HK1160880A - A bile resistant bacillus composition secreting high levels of essential amino acids - Google Patents

Description

Bile resistant bacillus compositions secreting high levels of essential amino acids

Technical Field

The present invention relates to a bacillus composition characterized by rapid germination and growth in bile salts (simulated intestinal environment) and secretion of high levels of essential amino acids. The bacillus composition can be used as an animal feed supplement, in which it has a prebiotic (health promoting) effect and increases the digestion and availability of nutrients in the animal feed.

Background

Probiotics such as Bacillus subtilis and Bacillus licheniformis are used as dietary supplements in the animal feed industry. Its use is related to the ability of the bacillus to replace or reduce the use of antibiotics used as growth promoters in the animal feed industry.

An example of such a probiotic growth promoting product is known under the trade name Christian Hansen A/S (Denmark)Commercialization was achieved (accession number DSM 17231).Is a bacillus subtilis spore cell composition.

In addition to the recommended mode of action (e.g., immunomodulation, gut flora modulator), probiotic bacillus are capable of producing many beneficial components, such as enzymes, which are secreted into the gastrointestinal tract (GIT) when used as animal feed supplements. Enzymes such as phytase are secreted and improve digestion and better uptake (higher digestibility) of animal feed. The diet (feed) is mainly of plant origin such as cereals, maize, soya oil and amino acids. All of these effects contribute to the production of low cost animal products.

Probiotic bacillus (probiotic bacillus) is also capable of producing other beneficial components such as essential amino acids.

Bacillus spores can pass through the acidic gastric barrier and bud and grow in the gastrointestinal tract (GIT) of animals. This property is of great advantage because when ingested, they can secrete many types of beneficial components, e.g., bacteriocins, and also secrete useful essential amino acids. Moreover, during feed pelleting, bacillus spores are thermostable, so they are excellent delivery systems for both bacteriocins and e.g. essential amino acids into the GIT (gastrointestinal tract).

The role of bile is important during the survival and proliferation of bacillus in the gastrointestinal tract. Bile is produced in the liver and stored in the gallbladder. Bile contains water, lecithin, bilirubin and biliverdin and bile salts.

It is known from the literature that bile has some negative effects on the survival and budding and growth of bacillus spore cells into vegetative cells in the gastrointestinal tract of animals. Studies are therefore being conducted to find probiotic bile-resistant bacillus strains.

The article (Antonie Van Leeuwenhoek.2006Aug; 90 (2): 139-46.Epub 2006, 7/4) describes the isolation of a number of Bacillus specimens/cells directly from chicken intestines. Detecting the probiotic activity of the isolated bacillus cells. The 6 bacillus species with the highest probiotic activity were tested for bile salt tolerance and a particular probiotic bacillus species was found to have a relatively high level of bile salt tolerance.

No particular emphasis has been placed in this article on any period of testing for bile resistance. In the experimental part, the tolerance of bacillus spore cells was only tested after 5 days in the presence of bile salts (see paragraph "Simulated small intestinal fluid tolerance test" on page 141).

US2003/0124104a describes that probiotic conventional bacillus endospores are sensitive to low concentrations of bile salts, i.e. the presence of even low concentrations of bile salts inhibits spore germination and/or rehydration. This is in contrast to other bacteria such as intestinal pathogens, such as E.coli or S.aureus (see paragraphs [0014] to [0015 ]). In this regard, it is proposed to screen/select for bacillus spores that are resistant to the inhibitory activity of bile salts and therefore are able to bud into vegetative cells and then colonize the colon (see [0019 ]).

All provided in this specification are working examples, and do not provide actual experimental data for the particular bacillus cells actually screened.

Furthermore, the description of the bile salt screening conditions is relatively general. In particular, no period of bile resistance is indicated to be selected. In other words, based on a rough or generalized illustration of this document, it is possible to select only slowly growing (germinating) bacillus cells, i.e. bacillus cells which are capable of germinating from spores to vegetative cells after, for example, 20 hours in the presence of a corresponding amount of bile salts.

In this document, there is no description or suggestion of selecting bacillus cells that grow (germinate) rapidly, i.e. that germinate from spores and grow into vegetative cells, which are capable of reaching a defined growth point within a specific time interval in the presence of a corresponding amount of bile salts.

In summary, prior art references to the selection/screening of bile resistant bacillus cells have not focused on the rapid growth/budding from spore cells into bacillus vegetative cells.

International PCT application No. PCT/EP2008/057296 was filed 11/06/2008. Hansen A/S and which was not published at the time of filing of the present application.

PCT/EP2008/057296 describes novel bacillus spores characterized by an increased/rapid rate of germination and growth from spores into vegetative cells in the presence of bile salt media.

The bacillus spores described herein have the same increased/rapid rate of germination and outgrowth from the spores into vegetative cells as described in PCT/EP 2008/057296.

PCT/EP2008/057296 describes only Bacillus vegetative cells that produce an increased amount of phytase compared to the reference Bacillus cell DSM 19467. There is no description and no suggestion to screen for a bacillus vegetative cell producing a higher amount of essential amino acids than the reference bacillus cell DSM 19467.

When the following description refers to the prior art, it should be understood that it is prior art (e.g., a published article/published patent) available to the public at the time of filing this application.

