CA2958602A1 - Essential amino acids provided by bacillus in liquid feed - Google Patents

Abstract

The present invention relates to a process for increasing the amount of at least one essential amino acid in liquid feed, the process comprising inoculating a Bacillus strain into a liquid feed and to liquid feed obtained by said process as well as to specific Bacillus subtilis strains suitable for this purpose.

Description

TITLE: Essential amino acids provided by Bacillus in liquid feed FIELD OF THE INVENTION
Liquid feed on farms is prepared by mixing feed, additives and water in a tank before feeding to the animals. Some farms ferment the feed before feeding to the animals to improve the feed quality and the gastro-intestinal balance of the animals.
Typical-ly the feed is fermented by naturally occurring or added lactic acid bacteria produc-ing lactic acid to reduce the pH which will kill pathogenic microorganisms that often have good growth conditions in liquid feed.
On the farm liquid feed is typically prepared in a tank and then pumped out through a pipeline (pipelines) to the different pens where the animals are housed.
Such a system typically includes many pipelines to ensure the supply to all animals.
In most systems some liquid feed will stay in the pipelines until the next feeding.
The present invention relates to inoculation of liquid feed with at least one Bacillus strain capable of producing a high amount of essential amino acids during a fermen-tation process by synthesizing these amino acids or by degrading proteins to amino acids by protease activity.
Amino acid production by Bacillus can be used in both fermented and non-fermented liquid feed. In non-fermented feed Bacillus will grow and produce amino acids in the feed left in the pipelines in the period from one feeding to the next feeding.
BACKGROUND OF THE INVENTION
Today feed often contains large amounts of protein to ensure the animals' require-ment for essential amino acids to ensure optimal growth. However, high protein con-tent in the feed also involves loss of nitrogen as the animals cannot utilize all the ni-trogen supplied to them. Typically only 40% of the supplied protein is utilized while 60% is excreted through the urine and feces. This may result in nitrogen wash out to lakes and streams with subsequent negative effects on animals and groundwater due to oxygen depletion. Moreover, high protein content in the feed may cause a higher risk for diarrhea, higher consumption of antibiotics as well as a high feed price and thus poor economy for the farmer.

2 Depending on the animal species different amino acids are considered as essential.
The amino acids in general regarded as essential are phenylalanine, valine, threo-nine, tryptophan, isoleucine, methionine, leucine, lysine, tyrosine and histidine. In addition, the amino acids arginine, cysteine (or sulphur-containing amino acids), gly-cine, glutamine, proline and serine are considered essential for some animal species.
Essential amino acids are "essential" not because they are more important to life than the others, but because the animal species does not de novo synthesize them and consequently they must be added with the diet to secure optimal farm animal performance.
Provision of available limiting essential amino acids would make it possible to formu-late feed rations with reduced protein content. A higher provision of available limit-ing amino acids would result in reduced nitrogen loss to the environment, improved production economy, healthier animals and subsequently, a reduced use of antibiot-ics. Currently, it is only possible to produce a few essential amino acids synthetically such as i.e. lysine, methionine and threonine and those are already used to reduce crude protein in animal diets. However, further use of essential amino acids is not possible due to the very high cost.of the amino acids and the fact that only a few of the essential amino acids are produced synthetically and thus are commercially available. Adding synthetic amino acids to the diet may also have application issues as many amino acids have to be sprayed onto the feed pellets after the heating pro-cess. The spraying process results in higher cost of the feed manufacturing process.
SUMMARY OF THE INVENTION
The problem to be solved by the present invention is to provide a sufficient amount of essential amino acids in liquid feed.
The solution is a process for increasing the amount of at least one essential amino acid in liquid feed, the process comprising inoculating a liquid feed with a Bacillus strain, which is capable of producing at least one essential amino acid in an amount of at least 600 mg/kg dry matter (DM) of at least one of the essential amino acids lysine, methionine, threonine and valine and/or at least 200 mg/kg dry matter (DM) of at least one of the essential amino acids tryptophan, isoleucine, histidine, phenyl-alanine, leucine, tyrosine, arginine, glycine, glutamine, proline and serine, when in-oculated into a liquid feed, which is fermented at a temperature of at least 30 C un-der good aerobic conditions for 24 hours.

