TY - JOUR
AU - Wiese,, Maria
AB - ABSTRACT The increasing efforts to substitute antibiotics and improve animal health combined with the acknowledgement of the role of gut microbiota in health have led to an elevated interest in the understanding on how fibre with prebiotic potential, such as pectin, can improve animal growth and health via direct or gut microbiota mediated effects. Various reports exist on the antiviral and antibacterial effects of pectin, as well as its potency as a modulator of the immune response and gut microbial community. Comprehensive insights into the potential of pectin to improve animal growth and health are currently still hampered by heterogeneity in the design of studies. Studies differ with regard to the dosage, molecular structure and source of the pectin implemented, as well as concerning the set of investigations of its effects on the host. Harmonisation of the study design including an in-depth analysis of the gut microbial community and its metabolome will aid to extract information on how pectin can impact growth and overall animal health. Studies with an increased focus on pectin structure such as on pectin-derived rhamnogalacturonan I (RG-I) are just starting to unravel pectin-structure-related effects on mammalian health. Gut Microbiome, Pig, Pectin, Short Chain Fatty Acids, Feed, Health PECTIN IN ANIMAL NUTRITION AND HEALTH Plant food has been consumed in the animal kingdom for millions of years and the mammalian gastrointestinal tract (GIT) co-evolved and adapted to pectin exposure. Pectin is a major building block of the cell wall of all land plants and displays a plethora of molecular structures. The three major pectic polysaccharides are homogalacturonan (HG), rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) (Willats et al. 2001). The structure to function related mode of action of pectin in the GIT remains poorly understood. Addition of pectin to modern animal feed formulations has been studied for various animals such as fish, (Volpe et al. 2015; Irvin et al. 2016; Goulart et al. 2017) horses (reviewed in (Silva, de Rezende and da Silva Inácio 2016)), pigs (reviewed in (Drochner, Kerler and Zacharias 2004)), broilers (Wang et al. 2016), cows (Huhtanen 1988; Gressley and Armentano 2005), as well as dogs (Biagi et al. 2010) and cats (Barry et al. 2010). This review focuses on study examples of the impact of pectin on pig nutrition and health, performed in vivo, ex vivo and in vitro. Within this context, the review explores the potential of pectin to improve growth and health via direct and indirect mechanisms related to the modulation of the gut microbiome, the immune system and the elimination of pathogens and viruses. Existing studies on pectin in animal feed display a vast heterogeneity in study design, which hampers the evaluation of its efficacy, as a feed ingredient for improved growth and health. Study variability relates to the specific pectin implemented, its source, molecular characteristics such as the degree of esterification (DE), the degree of acetylation, fine branching of sugar structures and molecular weight. Further variability exists concerning the feed matrix and, its protein, fat and fibre ratios. The level of information provided on these aspects also differs between studies, ranging from none to relatively detailed information on a few of the substrates (see Table 1 for an overview). The inclusion level of pectin, the adaptation and study period, the experimental hygienic conditions and general farm practices are additional factors that differ between studies (Larsen 1980; Lagreca and Marotta 1985; Baekey et al. 1988; Potkins, Lawrence and Thomlinson 1991; Langhout et al. 1999; Laerke et al. 2001; Dongowski, Lorenz and Proll 2002; Zhu, Rademacher and De Lange 2005; Buraczewska et al. 2007; Zhu, Rademacher and de Lange 2007; Strube et al. 2015a; Tarasenko and Rud 2016; Wu et al. 2016; Tian et al. 2017). Table 1. Pectin in pig feed in vivo study examples Pectin in feed Animal and or model Observations, parameters under investigation Reference LMP, HMP citrus (Classic CU-L 020/13, Classic CU-L 021/13 Herbstreith & Fox, Neuenburg, Germany) at 3% w/w of basal diet, aSBM soy bean meal, control diet (CONT) Piglet (Dutch Landrace × Large White) (mean 6.1 ± 0.02 kg) at weaning (age of 3 weeks) 4 piglets per diet for 28 days. Piglets were housed ad libitum in metabolic cages. The LMP, HMP and aSBM, differently affected the digestion processes and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominated community. Pectin supplemented diets led to a higher relative abundance of Bacteroidetes, and reduced abundance of Firmicutes in the colon and feces, but not in the ileum. A LMP related increase in the relative abundance of the genus Dialister was detected in the colonic digesta. LMP was more efficiently fermented in the ileum than HMP, which was mainly fermented in the proximal colon. The protein digestibility was lower for LMP and HMP fed pigs than for CONT fed pigs throughout the ileum and the large intestine. In the large intestine, lowest digestibility of NSP was observed for aSBM-fed pigs, followed by those for LMP- and HMP-fed pigs. Overall, the SCFA patterns in the pig feces were not significantly affected by the diet and intervention time. Effects of pectin on fermentation characteristics, carbohydrate utilisation and microbial community composition in the gastrointestinal tract of weaning pigs (Tian et al. 2017). 73, 104 or 145 g per/kg of DM (Genu pectin type B Rapid Set, CP Kelco) citrus pectin 50 pigs from 10 L weaned at 4 weeks of age (BW 8.6 ± 1.4 kg) and divided into 5 treatment groups. Pigs fed for 9 days, and were then euthanised, entire GIT was removed. Pectin decreased ADFI and ADG, pectin decreased the area of mucin in the crypts of the small intestine. Shorter villi and crypts, unaltered villous height/crypt depth ratio, no dosage effect. Maybe indirect effect of ADFI, but could be a direct effect too. No effect on crypt depth in the colon. Increased luminal viscosity and water binding capacity (J. E. Lindberg, unpublished data), which may have slowed down digesta passage and increased the satiety of the pigs, leading to lower feed intakes. Citrus pectin included in the diets may have had a negative impact on taste, mouth feel of the diets or both. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties (Hedemann et al. 2006). 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp 20 piglets (30 days of age) 2 days after weaning, the animals were of mixed breed and had been given a wheat-based creep feed since 7 days of age. Using low doses, 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp, a low-value industrial by-product, Strube et al. show that high molecular weight RG-I can be solubilised in the stomach of weaning piglets. The release of this fibre is in the order of 22%–38% of the theoretical amount, achieved within 20 min. The catalysis takes place mainly in the stomach of the animal and is then followed by distribution through the small intestines. In situ prebiotics: enzymatic release of galacto-rhamnogalacturonan from potato pulp in vivo in the gastrointestinal tract of the weaning piglet (Strube et al. 2015a) Cornstarch–SBM (Control) or Control with 12% pectin. Intravenously 1.5 mmol/kg BW of L-1- /react-text 13 react-text: 172 C valine (40 mol%) 12 barrows (21 kg mean BW) Dietary pectin inclusion increased plasma levels of glucose, isoleucine and glutamine but had no effect on insulin or urea nitrogen. There were no differences in FSR (Fractional Synthesis Rate) and ASR (Absolute Synthesis Rate) of whole intestinal protein in jejunum and colon. The FSR of mucosal proteins in colon, not in jejunum, was increased with dietary pectin supplementation. Assuming mucosal protein mass is constant, these results imply that the higher protein synthesis in colon mucosa contributes to the reduced THR efficiency observed in pectin-supplemented diet. Effect of feeding fermentable fiber on synthesis of total and mucosal protein in the intestine of the growing pig (Libao-Mercado et al. 2007). Two experiments were carried out using a balanced feed mixture composed of wheat, corn, soyabean meals, casein, and crystalline amino acids (AA) and supplemented with 0, 4, or 8% of apple pectin. Pigs (25–40 kg BW) with the post valve T-caecum (PVTC) cannula to determine AA digestibility and ileal digesta viscosity. Second experiment, the N-balance on pigs 20 and 28 kg BW six animals per diet and DFI and BW measured twice during the 28, 40 day The added pectin decreased standardized ileal AA digestibility (on average up to 5% dig. units) and increased digesta viscosity from about 1 to 88 mPas. The higher pectin level did not affect motility of the duodenum and mid-jejunum, measured as responses to electrical field stimulation and to acetylcholine. However, the response of the duodenum to electrical field stimulation was after feeding the diet with 4% pectin increased compared with other diets. Pectin supplementation