SEAWEED BLEND FEED SUPPLEMENT

Abstract

Use of a seaweed blend to modify gastrointestinal (GI) microbiota of an animal host. Modifying the GI microbiota may comprise modifying a community of bacteria, wherein the community of bacteria comprises Firmicutes and Bacteriodetes and the seaweed blend modifies the ratio of Firmicutes to Bacteriodetes, such as increasing the ratio of Firmicutes to Bacteriodetes. Further, the seaweed blend modifies specific families of commensal bacteria families including Ruminococceae and Lachnospiraceae within the Phyla Firmicutes. The seaweed blend may comprise (i) 65-80 wt % Ulva; (ii) 3-8 wt % Gracilaria; and (iii) 15-25 wt % Sargassum and/or Ascophyllum.

Claims

1. (canceled)

2. A method comprising providing a seaweed blend to an animal host, the animal host having a gastrointestinal (GI) microbiota; and modifying the gastrointestinal (GI) microbiota of the animal host.

3. The method of claim 2, wherein the seaweed blend has a composition and is delivered to the animal host at a dosage; and the composition and/or the dosage of the seaweed blend is selected in order to target a specific modification of the GI microbiota.

4. The method of claim 3, comprising an initial step of analysing a sample from the host to determine the relative abundance and/or population of at least one microorganism.

5. The method of claim 2, wherein modifying the GI microbiota comprises modifying a community of bacteria.

6. The method of claim 5, wherein the community of bacteria comprises Firmicutes and Bacteriodetes and the seaweed blend modifies the ratio of Firmicutes to Bacteriodetes, such as increasing the ratio of Firmicutes to Bacteriodetes.

7. The method of claim 5, wherein the seaweed blend modifies the relative abundance of bacteria of one or more of Bacteroides, Clostridium, Faecalibacterium, Eubacterium, Ruminococcus, Peptococcus, Peptostreptococcus, Bifidobacterium, Prevotella, Butyricicoccus, Coprococcus, Prevotella, Escherichia and Lactobacillus, such as increasing the relative abundance of one or more of (i) Faecalibacterium; (ii) Bifidobacterium; (iii) Butyricicoccus; (iv) Coprococcus; and (v) Lactobacillus.

8. The method of claim 5, wherein the seaweed blend modifies the relative abundance of bacteria of one or more of Ruminococcaceae (such as Faecalibacterium, Ruminococcus, Lachnospiraceae, and Coprococcus and, Blautia); Peptococcus, Peptostreptococcus, Bifidobacteriaceae (such as Bifidobacterium); Lactobacillaceae (such as Lactobacillus); Clostridiaceae (such as Butyricicoccus); Prevotelleceae (such as Prevotella), Enterobacteriaceae (such as Escherichia).

9. The method of claim 5, wherein the seaweed blend increases the relative abundance of one or more of (i) Faecalibacterium; (ii) Bifidobacterium; (iii) Butyricicoccus; (iv) Coprococcus; and (v) Lactobacillus.

10. The use-OF method of claim 5, wherein the seaweed blend reduces the relative abundance of Prevotella and/or Escherichia

11. The method of claim 2, wherein the seaweed blend comprises a blend of green seaweed, brown seaweed and red seaweed.

12. The method claim 2, wherein the seaweed blend comprises 60 to 75 wt % green seaweed; 15 to 25 wt % brown seaweed; and 3 to 10 wt % red seaweed.

13. (canceled)

14. The method claim 2, wherein the seaweed blend comprises seaweed of at least three different genera.

15. The method claim 2, wherein the seaweed blend comprises (i) Ulva; (ii) Gracilaria; and (iii) Sargassum and/or Ascophyllum.

16. The method of claim 2, wherein the seaweed blend comprises (i) 65-80 wt % Ulva; (ii) 3-8 wt % Gracilaria; and (iii) 15-25 wt % Sargassum and/or Ascophyllum.

17. The method claim 2, wherein the seaweed blend comprises Recipe A′, Recipe B′, Recipe C′ and/or Recipe D′: TABLE-US-00007 Recipe A’ Recipe B’ Recipe C’ Recipe D’ (wt %) (wt %) (wt %) (wt %) Ulva lactuca 70-80 70-80 65-75 55-70 Ascophyllum and  2-20  3-20  2-25  2-25 Sargassum Gracilaria 3-8 3-8  2-15  3-10 Maerl 0-1 0-3 0-7 0-7

18. The method claim 2, wherein the host is a non-human animal.

19. The method of claim 2, wherein the host is selected from a bird, a pig, a sheep, a cow, a horse, and a companion animal.

20. The method of claim 2, wherein the host is a bird and the seaweed blend modifies (i) a ratio of Firmicutes to Bacteroidetes; (ii) relative abundance of Faecalibacterium; and/or (iii) relative abundance of Bifidobacterium.