Disclosure of Invention

The problem to be solved by the present invention is to provide a bacillus composition which secretes a large amount of essential amino acids in the gastrointestinal tract (GIT) of an animal.

The technical scheme is based on: the present inventors have developed a new selection method for identifying new improved bacillus compositions.

A new important step of the new selection method described herein is the specific screening/selection of bacillus spore cells with increased/rapid germination from spores and growth into vegetative cells in the presence of bile salts.

As described above, the prior art has described methods for screening for bacillus capable of growing in the presence of bile salts, but the prior art screening/selection methods have not focused on the speed of germination and growth in the presence of bile salts. Thus, the bile-resistant cells screened in the prior art did not bud and grow fast enough to meet the speed criteria for sprouting and growth described herein. For example, bacillus cells isolated directly from, for example, chicken intestine in the gut environment (as described, for example, in the Antonie Van leewenhoek article discussed above) have not been selected (at atmospheric pressure) to rapidly bud and grow in the gut.

As shown in the working examples herein, for a commercial Bacillus compositionAlso, in the presence of physiological levels of bile saltsIt germinated and grew too slowly and failed to reach the defined growth point within the first 20 hours, not meeting the germination and growth rate criteria described herein.Is a commercially successful bacillus subtilis composition. By usingThe novel DSM19467 described herein was selected as the starting strain and the strain that rapidly buds out from spores and grows into vegetative cells in the presence of bile salts by selective pressure methods and subsequent isolation as described herein.

For further details see Table 1 (herein)Also known as DSM 17231).

This is schematically illustrated in fig. 1 herein.

In summary, it is believed that the prior art does not teach an isolated Bacillus composition comprising 10⁵-10¹²CFU/g bacillus cell, wherein the cells of the bacillus composition meet the criteria for rapid germination and growth in the presence of bile salts as described herein.

Without being bound by theory, the inventors have identified that rapid germination and growth is a very important aspect of the present invention, as bacillus spores are bile resistant, but do not germinate and grow fast enough to be excreted before the bacillus vegetative cells give rise to any significant positive properties in large quantities such as production of essential amino acids. Spores of bacillus that germinate too slowly simply pass through the gastrointestinal tract (GIT) before the bacteria can produce any significant amount of, for example, essential amino acids.

After extensive and exhaustive testing and analysis, the inventors therefore chose to work in a time frame of up to 20 hours and to select the spores that sprout and grow most rapidly in the presence of very high physiological concentrations of bile salts over this period. Without being bound by theory and based on experimental work disclosed in detail herein, the present inventors have identified that rapid budding and growth in the presence of 4 and 6mM bile salts, within the first 18 and 19 hours, respectively, is important.

The inventors then identified bacillus cells that, once selected for rapid germination and growth in bile salt medium, were very useful as starting cells for mutagenesis to obtain new cells with increased production of essential amino acids.

As schematically illustrated in fig. 1 and example 4, the fast-growing bile-resistant selection strain DSM19467 was used as the starting strain for typical mutations and strains with high essential amino acid production were selected. As can be seen in example 4, the essential amino acid leucine produced by some of the selected strains is DSM19467 andat least 5 times more.

Thus the novel probiotic bacillus cell disclosed herein is a bile-resistant, fast-germinating and growing bacillus cell which secretes large amounts of essential amino acids. The obtained strains are very useful for use as probiotic bacillus compositions for addition to animal feed. It combines all the beneficial abilities of probiotics to survive and proliferate in the intestine of animals (high bile salt levels are present), to inhibit pathogenic bacteria (bacteriocin production), and additionally to secrete large amounts of beneficial essential amino acids.

Accordingly, a first aspect of the present invention relates to a bacillus composition comprising 10⁵-10¹²CFU/g Bacillus spore cells, wherein the Bacillus composition is characterized by:

(i) the method comprises the following steps The bacillus spores are rapidly germinated and grown into vegetative cells from the spores in the presence of a bile salt medium comprising 4 and 6mM bile salts, wherein the rapid germination and growthDefined as the bacillus spore reaching 0.4OD in less than 18 hours and 19 hours, respectively₆₃₀Wherein the vegetative cell growth point is the point in the growth curve at which the OD value begins to increase in a continuous manner (caused by the growth of vegetative cells) and reaches an OD630 of 0.4;

(I) the method comprises the following steps Wherein the bile salt medium is a standard known nonselective veal leach broth (VIB) medium supplemented with a bile salt mixture comprising the bound bile salts taurodeoxycholate and glycodeoxycholate and the unbound bile salt deoxycholate in the proportions 60% taurodeoxycholate, 30% glycodeoxycholate and 10% deoxycholate, as in example 1 herein; and

wherein the OD determination analysis is performed by the following steps:

(a) the method comprises the following steps The wells of the microtiter plate were filled with 0.150ml of 10ml of medium per ml⁸Bile salt medium of bacillus spores (i.e., this is time zero); and

(b) the method comprises the following steps The plate was incubated at 37 ℃ under atmospheric conditions and OD was measured using a spectrophotometer₆₃₀Values and agitation prior to each reading to obtain a representative time-varying growth curve;

and

(ii) the bacillus vegetative cells produce at least one essential amino acid in an amount higher than the reference bacillus cell DSM19467, wherein the amount of essential amino acid produced is determined by the standard GC-MS based amino acid assay of example 2 herein after 2 days of Growth in standard known low salt Growth Medium (minimum Salts Growth Medium) of example 2 herein at 37 ℃.

