WO2019038153A1

WO2019038153A1 - Bacillus subtilis strain with probiotic activity

Info

Publication number: WO2019038153A1
Application number: PCT/EP2018/072114
Authority: WO
Inventors: Stefan Pelzer; Daniel Petri; Christos GIATSIS; Stella Molck; Maike KIPKER; Jessica KLEINBÖLTING; Lorena STANNEK-GÖBEL; Kiran DORANALLI; John Khun Kyaw HTOO; Claudia BORGMEIER; Sandra HERBOLD
Original assignee: Evonik Degussa Gmbh
Priority date: 2017-08-24
Filing date: 2018-08-15
Publication date: 2019-02-28

Abstract

The current invention concerns a new B. subtilis strain with strong inhibition of swine and poultry related pathogens and its use as probiotic.

Description

Bacillus subtilis strain with probiotic activity The current invention concerns a new B. subtilis strain with strong inhibition of swine and poultry related pathogens and its use as probiotic.

The use of B. subtilis strains as probiotic ingredient in the feed industry has been disclosed before in the state of the art. The function of probiotics (also called "direct- fed microbials" or "DFM") is to influence the gut microflora in a positive way by supporting the growth of beneficial bacteria and/or the suppression of the growth of pathogenic bacteria. Ideally, by using probiotics the use of antibiotic growth promotors (AGPs) becomes redundant. But besides that, it is desirable that the probiotic fulfills further functions like helping in the digestion of specific feed ingredients. Thus in view of the state of the art, there is a need for probiotics which influence the gut microflora in a positive way and beyond that desirably fulfill at least one further function.

Surprisingly it was found that the bacteria according to the current invention exhibit many advantageous features. Besides their ability to inhibit growth of C. perfringens, C. difficile, S. gallinaceus, S. suis, C. coli, C. jejuni and E. cecorum, the main commercially relevant pathogens of swine and poultry, they in particular show a very high proliferation rate in presence of bile and help to digest cellulose in a very effective way.

Bacillus subtilis DSM 32592 has been identified by targeted screening of naturally occurring isolates. It has been deposited with the DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, InhoffenstraBe 7B, 38124 Braunschweig, Germany) on August 16, 2017 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure under the Accession Number as mentioned before in the name of Evonik Degussa GmbH.

Thus a first subject of the current invention is a Bacillus subtilis strain and/or a preparation of said Bacillus subtilis strain selected from the following group:

a) The Bacillus subtilis strain as deposited under DSM 32592 at the DSMZ;

b) a mutant of the Bacillus subtilis strain as deposited under DSM 32592 having all identifying characteristics of the strain DSM 32592, wherein said mutant preferably has a DNA sequence identity to the strain DSM 32592 of at least 95%, preferably at least 96, 97 or 98 %, more preferably at least 99 or 99.5 % and/or wherein that mutant preferably has a genomic DNA sequence identity to the strain DSM particular of 100 %;

c) a preparation of (a) or (b);

d) a preparation containing an effective mixture of metabolites as contained in (a), (b) or (c).

The Bacillus subtilis strain as deposited under DSM 32592 at the DSMZ exhibits the following characterizing sequences:

a) a 16S rDNA sequence with a sequence identity of at least 99 or 99.5 %, in particular 100 %, to the polynucleotide sequence according to SEQ ID NO: 1;

b) a yqfD sequence with a sequence identity of at least 99 or 99.5 %, in particular 100 %, to the polynucleotide sequence according to SEQ ID NO: 2;

c) a gyrB sequence with a sequence identity of at least 99 or 99.5 %, in particular 100 %, to the polynucleotide sequence according to SEQ ID NO: 3;

d) an rpoB sequence with a sequence identity of at least 99 or 99.5 %, in particular 100 %, to the polynucleotide sequence according to SEQ ID NO: 4;

e) a groEL sequence with a sequence identity of at least 99 or 99.5 %, in particular 100 %, to the polynucleotide sequence according to SEQ ID NO: 5.

Thus, a further subject of the current invention is a Bacillus subtilis strain, in particular a B. subtilis strain with the characteristics as mentioned before, or a preparation thereof, wherein the B. subtilis strain exhibits at least one, preferably all, of the following characteristics:

a) a 16S rDNA sequence with a sequence identity of at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 1;

b) a yqfD sequence with a sequence identity of at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 2;

c) a gyrB sequence with a sequence identity of at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 3;

d) an rpoB sequence with a sequence identity of at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 4;

e) a groEL sequence with a sequence identity of at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 5.

Thus, a further subject of the current invention is a Bacillus subtilis strain or a preparation thereof, in particular a B. subtilis strain with the characteristics as mentioned before, exhibiting two, three or four, preferably all, of the following characteristics:

a) a 16S rDNA sequence with a sequence identity of at least 99 %, preferably at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 1; b) a yqfD sequence with a sequence identity of at least 99 %, preferably at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 2; c) a gyrB sequence with a sequence identity of at least 99 %, preferably at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 3; d) an rpoB sequence with a sequence identity of at least 99 %, preferably at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 4;

e) a groEL sequence with a sequence identity of at least 99 %, preferably at least 99.5 %, more preferably at least 99.8 or 99.9 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 5.

Thus, a particular subject of the current invention is also a Bacillus subtilis strain, exhibiting the following characteristics:

a) a 16S rDNA sequence according to SEQ ID NO: 1;

b) a yqfD sequence according to SEQ ID NO: 2;

c) a gyrB sequence according to SEQ ID NO: 3.

Preferably, this B. subtilis strain exhibits the following further characteristics:

d) an rpoB sequence according to SEQ ID NO: 4;

e) a groEL sequence according to SEQ ID NO: 5.

The strains of the current invention are preferably characterized by at least one, more preferably by all, of the following further features:

They are preferably able to grow under anaerobic conditions. Further, they are preferably able to degrade water-insoluble cellulose as well as protein under such anaerobic conditions.

Further, the strains of the current invention are preferably further able to degrade water-insoluble cellulose as well as protein under aerobic conditions, in particular in the presence of 2 mM bile. They preferably inhibit infectious bacteria very effectively, in particular at least one strain, preferably all strains, selected from C. perfringens BB-081_Cpe; C. perfringens BB_031_Cpe; C. difficile DSM 1296; S. gallinaceus DSM 15349; S. suis DSM 9682, C. coli DSM 4689, C. jejuni DSM 24129 and ^. cecorum DSM 20683. In particular they are preferably characterized by a pathogen clearance of at least 15 mm, more preferably at least 20 mm, in a well diffusion antagonism assay on LBKelly agar plates with respect to C. perfringens strains BB-08 l_Cpe and BB 03 l_Cpe, respectively. The strains according to the invention are preferably further characterized by exhibiting at least one, preferably all, of the following enzymatic activities: cellulase activity; xylanase activity; protease activity; catalase activity; superoxide dismutase activity.

In particular, they are preferably characterized by a xylanase activity of at least 15 mU/mL, more preferably at least 20 mU/mL, in particular about 26 mU/mL, and/or by a protease activity of at least 12 mU/mL, more preferably at least 16 mU/mL, in particular about 19 mU/mL, and/or by a cellulase activity of at least 500 mU/mL, more preferably at least 700 mU/mL, above all about 790 mU/mL.

The strains of the current invention preferably furthermore produce lactate and they are preferably further able to degrade mycotoxins. The B. subtilis strains according to the invention are preferably further characterized by being able to grow in presence of 2 mM bile, more preferably in presence of 4 mM bile, and/or in presence of 0.3 w - % porcine bile and/or in presence of 0.3 wt.-% chicken bile. In particular they are preferably characterized by being able to proliferate fast under such high bile concentrations. In addition, the strains are preferably able to grow under high salt conditions, in particular in presence of 5 wt.-% of NaCl, for at least one day.

Further the strains of the current invention, i.e. a significant part, in particular at least 80 %, of a respective sample, preferably survive the high temperatures necessary for pelleting animal feed, in particular they preferably survive a temperature of 80°C, more preferably of 95 or 99°C, for at least 20 minutes.

Without wishing to be bound by any theory, it is thought that the Bacillus subtilis strains according to the current invention enhance animal health, in particular gut health, by a multifaceted mode of action, including the production of antibacterial metabolites with selective efficacy and the competition with pathogenic bacteria by better consuming the available nutrients, thereby suppressing effective establishment of pathogenic bacteria in the gut. Hereby the enzymes produced by Bacillus subtilis may help to establish a balanced gut microbiota by providing predigested nutrients. It is a big advantage of probiotics in comparison to antibiotics, that they do not attack bacteria indiscriminately nor do they lead to antibiotic resistant strains of pathogenic bacteria. Normally they are able to selectively compete with pathogenic bacteria by production of antimicrobial substances with specific efficacy, and are ideally able to simultaneously enhance the growth and viability of beneficial gut microflora. Further, they are preferably able to stimulate a systemic immune response in the treated animals.

