HK1138036B - A bile resistant bacillus composition secreting high levels of phytase - Google Patents

Description

Bile-resistant bacillus compositions secreting high levels of phytase

Technical Field

The present invention relates to a bacillus composition characterized by rapid germination and outgrowth in bile salts (simulated intestinal environment) and secretion of high levels of phytase. 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 bacillus to replace or reduce the use of antibiotics, which are 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.

One of the enzymes widely used in the animal feed industry is phytase. Phytase is used to improve the digestibility of phosphorus in the diet of animals. Phytate is the predominant form of phosphorus in cereals, oilseeds and legumes. However, monogastric animals such as pigs, poultry and fish have poor utilization of this phosphate source because they lack the necessary gastrointestinal enzymes for releasing phosphate from the organic complex of phytate. Thus, most of the phytate in the consumed feed passes through the gastrointestinal tract and is excreted in the faeces. The catalytic release of phosphate occurs in soil and water environments and phytate in manure constitutes a serious phosphorus pollution problem leading to eutrophication of surface water. In addition, producers have to use expensive phosphorus supplement feeds to meet the needs of the animal diet. In addition, phytate has anti-nutritional properties including the formation of complexes with proteins and divalent cations, thereby reducing its bioavailability.

It is well documented in the literature that supplementation with phytase improves phytate utilization in monogastric animal feeding and has a positive effect on the bioavailability of minerals.

Bacillus spores can pass through the acidic gastric barrier and bud and proliferate within 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 enzymes such as phytases. Moreover, during feed pelleting, bacillus spores are heat resistant and therefore are excellent delivery systems for both bacteriocins and enzymes into the 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 of bacillus spore cells in the gastrointestinal tract of animals as well as on sprouting and proliferation into vegetative cells. Studies are therefore being conducted to find probiotic bile-resistant bacillus strains.

A paper (Antonie Van Leeuwenhoek.2006 Aug; 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 resistance and bacillus species with particularly high probiotic activity were found to have a relatively high level of bile salt resistance.

No particular emphasis has been placed in this paper on any period of testing for bile resistance. In the experimental part, the tolerance of the spore cells of Bacillus was simply tested after 5 days in the presence of bile salts (see paragraph "Simulated small intestinal fluid resistance 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 connection, it is proposed to screen/select for Bacillus spores which are resistant to the inhibitory activity of bile salts and which are therefore 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 description of this document, only bacillus cells can be selected that can only grow (germinate) slowly, i.e. that are capable of germinating from spores to vegetative cells after e.g. 20 hours in the presence of an appropriate amount of bile salts.

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

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

The prior art describes a number of detection/screening systems for selecting strains of bacillus which produce phytase.

In one example in US6255098 a phytase producing bacillus strain was identified. There is no mention of bile resistance of the identified bacillus strains.

Disclosure of Invention

The problem to be solved by the present invention is to provide a bacillus composition which secretes large amounts of phytase in the gastrointestinal tract (GIT) of animals.

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 novel selection method described herein is the specific screening/selection of bacillus spore cells that germinate faster/faster from spores and proliferate into vegetative cells in the presence of bile salts.

As mentioned above, the prior art has described many methods for screening for bacillus capable of growing in the presence of bile salts, but the existing screening/selection methods do not focus on the speed of budding and proliferation in the presence of bile salts. Thus, the rate of sprouting and proliferation of bile-resistant cells screened in the prior art does not meet the rate criteria for sprouting and proliferation described herein. For example, bacillus cells isolated directly from, for example, chicken intestine in the gut environment (as described, for example, in the paper by Antonie van leewenhoenhoek discussed above) have not been selected (at atmospheric pressure) to rapidly bud and proliferate in the gut.

As shown in the working examples herein, for a commercial Bacillus compositionAlso, in the presence of physiological levels of bile salts, sprouting and proliferation is too slow and does not reach the defined growth point within the first 20 hours, not meeting the sprouting and proliferation rate criteria described herein.Is a commercially successful bacillus subtilis composition.