3 Further, the invention relates to novel Bacillus strains which are capable of supplying at least one essential amino acid when inoculated into a liquid feed.
DETAILED DISCLOSURE OF THE INVENTION
The present invention is based upon the surprising finding that certain Bacillus strains can produce a high amount of at least one essential amino acid when inocu-lated into a liquid feed.
Liquid feed has advantages compared to dry feed due to several reasons: 1) the use of cheap liquid by-products from several sources makes liquid feed more economical for the farmer, 2) reduced environmental load due to the (re)use of resources such as liquid by-products is an advantage for the society, and 3) improved animal health including reduced number of pathogens in the GIT resulting in better animal produc-tion and reduced antibiotic use is an advantage for as well the farmer, the society and the animals. Further, liquid feeding increases digestibility of phytate bound phosphorus leading to reduced pollution (Lyberg K et al., 2006 and Blaabjerg, K et al., 2010).
Finally, weaned piglets can take advantage from liquid feed as they do not need to get used to dry feed because the liquid feed is similar to sow milk in consistence and their high feed intake can thereby be ensured.
A disadvantage of fermented feed is a reduction of amino acids during the fermenta-tion process resulting in a reduced nutritional value of the feed. This happens espe-cially when liquid feed stays in the feed pipeline and the amino acids are degraded (Canibe, N. & Jensen, B., 2012). The present invention provides a solution to this challenge.
Liquid feed can be based on cereals such as corn, barley or wheat, protein rich raw materials such as soy bean meal, rape seed meal, fish meal or dried distiller grains solubles (DDGS) as well as different additives such as organic acids, probiotics, ami-no acids etc. Different byproducts from the grain- , animal- , vegetable - or sugar and starch industry can be included such as i.e. bakery waste, liquid whey, corn glu-ten meal, and wheat bran. These are only examples and other ingredients can be included in the liquid feed as well.
By the term "liquid feed" is meant feed ingredients such as barley, corn and soybean meal, premixes or additives such as organic acids, vitamins and amino acids mixed together with water. Liquid feed may refer to a feed with a high water content (e.g.,

4 2:1 water:feed). Liquid feed can be fed at once to the animals or it can be fermented for a given period before being fed to the animals.
By the term "a high amount of at least one essential amino acid" is meant that the amount of a given amino acid has increased with at least 600 mg/kg dry matter (DM) of at least one of the essential amino acids lysine, methionine, threonine and valine and/or at least 200 mg/kg dry matter (DM) of at least one of the essential amino ac-ids tryptophan, isoleucine, histidine, phenylalanine, leucine, tyrosine, arginine, gly-cine, glutamine, proline and serine when inoculated into a liquid feed which is fer-mented at a temperature of at least 30 C under good aerobic conditions for 24 hours and measured by HPLC or GC-MS.
Preferably, the strain is able to produce an even higher amount such as at least 900 mg/kg dry matter (DM) of at least one of the essential amino acids lysine, methio-nine, threonine and valine, preferably at least 1200 mg/kg dry matter (DM) of at least one of the essential amino acids lysine, nnethionine, threonine and valine.
Most preferably, the strain is able to produce more than one, such as two, three or four of the amino acids in an amount of at least 600 mg/kg dry matter (DM).
Similarly, the amount of a given amino acid may increase by at least 200 mg/kg/dry matter (DM), such as at least 300 mg/kg, preferably at least 400 mg/kg, of at least one of the essential amino acids tryptophan, isoleucine, histidine, phenylalanine, leucine, tyrosine, arginine, glycine, glutamine, proline and/or serine when inoculated into a liquid feed which is fermented at a temperature of at least 30 C under good aerobic conditions for 24 hours. Most preferably, the strain is able to produce more than one, such as two, three, four, five, six, seven, eight, nine, ten, or eleven of these amino acids in an amount of at least 200 mg/kg dry matter (DM).
Some strains according to the invention are able to produce amino acids from both lists.
In this manner, the requirements for at least some of the essential amino acids will be fulfilled or at least to a certain extent meaning that it will only be necessary to add a minor amount or only some amino acids.
The term "inoculation", as used herein, refers to the act of introducing microorgan-isms or a suspension of microorganisms (e.g. Bacillus) into a culture medium (e.g.
liquid feed). One example of an inoculation is the addition of a liquid fermentation =
starter culture. Such starter culture is a microbiological culture which performs fer-mentation. A starter culture usually consists of a cultivation medium, such as grains, seeds, or nutrient liquids, which has been well colonized by microorganisms used for the fermentation. Some starter cultures include various enzymes in addition to ml-

