Food and/or feed compositions for preventing and treating inflammatory diseases

Abstract

This invention relates generally to compositions and methods for combating inflammatory disease and particularly to the use of food and/or feed compositions for preventing, reducing and/or treating inflammatory disorders, diseases, or discomforts, wherein the compositions comprising at least one specific pectin.

Claims

1. A method to ameliorate or to lessen immune-mediated diseases or inflammatory diseases caused by activation of TLR2, wherein the method comprises administering by ingestion to a patient in need thereof a mixture of esterified pectins comprising an effective amount between 0.01 and 5 g per kg body weight of the patient of a low esterified pectin having a degree of esterification (DE) of less than 55% obtained from de-esterification of methylated pectin, wherein the patient is an animal selected from the group consisting of human, avian, bovine, canine, equine, galline, feline, hircine, lapine, musteline, ovine, piscine, porcine and vulpine animals.

2. The method according to claim 1, wherein the DE of the low esterified pectin is less than 50%.

3. The method according to claim 1, wherein the DE of the low esterified pectin is at least 1%.

4. The method according to claim 1, wherein the DE of the low esterified pectin is at least 2%.

5. The method according to claim 1, wherein the DE of the low esterified pectin is at least 3%.

6. The method according to claim 1, wherein the administering is practiced by feeding a food or feed formulation which comprises the mixture of esterified pectins to the patient.

7. The method according to claim 1, wherein the DE of the low esterified pectin is less than 33%.

8. The method according to claim 1, wherein the DE of the low esterified pectin is less than 22%.

9. The method according to claim 7, wherein the DE of the low esterified pectin is at least 1%.

10. The method according to claim 8, wherein the DE of the low esterified pectin is at least 1%.

Description

EXAMPLES

Example 1

Pectin Inhibition of TLR2-Mediated NFkB Activation

(1) Cell Lines and Cultivation

(2) Cell lines were cultured in DMEM culture media (Lonza, Basel, Switzerland) with 10% decomplemented Fetal Calf serum, 50 U/ml Penicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma, St. Louis, Mo., USA) and 100 μg/ml Normocin (InvivoGen, Toulouse, France).

(3) HEK-Blue™ TLR2-CD14 cells (InvivoGen, Toulouse, France) expressing human TLR2 and SEAP (Soluble Embryonic Alkaline Phosphatase) were used. NFκB and AP1 are stimulated to move to the nucleus in these cell lines when TLR2 is activated by an agonist. Here, the SEAP gene is under the control of a NFκB/AP-1 responsive promoter. Upon expression, the SEAP gene product is secreted in media and can be quantified using the Quantiblue (InvivoGen, Toulouse, France) solution. The enzyme converts the pink color into blue depending on the activity, and this can be measured with a spectrophotometer.

(4) 1× HEK-Blue™ Selection (InvivoGen, Toulouse, France) was added to the cultivation medium to control only the growth of the HEK-Blue™ TLR2-CD14 cells.

(5) Pectin Sources

(6) All pectins are isolated from citrus. Pectin with a DE of 0 was obtained from MP Biomedicals, LLC. The pectins with a DE of 7, 22, 45, 60 and 75 were obtained from CP Kelko.

(7) Pectin Inhibition TLR2-1

(8) HEK-Blue™ TLR2-CD14 cells were activated with the TLR2-1 specific agonist Pam3CSK4. Pectins of different DE values (0, 7, 22, 45, 60 and 75) were added at 0.5, 1 and 2 mg/ml. As a control HEK-Blue™ TLR2-CD14 cells were incubated with only pectin.

(9) HEK-Blue™ TLR2-CD14 cells were seeded at 500,000 cell/ml in a 96 well plate with 100 μl volume per well. Cells were allowed to grow overnight. The following day, cells were treated with different pectins at different concentrations to study the effect on TLR2. After one hour of incubation with pectin, Pam3CSK4 was added at a concentration of 100 ng/ml. After 24 hrs of incubation at 37° C. with pectin and Pam3CSK4, the expression of the SEAP gene was determined. Supernatant of incubated cells was mixed with QUANTI-Blue solution in a ratio of 1:10. Presence of SEAP makes QUANTI-Blue turn blue. The NFκB activation was quantified by measuring the color intensity at 650 nm using an ELISA plate reader Versa Max (Molecular devices, Sunnyvale, Calif., USA). The assay was performed in 96 well plates with 8 technical repeats. Each experiment was repeated three times.

