Composition to improve gut health and animal performance and methods of making the same

Abstract

The present invention relates to compositions comprising at least about 8% -glucans and/or at least about 8% -fucans, which has a prebiotic effect and act as a replacement for in-feed antibiotics. The present invention also relates to extraction methods to obtain such extracts and various uses for the compositions comprising -glucans and/or -fucans.

Claims

1. A method of treating animals or humans comprising: feeding the animals or humans a composition comprising: at least 8% w/w partially hydrolysed -glucans, and at least 8% w/w partially hydrolysed -fucans, in a synergistic amount to at least one of: increase growth of beneficial microbes, reduce levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption, improve growth performance, or reduce gut infection and inflammation.

2. The method of claim 1, wherein the -glucans comprise at least 8% w/w.

3. The method of claim 1, wherein the -fucans comprise at least 8% w/w.

4. The method of claim 1, wherein the -glucans comprise -(1, 3)(1, 6)-glucans.

5. The method of claim 1, wherein the -glucans or the -fucans are derived from a seaweed.

6. The method of claim 5, wherein the seaweed is selected from Laminariaceae, Fucaceae and Gigartinaceae, Ascophyllum, Laminaria, Durvillea, Macrocystis, Chondrus, Ecklonia or any combinations thereof.

7. The method of claim 1, wherein the -glucans comprise laminarin.

8. The method of claim 1, wherein the -fucans comprise fucoidan.

9. The method of claim 1, further comprises mannitol, lactose or combinations thereof.

10. The method of claim 1, wherein the -glucans or -fucans are derived from seaweed by acid-extraction.

11. The method of claim 1, further comprising hydrolyzing the composition with one or more acids selected from lactic acid, hydrochloric acid, sulfuric acid, citric acid, propionic acid, or any combinations thereof.

12. The method of claim 1, wherein the composition is in a powder form or a liquid form.

13. The method of claim 1, wherein the composition further comprises one or more probiotic cultures.

14. The method of claim 13, wherein the one or more probiotic cultures are selected from Bifidobacteria or Lactobacillus.

15. The method of claim 13, wherein the one or more probiotic cultures is selected from the group consisting of Bifidobacterium adolescentis, Lactobacillus leichmannii, Lactobacillus plantarum, Lactobacillus cellobiosius, Lactobacillus acidophilus, and any combinations thereof.

16. The method of claim 1, wherein the composition is mixed with one or more probiotic cultures to form a tablet, or capsule.

17. The method of claim 1, wherein the composition comprises a synergistic amount of -glucans and -fucans.

18. The method of claim 1, wherein the -glucans comprise an amount between 8% and 30% by weight.

19. The method of claim 1, wherein the -fucans comprise an amount between 8% and 30% by weight.

20. The method of claim 1, wherein the composition consists essentially of 8% to 30% w/w partially acid treated -glucans, and 8% to 30% w/w partially acid treated -fucans in a synergistic amount that at least one of: promotes the growth of beneficial microbes, reduces the levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, improves gut structure, reduces gut inflammation, increases nutrient digestibility, increases mineral absorption, increases growth performance in animals or humans, or reduces in-feed antibiotics and treatment for gut infection and inflammation, wherein the composition is formulated for oral delivery.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) For a more complete understanding of the features and advantages of the present invention, reference is now made to the detailed description of the invention along with the accompanying figures and in which:

(2) FIG. 1 is a table, designated as Table 1, which represents the composition and chemical analysis of starter diets.

(3) FIG. 2 is a table, designated as Table 2, which represents the effect of dietary treatment on faecal DM, faecal pH, faecal score, apparent digestibility coefficients and digestible energy content of start diets.

(4) FIG. 3 is a table, designated as Table 3, which represents Composition and chemical analysis of starter diets (as fed).

(5) FIG. 4 is a table, designated as Table 4, which represents effect of lactose level and GutCare concentration on pig performance after weaning (day 0).

(6) FIG. 5 is a table, designated as Table 5, which represents the effect of GutCare on selected microbial populations in the caecum and colon of the piglet.

(7) FIG. 6 is a table, designated as Table 6, which represents the composition and analysis of experimental diets.

