A feed composition and use thereof

Abstract

The present invention provides composition comprising Lactobacillus postbiotic and one or more muramidase(s) for improving growth performance and / or health performance of an animal.

Description

[0001] 16322-WO-PCT

[0002] A feed composition and use thereof

[0003] Technical Field

[0004] The present invention relates to a feed composition for improving growth performance and / or 5 health performance of animals.

[0005] Background of the Invention

[0006] Lactobacillus is a genus of gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria. Lactobacillus currently contains over 180 species and 10 encompasses a wide variety of organisms. It has been reported that Lactobacillus postbiotic is capable of stimulating the growth of gut bifidobacteria and thereby improving gut health of animals (see WO 2021 / 219846 A1).

[0007] Muramidase is an O-glycosyl hydrolase produced as a defensive mechanism against bacteria 15 by many organisms. The enzyme causes the hydrolysis of bacterial cell walls by cleaving the glycosidic bonds of peptidoglycan, an important structural molecule in bacteria. Muramidase has been classified into five different glycoside hydrolase (GH) families (www.cazy.org): hen egg-white muramidase (GH22), goose egg-white muramidase (GH23), bacteriophage T4 muramidase (GH24), Sphingomonas flagellar protein (GH73) and Chalaropsis muramidases 20 (GH25). Muramidase extracted from hen egg white (a GH22 muramidase) is the primary product available on the commercial market and traditionally has just been referred to as muramidase even though nowadays there are many other known muramidases.

[0008] Surprisingly, the inventors of the present invention discovered that Lactobacillus postbiotic in 25 combination with a muramidase provides additional benefits in improving performance of animals.

[0009] Summary of the Invention

[0010] The present invention provides a feed composition comprising Lactobacillus postbiotic and one or more muramidase(s) for improving growth performance and / or health performance of an 30 animal.

[0011]

[0012] SEQ ID NO:1 is a GH25 muramidase obtainable from Sodiomyces alcalophilus

[0013] 1 Detailed description of the Invention

[0014] The present invention relates to a feed composition comprising Lactobacillus postbiotic and one or more muramidase(s) for improving growth performance and / or health performance of an animal.

[0015] 5

[0016] A postbiotic refers to fermentate or microbial biomass of non-living microorganisms and microbial cell fragments and furthermore typically comprises the bioactive compounds that are produced when probiotics (live microorganisms) metabolize materials, include metabolic byproducts such as enzymes, short-chain fatty acids (SCFAs), peptides, polysaccharides, 10 vitamins.

[0017] The present invention also relates to a novel use of Lactobacillus postbiotic in combination with one or more microbial muramidase(s) in the preparation of a feed composition for improving growth performance and / or health performance of an animal.

[0018] 15

[0019] The present invention also relates to a method for improving growth performance and / or health performance of an animal, comprising administering to the animal an effective amount of the feed composition as defined herein.

[0020] 20 In the present invention, the growth performance of an animal is characterized by one or more of the following parameters: body weight (BW), average daily weight gain (ADWG), feed intake (FI), average daily feed intake (ADFI), feed conversion ratio (FCR); and the health performance of an animal is characterized by withdraw ratio, reduced mortality, reduced disease frequency, improved faecal score, diarrhea incidence, diarrhea severity and / or times of medical 25 treatments administered to the animal.

[0021] An aspect of the invention is directed to a composition comprising a Lactobacillus and a polypeptide having muramidase activity for use in improving growth performance and / or health performance in an animal, said growth performance feature selected from the group consisting 30 of improved body weight gain, improved average daily weight gain, improved feed conversion ratio; and said health performance selected from the group consisting of reduced mortality, reduced disease frequency, improved intestinal health, reduced diarrhea incidence and reduced diarrhea severity, typically for improved intestinal health, reduced diarrhea incidence or reduced diarrhea severity.

[0022] 35

[0023] In the present invention, the improvement is compared to an animal feed wherein the Lactobacillus postbiotic and the muramidase are not included (herein referred to as the control.

[0024] 2 Preferably, one or more of the parameters on growth performance and / or health performance of animals is changed in a desired direction by at least 0.5%, such as by at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0% or at least 8.0%, compared to the control.