By usingAs starting strains and as described herein andsubsequent isolation of the strain that rapidly germinated from spores and proliferated into vegetative cells in the presence of bile salts selected the novel DSM19467 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 cells, wherein the cells of the bacillus composition meet the criteria for rapid germination and proliferation in the presence of bile salts as described herein.

Without being bound by theory, the inventors have identified that rapid germination and proliferation is a very important aspect of the present invention, as bacillus spores are bile-resistant, but do not germinate and proliferate rapidly enough to be excreted before the bacillus vegetative cells exert any significant positive properties such as production of significant amounts of phytase.

Spores of bacillus that bud too slowly pass only through the gastrointestinal tract (GIT) before the bacteria can produce any significant amount of, for example, phytase.

After extensive and exhaustive testing and analysis, the inventors therefore chose to work in the time frame of up to 20 hours and select the fastest germinating and proliferating spores in the presence of high physiological concentrations of bile salts over this period. Without being bound by theory and based on experimental work detailed herein, the present inventors have identified that rapid sprouting and proliferation 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 proliferation in a bile salt medium, were very useful as starting cells for mutagenesis to obtain new cells with improved phytase production capacity.

As shown in figure 1 and table 2, the rapidly proliferating bile resistant selection strain DSM19467 was used as the starting strain for classical mutation and DSM19489 strain with high phytase production was selected. Similarly, Genetically Modified Organism (GMO) strain DSM19466 was prepared by using DSM19467 as the starting strain. As can be seen from Table 2 and the associated description of example 4, DSM19489 and DSM19466 are compared to DSM19467 and DSM19467Significantly more phytase was produced. The high phytase producing strains DSM19489 and DSM19466 were rechecked for their ability to rapidly bud and proliferate as described herein, which maintained the rapid germination and proliferation properties of the rapidly proliferating bile resistant selection strain DSM19467 (see example 5 herein).

This is schematically illustrated in fig. 1 herein.

Thus the novel probiotic bacillus cell disclosed herein is a bile resistant, rapidly budding and proliferating bacillus cell that secretes large amounts of phytase. 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 phytase beneficial and useful for digestion and uptake of phosphorus obtained from phytate.

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 proliferated from the spores to vegetative cells in the presence of a bile salt medium comprising 4 and 6mM bile salt, wherein rapid germination and proliferation are defined as bacillus spores that germinate and proliferate, respectively, in less thanOD reached within 18 and 19 hours₆₃₀A vegetative cell growth point of 0.4, wherein the vegetative cell growth point is the point in the growth curve at which the OD begins to increase (caused by the growth of vegetative cells) in a continuous manner and reaches the OD₆₃₀A point of 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 supplemented with a bile salt mixture comprising bound bile salts taurodeoxycholate and glycodeoxycholate and unbound bile salts deoxycholate in proportions of 60% taurodeoxycholate, 30% glycodeoxycholate and 10% deoxycholate, as described 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 0); 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 produced at least 1.25 times more phytase than the reference bacillus cell DSM19467, wherein the amount of phytase produced is determined by the standard phytase assay of example 2 herein after 4 hours of growth at 37 ℃ in the standard known non-selective Heart Infusion Broth (HIB) medium of example 2 herein; and

wherein the phytase assay is performed by the following steps:

(a) the method comprises the following steps Preparing an overnight culture of vegetative cells of bacillus in a enrichment medium; and

(b) the method comprises the following steps From the overnight culture, 1% of the inoculum was transferred to HIB medium (i.e., this was time 0) and incubated at 37 ℃ until phytase activity was determined.

By using, as described aboveThe reference bacillus cell DSM19467 was chosen as the starting strain to bud and proliferate rapidly in the presence of bile salts. No increase in phytase production was observed with DSM 19467. Without being bound by theory, it is believed that the relative phytase production of DSM19467 is related to that of DSM19467And (4) the equivalent.