(8) FIG. 7 is a table, designated as Table 7, which represents the effect of GutCare concentration on microbial ecology in the colon.

(9) FIG. 8 is a table, designated as Table 8, which represents the effect of GutCare concentration on total tract digestibility coefficients.

(10) FIG. 9 is a table, designated as Table 9, which represents the effects of dietary treatment on faecal dry matter (DM), faecal pH and faecal score in piglets.

EXAMPLES

Example 1

Production of the Composition

(11) 5000 kg of the Raw material (Ascophyllum nodosum, but raw material could be any seaweed selected from the group mentioned above) in a wet state was washed, milled to approximately 10 mm and washed again. 5000 L of Water was added to a 15,000 L vessel and heated to 80 C. Ten litres of 36% Hydrochloric acid was added followed by the addition of the milled weed. The temperature was adjusted to between 75-80 C. by the addition of live steam and the pH adjusted to pH 4 with additional HCl. The vessel was then agitated for three hours followed by cooling to 50 degrees. The mixture in the vessel was then pumped to a press. The solids retained in the press were recycled and the procedure described above was repeated. The liquid from the press was then clarified, evaporated, and spray dried. A cream coloured product with the following composition was obtained. 1. Total Solids. 96.335% weight. 2. Ash Content. 33.210% weight. 3. Total Protein Content. 5.775% weight. 4. Total Fat Content. 2.876% weight. 5. Total Phenols. 37.500 mg/Kg. 6. Total Alginate Content. <5% 7. Laminarin Content. 9.850% weight. 8. Total Mannitol. 4.175% weight. 9. Fucoidin Content. 12.936% weight. 10. Total Carbohydrate Content. 59.335% weight. 11. Reducing Sugars Content. 3.500% weight. 12. Fibre Content. 4.580% weight. 13. Methypentosans. 4.775% weight 14. Antioxidant Analysis BHA 3.558 mg/Kg BHT 5.195 mg/Kg Ethoxyquin 1.886 mg/Kg Vitamin C 14.505 mg/Kg Tocopherols Vitamin E 2 mg/mg/Kg 15. Growth Hormones Cytokinin Content. 16.500 ppm. Auxin Content. 10.176 ppm. Gibberellin Content. 5.800 ppm. Betaine Content. 26.555 ppm.

(12) A small amount of a food-grade preservative, sodium benzoate was also added to the above composition to maintain the integrity of the composition, hereinafter called GutCare. GutCare is a trademark.

Example 2

Digestibility

(13) Experimental Diets

(14) The experiment was designed as a 32 factorial (3 lactose levels2 GutCare levels) consisting of 6 dietary treatments. The treatments were as follows (T1) 65 g/kg lactose with no supplementation, (T2) 170 g/kg lactose with no supplementation, (T3) 275 g/kg lactose with no supplementation, (T4) 65 g/kg lactose+5 g/kg GutCare, (T5) 170 g/kg lactose+5 g/kg GutCare and (T6) 275 g/kg lactose+5 g/kg GutCare. The starter diets were fed in meal form for 27 days. The compositions and chemical analysis of the experimental diets are shown in Table 1. The diets were formulated to have identical digestible energy (16 MJ/kg) and total lysine (16 g/kg) contents by adjusting soya oil and synthetic amino acids. Amino acid requirements were met relative to lysine (Close, 1994). All diets were milled on site. Chromic oxide (Cr.sub.2O.sub.3) was added to the diet during milling at a concentration of 150 ppm to determine nutrient digestibility.

(15) Animals & Management

(16) 165 piglets (progeny of Large White(Large WhiteLandrace sows)) were weaned at 24 days of age and had an initial live weight of 5.90 kg. The piglets were blocked on the basis of live weight and within each block were randomly assigned to one of six dietary treatments. The pigs were housed on fully slatted pens (1.68 m1.22 m). There were six replicates/treatment. Temperatures of the houses were kept at 30 C. during the first week and were then reduced by 2 C. per week. Each pig was weighed initially and on day 8, day 15, day 21 and day 27. The pigs were fed ad libitum and care was taken to avoid any wastage. Feed was available up to weighing but after weighing all the remaining feed in the trough was weighed back. Throughout the experiment samples of the feed were taken for chemical analysis. Fresh fecal samples were collected from each pen on a daily basis from days 10-14 to measure digestibilities. Feces samples were also collected from each pen every second week to measure fecal pH.