(10) TABLE-US-00001 NFκB activation through TLR2-1 by Pam3CSK4 with and without pectin Control 0.5 mg/ml 1 mg/ml 2 mg/ml (no Pam3CSK4) Pectin NFκB % NFκB % NFκB % NFκB type activation inhibition activation inhibition activation inhibition activation — 1.89 0 1.89 0 1.89 0 n.a.  0DE 1.22 35.51 1.27 32.59 1.23 35.13 0.01  7DE 0.99 47.64 0.77 59.15 0.81 57.05 0.15 22DE 1.62 14.00 1.20 36.67 0.57 69.64 0.04 45DE 1.75 7.33 1.32 30.12 0.64 66.22 0.03 60DE 1.95 0 1.56 17.36 0.88 53.22 0.05 75DE 1.96 0 1.88 0.52 0.85 54.93 0.13

(11) Surprisingly, pectins are inhibiting TLR2, as determined by the color change of Quanti-blue at 650 nm through the level of SEAP activity after NFκB activation of the SEAP gene expression. The inhibition was highest with pectins with a low DE, but the inhibition was found with all DE values depending of the concentration of the respective pectin. The inhibiting effect of pectins with higher DE values (45, 60 and 75) was clearly concentration dependent, suggesting that at higher concentrations the absolute number of intra- or intermolecular regions with a local DE<65% (due to the possible irregular or heterogeneous distribution of esterified GalA residues throughout the pectin molecules and/or sample) becomes higher and therefore the inhibition of TLR2—through those regions with DE lower than 65—increases.

(12) The viability of the HEK-Blue™ TLR2 CD14 was not affected by the additions of pectin (tested using WST-1 reagent).

Example 2

Pectin Binds to TLR2

(13) Construction of the TLR2Ectodomain-HA Expression Plasmid

(14) RNA was extracted from HEK-Blue™ hTLR2-CD14 cells using the RNeasy®Plus Mini kit (Qiagen, Venlo, Netherlands). cDNA was synthesized using OligodT primers (Life technologies, Carlsbad, Calif., USA), dNTP mix (Life technologies, Carlsbad, Calif., USA) and Superscript™III Reverse Transcriptase (Life technologies, Carlsbad, Calif., USA) according to the suppliers' manuals. TLR2 from codon1 to codon586 was synthesized using cDNA from HEK-Blue™ hTLR2-CD14 using the forward primer 5′-GCGCACCGGTATGCCACATACTTTGTGGATGG-3′ (SEQ ID NO: 1), the reverse primer 5′-GCGCGGATCCGTGACATTCCGACACCGAGAG-3′ (SEQ ID NO: 2) and Pfu DNA polymerase (Thermo scientific, Waltham, Mass. USA). Primers were flanked by a GC doublet at the 5′ end for restriction enzyme recognition. AgeI and BamHI restriction sites were included in the forward and reverse primer, respectively. The PCR product was digested with AgeI and BamHI restriction enzymes (Thermo scientific, Waltham, Mass. USA) and the plasmid pSELECT-CHA-blasti (InvivoGen, Toulouse, France) to create sticky ends. The PCR-amplified TLR2 ectodomain fragment and linear plasmid were ligated using T4 DNA Ligase (Thermo scientific, Waltham, Mass. USA). The ligated plasmid was used to transform One Shot TOP10 Chemically Competent E. coli (Life technologies, Carlsbad, Calif., USA). Transformed E. coli cells were selected using Blasticidin agar media (InvivoGen, Toulouse, France). Obtained colonies were screened for correct orientation of the gene in the plasmid. Selected correct colonies were grown in blasticidin liquid media (InvivoGen, Toulouse, France) and the plasmid was isolated using the Qiagen Midi prep kit. Plasmid was then sequenced for selection of non-mutated clones (Baseclear, Leiden, Netherlands).