5 croorganisms. Another example of inoculation of culture medium (e.g.
liquid feed) is the addition of dried microorganisms (e.g. spray dried) cells, powder, granula or the addition of frozen cells.
A starter culture comprising one or more Bacillus may be used for the inoculation of the liquid feed. In one embodiment the starter culture is incubated with or without stirring at an appropriate temperature (e.g. from 15 C - 37 C) for a sufficient period of time (e.g. at least 4 hours and no more than 7 days) before being mixed with an appropriate amount of non-fermented feed and fed to the farm animals.
Generally, the incubation will last for a period of at least 8, 12, 18, 24, 48, or 72 hours. Although the temperature may be as low as 10 C, it is more likely to be at least 25 C, such as at least 30 C, e.g. 37 C.
A mixture of fermented (e.g. the starter culture) and non-fermented feed may be incubated with or without stirring at an appropriate temperature (e.g. from 15-25 C) for a sufficient period of time (e.g. more than 2 hours and less than 2 days) before being fed to the animals.
In one embodiment of the invention, the process comprises the steps of i. mixing a liquid feed and at least one strain of Bacillus, ii. incubating said mixture at 10-30 C for 1-96 hours, and iii. feeding said mixture to an animal, wherein the incubated liquid feed gains an elevated level of at least one essential amino acid.
In a further embodiment the process comprises the steps of i. mixing a liquid feed and at least one strain of Bacillus, ii. incubating said mixture at 15-25 C for 3-72 hours, and iii. feeding said mixture to an animal, wherein the incubated liquid feed gains an elevated level of at least one essential amino acid.
Bacillus cells exist as bacillus spore cells and bacillus vegetative cells.
When refer-ence is made herein to Bacillus cells, this relates to both.

6 The term "Bacillus spore" in relation to a Bacillus spore cell relates herein to a spore that according to the art may be characterized as a dormant, tough, non-reproductive structure produced by Bacillus bacteria. The primary function of spores is generally to ensure the survival of a bacterium through periods of environmental stress. They are therefore resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, starvation, and chemical disinfectants. Spores are commonly found in soil and water, where they may survive for long periods of time. The spore coat is impermeable to many toxic molecules and may also contain enzymes that are involved in germination. The core has normal cell structures, such as DNA and ribo-somes, but is metabolically inactive. When a bacterium detects that environmental conditions are becoming unfavorable it may start the process of sporulation, which takes about eight hours.
The term "Bacillus vegetative cell" relates to functional vegetative Bacillus cells, which can divide to produce more vegetative cells.
The strain of the invention is of the genus Bacillus, preferably one of the species Ba-cillus amyloliquefaciens, such as Bacillus amyloliquefaciens subsp.
amyloliquefaciens or Bacillus amyloliquefaciens subsp. plantarum, Bacillus simplex, Bacillus licheniform-is, Bacillus megaterium, Bacillus mojavensis, Bacillus pumilus, Bacillus safensis, Ba-cillus simplex, Bacillus subtilis, Bacillus atrophaeus, Bacillus lentus, Bacillus methylotrophicus, Bacillus siamensis, Bacillus vallismortis or Bacillus tequilensis.
A bacterial "strain" as used herein refers to a bacterium which remains genetically unchanged when grown or multiplied. The multiplicity of identical bacteria are in-cluded.
"Wild type strain" refers to the non-mutated form of a bacterium, as found in nature.
Such Bacillus cells have not been under any selective pressure to provide a high amount of amino acids neither by synthesis nor degradation of proteins when inocu-lated into liquid feed. It is therefore believed that "wild-type" Bacillus cells do not provide a high amount of amino acids.
A "mutant bacterium" or a "mutant strain" refers to a natural (spontaneous, naturally occurring) mutant bacterium or an induced mutant bacterium comprising one or more mutations in its genome (DNA) which are absent in the wild type DNA. An "in-duced mutant" is a bacterium where the mutation was induced by human treatment, such as treatment with any conventionally used mutagenization treatment including

