Novel Composition to Improve Gut Health and Animal Performance and Methods of Making the Same
20210346443 · 2021-11-11
Inventors
Cpc classification
A61K2236/00
HUMAN NECESSITIES
A61K36/03
HUMAN NECESSITIES
Y02A50/30
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
International classification
Abstract
The present invention relates to a composition and methods comprising an amount of β-glucans and α-fucans, wherein the combination is synergistic to at least one of: increase growth of beneficial microbes and reduced 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 and growth performance, or reduce gut infection and inflammation.
Claims
1. A method of treating animals or humans comprising: feeding the animals or humans a composition comprising an amount of β-glucans and α-fucans wherein the combination is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation.
2. The method of claim 1, wherein the β-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
3. The method of claim 1, wherein the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
4. The method of claim 1, wherein the β-glucans comprise β-(1, 3)(1, 6)-glucans.
5. The method of claim 1, wherein the β-glucans or the α-fucans are derived from a seaweed.
6. The method of claim 1, wherein the β-glucans comprise laminarin.
7. The method of claim 1, wherein the α-fucans comprise fucoidan.
8. The method of claim 8, wherein the seaweed is selected from Laminariaceae, Fucaceae, Gigartinaceae, Ascophyllum, Laminaria, Durvillea, Macrocystis, Chondrus, Ecklonia or any combinations thereof.
9. The method of claim 1, further comprising adding to the composition mannitol, lactose, one or more probiotic cultures or combinations thereof.
10. The method of claim 1, wherein the β-glucans or α-fucans are derived from seaweed by acid-extraction.
11. The method of claim 1, wherein an acid extraction for hydrolysis uses 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 formulated into a powder form, a liquid form, a tablet, or capsule.
13. The method of claim 1, wherein the one or more probiotic cultures are selected from Bifidobacteria or Lactobacteria including Lactobacilli-leichmannii, L. plantarum, L. cellobiosius, Bifidobacterim, adolescentis, L. acidophilus, or any combinations thereof.
14. The method of claim 1, wherein the composition comprises a synergistic amount of (3-glucans and α-fucans.
15. The method of claim 1, wherein the composition consist essentially of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w β-glucans, and at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w α-fucans in a synergistic amount that that at least one of: promote the growth of beneficial microbes and reduce the levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, gut inflammation, nutrient digestibility, mineral absorption and growth performance in animals or humans, or act as a replacement for in-feed antibiotics and treatment for gut infection and inflammation, wherein the composition is formulated for oral delivery.
16. A method of treating animals or humans comprising: providing the animal or human a composition that consist essentially of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w β-glucans, and at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w α-fucans in a synergistic amount that that at least one of: promote the growth of beneficial microbes and reduce the levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, gut inflammation, nutrient digestibility, mineral absorption and growth performance in animals or humans, or act as a replacement for in-feed antibiotics and treatment for gut infection and inflammation, wherein the composition is formulated for oral delivery.
17. A composition comprising: an amount of β-glucans and α-fucans, wherein the amount is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation.
18. The composition of claim 17, wherein the composition consists essentially of at least one of: β-glucans in an amount between 0.1% and 60% by weight, or α-fucans in an amount between 0.1% and 60% by weight.
19. The composition of claim 17, wherein the 3-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
20. The composition of claim 17, wherein the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
21. A composition consisting essentially of: an amount of β-glucans and α-fucans, wherein the amount is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation.
22. The composition of claim 21, wherein the composition consists of β-glucans in an amount between 0.1% and 60% by weight, or α-fucans in an amount between 0.1% and 60% by weight.
23. The composition of claim 21, wherein the β-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
24. The composition of claim 21, wherein the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0079] 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:
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DETAILED DESCRIPTION OF THE INVENTION
[0100] While the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated that the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the invention and do not delimit the scope of the invention.
[0101] To facilitate the understanding of this invention, a number of terms are defined below. Terms defined herein have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as “a”, “an” and “the” are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
EXAMPLES
Example 1
[0102] Production of the Composition. 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.
[0103] 1. Total Solids. 96.335% weight.
[0104] 2. Ash Content. 33.210% weight.