The mutant strains of DSM 32592 of the current invention are preferably spontaneous mutants. The term "spontaneous mutant" refers to mutants that arise from DSM 32592 without the intentional use of mutagens. Such spontaneous mutants may be obtained by classical methods, such as growing the Bacillus subtilis strain in the presence of UV light and/or by applying high temperature or protoplast formation and/or in the presence of a certain antibiotic to which the parent strain is susceptible and testing any resistant mutants for improved biological activity or improved ability to enhance one or more of the indicia of animal health, in particular gut health. Other methods for identifying spontaneous mutants are known to those of ordinary skill in the art. But besides these preferred spontaneous mutants all other kinds of mutants of DSM 32592, like mutants obtained by genetic engineering, are also comprised by the current invention. One particular embodiment of the current invention are naturally non-occurring mutants, in particular spontaneous mutants as defined before, of the strain DSM 32592, characterized by the features as mentioned above.

In a preferred embodiment of the current invention, the strains and preparations of the present invention are preferably administered orally to animals or human beings.

Thus, a further subject of the current invention are compositions, such as feedstuffs, foodstuffs, drinking and rearing water as well as therapeutic compositions, containing a B. subtilis strain and/or a preparation of the current invention.

A further subject of the current invention is also the use of & B. subtilis strain and/or a preparation of the current invention as a probiotic ingredient (DFM) in feed or food products.

Preferred foodstuffs according to the invention are dairy products, in particular yoghurt, cheese, milk, butter and quark.

The cells of the strains of the current invention may be present, in particular in the compositions of the current invention, as spores (which are dormant), as vegetative cells (which are growing), as transition state cells (which are transitioning from growth phase to sporulation phase) or as a combination of at least two, in particular all of these types of cells. In a preferred embodiment, the composition of the current invention comprises mainly or only spores. In addition or as alternative the cells of the strains may also be used in non-living, inactivated form, as also the non-living cells are expected to still have a probiotic effect. Ways to inactivate the cells are known to those skilled in the art. The Bacillus subtilis strains of the current invention and compositions containing them, when administered to animals, preferably enhance the health of such animals and/or improve the general physical condition of such animals and/or improve the feed conversion rate of such animals and/or decrease the mortality rate of such animals and/or increase the survival rates of such animals and/or improve the weight gain of such animals and/or increase the productivity of such animals and/or increase the disease resistance of such animals and/or increase the immune response of such animals and/or establish or maintain a healthy gut microflora in such animals and/or reduce the pathogen shedding through the feces of such animals. In particular the strains and compositions of the current invention might be used to assist in re-establishing a healthy balance of the gut microflora after administration of antibiotics for therapeutic purposes.

A further subject of the current invention is therefore a method of enhancing the health of animals and/or of improving the general physical condition of animals and/or of improving the feed conversion rate of animals and/or of decreasing the mortality rate of animals and/or of increasing the survival rates of animals and/or of improving the weight gain of animals and/or of increasing the productivity of animals and/or of increasing the disease resistance of animals and/or of increasing the immune response of animals and/or of establishing or maintaining a healthy gut microflora in animals and/or of reducing the pathogen shedding through the feces of animals, wherein the strains and/or preparations of the current invention or the compositions of the current invention, which comprise such strain(s), are administered to animals.

A further subject of the current invention is therefore also the use of strains and/or preparations and/or compositions of the current invention for enhancing the health of animals and/or for improving the general physical condition of animals and/or for improving the feed conversion rate of animals and/or for decreasing the mortality rate of animals and/or for increasing the survival rates of animals and/or for improving the weight gain of animals and/or for increasing the productivity of animals and/or for increasing the disease resistance of animals and/or for increasing the immune response of animals and/or for establishing or maintaining a healthy gut microflora in animals and/or for reducing the pathogen shedding through the feces of animals, wherein the strains and/or preparations of the current invention or the compositions of the current invention, which comprise such strain(s), are administered to animals.

A further subject of the current invention are therefore also the strains and preparations of the current invention as mentioned before and the compositions of the current invention, containing those strains, for enhancing the health of animals and/or for improving the general physical condition of animals and/or for improving the feed conversion rate of animals and/or for decreasing the mortality rate of animals and/or for increasing the survival rate of animals and/or for improving the weight gain of animals and/or for increasing the productivity of animals and/or for increasing the disease resistance of animals and/or for increasing the immune response of animals and/or for establishing or maintaining a healthy gut microflora in animals and/or for reducing the pathogen shedding through the feces of animals.

"Increasing the productivity of animals" refers in particular to any of the following: production of more or higher quality eggs, milk or meat or increased production of weaned offspring.

The methods and uses of the strains, preparations and compositions of the current invention can be therapeutic or non- therapeutic. In a particularly preferred embodiment of the current invention, the methods and uses are non-pharmaceutic, in particular feeding applications.

As the untreated manure of animals due to pathogenic bacteria and other ingredients may have a detrimental environmental effect, in particular with respect to the animals themselves and/or with respect to human beings getting in contact with the manure, which can be avoided by either feeding the animals or directly treating the manure or the bedding of the animals with the strains, compositions or preparations of the current invention, therefore a further subject of the current invention is a method of controlling and/or avoiding detrimental environmental effects of manure or contaminated liquids, the method comprising the step of applying to manure, contaminated liquids, litter, a pit, or a manure pond at least one strain, one preparation and/or one composition according to the current invention.

Preferably the composition is applied in liquid form, for example by spraying, or as a powder, for example by strewing.

As detrimental bacteria may have a negative influence on the consistency of litter and in particular may effect a rather fluid or highly fluid litter, which might lead to foot pad lesions of poultry and which can be avoided by feeding the animals with the strains, compositions or preparations of the current invention, therefore a further subject of the current invention is a method of controlling and/or improving the consistency of litter, in particular a method of ensuring a solid consistency of litter and/or a method of avoiding foot pad lesions, the method comprising the step of feeding animals, in particular poultry, with at least one strain, one preparation and/or one composition according to the current invention.

The strains and preparations according to the invention can also be used for improving the quality of water. A further subject of the current invention is therefore also a method of controlling and/or improving the quality of water or aqueous solutions, in particular of drinking water and/or rearing water, comprising the step of applying to water or an aqueous solution at least one strain and/or at least one preparation and/or at least one composition of the current invention.

Further, the strains and preparations according to the invention can also be used for treating microbial diseases of plants. A further subject of the current invention is therefore also a method of treating and/or preventing microbial diseases of plants, in particular of cultivated plants, comprising the step of applying to the plants at least one strain and/or at least one preparation and/or at least one composition of the current invention. The application may be carried out in liquid form, such as by spraying, or in solid form, in particular as a powder, preferably as a formulated powder.

By using the strains, preparations and compositions of the current invention preferably an improvement of at least one of the features as mentioned before is realized, wherein realization of the feature preferably means an improvement of at least 1 %, more preferably of at least 3 or at least 5 %, in comparison to an adequate negative control. As negative control averages known in the animal husbandry field may be used, but preferably as negative control animals which are subjected to the same treatment like the animals tested are used, but without administration of the strains and/or preparations of the current invention.

In particular, the strains, preparations and compositions of the current invention may be administered or fed to an animal in an amount effective to inhibit and/or decrease the growth of pathogenic bacteria in the animal gut. Such pathogenic bacteria include Clostridia, Listeria, Salmonella, Enterococci, Staphylococci, Aeromonas, Streptococci, Campylobacter, Escherichia coli, Shigella, Haemophilus, Brachyspira and Vibrio. Relatedly, the methods of the present invention may be used to decrease the amount of pathogenic bacteria, viruses and protozoans shed in animal feces. The methods of the present invention may also be used to maintain or increase the growth of beneficial bacteria, such as lactic acid bacteria, in the animal gut. By decreasing pathogenic bacteria and/or increasing or maintaining beneficial bacteria, the compositions of the present invention are able to maintain an overall healthy gut microflora. Thus, a further subject of the current invention are also the strains, preparations and compositions of the current invention for inhibiting and/or decreasing the growth of pathogenic bacteria and/or for maintaining and/or increasing the growth of beneficial bacteria in an animal gut, wherein the pathogenic bacteria are preferably selected from Clostridia, in particular from C. perfringens, C. difficile, C. novyi, C. septicum and C. colinum, from Listeria, in particular from L. monocytogenes, L. seeligeri and L. welshimeri, from Salmonella, in particular S. enterica including the subspecies enterica, arizonae, bongori and in particular the serovars, S. gallinarum, S. pullorum, S. typhimurium, S. enteritidis, S. cholerasuis, S. heidelberg and S. infantis, from Enterococci, in particular E. faecalis, E. faecium and E. cecorum, from Staphylococci, in particular S. aureus, from Aeromonas, from Streptococci, in particular S. suis and S. gallinaceus, from Campylobacter, in particular C. jejuni and C. coli, from Escherichia coli, from Haemophilus, in particular Haemophilus parasuis, from Brachyspira, in particular

Brachyspira hyodysenteriae and from Vibrio, in particular V. parahemolyticus and V. harveyi, and the beneficial bacteria are preferably selected from lactic acid bacteria, in particular from lactobacilli and bifidobacteria.