7 treatment with chemical mutagens, such as a chemical mutagen selected from (i) a mutagen that associates with or become incorporated into DNA such as a base ana-logue, e.g. 2-aminopurine or an interchelating agent such as ICR-191, (ii) a mutagen that reacts with the DNA including alkylating agents such as nitrosoguanidine or hy-droxylamine, or ethane methyl sulphonate (EMS) or N-methyl-N'-nitro-N-nitroguanidine (NTG), UV- or gamma radiation etc. In contrast, a "spontaneous mu-tant" or "naturally occurring mutant" has not been rnutagenized by man.
A mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screen-ing/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out. In a presently preferred mutant less than 1%, less than 0.1, less than 0.01, less than 0.001% or even less than 0.0001% of the nucleotides in the bacterial genome have been replaced with another nucleotide, or deleted, compared to the mother strain.
Mutant bacteria as described above are non-GMO, i.e. not modified by recombinant DNA technology. As an alternative to above preferred method of providing the mu-tant by random mutagenesis, it is also possible to provide such a mutant by site-directed mutagenesis, e.g. by using appropriately designed PCR techniques or by using a transposable element which is integratable in bacterial replicons.
When the mutant is provided as a spontaneously occurring mutant the above wild-type strain is subjected to the selection step without any preceding mutagenization treatment.
Several species of Bacillus have GRAS (Generally Recognized As Safe) and QPS
(Qualified Presumption of Safety) status, i.e., they are generally recognized as safe.
All Bacillus subtilis strains are GRAS and on the QPS list. The Bacillus strains de-scribed herein are aerobe or facultative anaerobe as well as spore formers.
Bacillus species are the only spore formers that are considered GRAS. Feeding microorgan-isms that have GRAS status to livestock is an acceptable practice amongst produc-ers, veterinarians, and others in the livestock industry.
As evidenced in the examples the Bacillus subtilis deposited as DSM 25483 can pro-duce the amino acid valine in a liquid feed resulting in a production of 250 mg/kg liquid feed with 16% dry matter corresponding to 1560 mg/kg feed dry matter.
This amount corresponds to an amino acid content in the liquid feed of 1.5%. This amount

8 is significant compared to the amount needed in the pig feed industry for growers and piglets (0.1%). Both liquid feed based on barley as well as standard feed with cereals and protein sources have been tested with good results.
It is contemplated that similar results will be obtained with DSM 26668 and DSM
26669 which are also developed as mutants of DSM 19467 and which have been found to provide a significant amount of valine and tryptophan, respectively, when inoculated into liquid feed, as well as with other mutants developed from DSM

or the mother strain of this strain, DSM 17231.
DSM 26668, DSM 26669 and DSM 25483 have structural genomic changes compared to DSM 19467. Genomic DNA was prepared from the four strains and DNA was se-quenced using an Illumina HiSeq platform. The genome sequences were analyzed using the CLC genome workbench (CLC Bio, Arhus, Denmark) by reference assembly of reads from one strain towards de novo assembly of another strain and lists of mu-tations or DNA sequence variations also called Single Nucleotide Polymorphism (SNP's) were generated. DSM 25483 and DSM 26668 had 5 and 7 unique SNP's re-spectively compared to the mother strain DSM 19467 and 2 in non-coding regions.
DSM 26669 had 3 unique SNP compared to the mother strain DSM 19467 in a non-coding region. These results show that the mutants were genomically different from the mother strain and that the mutants were not wild strains.
A mutant strain of the Bacillus subtilis strains with accession numbers DSM
25483, DSM 26668, or DSM 26669 can be obtained by subjecting the strain to mutageniza-tion treatment as described to obtain mutant strains and selecting for mutant strains having the desired properties. Alternatively, a selection is performed for spontane-ously occurring mutants.
Accordingly, in a further aspect the invention relates to a Bacillus composition corn-prising cells of a Bacillus strain of the invention. The composition may comprise cells of at least one, at least two, at least three, at least four or even more Bacillus strains chosen from at least one of the strains of the invention. Preferably, the cells of the Bacillus composition are spore cells.
The relevant Bacillus strains of the composition may be present in a commercially relevant form known to the skilled person. Accordingly, in an embodiment the Bacil-lus strains of the composition are present as dried (e.g. spray dried) cells or as fro-zen cells. The composition may be provided in any suitable form such as in the form of a liquid, a slurry, a powder or a pellet.