[0105] 3. Total Protein Content. 5.775% weight.
[0106] 4. Total Fat Content. 2.876% weight.
[0107] 5. Total Phenols. 37.500 mg/Kg.
[0108] 6. Total Alginate Content. <5%
[0109] 7. Laminarin Content. 9.850% weight.
[0110] 8. Total Mannitol. 4.175% weight.
[0111] 9. Fucoidin Content. 12.936% weight.
[0112] 10. Total Carbohydrate Content. 59.335% weight.
[0113] 11. Reducing Sugars Content. 3.500% weight.
[0114] 12. Fibre Content. 4.580% weight.
[0115] 13. Methypentosans. 4.775% weight
[0116] 14. Antioxidant Analysis [0117] BHA 3.558 mg/Kg [0118] BHT 5.195 mg/Kg [0119] Ethoxyquin 1.886 mg/Kg [0120] Vitamin C 14.505 mg/Kg [0121] Tocopherols Vitamin E 2 mg/mg/Kg
[0122] 15. Growth Hormones [0123] Cytokinin Content. 16.500 ppm. [0124] Auxin Content. 10.176 ppm. [0125] Gibberellin Content. 5.800 ppm. [0126] Betaine Content. 26.555 ppm.
[0127] 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
[0128] Digestibility. Experimental Diets. The experiment was designed as a 3×2 factorial (3 lactose levels×2 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, (TS) 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.
[0129] Animals & Management. 165 piglets (progeny of Large White×(Large White×Landrace 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 m×1.22 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.
[0130] Feces Scoring. 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.
[0131] Laboratory Analysis. 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 N×6.25 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).
[0132] 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).
[0133] Statistical Analysis. The experimental data was analysed as a 3×2 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.
[0134] Nutrient Digestibility & Fecal Analysis. 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.
Discussion of Example 2
[0135] 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 GutCare.
[0136] 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.
[0137] 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.
[0138] 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.
[0139] 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
[0140] A process for producing the composition from algae comprises the following:
[0141] 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.
[0142] 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.
[0143] 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.
[0144] 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.
[0145] 4) The processed material can then be cooled to 10-50° C. thus protecting sensitive compounds and/or making the material safe to press.
[0146] 5) Ideally, the mixture may then be decanted or pressed.
[0147] 6) The insoluble material may be collected and reprocessed using the same procedure outlined above.
[0148] 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.
[0149] 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.
[0150] 9) If the application requires a high degree of desalination the product can be processed by either Electrodialysis or Ion Exchange.
[0151] 10) The concentrated, nanofiltered or demineralised product can be evaporated and a preservative such as sodium benzoate added if required in liquid form.
[0152] 11) As a further embodiment to make a purified β-glucan & α-fucan evaporated product, the product can be crystallised 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 β-glucans and α-fucans available to be dried in a spray dryer.
[0153] 12) The evaporated product can be dried in a spray dryer. The resultant powder is a cream colour.
[0154] 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
[0155] Performance. Diets. The experiment was arranged as a 4×3 factorial (4 GutCare levels and 3 lactose levels), over four consecutive runs. 384 piglets (progeny of Large White×(Large White×Landrace)) 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.
Management
[0156] 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.
[0157] Statistical Analysis. The experimental data were analysed as a 4×3 factorial as described in Example 2
[0158] Performance. 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.
[0159] 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.
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] Experiment 5
[0165] Anti-microbial. Animals and Experimental Diets. The experiment was designed as a 2×1 factorial. Ten piglets (progeny of Large White× (Large White×Landrace)) 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.
[0166] Management. The pigs were housed individually as described in Experiment 2.
[0167] Microbiology. 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.
[0168] 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.
[0169] 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
[0170] Prebiotic and Mineral Absorption. Experimental Design and Diets. 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.
[0171] The experimental treatments were as follows:
(1) 0 g/kg GutCare (control)
(2) 0.7 g/kg GutCare
(3) 1.4 g/kg GutCare
(4) 2.8 g/kg GutCare
(5) 5.6 g/kg GutCare
[0172] Animals and Management. Sixteen finishing boars progeny of Meat line boars×(Large White×Landrace 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.).