With regard to the feature (i),the vegetative cell growth point of (a) is at least 20 hours after incubation in 4 and 6mM bile salts, and for the new DSM19489 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 feature (i) of the first aspect. As forCommercially available productIs a bacillus subtilis composition used as a probiotic feed additive. For theVegetative cell under the conditions of feature (i) of the first aspectThe growth point was 20 hours or more in 4 and 6mM bile salts, respectively. This is well in excess of the 18 and 19 hours required in feature (i) and this illustrates that, to date, no one commercially available product can be selected for rapid sprouting and proliferation. As mentioned above, "native" Bacillus cells cannot bud and proliferate rapidly under any selective pressure. Without being limited to theory, it is therefore believed that "native" Bacillus cells do not meet the conditions of feature (i) of the first aspect.

The determination of the bile resistance [ characteristic (i) ] and the determination of the phytase [ characteristic (ii) ] are based on known, commercially available standard factors (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 DSM17231 (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 and the phytase assay of example 2 herein), the skilled artisan is able to routinely repeat these assays to objectively determine whether a particular bacillus cell of interest meets the bile resistance of [ feature (i) ] and the phytase level of [ feature (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 is directed 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 in combination 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 from a pool of single bacillus spore cells, new bacillus spore cells which are capable of budding and proliferating rapidly such that they reach the vegetative cell growth point in less than 18 and 19 hours under the conditions characteristic (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 newly selected and isolated cells to obtain a pool of new single bacillus vegetative cells;

(c) the method comprises the following steps Selecting and isolating from the pool of novel single bacillus vegetative cells of step (b) a novel bacillus vegetative cell capable of producing at least 1.25 times more phytase under the conditions of feature (ii) of the first aspect than the reference bacillus cell deposited under DSM deposit No. 19467; and

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

It will be apparent to the skilled person that once the inventors herein disclose relevant detection assays (in particular the assay for detecting rapid germination and outgrowth of example 1) plus the reference strain DSM19467, it will be routine work for the skilled person to select other new 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, which have been deposited, by using the new screening/selection methods as described herein.

Accordingly, a fourth aspect of the invention relates to a bacillus cell selected from the group consisting of:

(a) the bacillus subtilis cell of deposit number DSM 19467;

(b) the bacillus subtilis cell deposited under number DSM 19489; and

(c) bacillus subtilis cell with deposit number DSM 19466;

or a mutant strain thereof, wherein the mutant strain is obtained by using one of the deposited strains as a starting material, and wherein the mutant strain retains essential characteristics of the deposited strain.

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

Definition of

All definitions of terms used herein are consistent with those understood by 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-genital 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 proliferation" refers to the germination and proliferation of a bacillus spore into a bacillus vegetative cell. As is well known to the skilled person, the revival of spores occurs when conditions are favorable and involves germination and proliferation. 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. Proliferation occurs after budding and involves the spore core making new chemical components 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 ═ proliferative 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. In this contextWorking example from DSM17231Initially, it was typically mutated and screened/selected for rapid budding and proliferation in the presence of bile salts to give the new selection strain DSM 19467. DSM19467 was used as the starting strain for the typical mutation and DSM19489 strain with high phytase production was selected. Similarly, Genetically Modified Organism (GMO) strain DSM19466 was prepared by using DSM19467 as the starting strain.

Fig. 2a and 2 b: these figures clearly show that the bacillus spores of DSM19489 were germinated and proliferated 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 can include different types of bacillus cells (e.g., bacillus subtilis and bacillus licheniformis). In essence, the composition should simply comprise an amount of the bacillus spore cells specified in the first aspect herein, wherein the bacillus cells meet the criteria specified in the first aspect.

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

Suitable compositions, e.g. 10⁵-10¹²The CFU/g Bacillus cells may be suitably obtained from commercial sources known to those skilled in the artThe form exists.