did not alter the weight and the length of the small intestine, but induced changes in the intestinal morphology. Muscle layer width increased significantly in the duodenum, the mid-jejunum, and the ileum while villi length increased in the duodenum and ileum. The addition of pectin decreased N retention and increased the F/G ratio but did not affect significantly the ADG of the pigs. The effect of pectin on amino acid digestibility and digesta viscosity, motility and morphology of the small intestine, and on N-balance and performance of young pigs (Buraczewska et al. 2007). Daily infusion of either 60 g of pectin dissolved in 1.8 L of water or 1.8 L of water as a control infusion 2 × 8 barrows growing pigs (BW 30.1 ± 1.3 kg) (German Landrace × Piétrain), the pigs were surgically fitted at the distal ileum with a simple T-cannula In Pectin infusion reduced (P = 0.005) ileal gene copy number of the C. leptum cluster. Ileal bacterial populations and fermentation patterns are susceptible to changes in the intestinal availability of Ca and P as well as to the supply of pectin as a fermentable substrate. Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion (Metzler-Zebeli et al. 2010). Cereal-based diets with 0, 40 or 80 g of Apple pectin (P) or 270 g of rye (R) per kg Four groups (six pigs each), pigs of 15 kg BW Digesta viscosity was increased more by 80 g than by 40 g P or by R. Ileal digestibility of AA and nitrogen retention were negatively affected by P, whereas growth performance was decreased by R. The effect of the supplements on intestinal morphology was variable, except for the increase in myenteron thickness by P and crypt depth by R. The number of goblet cells containing acidic mucins was decreased by 40 g P in crypts in the mid-jejunum and by 40 g P and R in villi in the ileum. Fasting and postprandial plasma levels of free threonine and of threonine dehydrogenase activity in the liver and pancreas were not affected. In conclusion, feeding P or R negatively affects ileal AA digestibility and provokes irregular changes of small intestinal morphology. These effects cannot be attributed to the increase of digesta viscosity as the main factor. The effects of pectin and rye on amino acid ileal digestibility, threonine metabolism, nitrogen retention and morphology of the small intestine in young pigs (Święch et al. 2012). Citrus Pectin (Genu pectin type B Rapid Set, CP Kelco) 150 castrated piglets, five diets with varying contents of citrus pectin (soluble fibre) and barley hulls (insoluble fibre) There was no difference in SCFA caused by dietary treatment, but across treatments there was a correlation between concentration of SCFA and the proportion of butyrate. The correlation was lowest in the caecum and highest in the distal colon. Association between butyrate and short-chain fatty acid concentrations in gut contents and faeces in weaning piglets (Lærke et al. 2007). Mango pectin 30 large Whitemale pigs with an initial weight of ∼19 kg (10 pigs per group) The rheology of ingredients does not necessarily reflect the rheological effect when ingested. Viscous property of pectin hinders amylase activity in pig stomach. Rheological and microstructural properties of porcine gastric digesta and diets containing pectin or mango powder (Wu et al. 2016). Pectin (0%–12% inclusion level) CP Kelco, Wilmington, DE Two groups of eight Yorshire barrows, average BW of 14.3 ± 1.4 kg (Lysine study) and 17.2 ± 1.3 kg (Threonine study) Utilisation of ileal digestible threonine, but not of ileal digestible lysin intake, for PD (protein deposition) decreased linearly with dietary pectin level, and was not influenced by diet cellulose level. Increasing dietary pectin level reduces utilization of digestible threonine intake, but not lysine intake, for body protein deposition in growing pigs (Zhu et al. 2005). 0.5 and 10% pectin Weaned piglets three groups of six castrated male crossbred pigs with an average BW of 8 kg were fitted with T-cannulas at the caecum fed with diets supplemented with 0%, 5% and 10% pectin. Faeces were collected over a period of 3 days. Addition of 5% pectin to the diet lowered the content of dry matter and lactic acid in fecal matter. The pH and the digestibility of pectin, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine. The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs (Zacharias et al. 2004). 