9 In a preferred embodiment the Bacillus composition comprises from 105 to 1012 CFU/g, more preferably from 106 to 1012 CFU/g, and most preferably from 102 to CFU/g.
The term "CFU/g" relates to the gram weight of the composition as such, including suitable relevant additives present in the composition. As known to the skilled per-son a commercially relevant bacterial composition generally also comprises other relevant additives such as e.g. one carrier/ingredient of the group belonging to whey, whey permeate, calcium carbonate/limestone and anti caking agents such as aluminum silicates and kieselgur (diatomaceous earth). It does not include the weight of a suitable container used to package the Bacillus composition. One embod-iment of the invention relates to a composition packaged into a suitable container.
Compositions of the present invention may include a Bacillus strain of the invention including mutants, and carriers that make these compositions suitable for addition to animals' feed. In particular, the Bacillus strain of the invention including mutants may be formulated with ingredients for liquid feed, including feed protein and/or feed carbohydrates. Such combinations may be in the form of pellets that are extruded through standard pelleting processes.
The invention also provides a method for producing a liquid feed or premix compris-ing adding a Bacillus composition of the invention to an animal feed, and appropriate instructions for fermenting the liquid feed.
The invention relates to a Bacillus composition comprising cells of at least one Bacil-lus strain. The composition may comprise cells of at least one, at least two, at least three, at least four or even more Bacillus strains. If the composition comprises more than one strain, each of the strains may be present as 5%, 10%, 20%, 25%, 33%, 40%, 50%, 60%, 66%, 75%, 80%, 90% or 95% of the Bacillus cells.
As used herein the term "premix" refers to a Bacillus strain added to a carrier to make a premix which is then added to the feed at a desired inclusion rate and fed to the animal.
The invention further relates to a method of feeding animals comprising the steps of mixing a liquid feed and a Bacillus composition, premix or starter culture as de-scribed above, fermenting said mixture at 15 C to 37 C for 1 to 96 hours, and feed-ing said mixture to an animal, wherein the fermented liquid feed gains an elevated level of at least one essential amino acid.
The animal may be selected from the group consisting of poultry, ruminants, calves, 5 pigs, rabbits, horses, fish and pets. In a preferred embodiment, the animal is a farm animal, which is raised for consumption, such as pigs, broilers and turkey, or as food-producers, such as layers.
= The use of the terms "a" and "an" and "the" and similar referents in the context of

10 describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to,") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the prac-tice of the invention.
LEGENDS TO FIGURES
Figure la Vegetative cells and spores in liquid feed fermented 48 hours with and without DSM
25483 added to the feed rations classic or ideal as described in detail in experiment 1. Samples were taken at 0, 4, 24 and 48 hours. I = classic, I = classic + DSM

25483, III = ideal, and IV= ideal+ DSM 25483 Figure lb Spores in liquid feed fermented 48 hours with and without DSM 25483 added to the feed rations classic or ideal as described in detail in experiment 1. Samples were taken at 0, 4, 24 and 48 hours. I = classic, I = classic + DSM 25483, III =
ideal, and IV= ideal+ DSM 25483.

11 Figure 2 Valine content (ppm) in liquid feed fermented 48 hours with and without DSM

added to the feed rations classic or ideal as described in detail in experiment 1.
Samples were taken at 0, 4, 24 and 48 hours. I = classic, I = classic + DSM
25483, III = ideal, and IV= ideal+ DSM 25483.
Figure 3 Vegetative cells and spores in liquid feed fermented 48 hours with and without DSM
25483 added to the feed ration ideal as described in detail in experiment 2.
Samples were taken at 0, 4, 24 and 48 hours. I = ideal, and II = ideal+ DSM 25483.
Figure 4 Valine content (ppm) in liquid feed fermented 48 hours with and without DSM

added to the feed ration ideal as described in detail in experiment 2. Samples were taken at 0, 4, 24 and 48 hours. I = ideal, and II = ideal+ DSM 25483.
Figure 5 Lysine content (ppm) in liquid feed fermented 48 hours with and without DSM

added to the feed ration ideal as described in detail in experiment 2. Samples were taken at 0, 4, 24 and 48 hours. I = ideal, and II = ideal+ DSM 25483.
Figure 6 Methionine content (ppm) in liquid feed fermented 48 hours with and without DSM
25483 added to the feed ration ideal as described in detail in experiment 2.
Samples were taken at 0, 4, 24 and 48 hours. I = ideal, and II = ideal+ DSM 25483.
Figure 7 Threonine content (ppm) in liquid feed fermented 48 hours with and without DSM
25483 added to the feed ration ideal as described in detail in experiment 2.
Samples were taken at 0, 4, 24 and 48 hours. I = ideal, and II = ideal+ DSM 25483.
DEPOSITS and EXPERT SOLUTION
A sample of the novel Bacillus subtilis strain BacVall has been deposited at DSMZ
(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg lb, D-38124 Braunschweig) under the accession number DSM 25483 with a deposit date of December 14, 2011. The deposit has been made under the conditions of the