[0025] 5

[0026] In the present invention, the Lactobacillus postbiotic is a fermentate and microbial biomass of two Lactobacillus strains, i.e., Lactobacillus fermentus and Lactobacillus delbrueckii. The Lactobacillus postbiotic of the invention can be made by growing a culture of mixture of Lactobacillus fermentus and Lactobacillus delbrueckii to a particular cell density and, when the 10 desired cell density is reached, subjecting the culture to heat inactivation to kill the cells. The heat-inactivated culture comprises dead cells of the Lactobacillus strains and metabolites produced by the cells.

[0027] The amount of dead cells of the Lactobacillus strains in the Lactobacillus postbiotic may be at 15 least 1×106, 1×107, 1×108, 1×109, 1×1010, 1×1011, or 1×1012colony-forming unites (CFU).

[0028] Preferably, the amount of the dead cells of the Lactobacillus strains in the Lactobacillus postbiotic is between 1×108and 1×1014CFU, between 1×108and 1×1013CFU, between 1×109and 1×1013CFU or between 1×108and 1×1012CFU. More preferably the amount of the dead cells of the Lactobacillus strains in the Lactobacillus postbiotic is between 1×1010and 3×101020 CFU. The most preferably, the amount of the dead cells of the Lactobacillus strains in the Lactobacillus postbiotic is 2×1010CFU.

[0029] The weight ratio of Lactobacillus fermentus to Lactobacillus delbrueckii in the Lactobacillus postbiotic may be any suitable ratio from about 99: 1 to about 1:99, e.g. about 9: 1 to 1:9, 25 including 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9. Preferably, the weight ratio of Lactobacillus fermentus to Lactobacillus delbrueckii in the Lactobacillus postbiotic is ranges from 50:1 to 1:1, such as 20:1 to 1:1, typically about 10:1 to 1:1, such as from 10:1 to 5:1, typically 10:1, 9: 1, 8:1, or 7:1.

[0030] 30 The Lactobacillus postbiotic may be used in a solid form by drying the biomass with any process known in the art, such lyophilization, spray-drying and fluid-bed drying. Alternatively, the Lactobacillus postbiotic may be used in a liquid form by omitting the drying step or reconstituting the dried product with a suitable liquid such as water.

[0031] 35 An example of the Lactobacillus postbiotic is GutServTMBiotix (dsm-firmenich, Switzerland).

[0032] 3 In the present invention, the muramidase may be of microbial origin, preferably of fungal origin. Preferably, the muramidase is obtained or obtainable from the phylum Ascomycota, such as the sub-phylum Pezizomycotina. More preferably, the muramidase is obtained or obtainable from Acremonium alcalophilum or Trichophaea saccate. Even more preferably, the 5 muramidase is GH24 muramidase or GH25 muramidase. An example of the muramidase is Balancius® (dsm-firmenich, Switzerland). In a typically embodiment, the muramidase has at least 80% sequence identity to SEQ ID NO:1, such as at least 85%, at least 90%, at least 95% sequence identity, including at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO:1.

[0033] 10

[0034] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open 15 Software Suite, Rice et al., 2000, Trends Genet.16: 276-277), preferably version 6.6.0 or later.

[0035] The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0036] 20

[0037] (Identical Residues x 100) / (Length of Alignment – Total Number of Gaps in Alignment)

[0038] As anticipated by any person skilled in the art, the feed composition according to the present invention may be formulated as an animal feed additive. Accordingly, the feed composition of the 25 present invention may also include micro-ingredients.

[0039] The micro-ingredients include but are not limited to aroma compounds; antimicrobial peptides; polyunsaturated fatty acids (PUFAs); reactive oxygen generating species; at least one enzyme, and fat- and water-soluble vitamins, as well as minerals.

[0040] 30

[0041] Examples of antimicrobial peptides (AMP's) are CAP18, leucocin A, protegrin-1, thanatin, defensin, lactoferrin, lactoferricin, and ovispirin such as novispirin (Robert Lehrer, 2000), plectasins, and statins.

[0042] 35 Examples of polyunsaturated fatty acids are C18-, C20-and C22-polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.

[0043] 4 Examples of reactive oxygen generating species are chemicals such as perborate, persulphate, or percarbonate; and enzymes such as an oxidase, an oxygenase or a syntethase.

