Dietary Supplement Comprising Beta-Glucan and Casein Hydrolysate for Improving Health and Growth Performance in a Mammal

Abstract

A non-therapeutic method of improving growth performance of a weaning mammal comprises a step of administering to the weaning mammal or a maternal mammal an effective amount of beta-glucan and casein hydrolysate, wherein the weight ratio of beta-glucan to casein hydrolysate is 1:2 to 2:1. A dietary supplement suitable for oral administration to a mammal comprising beta-glucan and casein hydrolysate is also provided, wherein the weight ratio of beta-glucan to casein hydrolysate is 1:2 to 2:1.

Claims

1. (canceled)

2. A method of improving growth performance in a weaning mammal, comprising administering to a weaning mammal or maternal mammal, a combination of beta glucan and enzymatically-derived casein hydrolysate, in which the enzymatically-derived casein hydrolysate has a degree of hydrolysis of 5-15% degree of hydrolysis (DH), in which the enzymatically-derived casein hydrolysate is depleted in high molecular weight peptides, and wherein the ratio of beta-glucan to casein hydrolysate administered to the mammal is 1:2 to 2:1.

3. The method of claim 2, wherein the combination is administered to a maternal mammal during pregnancy, lactation, or both pregnancy and lactation.

4. The method of claim 2, wherein 1-100 mg beta-glucan per day per Kg body weight and 1-100 mg casein hydrolysate per day per Kg body weight is administered to the mammal.

5. The method of claim 2, wherein 10-100 mg beta-glucan per day per Kg body weight and 10-100 mg casein hydrolysate per day per Kg body weight is administered to the mammal.

6. The method of claim 2, wherein 10-60 mg beta-glucan per day per Kg body weight and 10-60 mg casein hydrolysate per day per Kg body weight is administered to the mammal.

7. The method of claim 2, wherein the combination of beta glucan and enzymatically derived casein hydrolysate is administered in a plurality of doses over a period of at least three days.

8. The method of claim 2, wherein improving growth performance comprises lowering faecal scores.

9. The method of claim 2, wherein improving growth performance comprises increasing average daily weight gain.

10. The method of claim 2, wherein the combination of beta glucan and enzymatically derived casein hydrolysate is provided as a food product formulated for a lactating or pregnant maternal human or a weaning infant human.

11. The method of claim 10, wherein the food product comprises a composition consisting of the beta glucan and enzymatically derived casein hydrolysate and said composition comprises 0.001 to 1% (w/w) of the food product.

12. The method of claim 2, wherein the combination of beta glucan and enzymatically derived casein hydrolysate is provided as an animal feed formulated for a lactating or pregnant maternal human or a weaning infant human.

13. The method of claim 12, wherein the animal feed comprises a composition consisting of the beta glucan and enzymatically derived casein hydrolysate and said composition comprises 0.005 to 0.1% (w/w) of the animal feed.

14. The method of claim 2, wherein the beta glucan is yeast-derived beta glucan.

15. A method of improving immune status in a mammal, comprising administering to a weaning mammal or maternal mammal, a combination of beta glucan and enzymatically-derived casein hydrolysate, in which the enzymatically-derived casein hydrolysate has a degree of hydrolysis of 5-15% degree of hydrolysis (DH), in which the enzymatically-derived casein hydrolysate is depleted in high molecular weight peptides, and wherein the ratio of beta-glucan to casein hydrolysate administered to the mammal is 1:2 to 2:1.

16. The method of claim 15, wherein the combination is administered to a maternal mammal during pregnancy, lactation, or both pregnancy and lactation.

17. A method of improving or maintaining intestinal microflora in a mammal, comprising administering to a weaning mammal or maternal mammal, a combination of beta glucan and enzymatically-derived casein hydrolysate, in which the enzymatically-derived casein hydrolysate has a degree of hydrolysis of 5-15% degree of hydrolysis (DH), in which the enzymatically-derived casein hydrolysate is depleted in high molecular weight peptides, and wherein the ratio of beta-glucan to casein hydrolysate administered to the mammal is 1:2 to 2:1.

18. The method of claim 17, wherein the combination is administered to a maternal mammal during pregnancy, lactation, or both pregnancy and lactation.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The foregoing will be apparent from the following more particular description of example embodiments, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments.

[0073] FIG. 1: Effect of feed additives on copy numbers of a panel of selected bacterial groups present in caecal digesta.