21. The method of claim 2, wherein the host is a pig and the seaweed blend modifies (i) ratio of Firmicutes to Bacteroidetes; (ii) relative abundance of Lactobaciillus; (iii) relative abundance of Butyricicoccus; (iv) relative abundance of Coprococcus and/or (v) relative abundance of prevotella.

22. The method claim 2, wherein (i) the seaweed blend is formulated as a tablet, a capsule or a pellet; (ii) the seaweed blend is provided to the host in an amount of at least 0.5 wt % relative to a regular feed; (iii) the seaweed blend is incorporated into a feed composition; (iv) the seaweed blend is provided to the host daily for at least 4 weeks; and/or (v) the host has a diet free of antibiotics and/or coccidiostat.

Description

[0071] The invention will now be described, in a non-limiting fashion, with reference to the following figures:

[0072] FIGS. 1 to 3 show the relative abundance of various bacteria in the caeca of broiler chickens fed diets with and without a seaweed blend.

[0073] FIGS. 4 to 7 show the relative abundance of various bacteria in the faeces of pigs fed diets with and without a seaweed blend.

[0074] FIGS. 8 and 9 show Shannon Entropy of Counts (Alpha Diversity metric indicating higher diversity of bacteria) and the Firmicutes to Bacteroidetes ratio as identified by 16S RNA gene sequencing of faecal samples from pigs fed a Control diet or the Control with added seaweed blend.

[0075] FIGS. 10, 11 and 12 show Relative Abundance of bacterial families Ruminococcaceae Prevotellaceae and Lachnospiraceae sampled at day 42 from the ceca of broiler chickens fed a control diet or the control diet supplemented with 0.5% Seaweed blend from 0-42 days of age. Significant increase in Ruminococcaceae (P=0.054).

[0076] FIG. 13 shows Firmicutes to Bacteroidetes ratio as identified by 16S RNA gene sequencing of the ceca of broiler chickens fed a control diet or the control diet supplemented with 0.5% seaweed blend from 0-42 days of age.

[0077] FIGS. 14 and 15 show Relative Abundance of bacterial families Ruminococcaceae and Lachnospiraceae sampled at day 42 from the ceca of broiler chickens fed a control diet or the control diet supplemented with 0.5% from 0-42 days of age. Significant increase in Ruminococcaceae (P=0.054).

BROILER CHICKEN TRIAL 1

[0078] Several studies have shown that growth performance, feed efficiency, and gut health in broiler chickens can be improved by dietary prebiotics, non-digestible carbohydrates selectively stimulating the growth of beneficial bacteria (Xu et al., 2003; Yusrizal & Chen, 2003; Yang et al., 2008). For example, feed supplementation with 0.4% fructo-oligosaccharides (FOS) in broiler chickens significantly increased body weight gain, feed efficiency, the activities of protease and amylase, ileal villus height, and the growth of Bifidobacterium and Lactobacillus (Xu et al., 2003).

TABLE-US-00003 Recipe B Wt % Ulva lactuca 70-80 Sargassum 10-20 Gracilaria 3-8 Ascophyllum nodosum 3-8 Maerl 0-3

[0079] A seaweed blend (above) was added to the feed (0.5 wt % of the feed) of commercial broilers from day 0 to 6 weeks of age, at which time caecal samples were collected to assess changes in the bacterial community. The cecum is an intraperitoneal pouch that is considered to be the beginning of the large intestine. Caecal samples were analysed by 16S RNA qPCR, cloning and sequencing (BaseClear NV).

[0080] Referring to FIG. 1, it can be seen that the relative abundance of bifidobacteria from the caeca of broiler chickens fed diets containing the seaweed blend was higher (27% compared to 25%) compared to control birds fed the same diet without the addition of the seaweed blend.

[0081] Referring to FIG. 2, Faecalibacterium, a butyrate producing bacterial genus associated with carbohydrate metabolism was also higher. Butyrate has been shown to be anti-inflammatory.

[0082] Referring to FIG. 3, the addition of the seaweed blend increased the ratio of Firmicutes to Bacteroides. Petersen et al., (2013) observed a positive correlation between weight gain and relative abundance of Firmicutes.

[0083] Swine (Pig) Trial 1

TABLE-US-00004 Recipe C wt % Ulva lactuca 65-75 Sargassum 15-25 Gracilaria  2-15 Ascophyllum nodosum  2-15 Maerl 0-7

[0084] A seaweed blend of brown, green and red seaweeds (above) was added to the feed (0.5 wt % of the feed) of commercial pigs post-weaning (pigs weaned at 21 days of age) for 6 weeks. Faecal samples were collected to assess changes in the bacterial community. Faecal samples were analysed by 16S RNA qPCR, cloning and sequencing (BaseClear NV).

[0085] As shown in FIG. 4, the relative abundance of lactobacillus species from the faeces of pigs fed diets containing the seaweed blend was higher (20% compared to 19%) compared to control pigs fed the same diet without the addition of the seaweed bend.