In a preferred embodiment of the invention the amount of at least one pathogenic bacterium, in particular the amount of C. perfringens, is reduced by at least 0.5 log, more preferably by at least 1 log, 2 log, or 3 log.

Thus, a further subject of the current invention are also the strains, preparations and compositions of the current invention for inhibiting and/or decreasing the growth of pathogenic bacteria and/or for maintaining and/or increasing the growth of beneficial bacteria in an animal gut, wherein the pathogenic bacteria are preferably selected from Clostridia, in particular from C. perfringens, C. difficile, C. novyi, C. septicum and C. colinum, from Listeria, in particular from L. monocytogenes, L. seeligeri and L. welshimeri, from Salmonella, in particular S. enterica including the subspecies enterica, arizonae, bongori and in particular the serovars, S. gallinarum, S. pullorum, S. typhimurium, S. enteritidis, S. cholerasuis, S. heidelberg and S. infantis, from Enterococci, in particular E. faecalis, E. faecium and E. cecorum, from Staphylococci, in particular S. aureus, from Aeromonas, from Streptococci, in particular S. suis and S. gallinaceus, from Campylobacter, in particular C. jejuni and C. coli, from Escherichia coli, from Haemophilus, in particular Haemophilus parasuis, from Brachyspira, in particular

The occurrence and/or increased growth of the pathogenic bacteria does or can lead to the outbreak of certain diseases. For example the occurrence and/or increased growth of Clostridium perfringens can lead to the outbreak of gut diseases, in particular to the outbreak of necrotic enteritis in swine and poultry. The occurrence and/or increased growth of C. perfringens can also lead to the outbreak of further diseases like bacterial enteritis, gangrenous dermatitis and cholangiohepatitis. Even the mildest form of infection by C. perfringens can already be accompanied by diarrhea, which results in wet litter and by that may lead to secondary diseases like foot pad dermatitis. While C. perfringens type C generally is considered to be the primary cause of necrotic enteritis and necrohemorrhagic enteritis in piglets, type A has been linked to enteric disease in suckling and feeding pigs with mild necrotic enterocolitis and villous atrophy.

Clostridium difficile is an important emerging pathogen that causes diarrhea primarily in neonatal swine. Affected piglets may have dyspnea, abdominal distention, and scrotal edema. E. cecorum is known to cause lameness, arthritis and osteomyelitis in broilers usually caused by an inflammation of a joint and/or bone tissue. Further E. cecorum can cause an inflammation of the pericardium.

S. gallinaceus can cause septicaemia in poultry. The gross lesions included splenomegaly,

hepatomegaly, renomegaly and congestion. Multiple areas of necrosis and/ or infarction in the liver and spleen associated with valvular endocarditis were also observed. C. coli is a foodborne bacterium, most people usually get infected by eating pig meat that contained the bacteria. It causes gastroenteritis and acute enterocolitis in humans, and also of acute diarrheal illnesses. Pigs are the main host, but it can also infect humans, avian species and a wide range of other animals.

Infection with Campylobacter jejuni is one of the most common causes of gastroenteritis worldwide [Acheson et al. 2001]. Infection results from the ingestion of contaminated food or water. C. jejuni is commonly associated with poultry, and it naturally colonises the digestive tract of many bird species [Colles et al. 2009] (20 to 100% of retail chickens are contaminated) as well as cattle especially calves. Some strains of C. jejuni have been reported to cause enteritis and death in newly hatched chicks and poults. S. suis is an important pathogen in pigs and one of the most important causes of bacterial mortality in piglets after weaning causing septicemia, meningitis and many other infections.

Pathogens can cause further diseases like polyarthritis, fibrinous polyserositis, post-weaning enteric disorders like post-weaning diarrhea and edema disease and swine dysentery.

A further subject of the current invention is therefore also a therapeutic composition comprising the strains and/or compositions of the current invention as mentioned before.

A preferred subject in this context is therefore a therapeutic composition for treatment and/or prevention of necrotic enteritis and necrohemorrhagic enteritis, in particular sub-clinical necrotic enteritis and necrohemorrhagic enteritis, in animals, preferably swine or poultry, comprising the strains and/or compositions of the current invention as mentioned before.

Another preferred subject in this context is therefore a therapeutic composition for treatment and/or prevention of bacterial enteritis, gangrenous dermatitis, cholangiohepatitis, clostridiosis, diarrhea, dyspnea, abdominal distention, scrotal edema, bumblefoot, foot pad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post-weaning enteric disorders like post- weaning diarrhea and edema disease, dysentery, osteomyelitis, inflammation of joints and/or bone tissue, inflammation of the pericardium, splenomegaly, hepatomegaly, renomegaly, congestion, necrosis, infarction in the liver or spleen, valvular endocarditis, septicemia and/or meningitis, in animals, preferably in swine or poultry, comprising the strains and/or compositions of the current invention as mentioned before.

A further subject of the current invention is therefore also the treatment and/or prevention of a disease, in particular of a gut disease, preferably of necrotic enteritis or necrohemorrhagic enteritis, in particular of sub-clinical necrotic enteritis or sub-clinical necrohemorrhagic enteritis, in swine or poultry, wherein a strain and/or composition and/or preparation of the current invention is administered to an animal in need thereof.

A further subject of the current invention is therefore also the treatment and/or prevention of a disease, preferably a disease of swine or poultry, selected from bacterial enteritis, gangrenous dermatitis, cholangiohepatitis, clostridiosis, diarrhea, dyspnea, abdominal distention, scrotal edema, bumblefoot, foot pad dermatitis, streptococcal mastitis, lameness, arthritis, polyarthritis, fibrinous polyserositis, post- weaning enteric disorders like post-weaning diarrhea and edema disease, dysentery, osteomyelitis, inflammation of joints and/or bone tissue, inflammation of the pericardium, splenomegaly, hepatomegaly, renomegaly, congestion, necrosis, infarction in the liver or spleen, valvular endocarditis, septicemia and/or meningitis, wherein a strain and/or composition and/or preparation of the current invention is administered to an animal in need thereof.

The strains and/or preparations and/or compositions of the current invention can be administered to animals in feed and/or drinking water over multiple days throughout the animal's life or during particular stages or portions of the animal's life. For example, the strains and/or compositions can be administered only in a starter diet or only in a finisher diet of farm animals.

A particular subject of the current invention is also a method of enhancing the health of human beings and/or of improving the general physical condition of human beings and/or of increasing the disease resistance of human beings and/or of increasing the immune response of human beings and/or of establishing or maintaining a healthy gut microflora in human beings, wherein the strains and/or preparations of the current invention or the compositions of the current invention, which comprise such strain(s), are administered to human beings.

A further subject of the current invention is therefore also the use of strains and/or preparations and/or compositions of the current invention for enhancing the health of human beings and/or for improving the general physical condition of human beings and/or for increasing the disease resistance of human beings and/or for increasing the immune response of human beings and/or for establishing or maintaining a healthy gut microflora in human beings, wherein the strains and/or preparations of the current invention or the compositions of the current invention, which comprise such strain(s), are administered to human beings.

The compositions of the present invention, in particular the feed, food and pharmaceutical compositions as well as the drinking or rearing water, preferably comprise the strains of the current invention and are administered to animals at a rate of about lxlO³ to about 2x10¹² CFU/g feed or ml water, in particular in a rate of about lxlO³ or about lxlO⁴ or about lxlO⁵ or about lxlO⁶ or about lxlO⁷ or about lxlO⁸ or about lxlO⁹ or about lxlO¹⁰ or about lxlO¹¹ or about lxlO¹² CFU/g feed or ml water, preferably in an amount of about lxlO⁴ to about lxlO¹⁰ CFU/g feed or ml water, more preferably in an amount of lxl 0⁴ to lx 10⁷ CFU/g feed or ml water.

Correspondingly, preferred amounts of the strains and/or preparations of the current invention in the feed, food and water compositions of the current invention range preferably from 0.1 wt.-% to 10 w - %, more preferably from 0.2 wt.-% to 5 wt.-%, in particular from 0.3 wt.-% to 3 wt.-%.

The methods of the present invention may be used for all kind of animals, in particular all kind of non- human and non-insect animals, more preferably all kind of vertebrates such as mammals, aquatic animals and birds. Animals that may benefit from the current invention include but are not limited to farm animals, pets, exotic animals, zoo animals, aquatic animals, animals used for sports, recreation or work.

Pets are preferably selected from dogs, cats, domestic birds and domestic exotic animals. Aquatic animals are preferably selected from finfish and crustaceans which are preferably intended for human nutrition. These include, in particular, carp, tilapia, catfish, tuna, salmon, trout, barramundi, bream, perch, cod, shrimps, lobster, crabs, prawns and crayfish. Preferred types of salmon in this context are the Atlantic salmon, red salmon, masu salmon, king salmon, keta salmon, coho salmon, Danube salmon, Pacific salmon and pink salmon.