[0074] FIG. 2: Effect of feed additives on relative quantity (RQ) of a panel of selected cytokine genes in porcine colonic tissues.

DETAILED DESCRIPTION

[0075] A description of example embodiments follows.

[0076] The teachings of all patents, published applications and references cited herein are incorporated by reference in their entirety.

Materials and Methods

[0077] All procedures described in this experiment were conducted under experimental license from the Irish Department of Health in accordance with the Cruelty to Animals Act 1876 and the European Communities (amendments of the Cruelty to Animals Act 1876) regulations, 1994.

Experimental Design and Dietary Treatments

[0078] This study was designed in a completely randomized block design with 5 dietary groups. The dietary groups are as follows: 1) control diet, 2) control diet+ZnO, 3) control diet+casein hydrolysate, 4) control diet+β glucan and 5) control diet+casein hydrolysate+β glucan. The control diet used in this study was based on previous work by Walsh et al. (2013). The levels of casein hydrolysate used were based on previous experiment and the levels of β glucan, derived from yeast (S. cerevisiae), used were based on a previous study by Sweeney et al. (2012). Diets were formulated to contain similar concentrations of digestible energy (DE) (14.5 MJ/kg) and standardized ileal digestible (SID) lysine (12.5 g/kg) contents. All amino acids requirements were met relative to SID lysine (NRC, 1998). All diets were milled on site and fed in meal form for 12 days after weaning. The composition and analysis of experimental diets is presented in Table 1.

TABLE-US-00001 TABLE 1 Composition of dietary treatments. casein casein hydrolysate + Ingredient (g/kg) Control ZnO hydrolysate β glucan β glucan Whey powder 50 50 50 50 50 Wheat 380 380 380 380 380 Barley 233.5 233.5 233.5 233.5 233.5 Soya bean meal 170 170 170 170 170 Full fat soybean 120 120 120 120 120 Soya oil 10 10 10 10 10 Vitamins and 3 3 3 3 3 minerals Salt 3 3 3 3 3 Dicalcium 12.5 12.5 12.5 12.5 12.5 phosphate Limestone 11 11 11 11 11 Lysine HCL 4 4 4 4 4 DL-methionine 1.5 1.5 1.5 1.5 1.5 L-threonine 1.5 1.5 1.5 1.5 1.5 Zinc Oxide 3.2 Casein 0.25 0.25 hydrolysate β glucan 0.25 0.25

Animals and Management

[0079] Forty weaned piglets [progeny of Landrace boars×(Large White×Landrace sow)] were weighed and blocked on the basis of initial live body weight (7.3±0.2 kg) and assigned to 1 of the 5 dietary treatment groups (n=8). Piglets were weighted individually at the beginning of the experiment (day 0=day of weaning), day 6 and day 12. The piglets were housed in groups of two on fully slatted floors (1.68×1.22 m). The ambient environmental temperature within the house was thermostatically controlled at 30° C. The feed was available ad libitum and water was available via nipple drinkers. Feed was available up to the final weighing and all remaining feed was weighed back for the purpose of calculating feed efficiency.

Feces Scoring

[0080] From day 0 of the experiment until day 12, fecal scores were assigned daily to individual pens. Each pen floor was observed daily and the feces were scored based on their consistency. The following scoring system was used to assign fecal scores: 1=hard feces, 2=slightly soft feces, 3=soft, partially formed feces, 4=loose, semi-liquid feces, 5=watery, mucous-like feces. This scoring system was used to indicate the presence and severity of diarrhea, as described in a previous study by Pierce et al. (2005).

Animal Sacrifice and Sample Collection

[0081] At day 12, all the pigs were sacrificed following a lethal injection of Euthatal (pentobarbitone sodium BP-Merial Animal Ltd, Sandringham House, Essex, UK) at a rate of 1 ml/1.Math.4 kg live body weight. In sterile conditions, the digestive tract was removed and tissue samples from colon were placed in sterile phosphate buffer saline (PBS) (Oxoid, Basingstoke, UK). The digesta samples were recovered aseptically from caecum and colon and transported on ice to proper storage conditions.

Extraction of Microbial DNA from Caecal and Colonic Digesta

[0082] To evaluate the effect of feed additives on the microbiota, samples of digesta contents (approximately 10 g) were recovered from the caecum and colon of each animal in sterile conditions immediately after slaughter. Digesta samples were stored in sterile containers (Sarstedt, Wexford, Ireland), placed on ice and transported to the laboratory within 2 h. Bacterial genomic DNA was extracted from digesta samples using a QIAamp DNA stool kit (Qiagen, West Sussex, UK) following manufacturer's instructions. Quantity and quality of DNA were assessed using a Nanodrop (Nanodrop, ND1000; Thermo Scientific, Wilmington, Del.).