By using, as described aboveSelection of reference Bacillus cells DSM19 for Rapid Germination and growth in the Presence of bile salts as starting Strain467. No increase in essential amino acid production was selected for DSM 19467. Without being bound by theory, it is believed that the essential amino acid production and the herein related essential amino acid production of DSM19467And (4) the equivalent.

With respect to the item (i) above,the vegetative cell growth point of (a) is at least 20 hours after incubation in 4 and 6mM bile salts, and for the new DSM19467 strain, the vegetative cell growth point is after 14 and 15 hours in 4 and 6mM bile salts, respectively, as described herein (see figure 2 and working example 3 herein).

It is noted here that the inventors also tested commercially available products(Calpis Co., Ltd., Japan) to determine the vegetative cell growth point under the conditions of item (i) of the first aspect. LikeCommercially available productIs a bacillus subtilis composition used as a probiotic feed additive. For theThe vegetative cell growth point under the conditions of item (i) of the first aspect is 20 hours or more in 4 and 6mM bile salt, respectively. This is well in excess of the 18 and 19 hours required in item (i) and this illustrates that, to date, no commercially available product can be selected for rapid germination and growth. As mentioned above, "native" Bacillus cells cannot bud and grow rapidly under any selective pressure. Without being limited to theory, it is therefore believed that "native" Bacillus cells do not meet the conditions of item (i) of the first aspect.

The determination of bile resistance [ item (i) ] and the determination of essential amino acids [ item (ii) ] are both based on known commercially available standard elements (such as, for example, standard medium, bile salts; standard OD assay and standard detection).

The reference bacillus cell was deposited as DSM19467 and is therefore publicly available.

Bacillus subtilis cellDeposited as DSM 17231 (named) And are therefore also publicly available.

Thus, based on the detailed assay description herein (see, e.g., the bile resistance assay of example 1 herein and the essential amino acid assay of example 2 herein), one skilled in the art can routinely repeat these assays to objectively determine whether a particular bacillus cell of interest meets the bile resistance of [ item (i) ] and the essential amino acid level of [ item (ii) ] of the first aspect of the invention.

The novel bacillus composition as described herein may be used as a probiotic supplement for animal feed. Dosage and administration can be according to the art, e.g., prior artThe bacillus composition is performed according to the protocol employed.

Accordingly, a second aspect of the present invention relates to a method of feeding an animal comprising administering the bacillus composition of the first aspect and the related embodiments described herein to the animal together with other animal feed ingredients.

A third aspect of the present invention relates to a method for screening and isolating a Bacillus neoformans cell comprising the steps of:

(a) the method comprises the following steps Screening and isolating Bacillus neoformans spore cells from a collection of Bacillus monoformans spore cells that are capable of rapid germination and growth such that they reach the vegetative cell growth point in less than 18 and 19 hours under the conditions of item (i) of the first aspect;

(b) the method comprises the following steps Preparing bacillus vegetative cells from the isolated spore cells of step (a) and mutating the selected and isolated new cells to obtain a pool of new bacillus mono-vegetative cells;

(c) the method comprises the following steps Selecting and isolating from the pool of Bacillus neoformans mono-vegetative cells of step (b) a Bacillus neoformans vegetative cell capable of producing a higher amount of at least one essential amino acid than the reference Bacillus cell DSM19467 under the conditions of item (ii) of the first aspect; and

(d) the method comprises the following steps Analyzing the high-yielding vegetative bacillus cells of step (c) to confirm that they maintain the rapid germination and growth of step (a), and isolating the selected cells.

It will be apparent to those skilled in the art that once the inventors have disclosed herein a relevant detection assay (in particular the assay for detecting rapid germination and growth of example 1) plus the reference strain DSM19467, it will be routine work for those skilled in the art to select other novel bacillus cells that meet the criteria of the first aspect herein.

As discussed herein, the present inventors have selected and isolated a number of new modified bacillus cells by using the new screening/selection methods as described herein.

Embodiments of the present invention are illustrated below by way of examples only.

Definition of

All definitions of terms used herein are consistent with those skilled in the art to which the relevant art pertains.

The term "Bacillus cell" as used herein refers to both Bacillus spore cells and Bacillus vegetative cells.

With respect to bacillus spore cells, the term "bacillus spore" as used herein refers to a spore that, according to the characteristics of the art, can be a dormant, tough, non-reproductive structure produced by bacillus. The main function of spores is generally to ensure the survival of bacteria during environmental stress. They are therefore resistant to ultraviolet and gamma radiation, desiccation, lysozyme, temperature, hunger and chemical disinfectants. Spores are commonly found in soil and water where they can survive for long periods of time. The spore coat is impermeable to many toxic molecules and also contains enzymes involved in budding. The core has normal cellular structures such as DNA and ribosomes, but is metabolically inactive. When the bacteria detect that environmental conditions become unfavorable, it can initiate the sporulation process, taking about 8 hours.

The term "bacillus vegetative cell" refers to a functional vegetative bacillus cell that can be split to produce more vegetative cells.