Further preferred aquatic animals are fanning fish which are subsequently processed to give fish meal or fish oil. In this connection, the fish are preferably herring, pollack, menhaden, anchovies, capelin or cod. In a further preferred embodiment, the animals are farm animals, which are raised for consumption or as food-producers, such as poultry, swine and ruminants. The poultry may be selected from productive or domestic poultry, but also from fancy poultry or wild fowl.

Preferred productive poultry in this context are chickens, turkeys, ducks and geese. The productive livestock in this context is preferably poultry optimized for producing young stock or poultry optimized for bearing meat.

Preferred fancy poultry or wild fowl are peacocks, pheasants, partridges, chukkars, guinea fowl, quails, capercaillies, grouse, pigeons and swans, with quails being especially preferred.

Further preferred poultry are ratites, in particular ostriches and emus, as well as parrots.

Ruminants according to the current invention are preferably selected from cattle, goat and sheep. In one embodiment, the compositions of this invention may be fed to preruminants to enhance their health and, in particular, to decrease the incidence of diarrhea in these animals. Preruminants are ruminants, including calves, ranging in age from birth to about twelve weeks.

The compositions of the current invention may comprise at least one carrier or typical feed ingredients or combinations thereof.

Suitable carriers are inert formulation ingredients added to improve recovery, efficacy, or physical properties and/or to aid in packaging and administration. Such carriers may be added individually or in combination. These carriers may be selected from anti-caking agents, anti-oxidation agents, bulking agents, and/or protectants. Examples of useful carriers include polysaccharides (in particular starches, maltodextrins, methylcelluloses, gums, chitosan and/or inulins), protein sources (in particular skim-milk powder and/or sweet- whey powder), peptides, sugars (in particular lactose, trehalose, sucrose and/or dextrose), lipids (in particular lecithin, vegetable oils and/or mineral oils), salts (in particular sodium chloride, sodium carbonate, calcium carbonate, chalk, limestone, magnesium carbonate, sodium phosphate, calcium phosphate, magnesium phosphate and/or sodium citrate), and silicates (in particular clays, in particular beolite clay, amorphous silica, fumed/precipitated silicas, zeolites, Fuller's earth, Baylith®, clintpolite, montmorillonite, diatomaceous earth, talc, bentonites, and/or silicate salts like aluminium, magnesium and/or calcium silicate). Suitable carriers for animal feed additives are set forth in the American Feed Control Officials, Inc.' s Official Publication, which publishes annually. See, for example Official Publication of American Feed Control Officials, Sharon Krebs, editor, 2006 edition, ISBN 1-878341-18-9. The carriers can be added after concentrating the fermentation broth and/or during and/or after drying. Preferred carriers according to the invention are selected from calcium carbonate, diatomaceous earth and vegetable oil. A preferred embodiment of the current invention are concentrate compositions, in particular feed additive compositions, i.e. compositions suitable for preparing a feed composition, which comprise at least one strain of the current invention and at least one carrier as mentioned before, wherein the at least one strain is preferably comprised in an amount of 0.1 to 10 wt.-%, more preferably in an amount of 0.2 to 5 wt.-%, in particular in an amount of 0.3 to 3 wt.-%, above all in an amount of 0.4 to 2.2 wt.-%, and the at least one carrier is preferably comprised in an amount of at least 90 wt.-%, preferably in an amount of 90 to 99.9 wt.-%, more preferably in an amount of 95 to 99.8 wt.-%, in particular in an amount of 97 to 99.7 wt.-%, above all in an amount of 97.8 to 99.6 wt.-%, and wherein the carrier consists preferably substantially of limestone, in particular of limestone with smaller parts of diatomaceous earth and/or vegetable oil.

These preferred compositions of the current invention, which contain stabilized strains, can be used for the preparation of feed and pharmaceutical compositions as well as drinking and rearing water which preferably comprise the strains according to the invention in an amount as mentioned in the specification above. In a preferred embodiment 50 to 1000 grams of such a concentrate composition, in particular 50, 100, 250, 500 or 1000 grams of such a concentrate composition, are used per ton of feed, drinking or rearing water to provide compositions which can be used for feeding animals. These concentrate compositions preferably comprise at least one strain of the current invention in an amount of lxlO⁹ to 2xlO^u CFU, in particular 2xl0⁹ to lxlO¹¹ CFU, per g of the concentrate composition.

Starting from these concentrate compositions, feed and food compositions can be prepared by mixing the concentrate compositions with typical feed or food ingredients, respectively.

Suitable typical animal feed ingredients which may be contained in the compositions according to the invention and/or used in the preparation of feed compositions starting from concentrate compositions according to the invention include one or more of the following: proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immune modulators, milk replacers, minerals, amino acids, coccidiostats, acid-based products and/or medicines, such as antibiotics. Carbohydrates containing components which may be used according to the invention are for example forage, roughage, wheat meal, sunflower meal or soya meal, and mixtures thereof.

Proteins containing components which may be used according to the invention are for example soya protein, pea protein, wheat gluten or corn gluten, and mixtures thereof.

Fats containing components which may be used according to the invention are in particular oils, of both animal and plant origin, like vegetable oils, for example soya bean oil, rapeseed oil, sunflower seed oil, flaxseed oil or palm oil, fish oil, and mixtures thereof. Proteins containing components which additionally contain fats which may be used according to the invention are for example fish meal, krill meal, bivalve meal, squid meal or shrimp shells, as well as combinations thereof. Further probiotics (DFM) which may be used according to the invention in combination with the strains and preparations of the invention are preferably bacteria selected from the species Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus pumilus, Bacillus laterosporus, Bacillus coagulans, Bacillus alevi, Bacillus cereus, Bacillus badius, Bacillus thurigiensis, Enterococcus faecium, and Pediococcus acidilactici. Preferred examples are Bacillus pumilus DSM 32539 and Bacillus subtilis DSM 32540 (both deposited with the DSMZ on June 14, 2017 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure) and derivatives thereof, Bacillus licheniformis DSM 32314 and Bacillus subtilis DSM 32315 (both deposited with the DSMZ on May 12, 2016 under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure) and derivatives thereof, Bacillus subtilis PB6 (as described in US Patent No. 7,247,299 and deposited as

ATCC Accession No. PTA-6737), which is sold by Kemin under the trademark CLOSTAT®, Bacillus subtilis C-3102 (as described in US Patent No. 4,919,936 and deposited as FERM BP- 1096 with the Fermentation Research Institute, Agency of Industrial Science and Technology, in Japan), sold by Calpis as CALSPORTN®, Bacillus subtilis DSM 17299, as sold by Chr. Hansen under the trademark GalliPro®, Bacillus licheniformis DSM 17236, as sold by Chr. Hansen under the trademark

GalliProTect®, a mixture of Bacillus licheniformis DSMZ 5749 and Bacillus subtilis DSMZ 5750 spores, as sold by Chr. Hansen under the trademark BioPlus®YC, B. subtilis DSM 29784, as sold by Adisseo/Novozymes under the trademark Alterion®, Bacillus subtilis, as sold by Chr. Hansen under the trademark PORCBOOST®, or Bacillus coagulans strains as described in US Patent No. 6,849,256. Other non-Bacillus probiotics, such as Saccharomyces cerevisiae, Pichia pastoris, Aspergillus niger, Aspergillus oryzae, or Hansenula, may also be used in compositions of the present invention. In particular in food compositions further probiotics which are known to be useful to the human health may be used such as lactic acid producing bacteria, in particular lactobacilli, or Bifidobacteria. If said further probiotics are not formulated as part of the compositions of the present invention, they may be administered together (either at the same time or at different times) with the compositions of the present invention.

Prebiotics which may be used according to the invention are preferably oligosaccharides, in particular selected from galactooligosaccharides, sialyloligosaccharides, lactulose, lactosucrose,

fructooligosaccharides, palatinose or isomaltose oligosaccharides, glycosyl sucrose,

maltooligosaccharides, isomaltooligosaccharides, cyclodextrins, gentiooligosaccharides, soybean oligosaccharides, xylooligosaccharides, dextrans, pectins, polygalacturonan, rhamnogalacturonan, mannan, hemicellulose, arabinogalactan, arabinan, arabinoxylan, resistant starch, mehbiose, chitosan, agarose, inulin, tagatose, polydextrose, and alginate.

Enzymes which may be used in feed compositions according to the invention and which may aid in the digestion of feed, are preferably selected from phytases (EC 3.1 .3.8 or 3.1.3.26), xylanases (EC

3.2.1.8), galactanases (EC 3.2.1 .89), galactosidases, in particular alpha-galactosidases (EC 3.2.1.22), proteases (EC 3.4), phospholipases, in particular phospholipases Al (EC 3.1 .1.32), A2 (EC 3.1.1.4), C (EC 3.1.4.3), and D (EC 3.1.4.4), lysophospholipases (EC 3.1 .1.5), amylases, in particular alpha- amylases (EC 3.2.1.1 ); lysozymes (EC 3.2.1 .17), glucanases, in particular beta-glucanases (EC 3.2.1.4 or EC 3.2.1.6), glucoamylases, cellulases, pectinases, or any mixture thereof.