4 Citrus pectins with DE (DM) 80, 60%, 46%, 5% Two citrus pectins classified as calcium sensitive (CAS) and calcium insensitive (CAN) and SBP were tested. Piglets (eight per group, n = 32 for control animals) were weaned at 4 weeks of age. Killed 7 weeks of age. Dissimilarity of viscosity between water and GI due to GI juice interaction with pectin structures. DM46 and DM 80 reduced digestibility of organic matter, no difference of digestibility was seen for DM 60, DM5 CAS and CAN compared to the control group. Concluding that the rheological characteristics of pectins in water differ in the GIT. 29 Isolated Pectins: vary in their Functional Properties in the Gut of Piglets (Laerke et al. 2001). Grapefruit pectin 18 female miniature Swine of the Pitman–Moore strain Grapefruit pectin supplementation inhibits hypercholesterolemia and appears to be proportionately protective against atherosclerosis. Grapefruit pectin inhibits hypercholesterolemia and atherosclerosis in miniature swine (Baekey et al. 1988). Control treatment: Corn-SBM basal diet + ZnO (phase 1: 0.05%; phase 2; 0.03%) and four different levels of sugar beet pulp were supplemented in Corn-SBM basal diet (3%, 6%, 9% or 12%). Two phase feeding programs (phase 1: 1–2 weeks; phase 2: 3–5 weeks) were used for 5 week of growth trial. 200 weaning pigs [(Yorkshire × Landrace) × Duroc], averaging 9.01 ± 1.389 kg. Each treatment was composed of four replicates with 10 pigs per pen. No significant differences in growth performance and incidence of diarrhea among treatments. Linear response was observed in Lactobacillus counts as sugar beet pulp supplementation increased (P < 0.05). In addition, IGF-1, IgA and IgG were not affected by dietary treatments. However, the BUN concentration was decreased when pigs were fed the treatments of diets with SBP compared to that of control treatment (P < 0.05). In nutrient digestibility, crude fiber and NDF digestibilities were improved as the sugar beet pulp increased (P < 0.05). However, digestibilities of crude ash, crude fat, crude fiber and nitrogen retention were not affected by dietary sugar beet pulp levels. Effect of Dietary sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal microflora, blood profiles and diarrhea incidence in weaning pigs (Yan et al. 2017). 40 days on one of four diets differing in the level and type of polysaccharide. Control diet with or without 4 or 8% pectin or a diet with rye instead of part of the wheat. 24 male growing pigs with a high lean gain potential (synthetic line 990, Poland), divided into four groups (six pigs each) initial BW of about 20 kg The influence of polysaccharidesonthe epithelial structure was evaluated in the duodenum, mid-jejunum and ileum. Increased crypt depth and mucosa thickness were found after feeding the diet with rye in all measured segments, whereas feeding the diets with 4% and 8% pectin caused an increase in myenteron thickness of the mid-jejunum and ileum, in comparison with other diets. The structure of small intestinal tissue in pigs as influenced by indigestible polysaccharides (Tusnio et al. 2006). Pectin was supplied at levels of 0 and 334 g dry matter day−1 together with a semi-synthetic basal diet to. In addition to Pectin treatments two different levels of thiamine, 0.66 and 2.57 mg day−1 were applied. 12 adult sows weighing 166 ± 17 kg The pectin fermentation rate was determined by three different methods s to account for 94%–96% of pectin intake. The apparent digestibility of nitrogen was reduced by more than 20% at an almost constant true N digestibility. The increased fecal nitrogen excretion with pectin caused a reduction in protein retention to zero, and was composed of 82% bacterial nitrogen. The changes in the proteinaceous water-soluble fraction of fecal nitrogen indicated an increase in the excretion of endogenous proteins. Effects of pectin supplementation on the digestion of different structural carbohydrate fractions and on bacterial nitrogen turnover in the hindgut of adult sows (Roth-Maier et al.1993). Sugar beet fibre Two groups of pigs/ 36 female weanling piglets (14 ± 2 kg) of the Large White breed After 4 week, total serum cholesterol and high-density-lipoprotein cholesterol concentrations were similar on both diets. By contrast, the fasting triacylglycerols were 21% lower (P < 0.05) and apparent feed-conversion efficiency was 47% higher (P < 0.01) on the SBF diet than on the control diet. Accordingly, the effect of SBF did not appear to be mediated by an impairing effect on dietary lipid absorption. The results suggest that the decreasing effect of SBF on triacylglycerols was due to a reduction in