(17) Feces Scoring

(18) The pigs were closely monitored for any signs of diarrhea and a scoring system was used to indicate the presence and severity of this. Feces scoring was carried out on Day 0 and continued up until day 27. The feces scoring applied was: 1=watery like feces, 2=semi-liquid feces, 3=soft but partially solid feces, 4=slightly soft feces, 5=solid feces.

(19) Laboratory Analysis

(20) Both concentrates and feces were analysed for nitrogen, dry matter, ash, gross energy, neutral detergent fibre and chromium concentration. After collection, the feces were dried at 100 C. for 72 hours. The concentrates and dried feces were then milled through a 1-mm screen (Christy and Norris hammer mill). The dry matter was determined after drying overnight (min 16 hours) at 103 C. Ash was determined after ignition of a known weight in a muffle furnace (Nabertherm, Bremen, Germany) at 550 for 4 hours. Crude protein was determined as Kjeldahl Nx6.25 using both a Buchi 323 distillation unit and a Buchi 435 digestion unit (Buchi, Flawil/Schweiz, Switzerland) according to AOAC (1980). Neutral detergent fibre and crude fibre was determined using a Fibertec extraction unit (Tecator, Hoganans, Sweden). The Neutral detergent fibre was determined according to Van Soest (1976).

(21) Gross energy of both the feed and fecal samples was determined using a Parr 1201 oxygen bomb calorimeter (Parr, Moline, Ill., USA). The chromium concentration was determined according to Williams et al. (1962).

(22) Statistical Analysis

(23) The experimental data was analysed as a 32 factorial using the General Linear Model procedure of Statistical Analysis System Institute (1985). The models for performance and digestibility analysis included the main effects of lactose level, the composition of the invention or GutCare and the interaction between lactose level and the composition of the invention or GutCare. Initial liveweight at weaning was included as a covariate in the model.

(24) Results

(25) Nutrient Digestibility & Fecal Analysis

(26) The effects of dietary treatment on fecal DM, fecal pH, fecal score and apparent nutrient digestibilities of the diets are presented in Table 2. Pigs offered diets containing GutCare had harder feces between days 15-21 than pigs offered diets without GutCare. There was a significant interaction between lactose level and GutCare for feces score during days 15-21. Pigs offered diets containing 275 g/kg lactose with GutCare had softer feces compared to pigs offered diets containing 275 g/kg without GutCare. This is probably due to the overloading effect of excess carbohydrates in the lower gut. Pigs offered diets containing GutCare had solider feces between days 21-27 than pigs offered diets without GutCare. Pigs offered diets containing GutCare had a significantly lower feces pH compared to pigs offered diets without GutCare. There was a significant interaction between lactose level and GutCare in dry matter (DMD) (P<0.01), organic matter (OMD) (P<0.01), neutral detergent fibre (NDF) (P<0.05), nitrogen (P<0.001) and gross energy (GE) (P<0.001) digestibilities. The inclusion of GutCare extract to 275 g/kg lactose significantly reduced apparent nutrient digestibilities of DMD, OMD, NDF nitrogen and gross energy compared to pigs offered 275 g/kg lactose without GutCare. However, the inclusion of GutCare to 65 g/kg lactose significantly improved apparent nutrient digestibilities of DMD, OMD, NDF, nitrogen and digestible energy compared to pigs offered 65 g/kg lactose without GutCare.

(27) Discussion of Example 2

(28) The results show that the inclusion of GutCare reduces the requirement for high lactose in antibiotic free piglet diets, and improve digestibility in low lactose diets. There was a significant interaction between lactose and GutCare in DMD, NDF, OMD, nitrogen and GE digestibility. The pigs offered the low level of lactose with the GutCare had significant improvements in DMD, NDF, OMD, nitrogen and digestible digestibility of 0.02, 0.06, 0.02, 0.03 and 0.03 respectively compared with pigs offered diets containing the low level of lactose without GtCare.