Examples of commercially available phytases include Bio-Feed™ Phytase (Novozymes), Ronozyme® P and HiPhos™ (DSM Nutritional Products), Natuphos™ (BASF), Finase® and Quantum® Blue (AB Enzymes), the Phyzyme® XP (Verenium/DuPont) and Axtra® PHY (DuPont). Other preferred phytases include those described in e.g. WO 98/28408, WO 00/43503, and WO 03/066847.

Examples of commercially available xylanases include Ronozyme® WX and G2 (DSM Nutritional Products), Econase® XT and Barley (AB Vista), Xylathin® (Verenium) and Axtra® XB

(Xylanase/beta-glucanase, DuPont). Examples of commercially available proteases include Ronozyme® ProAct (DSM Nutritional Products).

Vitamins which may be used according to the invention are for example vitamin A, vitamin D3, vitamin E, vitamin K, e.g., vitamin K3, vitamin B 12, biotin, choline, vitamin B l , vitamin B2, vitamin B6, niacin, folic acid and pantothenate, e.g. , Ca-D-pantothenate, or combinations thereof.

Immmune modulators which may be used are for example antibodies, cytokines, spray-dried plasma, interleukins, or interferons, or combinations thereof.

Minerals which may be used according to the invention are for example boron, cobalt, chloride, chromium, copper, fluoride, iodine, iron, manganese, molybdenum, selenium, zinc, calcium, magnesium, potassium, or sodium, or combinations thereof.

Amino acids which may be used according to the invention are for example lysine, alanine, threonine, methionine or tryptophan, or combinations thereof.

Thus, a further embodiment of the current invention is a method of preparing an animal feed composition comprising mixing at least one strain and/or at least one preparation and/or at least one concentrate composition of the current invention, in particular in an amount effective to enhance animal health, in particular gut health, with feed ingredients, such as proteins, lipids and/or carbohydrates, and optionally further beneficial substances, preferably as mentioned before, to provide a feeding product. This method may comprise for example also a pelleting step. Standard pelleting processes known to those of skill in the art may be used, including extrusion processing of dry or semi-moist feeds. Preferred pelleting temperatures are between about 65° C and about 120° C.

The strains and compositions of the present invention can be obtained by culturing the strains of the current invention according to methods well known in the art, including by using the media and other methods as described for example in US 6,060,051, EP0287699 or US2014/0010792. Conventional large-scale microbial culture processes include submerged fermentation, solid state fermentation, or liquid surface culture. Towards the end of fermentation, as nutrients are depleted, the cells of the strains begin the transition from growth phase to sporulation phase, such that the final product of fermentation is largely spores, metabolites and residual fermentation medium. Sporulation is part of the natural life cycle of these strains and is generally initiated by the cell in response to nutrient limitation.

Fermentation is configured to obtain high levels of colony forming units of the Bacillus subtilis cells and to promote sporulation. The bacterial cells, spores and metabolites in culture media resulting from fermentation may be used directly or concentrated by conventional industrial methods, such as centrifugation, tangential-flow filtration, depth filtration, and evaporation. The concentrated fermentation broth may be washed, for example via a diafiltration process, to remove residual fermentation broth and metabolites.

The fermentation broth or broth concentrate can be dried with or without the addition of carriers using conventional drying processes or methods such as spray drying, freeze drying, tray drying, fluidized-bed drying, drum drying, or evaporation. The resulting dry products may be further processed, such as by milling or granulation, to achieve a specific particle size or physical format. Carriers, as described above, may also be added post-drying. Preparations of the strains of the current invention may be cell-free preparations or preparations containing cell debris or preparations containing a mixture of intact cells and cell debris.

Cell-free preparations of the strains of the current invention can be obtained for example by centrifugation and/or filtration of fermentation broth. Depending on the technique used, these cell- free preparations may not be completely devoid of cells, but may still comprise a smaller amount of cells. As the cells secret compounds like metabolites, enzymes and/or peptides into the surrounding medium, the supernatant of the cells comprises a mixture of such compounds, in particular metabolites, enzymes and/or peptides, as secreted by the cells. Thus, in a preferred embodiment of the invention, the preparation of the strains is a supernatant of the fermentation broth. Compositions comprising cell debris of the strains may be obtained by rupturing the cells applying techniques as known to those of skill in the art, for example by mechanical means or by applying high pressure. Depending on the degree of force applied, a composition comprising only ruptured cells or a composition comprising a mixture of cell debris and intact cells is obtained. Homogenization of the cells may be realized for example by utilizing a French cell press, sonicator, homogenizer, microfluidizer, ball mill, rod mill, pebble mill, bead mill, high pressure grinding roll, vertical shaft impactor, industrial blender, high shear mixer, paddle mixer, and/or polytron homogenizer. Suitable alternatives are enzymatic and/or chemical treatment of the cells.

Cell-free preparations of the current invention comprise also preparations which are obtained by first rupturing the cells by applying techniques as mentioned before and subsequently removing the cell debris and the remaining intact cells. Removing of the cell debris and remaining intact cells can be carried out in particular by centrifugation and/or filtration.

The preparations of the strains of the current invention may comprise as active compounds at least one metabolite, preferably a mixture of metabolites, as further described below, and/or at least one enzyme selected from proteases, in particular subtilisin, xylanases and/or cellulases, and/or at least one peptide, and/or combinations thereof.

A preparation containing an effective mixture of metabolites as contained in the strains of the current invention and/or as contained in the cell preparations as mentioned before, can be obtained for example according to the methods set forth in US Patent No. 6,060,051. In particular the preparation can be obtained by precipitating the metabolites as contained in the preparations mentioned before by using organic solvents like ethyl acetate and subsequent redissolving of the precipitated metabolites in an appropriate solvent. The metabolites may subsequently be purified by size exclusion filtration that groups metabolites into different fractions based on molecular weight cut-off.

The preparation containing an effective mixture of metabolites of the current invention preferably comprises at least five, more preferably at least 6, 7, 8, 9, 10 or 12, in particular all metabolites of the strains of the invention. The content of metabolites of the strain DSM 32592 is depicted in Table 3.1. The metabolites possess preferably a molecular weight of between 500 and 4000 Dalton, more preferably of between 800 and 3500 Dalton. Preferably according to the invention always an effective amount of the strains and/or preparations and/or compositions of the current invention is used in the embodiments of the current invention. The term "effective amount" refers to an amount which effects at least one beneficial effect to an animal and/or to the environment, in particular with respect to the features as already mentioned before, in comparison to an animal that has not been administered the strains and/or preparations and/or compositions of the current invention, but besides that has been administered the same diet (including feed and other compounds). In case of therapeutic applications preferably a therapeutic amount of the strains and/or preparations and/or compositions of the current invention is used. The term "therapeutic amount" refers to an amount sufficient to ameliorate, reverse or prevent a disease state in an animal. Optimal dosage levels for various animals can easily be determined by those skilled in the art, by evaluating, among other things, the composition's ability to (i) inhibit or reduce pathogenic bacteria in the gut at various doses, (ii) increase or maintain levels of beneficial bacteria and /or (iii) enhance animal health, in particular gut health, at various doses.

Working Examples

Example 1. Strain characteristics relevant to survival in the gastrointestinal tract.

Bacillus subtilis strains were screened from various environmental samples in order to obtain a superior strain as animal direct-fed microbial / probiotic. As the strain is intended to reach its full potential in the intestine of the target animal, the strain was pre-screened to withstand various environmental and gut related conditions. Strain spores were generated (Nicholson and Setlow 1990), washed and incubated at 80 °C for 20 minutes (pasteurization), then titrated in logarithmic / 1 in 10 dilutions using veal infusion broth agar (VI, Difco™, no. 234420, Becton Dickinson GmbH, Heidelberg, Germany). The second highest dilution prior to no growth was stored at -80 °C and used as standardized starting point for all further assessments from spore state. To simulate gastric passage (Argenzio 2004a; Trampel and Duke 2004), survival of acid exposure was assessed based on Larsen et al. (2014). Growth of vegetative cells was furthermore assessed at low pH indicating growth under stomach/proventriculus and gizzard conditions, as well as in presence of up to 4 mM bile (B8631, CAS 8008-63-8, Sigma-Aldrich) at pH 7 in order to confirm strain growth at the proximal part of the small intestine right after clearance of the stomach or gizzard (Argenzio 2004b; Trampel and Duke 2004). Strain fitness in the anaerobe intestine (Argenzio 2004b; Trampel and Duke 2004) was assessed by inoculating standardized spore solutions under anaerobic conditions (AnaeroPak™, Thermo Fisher Scientific) in VI medium supplemented with 2.5 mM KNO3 (Glaser et al. 1995). Furthermore was the anaerobe proteolytic and cellulytic activity of strains assessed on VI agar plates supplemented with 1% skim milk powder (70166, Sigma-Aldrich) or 0.1% water insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland).