[0086] The inventors propose that an increase in Lactobaciillus provides a benefit. Lactobacillus species such as L. fermentum have been used as a growth-promoting feed supplement preventing and treating diarrhoea of weaned piglets and maximising average daily gain, crude protein apparent digestibility and serum specific IgG level.

[0087] As shown in FIG. 5, the relative abundance of Butyricicoccus species in the faeces of pigs fed diets containing the seaweed blend was higher than the control.

[0088] As shown in FIG. 6, the addition of the feed supplement increased the relative abundance of Firmicutes: Bacteroidetes. Petersen et al., (2013) observed a positive correlation between weight gain and relative abundance of Firmicutes.

[0089] Further Investigations

[0090] Studies of the GI microbiome (genetic footprint of the microbiota) have shown that domestic animals such as swine and poultry (chickens) possess a core microbiome, which changes in composition based on certain factors such as diet and other environmental factors. Liu et al., December 2015, BMC Complementary and Alternative Medicine 15(1):279 DOI: 10.1186/s12906-015-0802-5 investigated the prebiotic effects of cultivated red seaweed Chondrus crispus in diets fed to young rats using 16S rRNA sequencing to profile the colonic microbiome. This study successfully demonstrated that seaweed increased the relative abundance of Bifidobacterium breve and decreases the abundance of pathogenic species such as Clostridium septicum and Streptococcus pneumonia. However it does not demonstrate a consistent increase in a family of butyrate producing bacteria. This present invention uses 16S rRNA sequencing to gain a snapshot of the GI microbiome to consistently define changes at the bacterial family level.

[0091] The commensal bacteria in the GI of domestic swine and poultry are those that ferment complex carbohydrates in the colon and ceca, producing metabolic intermediaries such as short chain fatty acids (SCFA) as the end products of fermentation (Fouhse et al., Animal Frontiers, Volume 6, Issue 3, July 2016, Pages 30-36, https://doi.org/10.2527/af.2016-0031). One of these SCFAs, Butyric Acid or Butyrate is preferentially utilized by the GI mucosa cells, as a source of energy and as a metabolic signal resulting in several digestive and systemic effects on the hosts. The results explained below demonstrate consistent increases in bacterial families that have been shown to be the main butyrate producing families in the lower GI of pigs and poultry.

[0092] Investigating the Gastrointestinal Microbiome

[0093] The potential for using unique blends of red, brown and green seaweed species as natural prebiotic feed additives was investigated in swine and in poultry (broiler chickens). Unique blends of green, brown and red seaweeds were added to the daily ration at rates between 0.50 and 0.75% of the food.

[0094] Diets containing the seaweed blends were fed throughout the early growth stage to pigs and chickens. At the end of the test period fresh faecal samples (pigs) or ceca samples (chickens) were collected from several replicate pens into collection tubes containing a preservative buffer solution (RNA/DNA Shield® Zymo Research), which preserved the samples during storage and shipping. The microbiomes of the faecal and ceca samples were analysed by 16S RNA qPCR gene (V3-4), cloning and sequencing using the Illumina Miseq® platform (BaseClear NV).

[0095] These results clearly demonstrate that the use of seaweed blends of red, brown and green species, positively stimulated increases in commensal bacterial families that have been shown to be the dominant butyrate producing microorganisms in the gastrointestinal tract. The observed increases in these commensal bacteria may lead to increases in gastrointestinal health and digestive efficiency.

[0096] Summary of Findings: Swine (Pig) Trial 2

TABLE-US-00005 Recipe C’ wt % Ulva lactuca 65-75 Sargassum and Ascophyllum nodosum  2-25 Gracilaria  2-15 Maerl 0-7

[0097] Referring to FIG. 9, compared to the Control group, pigs consuming diets containing blend C′ had improved firmicutes to bacteroidetes ratio. Bacteria in the phyla Firmicutes are associated with a leaner and more feed-efficient pigs.

[0098] Referring to FIGS. 10 and 11, analysis of faecal bacterial community also revealed an increase in the abundance of the bacterial families Ruminoccocaceae and Lachnospiraceae, which are producers of butyrate, a short chain fatty acid with positive effects in the gastrointestinal tract.

[0099] Summary of Findings: Broiler (Chicken) Trial 2

TABLE-US-00006 Recipe B’ wt % Ulva lactuca 70-80 Ascophyllum and Sargassum  3-20 Gracilaria 3-8 Maerl 0-3

[0100] In a 42-day growth performance study, seaweed blend inclusion to a wheat-soyabean based diet at 0.5% of the feed significantly increased the Firmicutes to Bacteroidetes ratio in the ceca of broiler chickens. This ratio indicates whether the microbiome is healthy, displaying a higher abundance of Firmicutes or in dysbiosis (a higher or increased abundance of Bacteroidetes). The results are shown in FIG. 13.

[0101] Referring to FIGS. 14 and 15, the relative abundance of important, butyrate producing bacterial families including Ruminococcaceae and Lachnospiraceae were increased in the ceca.