(29) However, the inclusion of GutCare to the high lactose diets resulted in a decrease in DMD, NDF, OMD, nitrogen and GE digestibility of 0.02, 0.12, 0.02, 0.05 and 0.03 respectively compared to the high lactose diet unsupplemented with GutCare. The combination of the high lactose and GutCare resulted in an excessive quantity of carbohydrate entering the colon that exceeded the fermentation capacity of the piglet. Mul and Perry (1994) showed that an excess intake of oligosaccharides can result in excessive fermentation which may lead to undesirable conditions in the large intestine.

(30) The significant interaction between lactose level and GutCare in fecal consistency when pigs were offered diets containing the high level of lactose supplemented with GutCare resulted in softer feces than pigs offered diets containing the high level of lactose unsupplemented with GutCare indicating such an overload.

(31) The inclusion of GutCare to the low, medium and high lactose diets resulted in a significant reduction in fecal pH. This lowering of the pH is due to the increased production of VFAs in the hind gut and indicates a prebiotic effect for GutCare.

(32) In conclusion, the inclusion of the GutCare to the low lactose diets improved nutrient digestibility however, the inclusion of GutCare to the high lactose diets reduced nutrient digestibility due to an overloading of the gut as described above. However, the inclusion of GutCare in the diet lowered the pH of the feces, indicating a prebiotic effect.

Example 3

(33) A process for producing the composition from algae comprises the following:

(34) 1) Washing the sand and grit off the wet weed, chopping the wet weed to pieces, about 3 to about 10 mm followed by sand separation.

(35) 2) Extracting the chopped weed in water at a temperature ideally between about 70-80 deg C. for about 2-3 hours at a pH of about 3.5 to about 4.5 and preferably about pH 4. The seaweed is preferably combined with water. The water may be brought to, or is at, a temperature between about 0 C. to about 100 C., preferably between about 37 C. and 95 C., more preferably about 50 C. to about 80 C., and most preferably about 75 C.

(36) The process of the invention is ideally carried out at pH 1 to pH 7, more preferably pH 1 to pH 6, more preferably about pH 4 to about pH 5 and most preferably about pH 4.5. In one embodiment the pH of the solution can be adjusted to about pH 4.5 prior to the agitation step. While not wishing to be bound by theory, it is believed that a pH between about pH 4 and about pH 5 optimises yield, while lowering the requirement for addition of acids, minimising the hydrolysis effects and harm thereof. Ideally, the acid used is chosen from a group consisting of inorganic acids like hydrochloric acid, phosphoric acid and sulphuric acid and organic acids like lactic acid, formic acid and propionic acid, or any soluble inorganic or organic acid.

(37) 3) The process of extraction is further aided by continuous agitation. Ideally, the mixture is agitated for a period of time, preferably between about 1-10 hours, and most preferably about 3 hours. The agitation creates a slurry.

(38) 4) The processed material can then be cooled to 10-50 C. thus protecting sensitive compounds and/or making the material safe to press.

(39) 5) Ideally, the mixture may then be decanted or pressed.

(40) 6) The insoluble material may be collected and reprocessed using the same procedure outlined above.

(41) 7) The liquid plus small insoluble residue is pumped to a clarifier where the remaining insoluble fractions are removed. The product may be clarified to yield a liquid composition.

(42) 8) The clarified liquid can be pumped to a storage tank (direct to nanofiltration unit or evaporator) from where it can be pumped to evaporator for concentration or to nanofiltration (NF) plant where up to 70% of the chloride salts are removed and up to 30% of the sodium and potassium salts are removed. This desalting helps to remove the salty taste in the product. The operating pressure in the NF step is between 20-40 bar and preferably 25 bar and the membrane pore size is 10.sup.-3-10.sup.-2 um.

(43) 9) If the application requires a high degree of desalination the product can be processed by either Electrodialysis or Ion Exchange.

(44) 10) The concentrated, nanofiltered or demineralised product can be evaporated and a preservative such as sodium benzoate added if required in liquid form.