The term "germination and growth" refers to the germination and growth of a bacillus spore into a bacillus vegetative cell. As is well known to those skilled in the art, the revival of spores occurs when conditions are favorable and involves germination and growth. Budding means that the dormant spores turn on metabolic activity and thus interrupt dormancy. It is generally characterized by rupture or absorption of the spore coat, swelling of the spores, increased metabolic activity, and loss of tolerance to environmental stress. Growth occurs after budding and involves the spore core making new chemical constituents and breaking off the old spore coat to develop into functional vegetative bacterial cells that divide to produce more cells.

The growth curve (OD over time) of the bacterial cells shows different growth phases. When the spores are transferred to a nutrient-rich medium, budding begins, followed by a temporary drop in OD (phase I), due to the release of dipicolinic acid and thus hydration of the spore coat. In the second phase (phase II ═ growth phase), there is a period in which the change in OD is relatively small until the spores develop into functional vegetative bacterial cells which can divide to give rise to more cells, thereby allowing a continuous increase in OD value. The point when the OD value begins to increase continuously to reach an OD of 0.4 is referred to herein as the "vegetative cell growth point".

The term "optical density" is defined as an indicator of absorbance using a spectrophotometer. Optical Density (OD) is the absorbance of an optical element per unit distance for a given wavelength λ. If, for example, the OD is determined at a wavelength of 630nm, it can be referred to as OD₆₃₀。

Drawings

FIG. 1: in this figure, the steps to obtain the new improved strains herein are illustrated. Working example herein is from DSM 17231Initially, it was typically mutated and screened/selected for rapid budding and growth in the presence of bile salts to give the new selection strain DSM 19467. DSM19467 was used as a starting strain for typical mutations and strains with high essential amino acid production were selected.

Fig. 2a and 2 b: these figures clearly show that the bacillus spores of DSM 19647 according to the invention germinated and grew faster in the presence of 4 and 6mM bile salts as described herein compared to DSM 17231.

Detailed Description

The bacillus composition comprises the following components:

the term "bacillus composition" should be understood in the art. It is herein understood to be a bacillus composition comprising a plurality of bacillus spore cells having a property of interest.

The Bacillus composition may comprise different types of Bacillus cells (e.g. Bacillus subtilis) and Bacillus licheniformis(s) per se, the composition should simply comprise a quantity of Bacillus spore cells as specified in the first aspect herein, wherein the Bacillus cells meet the criteria specified in the first aspect.

As is well known to those skilled in the art, the relevant Bacillus spore cell compositions commercially available herein are typically prepared by fermentation. The obtained spore cells are concentrated, dried, mixed with a carrier and packaged in a suitable container.

Suitable compositions, e.g. 10⁵-10¹²CFU/g Bacillus cells can be present in a suitable form commercially available as known to those skilled in the art.

Thus, in one embodiment, 10 of the composition⁵-10¹²CFU/g bacillus spore cells exist as dried (e.g., spray dried) cells or as frozen spore cells.

In a preferred embodiment, the bacillus composition includes 10⁶-10¹²CFU/g Bacillus spore cell, more preferably 10⁷-10¹²CFU/g Bacillus spore cells.

The term "CFU/g" refers to the gram weight of the composition, such as including suitable relevant additives present in the composition. It does not include the weight of a suitable container used to package the bacillus composition.

One embodiment relates to the bacillus composition being packaged in a suitable container.

As is well known to those skilled in the art, commercially relevant bacterial compositions often also include other relevant additives such as, for example, a carrier/ingredient belonging to the group of whey, whey permeate, calcium carbonate/limestone and anti-caking agents such as aluminium silicates and diatomaceous earth.

In addition to the relevant bacillus cells herein, the compositions also comprise other relevant microorganisms of interest, such as, for example, lactic acid bacteria of interest.

Bacillus cell

The bacillus cell may be any related bacillus cell of interest.

In a preferred embodiment, the bacillus cell is at least one bacillus cell selected from the following bacillus species:

bacillus subtilis, Bacillus megaterium, Bacillus polymyxa, Bacillus licheniformis, Bacillus pumilus, Bacillus coagulans, Bacillus thermophilus, Bacillus mycosphatilis, Bacillus cereus and Bacillus circulans.

In a more preferred embodiment, the Bacillus cell is a Bacillus subtilis cell or a Bacillus licheniformis cell.

Most preferred are those wherein the Bacillus cell is a Bacillus subtilis cell.

Selection of assays for Rapid Germination and growth in the Presence of bile salts

As mentioned above, the bile resistance assay of item (i) of the first aspect is based on known commercially available standard elements (such as, for example, standard medium, bile salts; standard OD assay).

Thus, based on the assay specifications detailed herein (see, e.g., example 1 herein), one skilled in the art can routinely repeat this assay to objectively determine whether a particular bacillus spore cell of interest meets the criteria for rapid germination and outgrowth from spores into vegetative cells as described in item (i).

In item (i), it is explained that the vegetative cell growth point is 10 from the OD corresponding to around 0.2-0.3 in the growth curve⁸Spores/ml began until the OD value had increased in a continuous manner (due to vegetative cell growth) and reached the point of OD 0.4. As explained herein, it is consistent with how one skilled in the art understands this vegetative cell growth point, and based on the growth curve, one skilled in the art can routinely determine this growth point, within a limited error range of about ± 30 minutes.

Working example 1 herein provides a detailed description of a bile resistance assay suitable for selecting rapid germination and growth in the presence of bile salts. The detailed conditions of this example 1 are a preferred assay for determining whether or not the spore cells of Bacillus sp.