[0173] Apparent Digestibility Study. 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.
[0174] Microbiology. 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).
[0175] Laboratory Analysis of Samples. 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).
[0176] Microbiology Study. The effect of dietary treatment on selected microbial populations and pH in the colon are presented in Table 7.
[0177] 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.
[0178] Total Tract digestibility. 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.
[0179] 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.
Example 7
[0180] PCV-2 Trial. Direct application of SWE to enhance immune response to PCV-2 in newly weaned pigs. Dry Matter: 44.25 (% w/w)
[0181] Glucan: 31.87 g/l (5.83% w/w), Fucan: 24.1 g/l (4.41% w/w), Dosage rate: 6 L/t.
[0182] Gain to feed ratio: improved feed efficiency between days 49 and 62. Porcine circovirus type 2 (PCV-2) antibodies: Glucan+fucan increased antibodies from day 13 post-infection onwards. Significant on day 25. Porcine parvovirus (PPV) viral load in sera: Glucan+fucan reduced viral load on day 4.
[0183] Cryostat staining: Lower infectious virus detected in the glucan+fucan treatment group on day 27 in all tissues but not statistically significant.
[0184] Mortality: Glucan+fucan reduced mortality when compared with the control (0.20 vs. 0.05; no statistics performed).
Example 7
[0185] PCV-2 Trial. Direct application of SWE to enhance immune response to PCV-2 in newly weaned pigs. Dry Matter: 44.25 (% w/w)
[0186] Glucan: 31.87 g/l (5.83% w/w), Fucan: 24.1 g/l (4.41% w/w), Dosage rate: 6 L/t.
[0187] Gain to feed ratio: improved feed efficiency between days 49 and 62. Porcine circovirus type 2 (PCV-2) antibodies: Glucan+fucan increased antibodies from day 13 post-infection onwards. Significant on day 25. Porcine parvovirus (PPV) viral load in sera: Glucan+fucan reduced viral load on day 4.
[0188] Cryostat staining: Lower infectious virus detected in the glucan+fucan treatment group on day 27 in all tissues but not statistically significant.
[0189] Mortality: Glucan+fucan reduced mortality when compared with the control (0.20 vs. 0.05; no statistics performed).
TABLE-US-00001 TABLE 10 Effect of seaweed extract (SWE) supplementation on growth performance, Porcine Circovirus type 2 (PCV2) antibody levels, Porcine Parvovirus (PPV) viral load and mortality rate of pigs after PCV2/PPV infection dual infection post-weaning. Dietary Treatments Sign- Item Control SWE SEM ificance Performance (Day 49-62) 0.57 0.70 ND ND *Gain to feed ratio Average PCV2 sera IPMA score Day 25 23720 37206 13467 0.011 Quantitative PCR for PPV in sera Day 4 1.01E+07 4.74E+06 3.46E+06 0.03 *Mortality rate 0.20 0.05 ND ND Abbreviations: IPMA, Immunoperoxidase monolayer assay; ND, Not determined; NS, Not significant (P > 0.05). [0190] No statistical analysis performed on this data.
[0191]
[0192]
[0193]
[0194]
Example 8
[0195] Following the teachings of the present invention, the inventors provided a direct application during lifetime of offspring to determine the effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status, Lab analysis: Dry Matter: 27.84 (% w/w), Glucan: 11.13 g/l (3.4% w/w). See post-filing publication: Leonard, S. G., Sweeney, T., Bahar, B., Lynch, B. P. & O'Doherty, J. V. 2011. Effects of dietary seaweed extract supplementation in sows and post-weaned pigs on performance, intestinal morphology, intestinal microflora and immune status. British Journal of Nutrition, 106, 688-699.
[0196] DO-21 (starter Period). Gain to feed ratio: Pigs weaned from basal-fed sows and offered glucan+fucan supplemented diets post-weaning had an improved feed efficiency.
[0197] D21-117 (Grower-Finisher Period). Average daily gain (ADG): Pigs weaned from basal-fed sows and offered glucan+fucan supplemented grower-finisher diets had a higher ADG. Gain to feed ratio: Pigs weaned from basal-fed sows and offered glucan+fucan supplemented grower-finisher diets had an improved feed efficiency.