(45) 11) As a further embodiment to make a purified -glucan & -fucan evaporated product, the product can be crystalised in crystallation tanks where the mannitol is converted to crystals and removed by centrifugation. This involves obtaining a highly concentrated liquid, followed by transfer to crystallation tanks, followed by seeding, followed by cooling at a predetermined time and temperature. When the crystals are formed the product is then centrifuged is a two step separation process separating the crystals for drying in a fluid bed dryer and the balance of the product which is high in -glucans and -fucans available to be dried in a spray dryer.

(46) 12) The evaporated product can be dried in a spray dryer. The resultant powder is a cream colour.

(47) 13) Solubilisation of the laminarin may be required if the starting raw material is Laminaria hyperborea. In this case the product after clarification is subjected to pH modification as per the process described for yeast glucans in US Pat. No 20040082539.

Example 4

Performance

(48) Diets

(49) The experiment was arranged as a 43 factorial (4 GutCare levels and 3 lactose levels), over four consecutive runs. 384 piglets (progeny of Large White(Large WhiteLandrace)) were selected after weaning at 21 days with an initial live weight of 7.43 kg. The pigs in run 1, 2, 3 and 4 had an initial live weight of 7.88 kg, 7.57 kg, 6.56 kg and 7.72 kg respectively. The pigs were blocked on the basis of live weight and within each block assigned to one of twelve dietary treatments. The dietary treatments consisted of (T1) 24.3 g/kg lactose with 0 g/kg GutCare, (T2) 24.3 g/kg lactose with 3 g/kg GutCare, (T3) 24.3 g/kg lactose with 6 g/kg GutCare (T4) 24.3 g/kg lactose with 12 g/kg GutCare, (T5) 15.3 g/kg lactose with 0 g/kg GutCare, (T6) 15.3 g/kg lactose with 3 g/kg GutCare. (T7) 15.3 g/kg lactose with 6 g/kg GutCare. (T8) 15.3 g/kg lactose with 12 g/kg GutCare. (T9) 6.3 g/kg lactose with 0 g/kg GutCare. (T10) 6.3 g/kg lactose with 3 g/kg GutCare. (T11) 6.3 g/kg lactose with 6 g/kg GutCare. (T12) 6.3 g/kg lactose with 12 g/kg GutCare. The starter diets were milled on site and offered in meal form for 21 days post weaning. Diets were formulated as described in Example 2 except chromium oxide was at a concentration of 200 p.p.m. The ingredient composition and chemical analysis of the dietary treatments are presented in Table 3. The composition of the invention used in this example was a liquid sample with 33% solids.

(50) Management

(51) Pigs were housed in groups of four (eight replicates per treatment) as described in Example 2. Pigs were weighed initially and on days 7, 14 and 21. Fresh fecal samples were collected once daily from all pens on days 10 to 15.

(52) Statistical Analysis

(53) The experimental data were analysed as a 43 factorial as described in Example 2

(54) Results

(55) Performance

(56) The effects of lactose level and GutCare concentration on average daily gain (ADG), food intake and food conversion ratio (FCR) are presented in Table 4. There was a significant interaction between lactose and GutCare (P<0.05) on average daily gain (ADG) between days 0-7. Pigs offered diets containing no GutCare supplementation and low lactose levels had lower ADGs than pigs offered diets containing GutCare and high lactose. There was significant lactose by GutCare interaction on ADG in the overall growing period (days 0-21). Pigs offered diets containing GutCare and low lactose levels had lower ADGs than pigs offered diets containing GutCare and high lactose. GutCare also had a significant independent linear effect on daily gain (P<0.01) and led to an improvement in daily gain, at all lactose levels.

(57) There was a significant interaction between lactose and GutCare (P<0.05) during the starter period (days 0-7) on average daily feed intake (ADFI). Pigs offered high lactose diets and 3 g/kg GutCare had the overall highest ADFI. The inclusion of 6 g/kg and 12 g/kg GutCare at the high levels of lactose decreased ADFI. However, pigs offered medium levels of lactose and 12 g/kg GutCare and pigs offered low lactose diets and 6 g/kg GutCare obtained higher ADFIs than the pigs offered the same levels of lactose but no GutCare.