The term "bile salts" refers to salts of bile acids. Bile acids are steroid acids found primarily in bile of mammals. They are produced in the liver by the oxidation of cholesterol, stored in the gallbladder and secreted in the form of salts into the intestine. They act as surfactants, emulsifying lipids and assisting in their absorption and digestion. Physiological concentrations and compositions mimicking porcine bile salts the bile salts used in example 1 were prepared. As is well known to those skilled in the art, porcine bile salt compositions may be considered herein as relatively "harsh" conditions compared to avian bile salt compositions.

The term "bile salt medium" refers to a medium comprising relevant bacillus growth components such as relevant nutrients and bile salts.

Vegetative cell growth Point-in bile salt assay item (i) of the first aspect

As described above, in relation to item (i) of the first aspect, the speed at which bacillus spore cells bud from spores and grow into vegetative cells is such that they reach a vegetative cell growth point of 0.4OD in less than 18 and 19 hours in 4 and 6mM bile salts, respectively, as described herein.

As described above, the new DSM19467 strain reached vegetative cell growth points after 14 and 15 hours of incubation in 4 and 6mM bile salts, respectively.

Thus, in a preferred embodiment, under the conditions of item (i) of the first aspect the bacillus spore reaches the vegetative cell growth point after 17 and 18 hours of incubation in 4 and 6mM bile salt, respectively, more preferably under the conditions of item (i) of the first aspect the bacillus spore reaches the vegetative cell growth point after 15 and 16 hours of incubation in 4 and 6mM bile salt.

As explained above and shown schematically in FIG. 1, the novel DSM19467 strain described herein is obtained by using the commercially available strainAs starting strains were selected by mutation in the presence of bile salts and selection for rapid growth as described herein.

Is a composition comprising Bacillus subtilis cells and the Bacillus subtilis is deposited as DSM 17231. Accordingly, in this contextCan be regarded as a reference strain.

As has been described above, in the above-mentioned,the starting point of vegetative cell growth of (b) is after incubation in 4 and 6mM bile salt for 20 hours under the conditions of item (i) of the first aspect. Thus, in one embodiment, under the conditions of item (i) of the first aspect, the ratio of bacillus spores is deposited as DSM 17231The reference Bacillus subtilis spore cell of (a) reaches the vegetative cell growth point at least 3 hours earlier, more preferably under the conditions of item (i) of the first aspect the Bacillus spore deposited as DSM 17231) The reference Bacillus subtilis spore cell of (a) reaches the vegetative cell growth point at least 4 hours earlier, and most preferably under the conditions of item (i) of the first aspect the Bacillus spore is deposited as DSM 17231 (ii)) The reference bacillus subtilis spore cells reached the starting point of vegetative cell growth at least 5 hours earlier.

Essential amino acids

As known to those skilled in the art, an essential amino acid can be an essential amino acid selected from the group consisting of: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, cysteine, tyrosine, histidine and arginine.

In a preferred embodiment, the essential amino acid is at least one essential amino acid selected from the group consisting of: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, and lysine.

In a more preferred embodiment, the essential amino acid is at least one essential amino acid selected from the group consisting of: valine, isoleucine and leucine.

One essential amino acid of great relevance herein is leucine.

As understood by those skilled in the art, a bacillus vegetative cell may produce a higher amount of more than one essential amino acid, such as, for example, a higher amount of two or three or more different essential amino acids.

Amino acid determination

As mentioned above, the amino acid determination of item (ii) of the first aspect is based on standard known commercial factors (such as, for example, standard culture medium, standard assay).

Thus, based on the specification of the assay detailed herein (see, e.g., example 2 herein), one skilled in the art can routinely repeat the assay to objectively determine whether a particular bacillus vegetative cell of interest meets the amount of essential amino acid production as described in item (ii).

Working example 2 herein provides a detailed description of the determination of essential amino acids.

The detailed conditions of this example 2 are herein preferred essential amino acid assays to determine whether the Bacillus vegetative cells of interest meet the criteria of item (ii) of the first aspect.

Production amount of essential amino acids-item (ii) of the first aspect

With respect to item (ii) of the first aspect, under the conditions of item (ii) of the first aspect, the bacillus vegetative cell preferably produces at least 2 times more of an essential amino acid than the reference bacillus cell DSM 19467.

In a more preferred embodiment in respect of item (ii) of the first aspect, under the conditions of item (ii) of the first aspect, the bacillus vegetative cell preferably produces at least 4 times more of one essential amino acid than the reference bacillus cell DSM 19467.

Method for feeding/administering bacillus spores to animals

As mentioned above, a second aspect of the present invention relates to a method of feeding an animal comprising administering the bacillus composition of the first aspect and the related embodiments described herein to the animal together with other animal feed ingredients.

The animal may be any animal of interest. Preferably, the animal is an animal selected from poultry, ruminants, cattle, pigs, rabbits, horses, fish and pets.

When administered according to the artAt a normal rate of about 10⁴-10⁸CFU/g feed, usually at a meal of 10⁵-10⁶The dose of CFU/g feed is done either at a dose equivalent to normal food intake/kg live weight of the animal.