very-low-density-lipoprotein synthesis without changes in the size of particles. The low-density-lipoprotein receptor activity of a liver plasma membrane-enriched fraction was not influenced by the dietary treatment; however, a significant negative relationship between cholesterol concentrations and the receptor activity was observed irrespective of the diet. Effects of sugar beet fiber feeding on serum lipids and binding of low-density lipoproteins to liver membranes in growing pigs (Fremont, Gozzelino and Bosseau 1993). Each group of piglets was fed with corn–soybean meal diets containing 5% apple pectin (PEC, purchased from Yuzhong Biotech Corporation, Henan, China) or cornstarch (purchased from Yufeng Cornstarch and Sugar Company, Hebei, China) as the control (CON) Male Duroc × Large White crossbred piglets with an average body weight of 11.05 ± 0.11 kg were randomly divided into 2 groups of 6 animals each and allowed to adapt for 1 week before the start of the 72-day experiment. Growth performance was not affected by the inclusion of 5% pectin in the conventional corn–soybean meal diet over the 72-d trial period n pigs fed with pectin, total cholesterol and low-density lipoprotein cholesterol were lowered but high-density lipoprotein was increased (P < 0.05). Serum triglycerides tended to be lower in the pectin-fed animals (P = 0.093), whereas no change was noted in serum total bile acid. Bile acid pool: In pigs fed with 5% pectin, only cecal ursodeoxycholic acid (P = 0.097) and hyocholic acid (P = 0.088) showed a decreasing tendency. In the ileum, pectin increased in-and-out BA transport on the apical membrane, but it increased the overall BA transport in the cecum. Effects of dietary pectin on the profile and transport of intetinal bile acids in young pigs (Fang et al. 2018). Pectin in feed Animal and or model Observations, parameters under investigation Reference LMP, HMP citrus (Classic CU-L 020/13, Classic CU-L 021/13 Herbstreith & Fox, Neuenburg, Germany) at 3% w/w of basal diet, aSBM soy bean meal, control diet (CONT) Piglet (Dutch Landrace × Large White) (mean 6.1 ± 0.02 kg) at weaning (age of 3 weeks) 4 piglets per diet for 28 days. Piglets were housed ad libitum in metabolic cages. The LMP, HMP and aSBM, differently affected the digestion processes and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominated community. Pectin supplemented diets led to a higher relative abundance of Bacteroidetes, and reduced abundance of Firmicutes in the colon and feces, but not in the ileum. A LMP related increase in the relative abundance of the genus Dialister was detected in the colonic digesta. LMP was more efficiently fermented in the ileum than HMP, which was mainly fermented in the proximal colon. The protein digestibility was lower for LMP and HMP fed pigs than for CONT fed pigs throughout the ileum and the large intestine. In the large intestine, lowest digestibility of NSP was observed for aSBM-fed pigs, followed by those for LMP- and HMP-fed pigs. Overall, the SCFA patterns in the pig feces were not significantly affected by the diet and intervention time. Effects of pectin on fermentation characteristics, carbohydrate utilisation and microbial community composition in the gastrointestinal tract of weaning pigs (Tian et al. 2017). 73, 104 or 145 g per/kg of DM (Genu pectin type B Rapid Set, CP Kelco) citrus pectin 50 pigs from 10 L weaned at 4 weeks of age (BW 8.6 ± 1.4 kg) and divided into 5 treatment groups. Pigs fed for 9 days, and were then euthanised, entire GIT was removed. Pectin decreased ADFI and ADG, pectin decreased the area of mucin in the crypts of the small intestine. Shorter villi and crypts, unaltered villous height/crypt depth ratio, no dosage effect. Maybe indirect effect of ADFI, but could be a direct effect too. No effect on crypt depth in the colon. Increased luminal viscosity and water binding capacity (J. E. Lindberg, unpublished data), which may have slowed down digesta passage and increased the satiety of the pigs, leading to lower feed intakes. Citrus pectin included in the diets may have had a negative impact on taste, mouth feel of the diets or both. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties (Hedemann et al. 2006). 