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

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

The term "CFU/g" refers to the gram weight of the composition, 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 the skilled person, commercially relevant bacterial compositions will usually also comprise 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 include 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 subtilis unifoflagellatus, Bacillus laterosporus BOD, Bacillus megaterium, Bacillus polymyxa, Bacillus licheniformis, Bacillus pumilus, Bacillus stearothermophilus, Bacillus coagulans, Bacillus thermophilus, Bacillus mycoides, 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 sprouting and proliferation in the Presence of bile salts

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

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

In feature (i), it is explained that the vegetative cell growth point is from 10 corresponding to an OD of around 0.2-0.3 in the growth curve⁸Spore/ml began until the moment when the OD value had increased in a continuous manner (due to the growth of vegetative cells) and reached the point of OD 0.4. As explained herein, i.e. based on how the skilled person understands this vegetative cell growth point and based on the growth curve, the skilled person 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 sprouting and proliferation 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 to simulate 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 that includes relevant bacillus growth components such as relevant nutrients and bile salts.

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

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

As described above, the new strain DSM19467 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 feature (i) of the first aspect, the bacillus spores reach the vegetative cell growth point after 17 and 18 hours of incubation in 4 and 6mM bile salts, respectively, more preferably under the conditions of feature (i) of the first aspect, the bacillus spores reach the vegetative cell growth point after 15 and 16 hours of incubation in 4 and 6mM bile salts.

As explained above and shown schematically in FIG. 1, the novel DSM19467 strain described herein is prepared by using the commercially available strainAs starting strains were selected as described herein for mutation and for rapid growth in the presence of bile salts.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 (a) is after incubation in 4 and 6mM bile salt for 20 hours under the conditions of feature (i) of the first aspect. Thus, in an embodiment, under the conditions of feature (i) of the first aspect, the ratio of bacillus spores is deposited as DSM17231The reference Bacillus subtilis spore cell of (a) reaches the vegetative cell growth point at least 3 hours earlier, more preferably under the conditions of feature (i) of the first aspect the Bacillus spore ratio deposited as DSM17231The 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 feature (i) of the first aspect the Bacillus spore ratio is deposited as DSM17231The reference bacillus subtilis spore cell reaches the vegetative cell growth start point at least 5 hours earlier.

Phytase assay

As mentioned above, the phytase assay of feature (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), the skilled artisan is able to routinely repeat the assay to objectively determine whether a particular bacillus vegetative cell of interest meets the amount of phytase production as described in feature (ii).

Working example 2 herein provides a detailed description of phytase assays.

The detailed conditions of this example 2 are herein preferred phytase assays to determine whether or not the vegetative cells of the Bacillus species of interest meet the criteria of feature (ii) of the first aspect.

Amount of Phytase produced-characteristic of the first aspect (ii)

As described above with respect to feature (ii) of the first aspect, under the conditions of feature (ii) of the first aspect, the bacillus vegetative cells produce phytase in an amount at least 1.25 times more than the reference bacillus cell DSM 19467.

In a preferred embodiment, the bacillus vegetative cell produces phytase in an amount of at least 1.5 times the amount of the reference bacillus cell DSM19467 under the conditions of feature (ii) of the first aspect, more preferably in an amount of at least 1.75 times the amount of the reference bacillus cell DSM19467 under the conditions of feature (ii) of the first aspect.

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 in combination 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 that time, normally about 10 times a meal⁴-10⁸CFU/g, usually in a meal of 10⁵-10⁶The dose of CFU/g or 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 feature (i) and feature (ii) of the first aspect.