Preparation of Standard Curves

[0083] A combined aliquot of bacterial genomic DNA from all animals were used for preparation of standard curves. Phylum, species and genus specific primers were selected to amplify a segment of the gene encoding 16s rRNA using bacterial DNA from the pooled DNA using PCR. Thermal cycling conditions used were similar to the condition described below for quantitative real-time PCR. Standard curves were subsequently generated by quantitative real-time PCR of serial dilutions of these amplicons using the same genus and species-specific primers to permit absolute quantification based on gene copy number (Lee et al., 2006). Estimation of selected bacterial groups in cecal and colonic digesta were performed using quantitative real-time PCR (ABI 7500 Fast Real-Time PCR System; Applied Biosystems Ltd., Warrington, UK). For bacterial groups, real-time PCR were performed in a final reaction volume of 20 μL containing 1 μL template DNA, 1 μL of forward and reverse primers (100 pM), 10 μL fast SYBR Green PCR Master Mix (Applied Biosystems Ltd.) and 8 μL nuclease-free water. The thermal cycling conditions involved an initial denaturation step at 95° C. for 10 min followed by 40 cycles of 95° C. for 15 s and 65° C. for 1 min. Dissociation analyses of the PCR product were performed to confirm the specificity of the resulting PCR products. The mean threshold cycle values from the triplicate of each sample were used for calculations. The dry matter (DM) of the digesta was determined after drying overnight at 103° C.

[0084] Estimates of gene copy numbers for select bacteria were log transformed and are presented as gene copy numbers per gram of DM of digesta.

RNA Extraction

[0085] Tissue samples were collected from the mesenteric side of the colon, rinsed with ice-cold sterile phosphate-buffered saline (Oxoid, Basingstoke, UK) and stripped of overlying smooth muscle. The samples were then cut into small pieces using a sterile scalpel blade and stored in 15 ml of RNAlater™ (Applied biosystems, Foster city, CA, USA) overnight followed by storage at −20° C. prior to RNA extraction. Total RNA was extracted from the colonic tissue samples using a GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich Corporation, St Louis, Mo., USA) according to the manufacturer's instructions. To eliminate possible genomic DNA contamination, total RNA samples were subjected to DNAse I (Sigma-Aldrich Corporation, St Louis, Mo., USA) treatment according to the manufacturer's instructions followed by further purification using a phenol-chloroform extraction method. The total RNA was quantified and assessed for purity using the NanoDrop-ND1000 Spectrophotometer (Thermo Fisher Scientific Inc. MA, USA). The quality of the total RNA was determined by visualizing on an ethidium bromide stained 1% agarose gel.

cDNA Synthesis

[0086] Total RNA (1 μg) was used for the synthesis of First Strand cDNA using the First Strand cDNA Synthesis Kit (Qiagen Ltd. Crawley, UK) using oligo dT primers following the manufactures instructions. After the cDNA synthesis the final volume adjusted to 120 μl with nuclease free water.

Quantitative Real-Time PCR (qPCR)

[0087] qPCR was carried out to quantify the following two sets of targets: the first set of target are a selected panel of cytokine genes; Interleukins (IL-1α, IL-1β, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-21), Interferon (IFN-γ), Tumor Necrosis Factor (TNFα), Transforming growth factor (TGF-β) and Forkhead box P3 (FOXP3). The second set of primers target the members of suppressors of cytokine signalling (SOCS) family which include SOCS1, SOCS2, SOCS3, SOCS4, SOCS5, SOCS6, SOCS7 and cytokine inducible Src Homology 2 protein (CIS). The primer efficiency was determined using a serial dilution (1:4 dilution series over 7 points) of a cDNA pool, prepared by pooling an equal quantity of cDNA from all of the experimental samples, the efficiency of all primers was between 90% to 110%. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), (32 microglobulin (B2M), Beta-actin (ACTB), Peptidylprolyl isomerase A (PPIA) and 14-3-3 protein zeta/delta (YWHAZ) were used as endogenous controls as described by Ryan et al. (2010). All primers were designed using Primer Express™ software and were synthesised by MWG Biotech (Milton Keynes, UK). This assay was carried out using 96 well fast optical plates on a 7500HT ABI Prism Sequence Detection System (PE Applied Biosystems, Foster City, Calif.) using fast SYBR Green PCR Master Mix (Applied Biosystems). All reactions were performed in triplicate in a total volume of 20 μl containing 10 μl SYBR PCR Master mix, forward and reverse primer (5 μM) (1 μl), 8 μl DEPC treated water and 1 μl of template cDNA. The thermal cycling conditions were as follows, 95° C. for 10 min, 40 cycles of 95° C. for 15s and 65° C. for 1 min. Dissociation analysis confirmed the specificity of the resulting PCR products.