[0198] Pigs offered post-weaning (PW) diets containing glucan+fucan had: Decreased E. coli populations on day 11. Increased Lactobacillus:E. coli ratio on day 11. Decreased colonic Enterobacteriaceae numbers on day 117. Increased mRNA abundance of MUC2 in the colon at day 11. Greater villous height:crypt depth ratio in the jejunum.
[0199]
[0200]
[0201]
Example 9
[0202] Following the teachings of the present invention, the inventors determined the effect of the interaction of seaweed extracts containing laminarin and fucoidan with zinc oxide on the growth performance, digestibility and faecal characteristics of growing piglets. See post-filing publication: O'Shea, C. J., McAlpine, P., Sweeney, T., Varley, P. F. & O'Doherty, J. V. 2014. Effect of the interaction of seaweed extracts containing laminarin and fucoidan with zinc oxide on the growth performance, digestibility and faecal characteristics of growing piglets. British Journal of Nutrition, 111, 798-807.
[0203] Lab analysis: Dry matter: 45.21 (% w/w), Glucan: 52.04 g/l (9.28% w/w), Fucan: 28.35 g/l (5.06 w/w), Dosage rate: 5.76 L/t.
[0204] Transition period (d21-40 post-weaning) Feed conversion ratio (FCR)-Glucan+Fucan improved feed efficiency.
[0205] Microbiology (d21-40): E. coli: Glucan+fucan reduced E. coli numbers when compared with control group.
[0206] Digestibility: Glucan+fucan improved gross energy (GE), nitrogen (N) and neutral detergent fiber digestibility (NDF) when compared with control group.
[0207] Summary effects of glucan+fucan: Dietary supplementation with glucan+fucan improved N, organic matter, ash and NDF digestibility during the starter diet period and feed efficiency during the transition diet period. Furthermore, the dietary inclusion of glucan+fucan reduced the faecal counts of E. coli during the transition diet period.
TABLE-US-00002 TABLE 11 Effect of seaweed extract (SWE) and zinc oxide (ZnO) inclusion on feed efficiency and total tract apparent digestibility (Least square means and SEM). Dietary Treatments P-Values Con- Combin- SWE × trol SWE ZnO ation SEM SWE ZnO Zno Gain to feed ratio (kg/kg) D 21 to 40 0.495 0.567 0.561 0.492 0.024 0.947 0.840 0.005 Coefficient of apparent total tract digestibility Organic 0.804 0.875 0.823 0.808 0.006 0.001 0.001 0.001 matter Nitrogen 0.807 0.879 0.821 0.812 0.005 0.001 0.001 0.001 Neutral 0.386 0.608 0.444 0.412 0.019 0.001 0.007 0.001 detergent fibre Ash 0.346 0.631 0.466 0.451 0.014 0.001 0.048 0.001 Gross 0.767 0.851 0.792 0.774 0.006 0.001 0.002 0.001 Energy
[0208]
[0209]
[0210]
Example 10
[0211] Following the teachings of the present invention, the inventors determined the effect of maternal and postweaning seaweed extract supplementation on gut health in pigs after weaning and response to enterotoxigenic Escherichia coli K88 challenge. See post-filing publication: BOUWHUIS, M. A., SWEENEY, T., MCDONNELL, M. J., DOYLE, D. N., THORNTON, K., MUKHOPADHYA, A. & O'DOHERTY, J. V. 2016. The effect of maternal and postweaning seaweed extract supplementation on gut health in pigs after weaning and response to enterotoxigenic Escherichia coli K88 challenge1. Journal of Animal Science, 94, 395-398
[0212] Direct application to weaned pigs. Lab analysis: Dry Matter: 44.25 (% w/w), Glucan: 31.87 g/l (5.83 w/w), Fucan: 24.1 g/l (4.41% w/w), Dosage rate: 7.82 L/t (250 ppm glucan).