As understood by the skilled person, the specific bile resistance and phytase amount measurements detailed herein (see, e.g., example 1 herein for a bile resistance assay and example 2 herein for a phytase assay) parameters may be altered to alternative screening methods which still achieve the main objective as described herein, i.e. a bacillus cell capable of fulfilling the conditions of feature (i) and feature (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 phytase 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 feature (i) and feature (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 feature (i) and feature (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 high phytase producing cells in step (c). As understood by the skilled person, this step can be performed, for example, by classical mutation of the gene-specific exchange (e.g. by chemical treatment or UV) to make a so-called Genetically Modified Organism (GMO).

For example, the novel GMO strain DSM19466 described herein is fromAnd was first made bile resistant as described in the working examples herein to obtain DSM 19467. Thereafter, the promoter of phytase in strain DSM19467 was exchanged with another Bacillus promoter to make it a phytase high producer, and DSM19466 was obtained therefrom.

Similarly, a novel high phytase producing strain DSM19489 was obtained by using a typical mutation starting from DSM 19467. See, for example, fig. 1.

Deposited strains

As mentioned above, the fourth aspect of the present invention relates to a bacillus cell selected from the group consisting of:

(a) the bacillus subtilis cell of deposit number DSM 19467;

(b) the bacillus subtilis cell deposited under number DSM 19489; and

(c) bacillus subtilis cell with deposit number DSM 19466;

or a mutant strain thereof, wherein the mutant strain is obtained by using one of the above-mentioned deposited strains as a starting material, and the mutant strain retains the essential characteristics of the above-mentioned deposited strain.

The fourth aspect of the present invention relates to the novel strain or "mutant thereof" described herein.

It will be apparent to the skilled person that by using the deposited strains as starting materials, the skilled reader may further obtain mutants or derivatives thereof which retain the relevant features and advantages described herein, conveniently by conventional mutagenesis or re-isolation techniques. Thus, the term "mutant thereof" of the first aspect relates to a mutant strain obtained by using the deposited strain as a starting material.

This can optionally be expressed as a method of obtaining a strain comprising using one of the strains deposited herein as a starting strain, preparing a mutant of the deposited strain and isolating a new strain, wherein the mutant retains the essential characteristics of the deposited strain.

A sample of a new Bacillus subtilis strain has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg1b, D-38124 Braunschweig) under accession number DSM19467, on deposit at 27.6.2007. Deposits have been made under conditions internationally recognized for the preservation of the budapest treaty on microorganisms used in the patent procedure.

A sample of a new Bacillus subtilis strain has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg1b, D-38124 Braunschweig) under accession number DSM19489, on deposit date 2007, month 6 and 27. Deposits have been made under conditions internationally recognized for the preservation of the budapest treaty on microorganisms used in the patent procedure.

A sample of the novel Bacillus subtilis strain DSM19466 has been deposited at DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, Maschroder Weg1b, D-38124 Braunschweig) under accession number DSM19466 with a date of 27.6.2007. 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 comprises 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 pig 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.

Bound bile salts were taurodeoxycholate (Sigma T-0875, USA) and glycodeoxycholate (Sigma G-9910, USA) and deoxycholate as unbound bile salt (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 pH7.4 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.

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

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. From the appropriate ten-fold dilution, 0.1ml was inoculated into duplicate tryptic moon 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 OD₆₃₀ 0.2-0.3。

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/corresponding 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 proliferation kinetics of Bacillus strains 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: phytase Activity assay

The method used in this study to determine and quantify the phytase units produced by the Bacillus cells was modified from Walsh et al, 2004, Biochemistry & Molecular Biology Educationvol.32 no 5 (336-340). Basically, the only significant improvement made is to standardize the method for the purpose of growth kinetics, the improvement being the determination of the phytase activity relative to live Bacillus cells using a spectrophotometer and the determination of the Optical Density (OD) at a wavelength of 600, if necessary with dilution water 1: 4. By using this method, a relatively limited standard deviation is obtained.

Growth of Bacillus cells

The bacillus cells are inoculated and grown in a bacillus enrichment growth medium at 37 ℃, the growth of the bacillus and the phytase activity being carried out at intervals of up to 24 hours.