(15) Transfection of HEK293T With the TLR2Ectodomain-HA Expression Fragment

(16) HEK293T cells were seeded at 500,000 cells/ml in 12 well culture plates and incubated overnight. The following day, transfection was performed by using Lipofectamine LTX® (Life technologies, Carlsbad, Calif., USA). The sequence verified plasmid was linearized with the restriction enzyme NotI (Fast digest, Thermo Scientific, Waltham, Mass. USA). Purified, 1 μg linear plasmid was diluted in low serum media Opti-MEM® (Life technologies, Carlsbad, Calif., USA) and mixed with 3.5 μl of Lipofectamine LTX® (Life technologies, Carlsbad, Calif., USA). This transfection mix was incubated for 30 min at room temperature and then added to the previously seeded cells in the culture media. Cells were incubated with transfection medium mix for 24 hrs and transfected cells were selected using blasticidin in DMEM culture media (Lonza, Basel, Switzerland) with 10% decomplemented fetal calf serum, 50 U/ml Penicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma, St. Louis, Mo., USA) and 100 μg/ml Normocin (InvivoGen, Toulouse, France). Single cell clones were isolated to form the HEK293T TLR2ectodomain-HA cell line.

(17) Cell Cultivation

(18) Cell lines were cultured in DMEM culture media (Lonza, Basel, Switzerland) with 10% decomplemented Fetal Calf serum, 50 U/ml Penicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma, St. Louis, Mo., USA), 100 μg/ml Normocin (InvivoGen, Toulouse, France) and 50 μg/ml blasticidin (InvivoGen, Toulouse, France).

(19) Protein Immunoprecipitation

(20) Overnight grown HEK293T TLR2ectodomain-HA cells were lysed using 1× RIPA lysis buffer (Merck Millipore, Billerica, Mass., USA) in the presence of a protease inhibitor cocktail consisting of AEB SF (4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride), Aprotinin, Bestatin, E-64, EDTA and Leupeptin (Sigma, St. Louis, Mo., USA) at 4° C. for 10 min followed by two times sonification for 5 seconds at 0% power. Supernatant was isolated after centrifugation at 14000 g for 10 min. TLR2ectodomain-HA tagged protein was immunoprecipitated using Pierce® Anti-HA Agarose (Thermo scientific, Waltham, Mass. USA) in a micro centrifuge tube. Protein was competitively eluted using HA synthetic peptide (Thermo scientific, Waltham, Mass. USA) by incubating two times for 15 min with a single bed volume of HA synthetic peptide at 30 C. Isolated protein was desalted and HA peptide was removed using Zeba Spin Desalting Columns and Devices, 40K MWCO (Thermo scientific, Waltham, Mass. USA). Isolated and desalted protein was quantified using Thermo scientific BCA protein assay kit.

(21) ELISA for Binding of TLR2 and Pectin

(22) The ELISA buffer was composed of 1 mM CaCl2 and 150 mM NaCl in 0.05M Tris buffer at pH 8.2. The buffer was used for washing as well as diluent for antibodies and pectin. The blocking buffer was made by dissolving 3% milk powder (FrieslandCampina, Amersfoort, The Netherlands) in ELISA buffer. For antibody solutions, a 1:2 dilution of blocking buffer with ELISA buffer was used for dissolving antibodies. ELISA plates (Corning, Tewksbury, Mass., USA) were treated with 50 μl of 50 μg/ml of Poly-L lysine for 1 hour at 37 C. Wells were washed once with 400 μl ELISA buffer. Pectins (0, 7, 22, 45 60 and 75DE) were dissolved in ELISA buffer at 1 mg/ml concentration and 50 μl was added to each well. The plates were incubated for 4 hours at 37 C to allow for binding of the pectin. Each well was then washed with 400 μl ELISA buffer and blocked overnight with 100 μl of blocking buffer at 4 C. After the blocking step, the ELISA plate was washed once with ELISA buffer. TLR2 ectodomain-HA fusion protein was applied to the pectin coated wells at 0.33 μg, 1 μg, 3 μg and 9 μg concentration per well. The ELISA plates were then incubated at 37 C for 3 hrs. The HA synthetic peptide was used as a negative control at 0.33 μg, 1 μg, 3 μg and 9 μg concentration per well, same as with the TLR2 ectodomaian-HA fusion protein. Pectin binding antibodies LM19 (specific for DE 0 and 7) and LM20 (specific for DE 22, 45, 60 and 75) from Plantprobes (Leeds, UK) were used as positive control for pectin binding at 1:100 dilutions. Afterwards, the wells were washed with 400 μl of ELISA buffer for 5 times and incubated with 50 μl primary detection antibody for the HA tag (Cell Signaling, Danvers, Mass., USA) in a 1:200 dilution. The primary antibody was incubated for 2 hours at 37 C. After primary antibody incubation, the plates were washed again 5 times with ELISA buffer. After washing, 50 μl of Biotin tagged secondary antibody (Southern Biotech, Birmingham, Ala., USA) was applied to each well at a 1:500 dilution. The Biotin tagged antibody was incubated for 1 hr at 37 C. This step was followed by 5 washings with 400 μl of ELISA buffer. Streptavidin-HRP (Dako, Heverlee, Belgium) (100 μl) was added to each well at a 1:1000 dilution. After incubation at 37 C for 1 hr, plates were washed 7 times with 400 μl of ELISA buffer. Finally, for detection, 100 μl of TMB substrate (Cell Signaling, Danvers, Mass., USA) was applied to each well and incubated at 37 C for 30 min. The reactions were stopped by adding 100 μl of stop solution (Cell Signaling, Danvers, Mass., USA). The ELISA plate was read at 420 nm in Plate reader Versa Max (Molecular devices, Sunnyvale, Calif., USA). For clarity sake the values of the negative control are subtracted from the values obtained with TLR2 ectodomain-HA.