TABLE-US-00003 TABLE 12 Effect of seaweed extract (SWE) inclusion on the heat-labile enterotoxin (LT; log gene copy number/g digesta) produced by enterotoxigenic E. coli (ETEC). Dietary Treatments Control SWE SEM Significance Caecum 5.78 4.98 0.22 0.003 LT
[0213]
Example 11. Direct Application to Weaned Pigs to Control Salmonella Infection
[0214] Following the teachings of the present invention, the inventors determined the effect of direct application of the composition to weaned pigs to control Salmonella infection.
[0215] Dry Matter (DM): 44.25 (% w/w), Glucan: 31.87 g/l (5.83% w/w), Fucan: 24.1 g/l (4.41% w/w), Dosage rate: 7.82 L/t (250 ppm glucan). Pre-Challenge, Post-Challenge & Overall Performance: NS. Faecal Score 2d post-challenge: Supplementation of glucan+fucan synergistically improved faecal consistency compared with control group.
TABLE-US-00004 TABLE 13 Effect of seaweed extract (SWE) supplementation on faecal scoring of pigs challenged with Salmonella typhimurium on day 10 post-weaning. Control SWE *Faecal score 2d post-challenge 3.48.sup.a 2.17.sup.b *= Faecal scores were measured on a 1-5 scale, 1 = hard faeces, 2 = slightly soft faeces, 3 = soft partially formed faeces, 4 = loose semi liquid faeces and 5 = watery mucous like faeces. .sup.a-b,Mean values with unlike superscript letters were significantly different (P < 0.05).
[0216] 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.
[0217] In one embodiment, the present invention includes a method of treating animals or humans comprising, consisting essentially of, or consisting of: feeding the animals or humans a composition comprising an amount of β-glucans and α-fucans wherein the combination is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation. In one aspect, the β-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w. In another aspect, the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w. In another aspect, the β-glucans comprise β-(1, 3)(1, 6)-glucans. In another aspect, the β-glucans or the α-fucans are derived from a seaweed. In another aspect, the β-glucans comprise laminarin. In another aspect, the α-fucans comprise fucoidan. In another aspect, the seaweed is selected from Laminariaceae, Fucaceae, Gigartinaceae, Ascophyllum, Laminaria, Durvillea, Macrocystis, Chondrus, Ecklonia or any combinations thereof. In another aspect, the method further comprises adding to the composition mannitol, lactose, one or more probiotic cultures or combinations thereof. In another aspect, the β-glucans or α-fucans are derived from seaweed by acid-extraction. In another aspect, an acid extraction for hydrolysis uses one or more acids selected from lactic acid, hydrochloric acid, sulfuric acid, citric acid, propionic acid, or any combinations thereof. In another aspect, the composition is formulated into a powder form, a liquid form, a tablet, or capsule. In another aspect, the one or more probiotic cultures are selected from Bifidobacteria or Lactobacteria including Lactobacilli-leichmannii, L. plantarum, L. cellobiosius, Bifidobacterim, adolescentis, L. acidophilus, or any combinations thereof. In another aspect, the composition comprises a synergistic amount of β-glucans and α-fucans. In another aspect, the composition consist essentially of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% β-glucans, and at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% α-fucans in a synergistic amount that that at least one of: promote the growth of beneficial microbes and reduce the levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, gut inflammation, nutrient digestibility, mineral absorption and growth performance in animals or humans, or act as a replacement for in-feed antibiotics and treatment for gut infection and inflammation, wherein the composition is formulated for oral delivery.
[0218] In another embodiment, the present invention includes a method of treating animals or humans comprising, consisting essentially of, or consisting of: providing the animal or human a composition that consist essentially of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w β-glucans, and at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w α-fucans in a synergistic amount that that at least one of: promote the growth of beneficial microbes and reduce the levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, gut inflammation, nutrient digestibility, mineral absorption and growth performance in animals or humans, or act as a replacement for in-feed antibiotics and treatment for gut infection and inflammation, wherein the composition is formulated for oral delivery.