[0044] Examples of enzyme are phytase (EC 3.1.3.8 or 3.1.3.26), galactanase (EC 3.2.1.89), alpha- 5 galactosidase (EC 3.2.1.22), phospholipase A 1 (EC 3.1.1.32), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), phospholipase C (EC 3.1.4.3), and / or phospholipase D (EC 3.1.4.4).

[0045] Examples of fat-soluble vitamins include but are not limited to vitamin A, vitamin D3, and vitamin 10 K, e.g. vitamin K3.

[0046] Examples of water-soluble vitamins include but are not limited to vitamin B12, biotin and choline, vitamin B1, vitamin B2, vitamin B6, niacin, folic acid and panthothenate, e.g. Ca-D-panthothenate.

[0047] 15 Examples of minerals include but are not limited to calcium, phosphorus, sodium, potassium, magnesium, chlorine, iodine, iron, manganese, copper, molybdenum, cobalt and zinc.

[0048] As also anticipated by any person skilled in the art, the feed composition according to the present invention may further be formulated as an animal feed. Accordingly, the feed composition of the 20 present invention may further include any components typical for an animal feed, such as proteins, carbohydrates as defined above, fats and additional additives, which are known in the art.

[0049] In the present invention, the Lactobacillus postbiotic may be provided at a level of from 0.001% 25 to 10%, preferably from 0.0015% to 5%, more preferably from 0.01% to 1%, such as 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.6% and 0.8% by weight of animal feed.

[0050] In the present invention, the muramidase may be provided at a level of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 300 to 800 mg, 400 to 700 mg, 500 to 600 30 mg enzyme protein per kg animal feed, or any combination of these intervals.

[0051] The term “animal” in the present invention is a non-ruminant animal. Examples of the non- ruminant animal include but are not limited to horses, rabbits, pig or swine (including but not limited to, piglets, growing pigs, and sows), poultry such as turkeys, ducks, chickens (including 35 but not limited to broiler chicks, layers), goose and pigeon. Especially preferably, the animal is swine. In a preferred embodiment, the animal is selected from a piglet, a pre-weaning piglet, a post-wearning piglet,

[0052] 5 In a typical embodiment, the invention relates a feed composition comprising a muramidase having at least 80% sequence identity to SEQ ID NO: 1 and a Lactobacillus postbiotic of Lactobacillus fermentus and Lactobacillus delbrueckii, wherein the weight ratio of Lactobacillus 5 fermentus to Lactobacillus delbrueckii is about 10:1 to 1:1, wherein the amount of the dead cells of the Lactobacillus strains in the Lactobacillus postbiotic is between 1×108and 1×1014CFU.

[0053] The present invention is further illustrated with reference to the following Examples.

[0054] 10

[0055] Examples

[0056] 1. Animals

[0057] 15 A total of 728 healthy weaning piglets [(LD x LW) x DUROC] with an initial average body weight of 5.61 ± 0.85 kg and an average age at weaning of 25 days, were used for the trial.

[0058] 2. Experimental Design

[0059] 20 The piglets were classified by body weight into groups of 13 animals (gender mix with the same ratio males / females in each department) and housed in the 56 experimental pens. Subsequently they were weight (individually) and randomly assigned to one of the 4 experimental treatments (CTR, BAL, BIO, B+B) based exclusively on body weight (14 replicates per group) (Table 1). Thereafter, each group received a 2-phase feeding plan with 25 the only differentiating element being the dietary supplementation of the test products as shown in Tables 2 and 3.

[0060]

[0061] *LBIOTIXTMis Lactobacillus postbiotic GutServTMBiotix supplied by dsm-firmenich, 30 Switzerland.

[0062] Balancius™ is a product comprising SEQ ID NO:1. LBIOTIX™ is a postbiotic of Lactobacillus fermentum and Lactobacillus delbrueckii.

[0063] 6 *LBIOTIXTMis Lactobacillus postbiotic GutServTMBiotix supplied by dsm-firmenich, Switzerland.

[0064] 7 *LBIOTIXTMis Lactobacillus postbiotic GutServTMBiotix supplied by dsm-firmenich, Switzerland.

[0065] 5 Although the 2-phase feeding plan (0-14d (Pre-Starter) and 14-40d (Starter)) was used, the experimental period was distributed in 3 stages: 0-7d, 7-14d, and 14-40d. At the end of each period, piglets were weighted (per pen, except for the last weighing (d40), individually), and the total feed consumption recorded in order to subsequently calculate the main productive parameters (growth rate, average daily feed intake and feed conversion ratio).