(23) TABLE-US-00002 TLR2 ectodomain binding to pectin (a.u.) 0.33 μg 1 μg 3 μg 9 μg Pectin type protein protein protein protein 0DE 0.03 0.15 0.45 1.43 7DE 0.45 0.98 2.41 2.34 22DE 0.03 0.16 0.49 0.81 45DE 0.13 0.25 0.58 0.26 60DE 0.07 0.09 0.16 0.07 75DE 0.00 0.03 0.06 0.03

(24) Surprisingly, pectins with low DE are directly binding to the TLR2 ectodomain, as indicated by the dose-dependent values obtained in the ELISA test.

Example 3

Pectin Inhibits Only the Pro-Inflammatory Pathway of TLR2 and Not Its Regulatory Pathway

(25) Cell Line

(26) HEK-Blue™ Null1 (InvivoGen, Toulouse, France) was seeded at 500,000 cells/ml in 12 well culture plates and incubated overnight. The following day, transfection was performed by using Lipofectamine LTX® (Life technologies, Carlsbad, Calif., USA). Plasmid pUNO3-hTLR2 (InvivoGen, Toulouse, France) was linearized with NotI (Thermo scientific, Waltham, Mass. USA). One μg of purified DNA was diluted in low serum media Opti-MEM® (Life technologies, Carlsbad, Calif., USA) and mixed with 3.5 μl of Lipofectamine LTX® (Life technologies, Carlsbad, Calif., USA). This transfection mix was incubated for 30 min at room temperature and then added to the previously seeded cells in the culture media. Cells were incubated with transfection medium mix for 24 hrs and transfected cells were selected using Zeocin (100 μg/ml) and Hygromycin B (150 μg/ml) in DMEM culture media (Lonza, Basel, Switzerland) with 10% decomplemented fetal calf serum, 50 U/ml Penicillin (Sigma, St. Louis, Mo., USA), 50 μg/ml Streptomycin (Sigma, St. Louis, Mo., USA) and 100 μg/ml Normocin (InvivoGen, Toulouse, France). Single cell clones were isolated. The thus obtained HEK-Blue™ Null1 TLR2 cell line expresses only TLR2 and not CD14.

(27) TLR2 Activation and Inhibition Assay

(28) HEK-Blue™ Null1 TLR2 cells were seeded at 500,000 cell/ml in a 96 well plate with 100 μl volume per well. Cells were allowed to grow overnight. The following day, cells were treated with different pectins to study the effect on TLR2. After one hour of incubation with pectin, the TLR2-6 specific agonist FSL-1 was added at a concentration of 100 ng/ml. After 24 hrs of incubation at 37 C with pectin and FSL-1, the expression of the SEAP gene was determined. Supernatant of incubated cells was mixed with QUANTI-Blue solution in a ratio of 1:10. Presence of SEAP makes QUANTI-Blue turn blue. The NFκB activation was quantified by measuring colorimetric readings at 650 nm using ELISA plate reader Versa Max (Molecular devices, Sunnyvale, Calif., USA). The assay was performed in 96 well plates with 10 technical repeats. Each experiment was repeated three times.