[0219] In another embodiment, the present invention includes a composition comprising, consisting essentially of, or consisting of: an amount of β-glucans and α-fucans, wherein the amount is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation. In one aspect, the composition consists essentially of at least one of: β-glucans in an amount between 0.1% and 60% by weight, or α-fucans in an amount between 0.1% and 60% by weight. In another aspect, the β-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w. In another aspect, the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
[0220] In another embodiment, the present invention includes a composition comprising, consisting essentially of, or consisting of: an amount of β-glucans and α-fucans, wherein the amount is synergistic to at least one of: increase growth of beneficial microbes and reduced levels of harmful microbes selected from E. coli or Salmonella in animal or human intestines, reduced viral load, improve gut structure, reduce gut inflammation, improved nutrient digestibility, improve mineral absorption and growth performance, or reduce gut infection and inflammation. In one aspect, the composition consists of β-glucans in an amount between 0.1% and 60% by weight, or α-fucans in an amount between 0.1% and 60% by weight. In one aspect, the (3-glucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w. In another aspect, the α-fucans comprise at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30% w/w.
[0221] The inventors have found that a particular composition, comprising, consisting essentially of, or consisting of: a high proportion of β-glucans and α-fucans is able to act as an anti-microbial, particularly in young mammals (reducing the threat of infections like E. coli and Salmonella) and improve animal performance. This composition also acts as a prebiotic in humans and other large mammals and selectively boosts the growth of beneficial microbes. It also improves mineral absorption in the hind gut and increases nutrient digestibility. This is a large improvement over the art as, currently, there is no single composition that can concomitantly achieve all these objectives.
[0222] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, kit, reagent, or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.
[0223] It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
[0224] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
[0225] The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
[0226] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In embodiments of any of the compositions and methods provided herein, “comprising” may be replaced with “consisting essentially of” or “consisting of”. As used herein, the phrase “consisting essentially of” requires the specified integer(s) or steps as well as those that do not materially affect the character or function of the claimed invention. As used herein, the term “consisting” is used to indicate the presence of the recited integer (e.g., a feature, an element, a characteristic, a property, a method/process step or a limitation) or group of integers (e.g., feature(s), element(s), characteristic(s), propertie(s), method/process steps or limitation(s)) only.
[0227] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
[0228] As used herein, words of approximation such as, without limitation, “about”, “substantial” or “substantially” refers to a condition that when so modified is understood to not necessarily be absolute or perfect but would be considered close enough to those of ordinary skill in the art to warrant designating the condition as being present. The extent to which the description may vary will depend on how great a change can be instituted and still have one of ordinary skilled in the art recognize the modified feature as still having the required characteristics and capabilities of the unmodified feature. In general, but subject to the preceding discussion, a numerical value herein that is modified by a word of approximation such as “about” may vary from the stated value by at least ±1, 2, 3, 4, 5, 6, 7, 10, 12 or 15%.
[0229] Additionally, the section headings herein are provided for consistency with the suggestions under 37 CFR 1.77 or otherwise to provide organizational cues. These headings shall not limit or characterize the invention(s) set out in any claims that may issue from this disclosure. Specifically and by way of example, although the headings refer to a “Field of Invention,” such claims should not be limited by the language under this heading to describe the so-called technical field. Further, a description of technology in the “Background of the Invention” section is not to be construed as an admission that technology is prior art to any invention(s) in this disclosure. Neither is the “Summary” to be considered a characterization of the invention(s) set forth in issued claims. Furthermore, any reference in this disclosure to “invention” in the singular should not be used to argue that there is only a single point of novelty in this disclosure. Multiple inventions may be set forth according to the limitations of the multiple claims issuing from this disclosure, and such claims accordingly define the invention(s), and their equivalents, that are protected thereby. In all instances, the scope of such claims shall be considered on their own merits in light of this disclosure, but should not be constrained by the headings set forth herein.
[0230] All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
[0231] To aid the Patent Office, and any readers of any patent issued on this application in interpreting the claims appended hereto, applicants wish to note that they do not intend any of the appended claims to invoke paragraph 6 of 35 U.S.C. § 112, U.S.C. § 112 paragraph (f), or equivalent, as it exists on the date of filing hereof unless the words “means for” or “step for” are explicitly used in the particular claim.
[0232] For each of the claims, each dependent claim can depend both from the independent claim and from each of the prior dependent claims for each and every claim so long as the prior claim provides a proper antecedent basis for a claim term or element.
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