12 Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
A sample of the novel Bacillus subtilis strain BacVal2 has been deposited at DSMZ
(Deutsche Sammlung von Mikroorganisnnen und Zellkulturen GmbH, Maschroder Weg lb, D-38124 Braunschweig) under the accession number DSM 26668 with a deposit date of November 27, 2012. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
A sample of the novel Bacillus subtilis strain BacTRP has been deposited at DSMZ
(Deutsche Samnnlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg lb, D-38124 Braunschweig) under the accession number DSM 26669 with a deposit date of November 27, 2012. The deposit has been made under the conditions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure.
For all of the above-identified deposited microorganisms, the following additional in-dications apply:
As regards the respective Patent Offices of the respective designated states, the ap-plicants request that a sample of the deposited microorganisms stated above only be made available to an expert nominated by the requester until the date on which the patent is granted or the date on which the application has been refused or withdrawn or is deemed to be withdrawn Embodiments of the present invention are described below, by way of non-limiting examples.
EXAMPLES
Two series of experiments were conducted to examine the effect of inoculating pig liquid feed with a Bacillus mutant that overproduces amino acids on the amount on amino acids in the liquid feed after inoculation. In the first series two different com-mercial pig diets, Svin Enhed Classic Heise U ("classic") and Svin Enhed Ideal Heise U ("ideal") were fermented with or without the inoculation of DSM 25483. In the sec-ond series of experiments five independent fermentations with the inoculation of DSM 25483 were carried out with the diet ideal.

13 Materials and methods Two different commercially pelleted feed rations for growing pigs were used with dif-.
ferences in composition and chemically analysis (Tables 1 and 2) Svin Enhed Classic Heise U, DLG Denmark Svin Enhed Ideal Heise U, DLG Denmark Antifoam Y-30 Emulsion (Sigma, A6457) Trypic-soy agar (TSA) (Oxoid, CMO 129) Colistinsulfat (Sigma, C4461) Strains: DSM 25483

14 Table 1 Composition and ingredients of feed rations Svin Enhed Classic and Ideal Heise U
Feed for finisher pigs Composition Classic Ideal unit Amount Amount Wheat 35.00 25.00 %
Rye 9.00 19.80 %
Barley 25.00 22.50 %
Rapeseed meal , 0 10.00 %
Soybean meal 9.80 5.00 %
Sunflower meal 8.00 8.00 %
Oat 4.60 5.00 %
Wheat bran 3.00 0 %
Palm fat 1.40 0.80 %
Calcium Carbonate 1.26 1.14 %
Sugar beet molasses 1.00 1.0 %
Vitalys, liquid 0.76 0.70 %
Rock salt 0.44 0.45 %
Monocalcium phosphate 0.36 0.27 %
Swinevit 448 (vitamins & minerals) 0.20 0.20 %
Threonine 98% 0.09 0.07 %
Xylanase enzyme, E4a11 0.04 0.05 %
6-phytase, EC3.1.3.26 (E4a12) 0.03 0.02 %
DL-Methionine 0.02 0 %
Ingredient added per kg Amount Amount Unit Copper (II) sulphate, pentahydrate (E4) 39.00 39.00 mg Zinc oxide (E6) 137.00 137.00 mg Iron(11) sulphate, monohydrate (El) 256.00 256.00 mg Calcium iodate anhydrous (E2) 1.31 1.31 mg Manganese (I1)oxide (E5) 54.00 54.00 mg Sodium Selenite (E8) 0.69 0.69 mg Vitamin A (E672) 4200 4200 IE
DL-alpha-tocopherol (3a700) 40.00 40.00 mg Vitamin 81 2.10 2.10 mg Vitamin 812 0.02 0.02 mg Vitamin B2 2.10 2.10 mg Vitamin B6 (3a831) 3.15 3.15 mg Biotin 0.05 0.05 mg D-pantothenic acid 10.50 10.50 mg Vitamin D3 (E671) 420 420 IE
Niacin (3a314) 21.00 21.00 mg L-lysine sulphate 2.4 2.2 g DL-methionine 0.2 0 g L-threonine 0.9 0.7 g Phytase 1000 1000 units Endo-1,4-beta-xylanase, EC3.2.1.8 3200 3200 units =
Table 2 Analytical constituents of the feed rations Analytical constituents Classic Ideal Unit Amount Amount Feed units for swine pr. 100kg 104 103 FEsv Crude protein 15.1 15.6 Crude fat 3.6 3.2 Crude fiber 4.9 5.4 cyo Ash 4.7 4.6 0/0 Water 14.4 14.4 Lysine 9.0 9.2 g/kg Methionine 2.8 2.8 g/kg Calcium 6.9 6.7 g/kg Phosphorus 4.7 4.7 g/kg Sodium 1.9 2.0 g/kg 5 A fermentation of liquid feed was done in 4 small lab-scale bioreactors of 2 liters.
The bioreactor containing the pH and oxygen probes were added 1250 ml tap water, the probes were calibrated and the whole system sterilized by steam sterilization at 121 C. After the temperature was stable at 37 C 250 gram feed and 0.085% anti-foam were added. The feed was used as is (without sterilization). The dissolved oxy-1 0 gen concentration was adjusted to 100% air saturation and the fermentations were initiated for experiment 1 in the 4 bioreactors with I) negative control feed classic, II) feed classic + DSM 25483, III) negative control feed ideal and IV) feed ideal +
DSM 25483. DSM 25483 was added at a concentration of 0.7 E+07 CFU/g. The fer-mentations were carried out at 37 C at a stirring rate of 500 rpm with continuous air