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp 20 piglets (30 days of age) 2 days after weaning, the animals were of mixed breed and had been given a wheat-based creep feed since 7 days of age. Using low doses, 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp, a low-value industrial by-product, Strube et al. show that high molecular weight RG-I can be solubilised in the stomach of weaning piglets. The release of this fibre is in the order of 22%–38% of the theoretical amount, achieved within 20 min. The catalysis takes place mainly in the stomach of the animal and is then followed by distribution through the small intestines. In situ prebiotics: enzymatic release of galacto-rhamnogalacturonan from potato pulp in vivo in the gastrointestinal tract of the weaning piglet (Strube et al. 2015a) Cornstarch–SBM (Control) or Control with 12% pectin. Intravenously 1.5 mmol/kg BW of L-1- /react-text 13 react-text: 172 C valine (40 mol%) 12 barrows (21 kg mean BW) Dietary pectin inclusion increased plasma levels of glucose, isoleucine and glutamine but had no effect on insulin or urea nitrogen. There were no differences in FSR (Fractional Synthesis Rate) and ASR (Absolute Synthesis Rate) of whole intestinal protein in jejunum and colon. The FSR of mucosal proteins in colon, not in jejunum, was increased with dietary pectin supplementation. Assuming mucosal protein mass is constant, these results imply that the higher protein synthesis in colon mucosa contributes to the reduced THR efficiency observed in pectin-supplemented diet. Effect of feeding fermentable fiber on synthesis of total and mucosal protein in the intestine of the growing pig (Libao-Mercado et al. 2007). Two experiments were carried out using a balanced feed mixture composed of wheat, corn, soyabean meals, casein, and crystalline amino acids (AA) and supplemented with 0, 4, or 8% of apple pectin. Pigs (25–40 kg BW) with the post valve T-caecum (PVTC) cannula to determine AA digestibility and ileal digesta viscosity. Second experiment, the N-balance on pigs 20 and 28 kg BW six animals per diet and DFI and BW measured twice during the 28, 40 day The added pectin decreased standardized ileal AA digestibility (on average up to 5% dig. units) and increased digesta viscosity from about 1 to 88 mPas. The higher pectin level did not affect motility of the duodenum and mid-jejunum, measured as responses to electrical field stimulation and to acetylcholine. However, the response of the duodenum to electrical field stimulation was after feeding the diet with 4% pectin increased compared with other diets. Pectin supplementation did not alter the weight and the length of the small intestine, but induced changes in the intestinal morphology. Muscle layer width increased significantly in the duodenum, the mid-jejunum, and the ileum while villi length increased in the duodenum and ileum. The addition of pectin decreased N retention and increased the F/G ratio but did not affect significantly the ADG of the pigs. The effect of pectin on amino acid digestibility and digesta viscosity, motility and morphology of the small intestine, and on N-balance and performance of young pigs (Buraczewska et al. 2007). Daily infusion of either 60 g of pectin dissolved in 1.8 L of water or 1.8 L of water as a control infusion 2 × 8 barrows growing pigs (BW 30.1 ± 1.3 kg) (German Landrace × Piétrain), the pigs were surgically fitted at the distal ileum with a simple T-cannula In Pectin infusion reduced (P = 0.005) ileal gene copy number of the C. leptum cluster. Ileal bacterial populations and fermentation patterns are susceptible to changes in the intestinal availability of Ca and P as well as to the supply of pectin as a fermentable substrate. Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion (Metzler-Zebeli et al. 2010). Cereal-based diets with 0, 40 or 80 g of Apple pectin (P) or 270 g of rye (R) per kg Four groups (six pigs each), pigs of 15 kg BW Digesta viscosity was increased more by 80 g than by 40 g P or by R. Ileal digestibility of AA and nitrogen retention were negatively affected by P, whereas growth performance was decreased by R. The effect of the supplements on intestinal morphology was variable, except for the increase in myenteron thickness by P and crypt depth by R. The number of goblet cells containing acidic mucins was decreased by 40 g P in crypts in the mid-jejunum and by 40 g P and R in villi in the ileum. Fasting and postprandial plasma levels of free threonine and of threonine dehydrogenase activity in the liver and pancreas were not affected. In conclusion, feeding P or R negatively affects ileal AA digestibility and provokes irregular changes of small intestinal morphology. These effects cannot be attributed to the increase of digesta viscosity as the main