc dryer, preferably a steam-heated thermal rotary disc dryer.

6. The method according to any one of claims 1-5, wherein the milling step comprises two consecutive milling sub-steps, preferably a first sub-step of pre-milling the material to themedian particle size of 1-2 mm and a second sub-step of fine milling the pre-milled material to the particle size of 100-400 pm.

7. The method according to any one of claims 1-6, wherein the milling step of b) is performed in a hammer mill.

8. The method according to any one of claims 1-7, wherein the pelletizing process is preceded with a conditioning step of mixing steam with milled material obtained in step b) in order to raise the temperature of the material, preferably to temperature of 60 - 65 °C.

9. The method according to any one of claims 1-8, wherein the pellets obtained in step d) have a diameter in the range of 2-8 mm.

10. The method according to any one of claims 1-9, wherein the pellets obtained in step d) have moisture content of less than 20 % w / w, preferably 5-15 % w / w.

11. The method according to any one of claims 1-10, wherein the pellets obtained in step d) are devoid of Mycobacterium.

12. The method according to any one of claims 1-11, wherein the length of the pellet obtained in step d) is in the range of 10 - 50 mm.

13. The method according to any one of claims 1-12 comprising a further step of e) crushing or granulating the pellets obtained from step d).

14. The method according to any one of claims 1-13 comprising further steps of i) providing a sample of a pellet or granulate product obtained in step d) or e); ii) contacting said sample with liquid broth in a sterilized container, wherein said liquid broth is suitable for growing Mycobacterium species and comprises one or more antibiotics effective against other bacteria than Mycobacterium,' iii) incubating said container at a temperature range of 30 - 37 °C, preferably for less than 28 days, more preferably 1-21 days; iv) taking the incubated liquid broth or an aliquot thereof from said container and performing a nucleic acid amplification reaction comprising nucleic acids isolated from said incubated liquid broth or said aliquot and an oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species; wherein the presence of pathogenic Mycobacterium nucleic acid or increased presence of pathogenic Mycobacterium nucleic acid in the incubatedliquid broth or said aliquot confirms the presence of pathogenic Mycobacterium in said sample.

15. A pellet suitable for animal feed comprising at least 60 % w / w of peat, preferably 80 % w / w of peat having moisture content of less than 20 % w / w, preferably 5-15 % w / w, wherein said pellet is produced by the method according to any one of claims 1-14 and is devoid of Mycobacterium.

16. A method for detection of the presence of live pathogenic Mycobacterium species in a sample comprising peat and / or moss, the method comprising the steps of: a) providing a sample comprising peat and / or moss including a sample of a processed product comprising peat and / or moss, or a sample of a processed product made of peat and / or moss; b) contacting said sample with liquid broth in a sterilized container, wherein said liquid broth is suitable for growing Mycobacterium species and comprises one or more antibiotics effective against other bacteria than Mycobacterium,' c) incubating said container at a temperature range of 30 - 37 °C, preferably for less than 28 days, more preferably 1 - 21 days; d) taking the incubated liquid broth or an aliquot thereof from said container and performing a nucleic acid amplification reaction comprising nucleic acids isolated from said incubated liquid broth or said aliquot and an oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species; wherein the presence of pathogenic Mycobacterium nucleic acid or increased presence of pathogenic Mycobacterium nucleic acid in the incubated liquid broth or said aliquot confirms the presence of pathogenic Mycobacterium in said sample.

17. The method according to claim 16, wherein said sample in step a) has been pretreated by one or more of the following steps selected from the group consisting of: i) pre-cultivating the sample in order to germinate spores of environmental bacteria, preferably by contacting said sample with PBS buffer and incubating at a temperature range of 30 - 37 °C overnight; ii) filtering the sample in order to reduce the amount of peat / moss fibres in the sample; and iii) decontaminating the sample from other bacteria than Mycobacterium.

18. The method according to claim 17, wherein said decontaminating in step hi) is performed by contacting the sample with a disinfectant, preferably NALC-NaOH.

19. The method according to claim 16 or 17, wherein said liquid broth suitable for growing Mycobacterium species comprises the ingredients of Middlebrook 7H9 broth.

20. The method according to any one of claims 16-19, wherein said liquid broth suitable for growing Mycobacterium species comprises ingredients selected from the group consisting of: ammonium sulfate, L-glutamic acid, sodium citrate, pyridoxine, biotin, disodium phosphate, monopotassium phosphate, ferric ammonium citrate, magnesium sulfate, calcium chloride, zinc sulfate, and copper sulfate.

21. The method according to any one of claims 16-20, wherein said liquid broth suitable for growing Mycobacterium species is supplemented with ingredients selected from the group consisting of: oleic acid, albumin, casein peptone, catalase, Mycobactin J, and dextrose.

22. The method according to any one of claims 16-21, wherein one or more of said antibiotics is / are selected from the group consisting of: vancomycin, polymyxin B, trimethoprim, azlocillin, and nalidixic acid.

23. The method according to any one of claims 16-22, wherein said liquid broth suitable for growing Mycobacterium species further comprises a fungicide, preferably cycloheximide or amphotericin B.

24. The method according to any one of claims 16-23, wherein the method comprises further steps of taking a sample of the biomass provided in step a) or an aliquot of the liquid broth from said container before step c) and performing a nucleic acid amplification reaction comprising said aliquot or nucleic acids isolated from said sample or aliquot and oligonucleotide primers specific to at least one pathogenic Mycobacterium species in order to detect the presence and / or amount of pathogenic Mycobacterium nucleic acid in the sample before performing step c).

25. The method according to claim 24, wherein the amount of pathogenic Mycobacterium nucleic acid detected in the sample before performing step c) is used in step d) for the detection of increased presence of pathogenic Mycobacterium nucleic acid in said liquid broth during the incubation step c) in an aliquot taken after the onset of step d).

26. The method according to any one of claims 16-23, wherein step d) is performed at least two times at different time points after the onset of step d).

27. The method according to claim 26, wherein the amplification results of pathogenic Mycobacterium nucleic acid from any aliquot from any time point during step c) is used in step d) for the detection of increased presence of pathogenic Mycobacterium nucleic acid in said liquid broth in a subsequent time point during the incubation step c).

28. The method according to any one of claims 16-27, wherein said container is incubated less than 28 days but at least 1-21 days at a temperature range of 30 - 37 °C.

29. The method according to any one of claims 16-28, wherein said oligonucleotide primer pair specific to at least one pathogenic Mycobacterium species in step d) is an oligonucleotide primer pair specific to a strain or strains of Mycobacterium avium complex (MAC), preferably to Mycobacterium avium subspecies avium, or Mycobacterium avium subspecies paratuberculosis.

30. The method according to any one of claims 16-29, wherein said sample of a processed product comprising peat in step a) is a sample of the pellet produced by the method according to any one of claims 1-13.