Furthermore the aerobe cellulytic activity of strains was assessed using VI agar supplemented with 0.1% water insoluble AZCL-HE cellulose (I-AZCEL, Megazyme International, Bray, Ireland). After 24 h under aerobe conditions, a blue coloration of agar indicated cellulase activity.

Osmotic stress, as also found in the gut (Argenzio 2004b; Trampel and Duke 2004), was assessed by determining growth on VI agar with addition of 10 wt.-% NaCl (den Besten et al. 2009). Finally, spore heat stability was assessed to determine pelleting stability by exposing spores to 99 °C for 20 min (Palop et al. 1996) and subsequent inoculation on VI agar.

Bacillus subtilis strain DSM 32592 survived simulated gastric passage, growth was observed starting at pH 6. Under aerobic conditions the strain DSM 32592 was able to degrade protein and water-insoluble cellulose in the presence of 2 mM bile. Strain DSM 32592 grew anaerobically and was able to degrade water-insoluble cellulose and protein under anaerobic conditions. Strain DSM 32592 was able to grow in presence of 2 and 4 mM bile, in presence of 0.3 wt.-% porcine bile and in presence of 0.3 wt.-% chicken bile as well as in presence of 5 wt.-% NaCl. Strain DSM 32592 reached an average spore count of 8.9 x 10^s CFU/mL, and spores of strain DSM 32592 were viable after exposure to 99°C for 20 min.

References:

Argenzio, R. A. (2004a). Secretion of the Stomach and Accessory Glands, p. 405 - 418. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 25; Cornell University Press, Ithaca, New York, USA.

Argenzio, R. A. (2004b). Digestive and Absorptive Functions of the Intestines, p. 419 - 437. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 26; Cornell University Press, Ithaca, New York, USA.

Dawson, R. M. C; Elliot, D. C; Elliot, W. H.; Jones, K. M. (1986). Data for Biochemical Research; 3^rd edition, Oxford Science Publishing, United Kingdom.

Den Besten HMW, Mols M, Moezelaar R, Zwietering MH, Abee T. (2009). Phenotypic and transcriptomic analyses of mildly and severely salt-stressed Bacillus cereus ATCC 14579 cells. Appl Environ Microbiol. 75:4111-9.

Glaser, P., A. Danchin, F. Kunst, P. Zuber, and M. M. Nakano. (1995). Identification and isolation of a gene required for nitrate assimilation and anaerobic growth of Bacillus subtilis. J. Bacteriol. 177: 1112- 1115

Nicholson W. L., Setlow P. Sporulation, germination and outgrowth. In: Harwood C R, Cutting S M, editors. Molecular biological methods for Bacillus. Chichester, England: John Wiley & Sons Ltd.; 1990. pp. 27-74.

Palop, A., Raso, J., Pagan, R., Condon, S. and Sala, F.J. (1996). Influence of pH on heat resistance of Bacillus licheniformis in buffer and homogenized foods. International Journal of Food Microbiology 29, 1-10.

Trampel, D. W. and Duke, G. E. (2004). Avian Digestion, p. 488 - 500. In: Reece, W. O. (ed.), Duke's Physiology of Domestic Animals; Twelfth Edition, Chapter 29; Cornell University Press, Ithaca, New York, USA. Example 2. Comparative strain performance relative to state of the art direct-fed microbial (DFM) / probiotic for animal nutrition - quantitative assessment of enzymatic activity.

Strains DSM 32592, DSM 17236 and DSM 17299 were compared evaluating the respective aerobe cellulytic, xylanolytic and proteolytic activity. Cellulase and xylanase activity were determined as described in Larsen et al. (2014). For proteolytic activity analysis, starter and main culture of strains were grown in VIB at 37°C as previously described. From main culture, 10 uL were added to 20 uL 0.5% Fluorescein Isothiocyanate Casein (FITC; C3777, Sigma-Aldrich) solution with 20 uL buffer consisting of 20mM sodium phospate (dibasic, anhydrous) with 150 mM sodium chloride (all components from Sigma-Aldrich), then incubated for 1 h at 37 °C. After addition of 150 uL of 10 % (v/v) trichlor acetic acid (Sigma-Aldrich) and another 30 min incubation at 37°C, samples were centrifuged at 19,000 rpm for 15 min, then 2 uL of supernatant transferred to 200 uL 500 mM TRIS HC1 Solution (Trizma BaseTRIS, Sigma-Aldrich). Fluorescence of soluble peptides due to proteolytic release were determined (TECAN GENios Microplate Reader, Tecan Group Ltd., Mannedorf, Switzerland) at excitation 494 nm, emission 518 nm. Analysis was performed in three independent runs, then averaged as milliunits per microliter solution. Results can be found in Table 2.1.

Table 2.1. Xylanase, protease and cellulase activity of strains DSM 32592, DSM 17299 and DSM 17236.

In direct comparison, strain DSM 32592 has a more than 2 fold increased xylanase activity, protease activity and cellulase activity compared to benchmark strains DSM 17236 and DSM 17299. In particular it has a more than 10 fold increased cellulose activity compared to benchmark strain DSM 17236.

Reference:

Larsen, N., Thorsen, L., Kpikpi, E. N., Stuer-Lauridsen, B., Cantor, M. D., Nielsen, B., Brockmann, E., Derkx, E. M. F. and Jespersen, L. (2014). Characterization of Bacillus spp. strains for use as probiotic additives in pig feed. Applied microbiology and biotechnology, 98(3), 1105-1118.

Example 3. Comparative strain performance relative to state of the art direct-fed microbial (DFM) / probiotic for animal nutrition - expression of metabolites and pathogen inhibition.

Strains DSM 32592 and wildtype strain DSM 10 were compared evaluating the respective number of metabolites expressed and pathogens inhibited in the respective media. For metabolite expression analysis, starter cultures were grown and tests performed as described in Scholz et al. (2011). From 10 mL Luria Bertami broth (LB, Thermo Fisher Scientific) culture grown for 24 h at 37°C and 160 rpm in 100 mL flask, 100 uL were transferred to main culture. Main culture was grown either in 10 mL LB containing 0.2 mL / L KellyT trace metal solution (LBKelly, Scholz et al. 2011), or 10 mL Trypticase Soy Broth (Oxoid, Thermo Fisher Scientific) with 0.6% yeast extract (Y1625, CAS 8013-1-2, Sigma- Aldrich; resulting broth abbreviated TSBYE), both for 24 h at 37°C at 160 rpm in 100 mL flask. Of the main culture, 4 mL were combined with 2 mL w-Butanol in 15 mL test tube, vortexed for 3 min, then sonicated for 15 min. After centrifugation for 1 min at 5000 rpm, organic phase was transferred, vacuum dried and analyzed using High-performance liquid chromatography - electrospray ionization mass spectrometry (HPLC-ESI-MS; Chen et al. 2006). Every sample was measured in two different modes, negative and positive mode, and mass spectra were acquired. Resulting peaks as similarly reported in Teo and Tan (2005) were converted to molecular mass in Da. Results for comparison can be found in Table 3.1. Table 3.1. (a) Comparison of metabolites expressed by strains DSM 32592 and wildtype strain DSM 10 in LB-Kelly and TSBYE, respectively (n/d means not detected)

Table 3.1. (b) Comparison of metabolites expressed by strains DSM 32592 and wildtype strain DSM 10 in LB-Kelly and TSBYE, respectively (n/d means not detected)

Strain 1050 Da 1460 Da 1462 Da 1476 Da 1504 Da 1505 Da 3401 Da

DSM 10 yes yes yes yes yes n/d yes

DSM 32592 yes yes yes yes yes yes yes

In addition, pathogen inhibition via Bacillus subtilis secondary metabolite production, as part of metabolites from table 3.1. but not closer investigated, was assessed using well diffusion antagonism tests (Parente et al. 1995).

A well diffusion antagonisms test with 7 different pathogens, Clostridium perfringens, Clostridium difficile DSM 1296, Enterococcus cecorum DSM 20683, Streptococcus gallinaceus DSM 15349,

Streptococcus suis ATCC 43765, Campylobacter coli ATCC 33559 (DSM 4689) and Campylobacter jejuni DSM 24129 was performed.

Two pathogenic C. perfringens candidates were tested being pathogenic C. perfringens field isolates from swine, obtained from RIPAC-LABOR GmbH, Potsdam-Golm, Germany. The C. perfringens type C-strains from Ripac describe as follows: Strains BB-081_Cpe and BB-031_Cpe were isolated from necrotic enteritis positive swine digestive tract. Strain BB-081_Cpe is cpb2 positive (Songer et al. 2005) and netB negative and Strain BB-03 l_Cpe tested positive for β2 -toxin (Allaart et al. 2012). Clostridium difficile is an important emerging pathogen that causes diarrhea primarily in neonatal swine (Songer et al. 2000). Affected piglets may have dyspnea, abdominal distention, and scrotal edema. Diarrhea may not be present in all pigs affected. DSM 1296 is a known type strain for C. difficile and produces cytotoxin.