Alternatively, the bacillus spores can be administered to the animal in one of the following ways:

(1): putting it into animal drinking water;

(2): spraying to the surface of the animal body; or

(3): applied via paste, gel or bolus.

Method for screening and isolating novel bacillus cells

As described above, the third aspect relates to a method for screening and isolating novel Bacillus cells.

In the method of the third aspect, a Bacillus cell is selected which satisfies the conditions of items (i) and (ii) of the first aspect.

As will be appreciated by those skilled in the art, the specific bile resistance and essential amino acid amount assays detailed herein (see, e.g., example 1 herein for bile resistance assays and example 2 herein for essential amino acid assays) parameters can be altered to alternative screening methods that still achieve the primary goals as described herein, i.e., bacillus cells that are capable of meeting the conditions of items (i) and (ii) of the first aspect.

In a preferred embodiment, the bile resistance assay of example 1 is used in step (a) of the screening method of the third aspect and the essential amino acid assay of example 2 is used in step (c) of the screening method of the third aspect.

In step (d) of the third aspect, the vegetative bacillus cells are isolated. The vegetative bacillus cell can be used to prepare bacillus spore forms.

Thus, in one embodiment, the screening method of the third aspect is followed by an additional step (e) wherein the isolated Bacillus vegetative cells of step (d) are fermented to produce 10⁵-10¹²Bacillus vegetative cells, and these 10⁵-10¹²The bacillus vegetative cell is prepared into 10⁵-10¹²Bacillus spore cells isolated to provide a bacillus composition comprising 10⁵-10¹²CFU/g Bacillus spore cells.

The end result of step (e) is a novel bacillus composition comprising 10⁵-10¹²CFU/g Bacillus spore cell, and wherein the Bacillus cell is capable of satisfying the conditions of items (i) and (ii) of the first aspect.

Thus, a separate aspect of the invention relates to a bacillus composition comprising 10⁵-10¹²CFU/g of Bacillus spore cells, and wherein the Bacillus cells are capable of satisfying the conditions of items (i) and (ii) of the first aspect, the cells obtainable by the screening method of the third aspect followed by the additional step (f) described above.

In step (b) of the screening method of the third aspect, the previously selected bile-resistant Bacillus cells are subjected to mutation to select for cells with high essential amino acid production in step (c). As understood by those skilled in the art, this step can be performed, for example, by classical mutation of gene-specific exchanges (e.g. by chemical treatment or UV) to make a so-called Genetically Modified Organism (GMO).

Deposited strains

A sample of a new Bacillus subtilis strain has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg 1b, D-38124Braunschweig) under accession number DSM19467, on day 2007, month 6 and day 27. Deposits have been made under conditions internationally recognized for the preservation of the budapest treaty on microorganisms used in the patent procedure.

Examples

Example 1: bile resistance assay

Culture medium:

the medium was standard non-selective commercial medium veal extract broth (VIB) (Difco, 234420).

At the time of filing of the present application, a product catalog ("Difco") from supplier BD Diagnostic Systems (www.bd.com)^TM/BBL^TMHandbook) the references are thus made to veal extract broth:

"leachate from lean veal and peptone provides nitrogen, microorganisms, carbon and amino acids in veal leachate. Sodium chloride maintains osmotic balance of the formulation "; and

the medium was prepared according to the manufacturer's instructions by the following steps: 25g veal extract bouillon powder was suspended in 1L of pure water (2.5% solution), heated and frequently agitated, and boiled for 1 minute to completely dissolve the powder.

2.5% veal extract broth solution contains per liter:

lean beef, leachate: 10g

Monthly peptone: 10g

Sodium chloride: 5g

The medium was dispensed into sterile bottles and autoclaved at 121 ℃ for 15 min.

Bile salt solution/medium:

a bile salt mixture was prepared simulating the physiological composition and concentration of bile salts in porcine bile, and the bile salts were dissolved in veal extract broth prepared as above to give a final concentration of bile salts of 8 mM.

The conjugated bile salts were taurodeoxycholate (Sigma T-0875, USA) and glycodeoxycholate (Sigma G-9910, USA) and unconjugated bile salt deoxycholate (Sigma D-5670 USA), and the final 8mM mixed bile salt solution contained 60% taurodeoxycholate, 30% glycodeoxycholate and 10% deoxycholate. The solution was adjusted to pH 7.4 using sodium hydroxide and then autoclaved at 121 ℃ for 15 minutes. The 8mM bile salt medium thus prepared was diluted to give bile salt concentrations of 0, 1, 2, 4, 6 and 8 mM.

The bile salts are added in concentrated form to the veal extract broth. Thus, the final amounts of lean veal extract, lunatose peptone and sodium chloride were essentially the same as the 2.5% veal extract broth prior to addition of bile salts.

Spore suspension

To distinguish vegetative cells from spores and to ensure that a pure spore product was used for inoculation, the spore count of the bacillus product was determined using a heat treatment at 80 ℃ +/-10 min. After heat treatment and subsequent cooling to room temperature, serial 10-fold dilutions were made in peptone salt aqueous solution. 0.1ml of the appropriate ten-fold dilution was inoculated into duplicate tryptic blood agar plates (Difco 0232-01). The plates were incubated at 37 ℃ for the next day. Spore suspensions were prepared in sterile distilled water to achieve the final calculated spore concentration of 10 based on prior product spore count determinations⁸CFU/ml. The counts of vegetative cells and spores in the final inoculum were determined using the methods described above. 10⁸The final concentration of CFU/ml corresponds to the starting OD₆₃₀0.2-0.3。

And (3) growth determination: optical Density measurement

Sterile flat-bottomed 96-well microtiter plates (Greiner Bio-one GmbH, Germany) were used. Each well was filled with 0.150ml of seeded spores (. about.1X 10)⁸Spores/ml equivalent/equivalent to starting OD₆₃₀0.2-0.3), the plate was incubated at 37 ℃ for 20 hours with shaking with a cycle of intensity 4 (high) for 1 minute before each reading.