[0066] 10

[0067] Piglets that showed clinical signs of disease or whose body condition deteriorated progressively were withdrawn from the trial. For these animals, the assigned feeding plan was maintained and the average weight at the end of the experimental period recorded. The weight and the apparent cause of the piglets died during the experimental period or were withdrawn 15 from the study, as well as the injectable medical treatments applied, were also recorded.

[0068] 3. Analysis

[0069] 8 The following variables were recorded for analysis:

[0070] • Average body weight at 0, 7, 14, and 40d, kg / piglet

[0071] • Variation coefficient of body weight at 0 and 40d, %

[0072] 5 • Average daily weight gain (ADWG) at 0-7, 7-14, 0-14, 14-40, and 0-40d, kg / day • Average daily feed intake (ADFI) at 0-7, 7-14, 0-14, 14-40, and 0-40d, kg / day

[0073] • Feed conversion ratio (FCR) at 0-7, 7-14, 0-14, 14-40, and 0-40d, kg / kg

[0074] • Withdrawn ratio at 0-7, 7-14, 0-14, 14-40, and 0-40d, %

[0075] • Individual medical treatment ratio at 0-7, 7-14, 0-14, 14-40, and 0-40d, n / initial piglet 10 • Average fecal score at 0-7, 7-14, 0-14d, 1-5.

[0076] During the first three weeks of the trial, the faecal score was evaluated on a scale of 1 to 5 on a daily basis (1: very severe diarrhea, 2: bad fecal consistency, 3: moderate fecal consistency, 4: optimal fecal consistency, 5: too dry feces).

[0077] 15

[0078] 4. Results

[0079] Table 4 shows the results obtained in the Pre-Starter overall period (0-14d post-weaning).

[0080] 20 The study showed that piglets receiving the BIO and B+B strategies experienced the highest growth rate and consequently reached a higher body weight at the end of the period (P=0.068). Animals in the B+B group showed a lower incidence of diarrhea than those in the CTR, BAL and BIO groups (P=0.031). In addition, animals in the B+B group showed a lower withdraw rate, a lower medical treatment and an improved faecal score compared to the rest treatments.

[0081] 25

[0082] Table 4: Effect of dietary supplementation with Lactobacillus postbiotic and BALANCIUS® on productive performance of nursery piglets in the 0-14d post-weaning period TREATMENTS VARIABLE CTR BAL BIO B+B Body Weight d0, 5.623 5.597 5.612 5.601 kg / piglet

[0083] Body Weight d14, 8.746 8.799 9.037 9.036 kg / piglet

[0084] Avg. Daily Weight 0.224 0.228 0.245 0.245 Gain, kg / d

[0085] Avg. Daily Feed 0.266 0.272 0.283 0.287 Intake, kg / d

[0086] 9 Feed Conversion 1.214 1.213 1.183 1.183 Ratio, kg / kg

[0087] Withdrawn ratio, % 1.648 2.200 1.648 1.099 Medical treatment, 1.139 1.058 0.782 0.624 n / Navg

[0088] Faecal score, 1-5 2.993 2.997 3.020 3.032 Diarrhea incidence, 0- 0.459a0.469a0.413a0.260b

[0089] 1

[0090] *Superscripts a, b show statistically significant differences (P<0.05) between experimental groups.

[0091] Table 5 shows the results obtained in the Pre-Starter overall period (0-40d post-weaning). 5

[0092] Statistical analysis shows that the growth rate and consequently the body weight at the end of the experimental period of the piglets in the B+B group was higher than those in the CTR group (+25 g / day; P=0.008). The group whose diets were supplemented with both Lactobacillus postbiotic and BALANCIUS® exhibited a lower feed conversion ratio than the CTR group (-10 0.058 kg / kg; P<0.001).

[0093] Supplementation of Lactobacillus postbiotic as well as BALANCIUS® had an improving effect on average daily weight gain and feed conversion ratio (P<0.05) and medical treatment in the overall nursery period.