Bacillus spores were propagated in Heart Infusion Broth (HIB) basal medium with the following composition:

HIB(Bacto 238400) 25g/l

0.5% Bacto Yeast extract (Difco 212750) 5g/l

2mM CaCl₂(Merck 1.02382) 0.294g/l

Autoclaved at 121 ℃ for 15min and sterile filtered 1% mannose and 1% glucose was added.

HIB is a well-known commercially available non-selective medium. At the time of filing of the present application, the catalog of products from the supplier BD Diagnostic Systems (www.bd.com) states Bacto per liter^TMThe heart infusion broth comprises the following components in percentage by weight:

from 500g bovine heart infusion: 10.0g

Trypsin display: 10.0g

Sodium chloride: 5.0g

The supplemented HIB is a low phosphate medium and is therefore suitable for use in phytase assays. After overnight incubation, 1% inoculum was used in fresh HIB medium and incubated at 37 ℃ until activity was determined (e.g. after 4, 6, 8 and 24 hours).

Incubation of the medium was done in blue-cap well plates (blue cap Nunc)50ml or in smaller amounts (0.150ml) in well-ventilated 96-well ELISA plates.

Phytase assay

The cell supernatants were assayed for phytase as the enzyme was secreted into the medium. The microtiter plates were centrifuged at 3600RPM for 15 min. The larger volume was centrifuged for 15min in an Eppendorf centrifuge at 2400-3600 rpm. The supernatant was carefully removed and the cells were removed before phytase assay.

Solution:

0.1M TRIS/malate ph7.0

Solution A: 0.1M TRIS/Malate (1-malic acid, Sigma M1000) pH7.0+0.1w/v sodium phytate from maize (Sigma P8810) +2mM CaCl₂Substrate (freshly prepared before measurement)

Solution B: 8g ammonium molybdate (Sigma A7302) +50ml H₂O+27ml 10M H₂SO₄+H₂Make up to 100 ml.

Solution C: 5g FeSO₄(sigma F 7002)+90ml H₂O (stirred until dissolved) +10ml of solution B (freshly prepared)

0.5M TCA(Merck 1.00807.1000)8％w/v

1mM KH₂PO₄

In 96-well microtiter plates containing 0.020ml of Bacillus supernatantAssay, wherein 0.080ml of a solution containing 0.1% phytic acid and 2mM CaCl was added₂0.1M Tris/malic acid pH7.0 buffer (solution A). Assay plates were incubated at 50 ℃ for 30min (plates were masked to avoid evaporation).

And (3) color development reaction:

0.100ml of 0.5M TCA trichloroacetic acid was added

0.100ml of Fe + + solution (solution C) was added

Left at room temperature for 5 minutes. A blue color will appear.

The absorbance was read at 600 nm.

This assay measures total free phosphate in the supernatant. In order to determine the background amount of free phosphate, phytase assays were also performed in the absence of phytase substrate (phytic acid). This means that solution A (see above) in the assay is replaced by a single TRIS/malate buffer pH 7.0.

Calculation of Phytase Activity

The absorbance measured in the assay represents both free phosphate in the medium and phosphate released by phytase activity, and therefore free phosphate in the medium needs to be subtracted. For this purpose, the samples were assayed in buffers with and without phytic acid, and the two were subtracted to obtain the pure phytase activity. For the correction of the cell density (OD600), the phytase activity of the Bacillus cultures is expressed as the activity (in absorbance units) calculated by the formula:

samples and buffers with and without phytic acid-samples and buffers without phytic acid

OD measurement before centrifugation

Example 3: selection of bile resistant Bacillus subtilis cell DSM19467

The starting bacillus cell is bacillus subtilis cell

Was mutagenized to obtain a new single bacillus cell bank. Spores were prepared and selected for rapid germination and outgrowth from spores to vegetative cells in the presence of a bile salt medium comprising 4 and 6mM bile salts as described in example 1 above.