E. cecorum is known to cause lameness, arthritis and osteomyelitis in broilers usually caused by an inflammation of a joint and/or bone tissue. Additional E. cecorum can cause an inflammation of the pericardium [Kense et al. 2011]. DSM 20683 was isolated from caecum of a chicken.

hepatomegaly, renomegaly and congestion. Multiple areas of necrosis and/ or infarction in the liver and spleen associated with valvular endocarditis were also observed [Collins et al. 2002]. S. suis is an important pathogen in pigs and one of the most important causes of bacterial mortality in piglets after weaning causing septicemia, meningitis and many other infections [Goyette-Desjardins et al. 2014]. ATCC 43765 belongs to Serological group: R; serovar 2 and was isolated from pigs.

C. coli is a foodborne bacterium, most people usually get infected by eating pig meat that contained the bacteria. It causes gastroenteritis and acute enterocolitis in humans, and also of acute diarrheal illnesses [Fitzgerald et al. 2007]. Pigs are the main host, but it can also infect humans, avian species and a wide range of other animals. ATCC 33559 was isolated from pig feces.

Infection with Campylobacter jejuni is one of the most common causes of gastroenteritis worldwide [Acheson et al. 2001]. Infection results from the ingestion of contaminated food or water. C. jejuni is commonly associated with poultry, and it naturally colonises the digestive tract of many bird species [Colles et al. 2009] (20 to 100% of retail chickens are contaminated) as well as cattle especially calves. It is generally nonpathogenic in birds. Some strains of C. jejuni have been reported to cause enteritis and death in newly hatched chicks and poults. DSM 24129 is isolated from chickens.

Bacillus strains were grown in 10 mL TSBYE (30 g/1 TSB + 6 g/1 Yeast extract) or LB-Kelly (LB- Media supplemented with trace elements solution of DSMZ media 1032) for 16 h at 37°C and 200 rpm in 100 mL shaking flask. The pathogenic strains were grown under suitable conditions as liquid culture to an optical density of 595 nm of at least 1, then 100 μΐ were spread with sterile spatula on the surface of agar plates. For S. gallinaceus BHI agar plates, all other pathogens TSBYE agar plates are used. Three 9 mm diameter wells were cut into the dried plates. 1^st well was used as non-inoculated media control without culture, 2^nd well was inoculated with 100 uL not-inhibiting Bacillus strain (B. cereus var. toyoi, NCIMB 40112), the 3^rd well was inoculated with 100 uL of Bacillus subtilis DSM 32592 or DSM 17299 culture. After 24 h incubation under suitable conditions at 37°C, the zone of clearance in mm was determined measuring from the edge of the cut well to the border of the cleared lawn. Each colony was measured twice (horizontally, vertically), then averaged. The results can be found in the following tables. Table 3.2: Comparison of Bacillus subtilis DSM 32592, DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Clostridium strains in well diffusion antagonism assays on LB Kelly medium, values in mm clearance of pathogen.

The data show that DSM 32592 is able to inhibit the growth of C. perfringens and S. difficile very effectively, in particular in comparison to DSM 17299.

Table 3.3: Comparison iBacillus subtilis DSM 32592 , DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Staphylococcus, Streptococcus and Campylobacter strains in well diffusion antagonism assays on LB Kelly medium, values in mm clearance of pathogen.

The data show that DSM 32592 is able to inhibit the growth of S. gallinaceus,

S. suis, C. coli and C. jejuni very effectively, in particular in comparison to DSM 17299. Table 3.4: Comparison of Bacillus subtilis DSM 32592, DSM 17236 and DSM 17299 inhibitory capacity on pathogenic Enterococcus cecorum in well diffusion antagonism assays on TSBYE medium, values in mm clearance of pathogen.

The data show that DSM 32592 is able to inhibit the growth of E. cecorum very effectively, in particular in comparison to DSM 17299.

References

Teo, A. Y.-L. and Tan, H.-M. (2005). Inhibition of Clostridium perfringens by a novel strain of Bacillus subtilis from the gastrointestinal tracts of healthy chickens. Appl. Environm. Microbiol., 71:4185-90. Paul McMullin (2004) Chapter: Staphylococcus aureus subsp. aureus infections in chickens. Book: A pocket guide to poultry health and disease. Publisher: 5m Publishing.

Parente, E., Brienza, C, Moles, M., & Ricciardi, A. (1995). A comparison of methods for the measurement ofbacteriocin activity. Journal of microbiological methods, 22(1), 95-108.

GA Contreras, JM Rodriguez (2011) Mastitis: Comparative Etiology and Epidemiology Journal of Mammary Gland Biology and Neoplasia, Volume 16, Issue 4, pp 339-356

(2016) U.S. Department of Health & Human Services

(https://www.cdc.gov/foodsafety/diseases/staphylococcal.html)

Allaart, J. G., de Bruijn, N. D., van Asten, A. J., Fabri, T. H., and Grone, A. (2012). NetB-producing and beta2 -producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands. Avian Pathol., 41 :541-546

JG Songer, FA Uzal (2005) Clostridial Enteric Infections in Pigs. Volume: 17 issue: 6, page(s): 528- 536 Journal of Veterinary Diagnostic Investigation

Songer JG, Post KW, Larson DJ, et al. (2000) Infection of neonatal swine with Clostridium difficile. Swine Health Prod. 2000;8(4): 185-189

F M Colles, N D McCarthy, J C Howe, C L Devereux, A G Gosler, and M C J Maiden (2009) Dynamics of Campylobacter colonization of a natural host, Sturnus vulgaris (European Starling) Environ Microbiol. 2009 January; 11(1): 258-267.

Acheson, Alios (2001) Campylobacter jejuni Infections: Update on Emerging Issues and Trends Clinical Infectious Diseases, Volume 32, Issue 8, 15 April 2001, Pages 1201-1206

MJ Kense, WJM Landman (2011) Enterococcus cecorum infections in broiler breeders and their offspring: molecular epidemiology. Avian Pathology, Volume 40, Issue 6

MD Collins, RA Hutson, E Falsen, E Ingana, M Bisgaard (2002) Streptococcus gallinaceus sp. nov., from chickens. International Journal of Systematic and Evolutionary Microbiology, 52, 1161-1164 G Goyette-Desjardins, J-P Auger, J Xu, M Segura, M Gottschalk (2014) Streptococcus suis, an important pig pathogen and emerging zoonotic agent— an update on the worldwide distribution based on serotyping and sequence typing. Emerg Microbes Infect. 2014 Jun; 3(6):e45.

C Fitzgerald, I Nachamkin (2007). Campylobacter and Arcobacter. In P. R. Murray (Ed.), Manual of Clinical Microbiology (9th ed., pp. 933-946). Washington D.C.: ASM Press. Example 4. Qualitative assessment of antioxidant enzymatic activity.

The presence of reactive oxygen species during oxidative stress, which can be caused by stressful conditions as for example heat stress (Lin et al., 2006), can lead to a damage of DNA, proteins or lipids. Probiotics can support the host's oxidative defense system by increasing the antioxidant enzyme activities (Aluwong et al., 2013, Mishra et al., 2015). Therefore, the strain DSM 32592 was screened for antioxidant enzyme activities, in particular for superoxide dismutase and catalase activity.

For the evaluation of catalase activity of grown bio films of the strain, the strain was grown in LB medium supplemented with glucose for 15 hrs at 37° C and 200 rpm in shaking flasks. The cultures were adjusted to an optical density Οϋβοο of 1.0 and 10 μΐ of the cultures were spotted onto TSBYE (30 g/1 TSB + 6 g/1 Yeast extract) or LB-Kelly (LB-Media supplemented with trace elements solution of DSMZ media 1032) agar plates, which were incubated at 37° C under aerobic conditions and at 37° C under 0.2 % oxygen for 15 hrs. 3 % H₂O₂ (hydrogen peroxide solution 3%, Sigma-Aldrich®) was dropped onto the colonies and catalase activity was analyzed. The read out parameter was the production of Ch and H2O from H2O2 resulting in the formation of foam on the colonies. Table 4.1. Assessment of catalase activity of colonies of Bacillus subtilis strain DSM 32592 grown as biofilm on LB-Kelly or TSBYE medium under aerobic conditions.

Bacillus subtilis strain DSM 32592 displayed catalase activity when grown under aerobic conditions.

Table 4.2. Assessment of catalase activity of colonies of Bacillus subtilis strain DSM 32592 grown as biofilm on LB-Kelly or TSBYE medium under 0.2 % oxygen. Strain ID TSBYE LB-Kelly

DSM 32592 Yes Yes

Bacillus subtilis strain DSM 32592 displayed catalase activity also when grown under 0.2% oxygen.