To avoid coagulation on the inside of the titer plate cap, the cap was contacted with a dilution solution of Triton X-100.

Germination and growth kinetics of Bacillus strains A spectrophotometer (Bio-tek instruments, Inc. VE) was used at a wavelength of 630nm (OD)₆₃₀) The following measurements were made. Readings were taken at 10 minute intervals and KC4 was used^TMSoftware (Bio-tek Instruments, inc., USA). After 20h, the data is output toSpreadsheet programs were entered for further analysis into SAS version 9.0 and statistically analyzed.

Example 2: amino acid determination

The method used in this study to determine and quantify the amino acids produced by bacillus cells is a standard GC-MS method for aqueous samples using methyl chloroformate as the derivatizing agent.

Growth of Bacillus cells

Bacillus cells were inoculated and grown in a low salt growth medium (minimum Salts grown Medium) at 37 ℃ and 150rpm for 2 days, and then the amount of amino acids in the supernatant was measured as described below.

Bacillus cells were propagated in a low salt medium according to Chapman (1972) with the following composition:

autoclaved at 121 ℃ for 15min and autoclaved glucose was added to a final concentration of 0.5%.

Incubation was completed for 2 days at 37 ℃ and 150rpm in a tube containing 10ml of medium.

Amino acid determination

Amino acid assays were performed on cell supernatants as the amino acids were secreted into the culture medium. The samples were filter sterilized and kept at-20 ℃ until analysis.

Reagent:

reagent 1: internal standard solution. Norvaline 1 mM: 0.0172g norvaline +100ml MQW

Reagent 2: methanol/pyridine 32/8(v/v) (catalyst)

Reagent 3: methyl Chloroformate (MCF) (derivatizing reagent) for analysis

Reagent 4: 1% MCF/CHCl₃(v/v) (extraction): 1ml of the assay was supplemented to 1000ml with methyl chloroformate + chloroform.

Sample preparation:

transfer 150. mu.l (25. mu.l + 125. mu. MQW) sample into 2ml syringe bottles.

Add 150. mu.l IS.

Add 200. mu.l of 1-methanol/pyridine 32/8% (v/v). And (4) fully mixing.

Add 25. mu.l MCF (methyl chloroformate). Mix well until gas generation occurs.

Add 500. mu.l of 1% MCF/CHCl₃(v/v), cap and mix vigorously. Phase separation occurred within a few minutes. If the phase separation is too slow, the flask is centrifuged (500rpm/10 min).

If norvaline IS used as an antimetabolite, the external standard or another suitable internal standard should be used instead and the MQW or sample substituted for 150. mu.l IS.

Samples were run on a GC-MS using standard amino acid columns and protocols.

Example 3: selection of bile resistant Bacillus subtilis cell DSM19467

The starting bacillus cell is bacillus subtilis cell

Are mutagenized to obtain a new pool of single bacillus cells. Spores were prepared and selected for rapid germination and growth from spores into vegetative cells in the presence of bile salt medium containing 4 and 6mM bile salts as described in example 1 above.

Bacillus subtilis cell DSM19467 was selected.

Table 1 below shows the germination and growth data.

From to 10⁸Time (hours) for OD 0.2-0.3 of CFU/ml to reach OD 0.4 (mean of 3 replicates).

Some data for this example was obtained by testing phytase overexpressing DSM 19489. But for the technical effect of this embodiment it is relatively irrelevant in this context, since DSM19467 has about the same germination and growth as DSM 19489. See PCT/EP2008/057296 for more details.

Conclusion

DSM19467 is a bile-resistant strain and is clearly comparableSprouting and growth are faster.

Example 4: selection of Bacillus cells overproducing amino acids from DSM19467

The starting bacillus cell was the bacillus subtilis cell DSM19467 selected in example 3.

Wild-type DSM19467 or a DSM19467 mutant produced by e.g. UV mutagenesis were grown on low salt medium agar described in example 2B above with the addition of 1, 5% agar containing the appropriate inhibitory amount of the amino acid analogue. Depending on the amino acid to be overexpressed, a variety of amino acid analogs can be used, for example, norvaline or 4-aza-DL-leucine for overproduction of leucine (Bardos, 1974, Topics in Current chemistry 52, 63-98). Colonies resistant to the amino acid analogue were picked, grown in low salt medium and amino acid production was determined. Vegetative cells producing high amino acid content were selected by using the GC-MS method described in example 2B above.

Selecting Bacillus subtilis cells with high amino acid yield.

Results of amino acid measurement

A number of strains were selected which produced the essential amino acid leucine in significantly higher amounts than the reference bacillus cell DSM 19467.

Many selected strains produced at least 5 times more leucine than DSM 19467.