15 sparking (500 ml/min) where pH and dissolved oxygen could be continuously deter-mined by use of pH and oxygen probes.
In experiment 2 five independent fermentations with the inoculation of DSM

(0.7 E+07 CFU/g) were carried out with the diet ideal compared to a negative control group feed ideal without DSM 25483. At time 0, 4, 24 and 48 hours after initiating of the fermentations samples were aseptically withdrawn from the bioreactors for measurements of Bacillus subtilis (vegetative cells and spores), free amino acids, and lactic acid bacteria (experiment 2).

16 Table 3 Overview of experiments and treatment groups Experiment Treatment group Feed DSM 25483 1 1 Ideal 1 II Classic 1 III Ideal 1 IV Classic 2 I Ideal 2 II Ideal Vegetative cells and spores were analyzed by spread plate technique using trypic-soy agar (TSA) added colistine to kill other bacteria. Spores were analyzed by spread plate technique using TSA and heat treatment at 80 C for 10 minutes to kill other bacteria.
The concentrations of the amino acids valine, lysine, methionine, and threonine in the fermentation broth were measured by HPLC as described by Lazanov et al., 2007.
Results and Discussion Experiment 1:
In the negative control groups the count of Bacillus subtilis vegetative cells as well as B. subtilis spores were close to the detection limit of log 4 CFU/g in both diets at all sampling times (Figures la + lb). This means that no contamination with Bacillus has occurred in the negative control samples and that the samples did not contain relevant levels of Bacillus. In the treatment groups II and IV with DSM 25483 added, the count of Bacillus spores were close to 1 E+07 spores per gram of fermentation broth for both diets at the beginning of the fermentations (Figure lb). This agrees very well with the amount of 0.7 E+07 DSM 25483 added. After 4 hours of fermenta-tion the count of Bacillus spores were below the detection limit of Log 4 CFU/g for both diets in treatment groups II and IV (Figure lb). In contrast the counts of vege-tative cells + spores have increased compared to the counts at the beginning of the fermentations, showing that in the fermentations with both diets, DSM 25483 has germinated and has been growing. A further increase in the number of Bacillus vege-tative cells and spores were detected after 24 hours of fermentation of both diets in-dicating a further growth of DSM 25483. A decrease in the number of total vegetative cells and spores were detected between 24 and 48 hours of fermentations for both diets. This decrease in the total number of vegetative cells and spores were accom-

17 panied by a slight increase in the number of spores, indicating that the growth of DSM 25483 had terminated and that some of the vegetative cells had sporulated.
A high amount of free valine was found after 24 and 48 hours of fermentation of both diets when DSM 25483 was added to the diets, reaching values above 400 mg/kg fermentation broth (liquid feed), while no increase in free valine was found in the negative control fermentations (Figure 2b).
Experiment 2:
A similar pattern was seen in experiment 2 concerning vegetative cells and spores in the 5 treatment groups with CFU at detection level in the negative control group and count of bacillus spores at E+07 spores + vegetative cells per gram of fermentation broth for the diets added DSM 25483 at the beginning of the fermentations (Figure 3). High counts of lactic acid bacteria at the end of the fermentation were observed indicating a good fermentative process.
Also in experiment 2 an increased valine production could be measured when DSM
25483 was added to the feed (300 mg/kg liquid feed (variation from 172 to 389 mg/
kg). An amount of 300 mg valine /kg liquid feed correspond to 1.9 g free valine per kg dry feed.
Also a net production of lysine (500 mg/kg liquid feed), methionine (200 mg/kg liq-uid feed) and threonine (140 mg/kg liquid feed) was found in the fermentations add-ed DSM 25483, indicating protease activity rather than only a de novo synthesis of valine.
REFERENCES
Can ibe, N. & Jensen, B. "Fermented liquid feed - Microbial and nutritional aspects and impact on enteric diseases in pigs", Animal Feed Science and Technology, 2012, 173,17-40 Lazanov, V., Benkova, B., Mateva, L., Petrov, S., Popov, E., Slavov, C. &
Mitev, V.
"Liquid chromatography method for simultaneous analysis of amino acids and biogen-ic amines in biological fluids with simultaneous gradient of pH and acetronitrile", Journal of Chromatography B, 2007, 860, 92-97