(58) There was also a linear increase in ADFI (P<0.05) as the level of GutCare increased between days 7-14 and again between days 14-21. There was a linear increase to both lactose level (P<0.05) and GutCare supplementation (P<0.05) on ADFI during the overall starter period (0-21) as the level of both increased.

(59) There was a significant interaction between lactose and GutCare (P<0.05) on food conversion ratio (FCR) during days 0-7. There was an improvement in FCR as the level of GutCare increased at both medium and low lactose level. However, at medium levels of lactose there was an improvement in FCR up to 6 g/kg GutCare where there was deterioration thereafter. There was a linear decrease (P<0.05) in FCR, representative of improved feed efficiency, during the overall starter period (days 0-21) as the level of GutCare increased. Likewise there was also a tendency of a significant lactose by GutCare interaction (P<0.09) during days 0-21. Overall GutCare improved the feed conversion ratio in most of the diets. Furthermore, GutCare also led to more solid feces, particularly during day 0-7 as shown in Table 9.

(60) There was also a numerical tendency (p=0.13) for a drop in fecal pH on addition of GutCare at low lactose levels, indicating a prebiotic effect.

(61) Discussion

(62) The above experiments show the benefit of GutCare on animal performance in the absence of in feed antibiotics. The effect is as strong as the effect of the antibiotics the formulation is meant to replace. There is an improvement in daily gain, intake as well as feed conversion ratio, which are the key performance parameters. The composition interacts with lactose, which is to be expected, as both consist primarily of carbohydrate components, and giving an excess of the two could result in an overloading effect on the digestive system. This is discussed in greater detail in Example 2. GutCare works best at low and medium levels of lactose, providing an effective means to reduce the level of lactose (an expensive component) needed in animal diets. The harder feces as observed in Day 0-7 are representative of a reduction in scouring, which is a key health parameter in young piglets, which are especially susceptible to diarrhea.

(63) Experiment 5

(64) Anti-Microbial

(65) Animals and Experimental Diets

(66) The experiment was designed as a 21 factorial. Ten piglets (progeny of Large White(Large WhiteLandrace)) were selected from four closely related sows at 24 days of age. The piglets had a weaning weight of 7.8 (s.d 0.83) kg. They were blocked on the basis of litter, weight and sex and within each block randomly assigned to one of two dietary treatments. The dietary treatments were as follows: T1) Standard Diet; T2) Standard Diet+1.8 g/kg GutCare. Diets were formulated as described in Experiment 2.

(67) Management

(68) The pigs were housed individually as described in Experiment 2.

(69) Microbiology

(70) The effect of GutCare on selected microbial populations in the caecum and colon are shown in Table 5. GutCare had a significant effect on the microbial populations in the caecum with a decrease in the E. coli (P<0.01), Bifidobacteria (P<0.05), and Lactobacilli (P<0.05) populations. GutCare had a significant effect on the E. coli population (P<0.01) and the Lactobacilli population (P<0.001) of the colon, causing a decline.

(71) Discussion

(72) The composition has a pronounced anti-microbial action, similar to in-feed antibiotics in piglets. This is beneficial from a performance perspective, as a lower microbial load will result in a lower energy cost to the pig. Also, the removal of harmful bacteria like E. coli helps control disease rates in piglets.

(73) As mentioned above, the composition plays a role similar to the antibiotics in small pigs, acting as a replacement for them. This behaviour is different to the behaviour in large pigs where the formulation plays more of a prebiotic role. The reason for the difference could be the inability of the small pigs to break down some of the components in the composition to a form in which they act as a prebiotic. This is in line with the objectives of the formulation to act as a substitute for antibiotics in the small pig.

Example 6

Prebiotic and Mineral Absorption

(74) Experimental Design and Diets

(75) The experiment was designed as a complete randomised design comprising of five dietary treatments. All diets were formulated to have identical concentrations of net energy (9.8 MJ/kg) and total lysine (10.0 g/kg). The amino acid requirements were met relative to lysine (Close, 1994). All diets were fed in meal form. GutCare was supplied by BioAtlantis Ltd. (Kerry Technology Park, Tralee, Ireland). The dietary composition and analysis are presented in Table 6.