[0094] 15

[0095] Table 5: Effect of dietary supplementation with Lactobacillus postbiotic and BALANCIUS® on productive performance of nursery piglets in the overall period (0-40d post-weaning)

[0096] TREATMENTS VARIABLE CTR BAL BIO B+B

[0097] Body Weight d0, 5.623 5.597 5.612 5.601 kg / piglet

[0098] Body Weight d40, 21.223b21.667ab21.735ab22.213akg / piglet

[0099] Avg. Daily Weight 0.390b0.401ab0.403ab0.415aGain, kg / d

[0100] Avg. Daily Feed 0.568 0.572 0.575 0.579 Intake, kg / d

[0101] 10 Feed Conversion 1.472a1.444ab1.452b1.414bRatio, kg / kg

[0102] Withdrawn ratio, % 2.750 4.950 3.850 3.300 Medical treatments, 1.301 1.236 1.064 0.772 n / Navg

[0103] *Superscripts a, b show statistically significant differences (P<0.05) between experimental groups.

[0104] 5. Conclusion

[0105] 5

[0106] Supplementation of Lactobacillus postbiotic as well as muramidase had an improving effect on growth performance such as body weight, average daily weight gain and feed conversion ratio, and / or health performance such as withdraw ratio, faecal score, diarrhea incidence and medical treatments of animals.

[0107] 10

[0108] 11

Claims

Claims1. A feed composition comprising Lactobacillus postbiotic and one or more muramidase(s) for improving growth performance and / or health performance of an animal.

52. The feed composition of claim 1, wherein the Lactobacillus postbiotic is a fermentate and microbial biomass of Lactobacillus fermentus and Lactobacillus delbrueckii.

3. The feed composition of claim 2, wherein the Lactobacillus postbiotic contains the 10 Lactobacillus fermentus and Lactobacillus delbrueckii in a weight ratio of from 9:1 to 1:9(Lactobacillus fermentus to Lactobacillus delbrueckii).

4. The feed composition of claim 1, wherein the Lactobacillus postbiotic contains dead cells of the Lactobacillus strains in an amount of between 1×108and 1×1014CFU, between 15 1×108and 1×1013CFU, between 1×109and 1×1013CFU or between 1×108and 1×1012CFU.

5. The feed composition of claim 4, wherein the Lactobacillus postbiotic contains dead cells of the Lactobacillus strains in an amount of between 1×1010and 3×1010CFU.

206. The feed composition of any one of claims 1-5, wherein the muramidase is of microbial origin.

7. The feed composition of any one of claims 1-5, wherein the muramidase is GH24 25 muramidase or GH25 muramidase.

8. The feed composition of any one of claims 1-5, which is formulated as an animal feed additive or an animal feed.30 9. The feed composition of any one of claims 1-5, wherein the Lactobacillus postbiotic is provided at a level of from 0.001% to 10%, preferably from 0.0015% to 5%, more preferably from 0.01% to 1%, such as 0.02%, 0.05%, 0.1%, 0.2%, 0.5%, 0.6% and 0.8% by weight of animal feed.35 10. The feed composition of any one of claims 1-5, wherein the muramidase is provided at a level of 100 to 1000 mg enzyme protein per kg animal feed, such as 200 to 900 mg, 30012to 800 mg, 400 to 700 mg, 500 to 600 mg enzyme protein per kg animal feed, or any combination of these intervals.

11. The feed composition of any one of claims 1-5, wherein the animal is a non-ruminant 5 animal.

12. The feed composition of any one of claims 1-5, wherein the animal is selected from the group consisting of horses, rabbits, pig or swine, poultry such as turkeys, ducks, chickens, goose and pigeon.1013. A method for improving growth performance and / or health performance of an animal, comprising administering to the animal an effective amount of the feed composition according to any one of claims 1-12.15 14. Use of Lactobacillus postbiotic in combination with one or more muramidase(s) in the preparation of a feed composition for improving growth performance and / or health performance of an animal.

15. A composition comprising a Lactobacillus and a polypeptide having muramidase activity 20 for use in improving growth performance and / or health performance in an animal, said growth performance feature selected from the group consisting of improved body weight gain, improved average daily weight gain, improved feed conversion ratio; and said health performance selected from the group consisting of reduced mortality, reduced disease frequency, improved intestinal health, reduced diarrhea incidence and reduced diarrhea 25 severity, typically for improved intestinal health, reduced diarrhea incidence or reduced diarrhea severity.13

Images