18 Lyberg K, Lundh T, Pedersen C and Lindberg JE, "Influence of soaking, fermentation and phytase supplementation on nutrient digestibility in pigs offered a grower diet based on wheat and barley. "Animal Science 2006, 82, 853-858 Blaabjerg K, Jorgensen H, Tauson A-H and Poulsen HD, "Heat-treatment, phytase and fermented liquid feeding affect the presence of inositol phosphates in ileal diges-ta and phosphorus digestibility in pigs fed a wheat and barley diet. Animal 4, 2010b,

Claims (15)

19

1. A process for increasing the amount of at least one essential amino acid in liquid feed, the process comprising inoculating a Bacillus strain into a liquid feed.

2. The process according to claim 1, wherein said Bacillus strain is capable of producing an essential amino acid in an amount of at least 100 mg/kg dry matter (DM) when 0.7 E+07 is inoculated into a liquid feed which is ferment-ed at a temperature of at least 30°C under good aerobic conditions for hours.

3. The process according to claim 1 or 2, wherein the liquid feed comprises in-gredients from the group consisting of cereals such as corn, barley or wheat;
protein rich raw materials such as soy bean meal, rape seed meal, fish meal or dried distiller grains solubles (DDGS), additives such as organic acids, pro-biotics, amino acids, and byproducts such as bakery waste, liquid whey, corn gluten meal, and wheat bran.

4. The process according to any one of claims 1 to 3, wherein the Bacillus strain produces more than one essential amino acid.

5. The process according to any one of claims 1 to 4, wherein the essential ami-no acid is at least one amino acid selected from the group consisting of phe-nylalanine, valine, threonine, tryptophan, isoleucine, methionine, leuane, ly-sine, histidine, arginine, cysteine or other sulphur-containing amino acids, glycine, glutamine, histidine, proline, serine and tyrosine or other aromatic amino acids.

6. The process according to any one of claims 1 to 5, wherein the Bacillus strain is a Bacillus subtilis strain.

7. The process according to claim 6, wherein the Bacillus subtilis strain is capa-ble of producing valine in amount of at least 600 mg/kg feed dry matter when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

8. The process for preparing a liquid feed according to any one of claims 1 to 7, wherein the Bacillus subtilis strain is capable of producing threonine in amount of at least 600 mg/kg feed dry matter when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

9. The process for preparing a liquid feed according to any one of claims 1 to 8, wherein the Bacillus subtilis strain is capable of producing methionine in amount of at least 600 mg/kg feed dry matter when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

10. The process for preparing a liquid feed according to any one of claims 1 to 9, wherein the Bacillus subtilis strain is capable of producing lysine in amount of at least 600 mg/kg feed dry matter when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

11. The process for preparing a liquid feed according to any one of claims 1 to 10, wherein the Bacillus subtilis strain is selected from the group consisting of DSM 25483, DSM 26668 and DSM 26669 and a mutant of any of these strains which is capable of synthesizing at least 100 mg/kg DM of at least one essen-tial amino acid when inoculated into a liquid feed and the liquid feed is fer-mented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

12. A liquid feed obtained by the process according to any of claims 1-11.

13. A Bacillus subtilis strain deposited as DSM 25483 or a mutant strain thereof which is capable of synthesizing at least 100 mg/kg DM of at least one essen-tial amino acid when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

14. A Bacillus subtilis strain deposited as DSM 26668 or a mutant strain thereof which synthesizes at least 100 mg/kg DM of at least one essential amino acid when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.

15. A Bacillus subtilis strain deposited as DSM 26669 or a mutant strain thereof which synthesizes at least 100 mg/kg DM of at least one essential amino acid when inoculated into a liquid feed which is fermented at a temperature of at least 30°C under good aerobic conditions for 24 hours.