(76) The experimental treatments were as follows:

(77) (1) 0 g/kg GutCare (control)

(78) (2) 0.7 g/kg GutCare

(79) (3) 1.4 g/kg GutCare

(80) (4) 2.8 g/kg GutCare

(81) (5) 5.6 g/kg GutCare

(82) Animals and Management

(83) Sixteen finishing boars progeny of Meat line boars(Large WhiteLandrace sow)) with an initial live weight of 51 kg (s.d=3.4 kg) were used in this experiment. The pigs were blocked on the basis of live weight and were randomly allocated to one of five dietary treatments. The pigs were allowed a 14-day dietary adaptation period after which time they were weighed and transferred to individual metabolism crates. The pigs were given a further 5 days to adapt to the metabolism crates before collections began. The collection period was sub-divided into two parts to facilitate studies on apparent digestibility (days 3 to 7). The daily feed allowance (DE intake=3.44(live weight) 0.54 (Close, 1994) was divided over two meals. Water was provided with meals in a 1:1 ratio. Between meals, fresh water was provided ad libitum. The metabolism crates were located in an environmentally controlled room, maintained at a constant temperature of 22 C. (1.5 C.).

(84) Apparent Digestibility Study

(85) During collections, urine was collected in a plastic container, via a funnel below the crate, containing 20 ml of sulphuric acid (25% H.sub.2SO.sub.4). The urine volume was recorded daily and a 50 ml sample was collected and frozen for laboratory analysis. Total feces weight was recorded daily and oven dried at 100 C. At the end of the collection period, the feces samples were pooled and a sub-sample retained for laboratory analysis. Feed samples were collected each day and retained for chemical analysis.

(86) Microbiology

(87) All animals remained on their respective dietary treatments until slaughter. Digesta samples (approximately 10 g+1 g) were aseptically removed from the colon of each animal immediately after slaughter, stored in sterile containers (Sarstedt, Wexford, Ireland) on ice and transported to the laboratory within 7 h. Bifidobacteria spp. and E. coli were isolated and counted according to the method described by O'Connell et al., (2005). Bifidobacteria spp was chosen because of its positive effect on gut health while E. coli species was chosen because of its negative effect on gut health (De Lange, 2000).

(88) Laboratory Analysis of Samples

(89) Proximate analysis of diets for dry matter (DM) and ash was carried out according to Association of Analytical Chemists, (1980). The dry matter of the feed was determined after drying overnight at 103 C. Ash was determined after ignition of a known weight of concentrates or feces in a muffle furnace (Nabertherm, Bremen, Germany) at 500 C. for four hours. The gross energy (GE) of feed and feces samples was measured using an adiabatic bomb calorimeter (Parr Instruments, Il, USA).

(90) Results

(91) Microbiology Study

(92) The effect of dietary treatment on selected microbial populations and pH in the colon are presented in Table 7.

(93) There was a significant response to GutCare on colonic E. coli and colonic bifidobacteria populations (quadratic P<0.05). There was a significant (quadratic) decrease in E. coli population while there was an increase in bifidobacteria populations, up to a certain level. At high concentrations, these populations decreased, indicating overloading of the gut.

(94) Total Tract Digestibility.

(95) The effect of dietary treatment on ash total tract digestibility is presented in Table 8. There was a significant linear increase (P<0.01) in total tract ash digestibility with increasing extract concentration.

(96) Discussion

(97) This experiment shows the effect of GutCare on the gut microflora in growers and on mineral absorption (represented as ash digestibility). As can been seen from the results, the composition resulted in an increase in beneficial bacteria levels and a reduction in the levels of harmful microbes. This response is typical of prebiotic formulations. At higher dosages, the levels of all microbes changed, indicating overloading of the gut, again a response typical of prebiotic formulations. Furthermore, the increase in ash digestibility (which consists of micro and macro nutrients) indicates increased absorption of these nutrients in the gut.

(98) The words comprises/comprising and the words having/including when used herein with reference to the present invention are used to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

(99) It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

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