Normalization of Data

[0088] Mean C.sub.t values were converted to relative quantities using the formula, Relative quantity=(PCR efficiency).sup.ΔCt, where ΔC.sub.t is the change in the C.sub.t values of the sample relative to the highest expression (minimum C.sub.t value). Relative quantities for the endogenous controls were input in to geNorm (Vandeseaomple, 2002), a normalization factor was obtained from the four most stable (M<1.5) reference genes (GAPDH, B2M, ACTB and PPIA) for both the ileum and the colon. The relative quantities for the target genes were then divided by the normalization value to give the final normalized value for each target gene in each sample.

Statistical Analysis

[0089] The current study was a complete randomised design experiment and data analysed using the general linear model procedure of the Statistical Analysis Institute (SAS, 2004). The individual pen represented the experimental unit for the performance analysis. All the data were checked initially for normality using the PROC univariate procedure in SAS (2004). The microbial counts were log transformed before statistical analysis. Probability values of less than 0.05 were used as the criterion for statistical significance. Intercorrelations between cytokines and cytokine regulatory genes was analysed in a correlation matrix using PASW 18 (SPSS for Windows, version 11.0; SPSS, Chicago, Ill.) and the Pearson's r value greater than 0.400 was considered to be positively correlated. All results are presented in the tables as least square means±standard error of the means (SEM).

Results

Fecal Scoring and Weight Gain Over a Period of 12 Days Post Weaning

[0090] The effect of dietary inclusion of ZnO, casein hydrolysate, yeast β glucan and a combination of the casein hydrolysate and β glucan on faecal scores and weight of animals is presented in Table 2. The fecal scores of piglets from ZnO, casein hydrolysate, β glucan and casein hydrolysate+β glucan dietary groups were not different compared to control diet group piglets from day 0-6. The ZnO group piglets were associated with a lower fecal scores on day 6-12 (2.0 vs 3.2±0.25, P<0.01) as well as overall fecal scores (1.9 vs 2.8±0.21, P<0.05) relative to control-diet group. Similarly, casein hydrolysate+β glucan group piglets were also associated with a lower faecal scores during day 6-12 period compared to control-diet group (2.2 vs 3.2±0.25). The casein hydrolysate and β glucan dietary treatment groups were not associated with any difference in fecal scores compared to control diet group during the overall experimental period.

[0091] The casein hydrolysate, β glucan and casein hydrolysate+β glucan dietary treatment groups were not associated with any difference in weights compared to control diet group of piglets. Only the ZnO group was associated with an increase in the weight of piglets at the end of experiment relative to the control-diet group (8.64 vs 7.49±0.36 kg, P<0.05).

TABLE-US-00002 TABLE 2 Effect of feed additives on faecal scores and weights of weaning piglets casein casein hydrolysate + Measurements Control ZnO hydrolysate β glucan β glucan SEM Sign. Faecal Day 0-6 2.6 1.9 2.3 2.3 2.0 0.35 ns Score Day 6-12 3.2 2.0.sup.b 3.0 3.4 2.2.sup.a 0.25 .sup.a P < 0.05, .sup.b P < 0.01 Overall 2.8 1.9.sup.a 2.7 2.8 2.3 0.21 .sup.a P < 0.01 Weight Weight 0-6 7.19 7.53 7.28 7.35 7.66 0.18 ns (kg) Weight 6-12 7.49 8.64.sup.a 7.63 7.47 8.13 0.36 .sup.a P < 0.05 ns: not significant, P > 0.05

Growth Performance

[0092] The effect of feed additives like ZnO, casein hydrolysate, yeast β glucan and casein hydrolysate+β glucan on measurements of growth performance parameters over a period of 12 days is presented in Table 3. The ZnO and casein hydrolysate+β glucan dietary groups were associated with an increase in the overall average daily gain relative to control-diet group (0.10, 0.10 vs 0.00±0.03 kg/day, P<0.05). The dietary groups ZnO, casein hydrolysate, β glucan and casein hydrolysate+β glucan were also associated with an increase in the overall feed intake in piglets relative to control-diet group (0.37, 0.34, 0.34, 0.36 vs 0.27±0.02 kg/day, P<0.05). The dietary groups of ZnO and casein hydrolysate+β glucan were also associated with an increase in gain to feed ratio relative to control-diet group (0.26, 0.27 vs −0.11±0.11 kg/kg, P<0.05).