(29) TABLE-US-00003 NFκB activation through TLR2-6 by FSL-1 with and without pectin 0.5 mg/ml 1 mg/ml 2 mg/ml Control (no FSL-1) Pectin NFκB NFκB NFκB NFκB type activation activation activation activation — 1.14 1.14 1.14 n.a. 0DE 1.17 1.18 1.15 0 7DE 1.05 1.05 1.08 0 22DE 1.10 1.12 1.02 0 45DE 1.13 1.11 1.03 0 60DE 1.13 1.17 1.17 0 75DE 1.13 1.13 1.20 0

(30) The data shows that pectin does not inhibit the TLR2-6 signaling pathway as the stimulus in HEK-Blue™ Null1 TLR2 cells through FSL-1 is not changed by the presence of pectins, whereas the stimulation by Pam3CSK4 was clearly showing that pectin inhibits the TLR2-1 signaling response (example 1).

Example 4

Pectin in the Diet of Piglets Leads to a Reduced Intestinal Permeability of the Tight Junctions

(31) Piglet Feeding Trial Set-Up

(32) The experimental farm for young piglets is located in Flanders (Belgium) and consists of 8 batteries, each containing 4 pens. The piglets under study are hybrids of Topigs Piétrains and are weaned at 21 days. The piglets are weighed individually at weaning and 2 and 4 weeks after weaning. Feed intake is registered per pen of 4 piglets at the moments of weighing. At arrival the piglets are earmarked with a new Sanitel-number. During the trial, a veterinarian and a Felasa D certified person supervise the performed piglet experiment according to the international guidelines described in law EC/86/609.

(33) Each pen (1.5 m×1.5 m) contains 4 piglets at the start of the trial. For each pen, one feeder (ad libitum) is installed for meal or pellets. One drinking nipple is installed per pen. The temperature at start is at 28±2° C. until 10 days after weaning. Afterwards, temperature is decreased to 25±2° C.

(34) Commercial non-medicated diets are given. Non-medicated means that the piglet doesn't receive any therapeutic antibiotics before and during the trial. The diets are given in the form of meal. All feed were analysed for their nutritional content.

(35) Four treatments were applied (diets A, B, C, D) on 7 replicates with 4 piglets per group. At the start of the trial, the piglets (around 7 kg body weight) are allocated to the different pens by weight. This allocation is made in order to have an equal average weight and an equal standard deviation around the average weight for each treatment and pen. For microbiological counting's and for taking biopsis, piglets receive an overdose of barbiturates (Nembutal) followed by sacrification. Afterwards, a section is performed on the piglets. Samples for microbial counts are immediately processed, while samples taken for histochemical experiments were fixed for later analysis. During the whole trial period the piglets are fed ad libitum, except for the period of microbiological countings. At that moment, three days before the microbiological countings are performed, the piglets are fed restricted. Piglets receive three times a day an amount of feed, which is carefully weighed and noted. The feed is given at 8.00, 13.00 and 18.00. When necessary, the sick piglets were treated individually (by injection). The following parameters were taken into account. (i) individual growth data, (ii) feed intake data per pen (corrected for eventual losses), (iii) feed conversion ratio during weaning, starter and whole trial period, (iv) fecal score and clinical score, (v) tight Junctions, (vi) microbial analysis, (vii) histochemical analysis.

(36) Diets

(37) Feed Compositions (in g/kg):

(38) TABLE-US-00004 Ingredient Feed A Feed B Feed C Feed D Corn 171.13 169.13 169.13 169.13 Grains (wheat and barley) 491.83 491.83 491.83 491.83 Protein sources (soy, potato) 227.72 227.72 227.72 227.72 Milk derivatives (whey) 52.65 52.65 52.65 52.65 Soy bean oil 14.19 14.19 14.19 14.19 Amino acids 10.80 10.80 10.80 10.80 Minerals & trace minerals 10.24 10.24 10.24 10.24 Limestone 10.63 10.63 10.63 10.63 Enzyme* 0.64 0.64 0.64 0.64 Premix** 10.17 10.17 10.17 10.17 Pectin DE33 — 2.00 — — Pectin DE55 — — 2.00 — Pectin soybean meal — — — 2.00 *Xylanase/beta-glucanase and phytase cocktail (BASF) **Premix includes aroma's, extra trace minerals, vitamins (Vitamex N.V.)

(39) Pectin Sources

(40) Pectins with a DE 33 and 55 were isolated from citrus and obtained from Herbstreith & Fox (Neuenbürg/Württingen, Germany). The Soy Bean Meal (SBM) was from South-American origin (mixture from Argentina, Brasil and/or Paraguay) and processed to extract the residing pectins by mixing the SBM at 33% dry matter with tapwater and autoclaving for 30 mins at 120 C. After cooling the obtained material was freeze dried and milled, and used as such in the diet.