factor. The effects of pectin and rye on amino acid ileal digestibility, threonine metabolism, nitrogen retention and morphology of the small intestine in young pigs (Święch et al. 2012). Citrus Pectin (Genu pectin type B Rapid Set, CP Kelco) 150 castrated piglets, five diets with varying contents of citrus pectin (soluble fibre) and barley hulls (insoluble fibre) There was no difference in SCFA caused by dietary treatment, but across treatments there was a correlation between concentration of SCFA and the proportion of butyrate. The correlation was lowest in the caecum and highest in the distal colon. Association between butyrate and short-chain fatty acid concentrations in gut contents and faeces in weaning piglets (Lærke et al. 2007). Mango pectin 30 large Whitemale pigs with an initial weight of ∼19 kg (10 pigs per group) The rheology of ingredients does not necessarily reflect the rheological effect when ingested. Viscous property of pectin hinders amylase activity in pig stomach. Rheological and microstructural properties of porcine gastric digesta and diets containing pectin or mango powder (Wu et al. 2016). Pectin (0%–12% inclusion level) CP Kelco, Wilmington, DE Two groups of eight Yorshire barrows, average BW of 14.3 ± 1.4 kg (Lysine study) and 17.2 ± 1.3 kg (Threonine study) Utilisation of ileal digestible threonine, but not of ileal digestible lysin intake, for PD (protein deposition) decreased linearly with dietary pectin level, and was not influenced by diet cellulose level. Increasing dietary pectin level reduces utilization of digestible threonine intake, but not lysine intake, for body protein deposition in growing pigs (Zhu et al. 2005). 0.5 and 10% pectin Weaned piglets three groups of six castrated male crossbred pigs with an average BW of 8 kg were fitted with T-cannulas at the caecum fed with diets supplemented with 0%, 5% and 10% pectin. Faeces were collected over a period of 3 days. Addition of 5% pectin to the diet lowered the content of dry matter and lactic acid in fecal matter. The pH and the digestibility of pectin, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine. The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs (Zacharias et al. 2004). 4 Citrus pectins with DE (DM) 80, 60%, 46%, 5% Two citrus pectins classified as calcium sensitive (CAS) and calcium insensitive (CAN) and SBP were tested. Piglets (eight per group, n = 32 for control animals) were weaned at 4 weeks of age. Killed 7 weeks of age. Dissimilarity of viscosity between water and GI due to GI juice interaction with pectin structures. DM46 and DM 80 reduced digestibility of organic matter, no difference of digestibility was seen for DM 60, DM5 CAS and CAN compared to the control group. Concluding that the rheological characteristics of pectins in water differ in the GIT. 29 Isolated Pectins: vary in their Functional Properties in the Gut of Piglets (Laerke et al. 2001). Grapefruit pectin 18 female miniature Swine of the Pitman–Moore strain Grapefruit pectin supplementation inhibits hypercholesterolemia and appears to be proportionately protective against atherosclerosis. Grapefruit pectin inhibits hypercholesterolemia and atherosclerosis in miniature swine (Baekey et al. 1988). Control treatment: Corn-SBM basal diet + ZnO (phase 1: 0.05%; phase 2; 0.03%) and four different levels of sugar beet pulp were supplemented in Corn-SBM basal diet (3%, 6%, 9% or 12%). Two phase feeding programs (phase 1: 1–2 weeks; phase 2: 3–5 weeks) were used for 5 week of growth trial. 200 weaning pigs [(Yorkshire × Landrace) × Duroc], averaging 9.01 ± 1.389 kg. Each treatment was composed of four replicates with 10 pigs per pen. No significant differences in growth performance and incidence of diarrhea among treatments. Linear response was observed in Lactobacillus counts as sugar beet pulp supplementation increased (P < 0.05). In addition, IGF-1, IgA and IgG were not affected by dietary treatments. However, the BUN concentration was decreased when pigs were fed the treatments of diets with SBP compared to that of control treatment (P < 0.05). In nutrient digestibility, crude fiber and NDF digestibilities were improved as the sugar beet pulp increased (P < 0.05). However, digestibilities of crude ash, crude fat, crude fiber and nitrogen retention were not affected by dietary sugar beet pulp levels. Effect of Dietary sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal microflora, blood profiles and diarrhea incidence in weaning pigs (Yan et al. 2017). 