TABLE-US-00003 TABLE 3 Effect of feed additives on overall average daily gain (OADG), overall feed intake (OFI) and gain to feed ratio in weaning piglets casein casein β hydrolysate + β Measurements Control ZnO hydrolysate glucan glucan SEM Significance OADG (kg/day) 0.00 0.10* 0.02 0.00 0.10* 0.03 * P < 0.05 (Day 0-12) OFI (kg/day) 0.27 0.37* 0.34* 0.34* 0.36* 0.02 * P < 0.05 (Day 0-12) Gain to feed (kg/kg) −0.10 0.26* 0.05 −0.03 0.27* 0.11 * P < 0.05 (Day 0-12)

Microbiology

[0093] The effect of ZnO, casein hydrolysate, yeast β glucan and casein hydrolysate+β glucan dietary treatment groups on the abundance of a selected panel of bacterial groups present in caecal and colonic digesta is presented in FIG. 1.

[0094] The casein hydrolysate dietary treatment group was associated with an increase in the abundance of total bacteria copy numbers compared to control-diet group (12.83 vs 12.40±0.17 log.sub.10 bacteria/g DM digesta, P<0.05) in caecal digesta. The ZnO and casein hydrolysate dietary treatment groups were associated with an increase in abundance of Bacteroidetes copy numbers compared to control-diet group (12.60, 12.56 vs 11.71±0.27 log.sub.10 bacteria/g DM digesta, P<0.05).

[0095] However, ZnO and β glucan treatment groups were also associated with a reduction in Bifidobacteria spp. numbers compared to control-diet group (8.94, 8.99 vs 9.37±0.11 log.sub.10 bacteria/g DM digesta, P<0.05). The β glucan group was associated with an increase in AEEC strains compared to control-diet receiving group (10.49 vs 9.32±0.22 log.sub.10 bacteria/g DM digesta, P<0.01). There was no effect on abundance of Firmicutes, Lactobacilli and Enterobacteria copy numbers observed in this experiment.

[0096] In colonic digesta, the ZnO, β glucan and casein hydrolysate+β glucan dietary treatment groups were associated with a reduction in abundance of Bifidobacteria copy numbers relative to control-diet group (8.96, 8.87, 8.95 vs 9.45±0.17 log.sub.10 bacteria/g DM digesta, P<0.05). No change in bacterial copy numbers of any other bacterial group was associated with the dietary treatment groups of ZnO, casein hydrolysate, β glucan and casein hydrolysate+β glucan.

Cytokine Gene Expression Analysis

[0097] The effect of dietary treatment groups of ZnO, casein hydrolysate, yeast β glucan and casein hydrolysate glucan on expression of a selected panel of cytokine genes in colonic tissues is presented in FIG. 2. The ZnO dietary treatment group was associated with a decrease in IL-1α (0.006 vs 0.016±0.002 RQ), IL-1β (0.010 vs 0.021±0.003 RQ), IL-8 (0.003 vs 0.010±0.001 RQ) and IL-17 (0.061 vs 0.168±0.024 RQ) expression compared to control group. While, ZnO treatment group was also associated with an increase in TGF-β expression (0.183 vs 0.110±0.022 RQ) compared to control group.

[0098] The casein hydrolysate group was associated with a decrease in IL-1α expression relative to control-diet group (0.005 vs 0.016±0.002 RQ). The dietary β glucan treatment group was associated with an increase in IL-6 expression relative to control diet group (0.235 vs 0.078±0.025 RQ). The casein hydrolysate +β glucan treatment group was associated with a decrease in IL-1α (0.007 vs 0.016±0.002 RQ) expression compared to control group. IL-4, IL-21 and FOXP3 expression was un-detectable in this study.

[0099] While example embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the embodiments encompassed by the appended claims.