(41) Mannitol-Lactulose Test

(42) The permeability of the ileum was quantified by performing a mannitol-lactulose test in the animals. Lactulose cannot pass an integer small bowel which is considered to be positive as there is a lower chance on systemic infections and immune issues.

(43) Mannitol is a metabolically inert monosaccharide, which is passively absorbed through the intestinal mucosa. Any absorbed mannitol is fully excreted in the urine within a couple of hours. Mannitol was administered to the piglets through a stomach-pump at 0.3 g mannitol/kg body weight (4 hours before dissection). Lactulose is a metabolically inert disaccharide, which normally is not absorbed unless the mucosal barrier is compromised. Any absorbed lactulose is fully excreted in the urine within 6 hours. Lactulose was administered to the piglets through a stomach-pump at 0.75 g lactulose/kg body weight (4 hours before dissection)

(44) During dissection piglet urine was then collected. In piglets with a healthy intestine, the mean absorption of lactulose is less than 1% of the administered dose. A recovery of >1% lactulose in the urine indicates a disaccharide hyperpermeability.

(45) In piglets with a healthy intestine, the mean absorption of mannitol is >14% of the administered dose.

(46) A recovery of <14% mannitol in the urine indicates a carbohydrate malabsorption.

(47) A lower lactulose/mannitol ratio (L/M ratio) indicates a positive effect of a diet.

(48) TABLE-US-00005 Permeability test of the gastrointestinal tract (average of three individual pigs per diet) Diet L/M ratio SD A 0.55 0.90 B 0.019 0.032 C 0.024 0.042 D 0.15 0.27

(49) As shown all pectins, and especially the pectins with DE 33 and 55 pectin, lead to a reduced L/M ratio and thus have a positive effect in the small intestine of the young pigs

Example 5

Pectin in Diets Lead to an Increase of the Villus to Crypt Ratio

(50) The piglet feeding trial set-up was as described in example 4.

(51) Sample Preparation for Histology

(52) Sampling: Take a sample of the duodenum and/or ileum and rinse with physiological water (0.9% NaCl). Store in 20 ml formalin buffer (1 ml formaldehyde (37%)/liter). 4.5 g NaH.sub.2PO.sub.4+10.4 g Na.sub.2HPO.sub.4)

(53) Bedding: Poor the paraffin solution in the recipient (not fully full) and place the intestinal sample vertical in the recipient. Let solidify at 4° C. and fill the recipient completely. Let solidify at −3° C.

(54) Biopt: Clean scalpel with 10% xylene and let dry. Transfer coupe with brush and needle to 50% alcohol. Cut in pieces and transfer to distilled water (65° C.) with brush and microscope slide. Place sample on the microscope slide and incubate one night at 60° C.

(55) Haematoxiline-eosine (H&E) staining steps at ambient temperature: 1). Deparaffinize Wash 3 times with 10% xylene (for 5 minutes) Wash 2 times with 100% ethanol (for 3 minutes) Wash once with ethanol 90% (for 3 minutes) Wash once with ethanol 70% (for 3 minutes) Wash once with water (for 3 minutes) Wash once with water (for 3 minutes) 2). staining Incubate in Mayers haematoxiline (for 6 minutes) Wash once with water (for 5 minutes) 3). counter-staining Incubate in 10% eosine (for 5 minutes) Wash 10 times with water (each for 30 seconds) 4). Dehydrate Wash 10 times with ethanol 90% (each for 30 seconds) Wash 10 times with ethanol 70% (each for 30 seconds) Wash 2 times with ethanol 100% (for 5 minutes) Wash 3 times with 10% xylene (for 5 minutes)

(56) Quantification of the Villi Length and Crypt Depth

(57) Above embedded histological samples are analysed by means of an Olympus microscope, and villi length (mm) and crypt (depth) are measured.