Bacillus subtilis cell DSM19467 was selected.

Table 1 below shows the sprouting and proliferation data.

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

The selection of bile-resistant and phytase overexpressing DSM19489 is illustrated in example 4 below. DSM19467 had approximately the same sprouting and proliferation as DSM 19489.

Conclusion

DSM19489 and DSM19467 are bile-resistant strains and are clearly comparableSprouting and proliferation are faster.

Example 4: selection of high Phytase production BacillusBacterial cells DSM19489 (classical) and DSM19466 (GMO).

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

DSM19467 was mutated by classical mutation to get a new single bacillus vegetative cell bank. Vegetative cells producing high amounts of phytase were selected by using the phytase assay described in example 2 above.

The Bacillus subtilis cell DSM19489 (classical) was selected for high phytase production.

The promoter of the phytase in strain DSM19467 was exchanged with another Bacillus promoter to make it a high producer of phytase, thus obtaining DSM19466 (GMO).

Results of the Phytase assay

Bacterial strains

DSM19489 Bacillus subtilis, bile tolerance and high phytase yield

DSM19467 B.subtilis, bile-resistant, DSM19489 mother strain

DSM19466 Bacillus subtilis, bile resistance and genetically modified gene encoding phytase (phytase high producer)

TABLE 2 results of phytase production by the selected strains determined as described in example 2 above.

Time (hours)	4	6	8	24
					DSM 19489	2.68	0.83	1.06	0.44
DSM 19467	1.10	0.57	0.68	0.59
					DSM 19466	2.30	1.07	1.37	0.44

DSM19489 strain produced 2.68 units of phytase, 1.10 units compared to DSM19467 (which is a reference bile-resistant mother strain). The genetically modified bile-resistant strain DSM19466 (2.30) reached a similar level as DSM19489, and it is therefore also a phytase high producer.

Conclusion

DSM19489 is a bile-resistant and phytase-highly productive bacillus cell, and in this example after 4 hours of growth in culture medium, 2 times more phytase was produced compared to DSM 19467.

DSM19466(GMO) is a bile-resistant strain in which the phytase gene is genetically modified to a phytase high producer, and similar to DSM19489, it produces 2 times more phytase than the mother strain (DSM 19467) after 4 hours of growth in the medium.

DSM19467 is derived fromAnd it was not selected for high phytase production. It is believed, therefore,the amount of phytase produced was approximately the same as DSM 19467.

Example 5: the bacillus cells DSM19489 (classical) and DSM19466(GMO) with high phytase production were "checked" for bile resistance.

The bacillus cells DSM19489 (classical) and DSM19466(GMO) with high phytase production selected in example 4 were rechecked for their ability to rapidly bud and proliferate from spores into vegetative cells as described in example 1.

As a result, DSM19489 and DSM19466 retained substantially the same good rapid germination and proliferation capacity as DSM19467, the starting cells used to obtain them.

Reference to the literature

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

2.US2003/0124104A

3.US6255098

Claims (7)

1.A Bacillus composition comprises 10⁵-10¹²CFU/g Bacillus spore cell, wherein the Bacillus spore cell is the Bacillus subtilis cell deposited under DSM 19489.

2. The bacillus composition of claim 1, wherein the bacillus spore cells of the composition are present as dried spore cells.

3. The bacillus composition of claim 2, wherein the dried spore cells are spray-dried spore cells.

4. A method of feeding an animal comprising administering the bacillus composition of any one of claims 1-3 to the animal in combination with other animal feed ingredients.

5. The method of feeding an animal of claim 4, wherein the animal is an animal selected from the group consisting of poultry, ruminants and pets.

6. The method of feeding an animal according to claim 4, wherein the animal is selected from the group consisting of cattle, swine, rabbits, horses, and fish.

7. The Bacillus subtilis cell of deposit number DSM 19489.