Catalase activity was analyzed in protein extracts obtained from planktonic cells grown under aerobic conditions as well. The strain DSM 32592 was grown for 15 hrs in LB medium containing glucose at 37° C and 200 rpm in shaking flasks. 8 ml of the cell cultures were harvested by centrifugation for 10 min at 4° C and 3000 rpm and the pellet was resuspended in PBS pH 7,3. Soluble cell extracts were obtained by using a ribolyser. The disrupted cells were centrifuged for 10 min at 4° C and 13000 rpm and the supernatant was used for further steps. The protein concentration of the protein extracts was determined by the method of Bradford with bovine serum albumin as a standard (Bradford, 1976). The concentration of the protein extracts was adjusted with PBS pH 7,3, the protein extracts were mixed with native sample loading buffer (2x, VWR) and native gel electrophoresis (10 % non- denaturing polyacrylamide gels, Biorad) was applied to separate the proteins at 4° C. Catalase activity in cell extrmolacts from planktonic cells was then detected by staining the gel in a staining solution of 1 % FeCh and 1 % K.3Fe(CN)6 (Woodbury et al., 1971). Catalase activity can be seen as bright bands.

Bacillus subtilis strain DSM 32592 displayed catalase activity also under these specific conditions as tested.

Superoxide dismutase activity was analyzed in protein extracts from cells grown under aerobic conditions as well. Cell extracts were obtained by the method described above and proteins were separated by performing native gel electrophoresis at 4° C. Superoxide dismutase activity was detected by staining the gel with a nitroblue tetrazolium staining method adapted from Beauchamp and Fridovich (1971).

Bacillus subtilis strain DSM 32592 displayed superoxide dismutase activity under the conditions tested.

References

Aluwong, T.; Kawu, M.; Raji, M.; Dzenda, T.; Govwang, F.; Sinkalu, V. and Ayo, J. (2013). Effect of Yeast Probiotic on Growth, Antioxidant Enzyme Activities and Malondialdehyde Concentration of Broiler Chickens. Antioxidants 2: 326-339 Beauchamp, C. and Fridovich, I. (1971). Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44: 276-287.

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of proteins utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248-254.

Lin, H.; Decuvpere, E. and Buyse, J. (2006). Acute heat stress incuces oxidative stress in broiler chickens. Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 144: 11-17.

Mishra, V.; Shah, C; Mokashe, N.; Chavan, R.; Yadav, H. and Prajapati, J. (2015). Probiotics as potential antioxidants: a systematic review. J. Agric. Food Chem 63: 3615-3626

Woodbury, W.; Spencer, A.K. and Stahmann, M.A. (1971). An improved procedure using ferricyanide for detecting catalase isozymes. Anal. Biochem. 44: 301-305.

Example 5. Detection of lactate production

It has been shown in in vitro studies that inhibition of pathogen replication can be mediated by low- molecular-weight substances (Oelschlager 2010). Top of this list are short chain fatty acids, e.g. lactic acid (Oelschlager 2010). Possible explanation for the inhibition of pathogens can be decreasing the pH by production of lactic acid (Fuller 1992). Lactic acid bacteria, which are also used as probiotic in animal feed are known to produce lactic acid as major end-product during fermentation of cabohydrates (Halasz 2009). It is shown that B. subtilis strain DSM 32592 can produce lactate in vitro. B. subtilis strain DSM 32592 and Bacillus toyonensis - a probiotic bacillus known to produce lactic acid - were compared evaluating the anaerobe lactate production. Lactate production was determined as follows: Precultures of the strains were grown stover night at 37 °C in 10 mL TSBYE (30 g/1 TSB + 6 g/1 Yeast extract) under aerobic conditions. Main culture was inoculated with an Οϋβοο of 0.2 in 10 ml TSBYE complemented with 25 g/1 sucrose and 5 mM KNO3 in 15 ml Falcon Tubes at 37 °C without shaking for 48 h. Lactate was measured in the supernatant with HPLC-UV.

Strain B. subtilis DSM 32592 produces almost 50 % more lactate compared to probiotic strain of Bacillus toyonensis.

References

A Halasz (2009) Book: Food quality and standards Volume III; EOLSS Publishers Co Ltd. Chapti Lactic acid bacteria. TA Oelschlager (2010) Mechanisms of probiotic actions - A review. International Journal of Medical Microbiology Volume 300, Issue 1, January 2010, Pages 57-62.

R Fuller 1992 Publisher: Springer science and business media Dordrecht Book: Probiotics: The scientific basis Chapter 9.8 Antimicrobial activity.

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Claims

30

Claims

1. A Bacillus subtilis strain or a preparation thereof selected from the following group:

a) The B. subtilis strain as deposited under DSM 32592 at the DSMZ;

b) a mutant of the B. subtilis strain as deposited under DSM 32592 having all identifying characteristics of the strain DSM 32592;

c) a preparation of (a) or (b);

d) a preparation containing an effective mixture of compounds as contained in (a), (b) or (c).

2. A Bacillus subtilis mutant of DSM 32592 according to claim 1 (b), wherein the mutant has a DNA sequence identity to the strain DSM 32592 of at least 95%.

3. A Bacillus subtilis strain or a preparation thereof, wherein the B. subtilis strain exhibits at least one, preferably all, of the following characteristics:

a) a 16S rDNA sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 1; b) a yqfD sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 2; c) a gyrB sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 3; d) an rpoB sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 4; e) a groEL sequence with a sequence identity of at least 99.5 %, preferably at least 99.8 %, above all 100 %, to the polynucleotide sequence according to SEQ ID NO: 5. 4. The B. subtilis strain according to any of the preceding claims, characterized by being able to grow anaerobically, in particular by being able to degrade water-insoluble cellulose and protein under anaerobic conditions.

5. The B. subtilis strain according to any of the preceding claims, characterized by being able to inhibit the growth of at least one, preferably all, of the strains selected from C. perfringens BB-

081_Cpe; C. perfringens BB_031_Cpe; C. difficile DSM 1296; S. gallinaceus DSM 15349; S. suis DSM 9682; C. coli DSM 4689; C. jejuni DSM 24129 and ^. cecorum DSM 20683.

6. The B. subtilis strain according to any of the preceding claims, characterized by at least one, preferably all, of the following enzymatic activities: cellulase activity; xylanase activity; protease activity; catalase activity; superoxide dismutase activity. 31

7. The B. subtilis strain according to any of the preceding claims, characterized by being able to grow in presence of 2 mM bile, preferably in presence of 4 mM bile, and/or in presence of 0.3 wt.-% porcine bile and/or in presence of 0.3 wt.-% chicken bile. 8. The use of a B. subtilis strain or a preparation thereof according to any of the preceding claims as a probiotic ingredient (DFM) in feed or food products.

A feed- or foodstuff composition containing a B. subtilis strain or a preparation thereof according to any one of claims 1 to 7 and at least one further feed or food ingredient, preferably selected from proteins, carbohydrates, fats, further probiotics, prebiotics, enzymes, vitamins, immune modulators, milk replacers, minerals, amino acids, coccidio stats, acid-based products, medicines, and combinations thereof.

10. A pharmaceutical composition containing a B. subtilis strain or a preparation thereof according to any one of claims 1 to 7 and a pharmaceutically acceptable carrier.

11. A composition according to claim 9 or 10 for improving the health status, in particular the gut health status, of an animal or a human being.

12. A method of feeding an animal, in particular swine or an avian, characterized by applying to the animal a B. subtilis strain or a preparation according to any of claims 1 to 7 and/or a feedstuff composition according to claim 9 and/or a pharmaceutical composition according to claim 10.

13. A method of improving the health status, in particular the gut health status, of an animal or a human being, comprising administering to the animal or human being a B. subtilis strain or a preparation according to any of claims 1 to 7 or a composition according to any of claims 9 to 11. 14. A method of enhancing the health of animals and/or of improving the general physical

condition of animals and/or of improving the feed conversion rate of animals and/or of decreasing the mortality rate of animals and/or of increasing the survival rates of animals and/or of improving the weight gain of animals and/or of increasing the disease resistance of animals and/or of increasing the immune response of animals and/or of establishing or maintaining a healthy gut microflora in animals and/or of reducing the pathogen shedding through the feces of animals, wherein at least one strain and/or at least one preparation according to any of claims 1 to 7 or at least one composition according to any of claims 9 to 11 are administered to animals. 32

A method of controlling and/or avoiding detrimental environmental effects of manure or contaminated liquids, the method comprising the step of applying to manure, contaminated liquids, litter, a pit, or a manure pond at least one strain and/or at least one preparation according to any of claims 1 to 7 and/or at least one composition according to any of claims 9 to 11.

A method of controlling and/or improving the quality of water or aqueous solutions, in particular of drinking water or rearing water, the method comprising the step of applying to water or an aqueous solution at least one strain and/or at least one preparation according to any of claims 1 to 7 and/or at least one composition according to any of claims 9 to 11.

A method of treating and/or preventing a microbial disease of cultivated plants, comprising the step of applying to a cultivated plant at least one strain and/or at least one preparation according to any of claims 1 to 7 and/or at least one composition according to any of claims 9 to 11.