(58) TABLE-US-00006 Histological morphology of the gastrointestinal tract (average of 3 pigs per diet) Diet SI Crypt (mm) Villus (mm) V/C ratio A Duodenum 23.5 ± 3.3 36.6 ± 7.3 1.6 ± 0.3 Ileum 15.0 ± 0.8 22.0 ± 3.0 1.5 ± 0.2 B Duodenum 23.7 ± 4.7 40.5 ± 3.5 1.8 ± 0.3 Ileum 15.2 ± 0.5 25.1 ± 0.7 1.7 ± 0.1 C Duodenum 23.4 ± 1.9 42.5 ± 8.1 1.8 ± 0.4 Ileum 15.3 ± 1.2 26.7 ± 4.1 1.8 ± 0.1 D Duodenum 23.8 ± 2.7 37.0 ± 3.9 1.6 ± 0.1 Ileum 15.2 ± 2.6 21.5 ± 1.7 1.5 ± 0.4

(59) Longer villi imply a higher absorption capacity while longer crypts imply the opposite. Thus a higher villus to crypt ratio is considered to be a positive effect of a diet, while a lower villus to crypt ratio is considered to be a negative drawback for using the respective diet. As shown, especially the diets with pectin DE 33 and 55 have an enhanced villi-to-crypt ratio in both the duodenum and ileum, which demonstrate the potential of both pectins to improve gastrointestinal health.

Example 6

Pectin Acts as Anti-Inflammatory Agent in Doxorubicin Induced Mucositis

(60) Mucositis, also referred to as mucosal barrier injury, is one of the most severe side effects of radiotherapy and chemotherapy treatment. Both inflammation and apoptosis of the mucosal barrier result in its discontinuity, thereby promoting bacterial translocation. Five phases are important in the pathophysiology of mucositis: (1) the formation of reactive oxygen species leading to the activation of nuclear factor kappa B (NFkB) during the initiation phase, (2) the induction of messenger molecules such as tumor necrosis factor alpha (TNFa), resulting in treatment-related tissue inflammation and apoptosis during the upregulation/message generation phase, (3) the amplification of messenger molecules in the amplification/signaling phase, leading to more inflammation and apoptosis, (4) discontinuity of the epithelial barrier resulting from apoptosis during the ulcerative phase, thereby promoting bacterial translocation, and (5) a spontaneous healing phase, characterized by cell proliferation (Sonis, 2004, Semin Oncol Nurs. 20(1):11-5); Van Vliet et al, 2010, PLoS Pathog. 6(5):e1000879).

(61) Mice

(62) C57B1L/6 female mice (7-10 weeks old) were purchased from Janvier laboratories, France. The experimental use of animals was approved by the Animal Ethical Committee of the University of Groningen. All the mice were acclimatized for 2.5 weeks prior to start of the experiment. Mucositis was induced by administration of doxorubicin (Sigma, St. Louis, Mo., USA).

(63) DietsMice were supplied with ad-libitum RMH-B diet (AB diets, Woerden, The Netherlands). The ingredients of diet specified by supplier are wheat, meat meal (80% sterilized), yellow dent corn, whole oats, wheat middlings, alfalfa, soya oil, dried yeast, dicalcium phosphate, calcium carbonate, NaCl, dl-methionine, vitamins and trace elements. Mice were supplied with drinking water from tap and the water bottles were changed once a week.

(64) Pectin Sources

(65) The pectin with a DE of 7 was obtained from CP Kelko.

(66) Induction of Mucositis and Readouts

(67) Doxorubicin was dissolved in sterile 0.9% sodium chloride and stored in aliquots at 4 C. Pectin (7DM) was dissolved in sterile water and administered by gavage to mice for 10 or 11 days, twice a day at 3 mg/day. On day 8, doxorubicin was injected intra-peritoneal at 10 mg/kg concentration. Mice were sacrificed on day 10 (48 hour doxorubicin) or day 11 (72 hour doxorubicin). Animals receiving water by gavage served as controls. After the collection of tissue samples, mice were sacrificed by cervical dislocation.

(68) TLR2 Blocking in Mice

(69) TLR2 blocking antibody, clone T2.5 (InvivoGen, Toulouse, France) was administered IP at 10 mg/kg one hour prior to doxorubicin treatment.

(70) Neutrophil Count

(71) Peritoneal lavage was collected with 2 ml PBS to collect peritoneal neutrophil influx. The total number of living cells in the peritoneal lavage was counted using a Z™ Series coulter Counter® (Beckman Coulter, Brea, Calif., USA). The lavage was diluted at 500,000 cells/ml and 100 μl of cell solution was applied for cytospin preparation. The cytospin slides were stained with Giemsa stain (Merck Millipore, Billerica, Mass., USA) for 1 hour at room temperature. The stained slides were scanned in a Hamamatsu slide scanner (Hamamatsu photonics, Japan) and neutrophils were counted using morphological features in 250 cells. The total number of neutrophils were calculated using the total cell count in peritoneum and the neutrophoil counts from cytospin preparations.