Conclusion

This example shows that based on the teachings herein one can routinely screen and identify strains that produce significantly higher amounts of at least one essential amino acid (herein leucine for example) than the reference bacillus cell DSM 19467.

DSM19467 is derived fromAnd which has not been selected for high essential amino acid production. It is believed, therefore, thatThe amount of essential amino acids produced was approximately the same as DSM 19467.

Example 5: bile resistance "test" of bacillus cells with high essential amino acid production.

The preferred high essential amino acid producing bacillus cells selected in example 4 were rechecked for their ability to rapidly bud from spores and grow into vegetative cells as described in example 1.

As a result, they retained approximately the same good rapid germination and growth as the starting cell DSM19467 used to obtain them, as expected.

Reference to the literature

Antonie Van Leeuwenhoek.2006Aug; 90(2): 139-46.Epub 2006, 7 months and 4 days

2.US2003/0124104A

3.US6255098

4.PCT/FP2008/057296

PCT/RO/134 Table

Claims (12)

1.A Bacillus composition comprising 10⁵-10¹²CFU/g Bacillus spore cells, wherein the Bacillus composition is characterized by:

(i) the method comprises the following steps The bacillus spores are rapidly germinated and grown from spores to vegetative cells in the presence of a bile salt medium comprising 4mM and 6mM bile salts, the rapid germination and growth being defined as the bacillus spores reaching 0.4OD in less than 18 hours and 19 hours, respectively₆₃₀Wherein the vegetative cell growth point is the OD value of the growth curveInitially increased in a continuous manner (caused by the growth of the vegetative cells) and reached a point where the OD630 was 0.4;

(I) the method comprises the following steps Wherein the bile salt medium is a standard known non-selective veal leach liquor broth (VIB) medium of example 1 herein supplemented with a bile salt mixture comprising the bound bile salts taurodeoxycholate and glycodeoxycholate and the unbound bile salt deoxycholate in proportions of 60% taurodeoxycholate, 30% glycodeoxycholate, and 10% deoxycholate; and

wherein the OD determination analysis is performed by the following steps:

(a) the method comprises the following steps The wells of the microtiter plate were filled with 0.150ml of 10 medium per ml⁸Bile salt medium of bacillus spores (i.e., this is time zero); and

(b) the method comprises the following steps The plate was incubated at 37 ℃ under atmospheric conditions and OD was determined using a spectrophotometer with agitation prior to each reading₆₃₀Values, thereby obtaining a representative growth curve over time;

and

(ii) the bacillus vegetative cells produce a higher amount of at least one essential amino acid than the reference bacillus cell DSM19467, wherein the amount of essential amino acid produced is determined by the standard GC-MS method based amino acid assay of example 2 herein after 2 days of growth in standard known low salt growth medium of example 2 herein at 37 ℃.

2. The bacillus composition of claim 1, wherein the bacillus spore cells of the composition are present as dried (e.g., spray-dried) spore cells.

3. The bacillus composition of claim 1 or 2, wherein the bacillus cell is a bacillus subtilis cell.

4. Claim 1-3, wherein the bacillus spores are larger than the reference bacillus subtilis deposited under DSM 17231 under the conditions of item (i) of claim 1 (c) (b)) Spore cells reached the vegetative cell growth point at least 3 hours in advance.

5. The bacillus composition of any one of claims 1-4, wherein said essential amino acid is an essential amino acid selected from the group consisting of: phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, cysteine, tyrosine, histidine and arginine.

6. The bacillus composition of claim 5, wherein said essential amino acid is at least one essential amino acid selected from the group consisting of: valine, isoleucine and leucine.

7. The bacillus composition of claim 6, wherein said essential amino acid is leucine.

8. The bacillus composition of any one of claims 1-7, wherein under the conditions of item (ii) of claim 1, the bacillus vegetative cells produce at least 4 times more of at least one essential amino acid than the reference bacillus cell DSM 19467.

9. A method of feeding an animal comprising administering the bacillus composition of any one of claims 1-8 to the animal along with other animal feed ingredients.

10. The method of feeding an animal according to claim 9, wherein the animal is an animal selected from the group consisting of poultry, ruminants, cattle, swine, rabbits, horses, fish, and pets.

11. A method for screening and isolating a novel bacillus cell comprising the steps of:

(a) the method comprises the following steps (ii) screening and isolating from a collection of individual bacillus spore cells, new bacillus spore cells that are capable of rapidly budding and growing under the conditions of item (i) of claim 1 such that they reach the vegetative cell growth point in less than 18 and 19 hours;

(b) the method comprises the following steps Preparing bacillus vegetative cells from the spore cells isolated in step (a) and mutating the selected and isolated new cells to obtain a pool of new bacillus single vegetative cells;

(c) the method comprises the following steps Selecting and isolating from the pool of Bacillus neoformans mono-vegetative cells of step (b) a Bacillus neoformans vegetative cell capable of producing a higher amount of at least one essential amino acid than the reference Bacillus cell DSM19467 under the conditions of item (ii) of claim 1; and

(d) the method comprises the following steps Analyzing the highly productive bacillus vegetative cells of step (c) to confirm that they maintain the rapid germination and growth of step (a), and isolating the selected bacillus cells.

12. The method for screening and isolating a novel bacillus cell of claim 11, wherein said bacillus cell is a bacillus subtilis cell.