(72) TABLE-US-00007 Effect of 48 hour doxorubicin treatment on neutrophil count Relative increase in Mice groups Total number of neutrophils neutrophils Control (n = 6) 3.97E+06 ± 2.39E+06 1.0 Pectin DE7 (n = 6) 2.97E+06 ± 1.29E+06 0.7 Doxorubicin (n = 6) 2.23E+08 ± 6.74E+07 56.2 Pectin DE7 + Doxorubicin 5.44E+07 ± 2.30E+07 13.7 (n = 5) TLR2 blocking + Doxorubicin 7.04E+07 ± 7.30E+07 17.7 (n = 6) TLR2 blocking (n = 5) 8.96E+06 ± 7.90E+06 2.3

(73) As can be seen from the data, doxorubicin induced mucositis increases the neutrophil count by 56 times. Blocking TLR2 either by the TLR2 blocking antibody clone T2.5 or pectin DE7 significantly reduced the neutrophil count, demonstrating that pectin acts as an anti-inflammatory agent.

Example 7

Pectin Has a Positive Effect on Health Beneficial Microbes

(74) Digesta Collection

(75) The pig fecal samples were collected on day 14 and 28 during the experimental diet feeding (example 4). After the fecal collection period, animals were anesthetized and euthanized. Digesta samples were collected from terminal ileum, proximal colon, mid colon and distal colon. Part of each digesta was stored in 1.5 mL Eppendorf tubes for analysis of microbiota composition and SCFA. These tubes were immediately frozen in liquid nitrogen and stored at −80° C. The remaining amount of digesta was immediately stored at −20° C. until further analysis.

(76) DNA Extraction and Microbiota Analysis

(77) Microbial DNA was extracted from 250 mg of digesta by using a fecal DNA extraction protocol (Salonen A, Nikkilä J, Jalanka-Tuovinen J, Immonen O, Rajilié-Stojanovié M, Kekkonen R A, Palva A & de Vos W M. 2010. Comparative analysis of fecal DNA extraction methods with phylogenetic microarray: Effective recovery of bacterial and archaeal DNA using mechanical cell lysis. Journal of Microbiological Methods, 81: 127-134). The DNA is isolated by sequential precipitations and finally purified by using the QIAamp DNA Stool Mini Kit columns (Qiagen, Hilden, Germany) according to the manufacturer's recommendations. 16S rRNA gene was amplified and sequenced in paired-end mode by using the MiSeq platform (Illumina).

(78) Sequence Analysis

(79) Raw Illumina fastq files were demultiplexed, quality-filtered and analyed using QIIME 1.9.0.

(80) TABLE-US-00008 Relative abundance of Prevotella species in the microbiota compostion of experimental fed pigs Relative Relative Diet Pectin Fecal sample abundance* increase** A control Terminal ileum 0 — A control Proximal colon 0.05 1   A control Mid colon 0.11 1   A control Distal colon 0.09 1   B DE 33 Terminal ileum 0 — B DE 33 Proximal colon 0.51 9.99 B DE 33 Mid colon 0.58 5.49 B DE 33 Distal colon 0.55 6.16 C DE 55 Terminal ileum 0 — C DE 55 Proximal colon 0.49 9.55 C DE 55 Mid colon 0.41 3.90 C DE 55 Distal colon 0.45 5.07 D Soy Bean Terminal ileum 0 — D Soy Bean Proximal colon 0.21 4.04 D Soy Bean Mid colon 0.22 2.09 D Soy Bean Distal colon 0.30 3.36 *The relative abundance is the % of 16S rRNA data of Prevotella in the total data set obtained through Illumina sequencing **The relative increase is the fold increase in % of Prevotella 16S rRNA in the total data set. i.e. the relative abundance determined for a specific pectin fed sample divided by the control sample.

(81) Surprisingly, addition of pectin to the diet leads to an increased prevalence of Prevotella species in the gut (which is an indicator of health, see Wu G D, Chen J, Hoffmann C, Bittinger K, Chen Y Y, Keilbaugh S A, Bewtra M, Knights D, Walters W A, Knight R, Sinha R, Gilroy E, Gupta K, Baldassano R, Nessel L, Li H, Bushman F D & Lewis J D. 2011. Linking long-term dietary patterns with gut microbial enterotypes. Science 334:105-108).

(82) All these examples clearly and surprisingly show that the specific pectins show improvements, which are significant.