40 days on one of four diets differing in the level and type of polysaccharide. Control diet with or without 4 or 8% pectin or a diet with rye instead of part of the wheat. 24 male growing pigs with a high lean gain potential (synthetic line 990, Poland), divided into four groups (six pigs each) initial BW of about 20 kg The influence of polysaccharidesonthe epithelial structure was evaluated in the duodenum, mid-jejunum and ileum. Increased crypt depth and mucosa thickness were found after feeding the diet with rye in all measured segments, whereas feeding the diets with 4% and 8% pectin caused an increase in myenteron thickness of the mid-jejunum and ileum, in comparison with other diets. The structure of small intestinal tissue in pigs as influenced by indigestible polysaccharides (Tusnio et al. 2006). Pectin was supplied at levels of 0 and 334 g dry matter day−1 together with a semi-synthetic basal diet to. In addition to Pectin treatments two different levels of thiamine, 0.66 and 2.57 mg day−1 were applied. 12 adult sows weighing 166 ± 17 kg The pectin fermentation rate was determined by three different methods s to account for 94%–96% of pectin intake. The apparent digestibility of nitrogen was reduced by more than 20% at an almost constant true N digestibility. The increased fecal nitrogen excretion with pectin caused a reduction in protein retention to zero, and was composed of 82% bacterial nitrogen. The changes in the proteinaceous water-soluble fraction of fecal nitrogen indicated an increase in the excretion of endogenous proteins. Effects of pectin supplementation on the digestion of different structural carbohydrate fractions and on bacterial nitrogen turnover in the hindgut of adult sows (Roth-Maier et al.1993). Sugar beet fibre Two groups of pigs/ 36 female weanling piglets (14 ± 2 kg) of the Large White breed After 4 week, total serum cholesterol and high-density-lipoprotein cholesterol concentrations were similar on both diets. By contrast, the fasting triacylglycerols were 21% lower (P < 0.05) and apparent feed-conversion efficiency was 47% higher (P < 0.01) on the SBF diet than on the control diet. Accordingly, the effect of SBF did not appear to be mediated by an impairing effect on dietary lipid absorption. The results suggest that the decreasing effect of SBF on triacylglycerols was due to a reduction in very-low-density-lipoprotein synthesis without changes in the size of particles. The low-density-lipoprotein receptor activity of a liver plasma membrane-enriched fraction was not influenced by the dietary treatment; however, a significant negative relationship between cholesterol concentrations and the receptor activity was observed irrespective of the diet. Effects of sugar beet fiber feeding on serum lipids and binding of low-density lipoproteins to liver membranes in growing pigs (Fremont, Gozzelino and Bosseau 1993). Each group of piglets was fed with corn–soybean meal diets containing 5% apple pectin (PEC, purchased from Yuzhong Biotech Corporation, Henan, China) or cornstarch (purchased from Yufeng Cornstarch and Sugar Company, Hebei, China) as the control (CON) Male Duroc × Large White crossbred piglets with an average body weight of 11.05 ± 0.11 kg were randomly divided into 2 groups of 6 animals each and allowed to adapt for 1 week before the start of the 72-day experiment. Growth performance was not affected by the inclusion of 5% pectin in the conventional corn–soybean meal diet over the 72-d trial period n pigs fed with pectin, total cholesterol and low-density lipoprotein cholesterol were lowered but high-density lipoprotein was increased (P < 0.05). Serum triglycerides tended to be lower in the pectin-fed animals (P = 0.093), whereas no change was noted in serum total bile acid. Bile acid pool: In pigs fed with 5% pectin, only cecal ursodeoxycholic acid (P = 0.097) and hyocholic acid (P = 0.088) showed a decreasing tendency. In the ileum, pectin increased in-and-out BA transport on the apical membrane, but it increased the overall BA transport in the cecum. Effects of dietary pectin on the profile and transport of intetinal bile acids in young pigs (Fang et al. 2018). View Large Table 1. Pectin in pig feed in vivo study examples Pectin in feed Animal and or model Observations, parameters under investigation Reference LMP, HMP citrus (Classic CU-L 020/13, Classic CU-L 021/13 Herbstreith & Fox, Neuenburg, Germany) at 3% w/w of basal diet, aSBM soy bean meal, control diet (CONT) Piglet (Dutch Landrace × Large White) (mean 6.1 ± 0.02 kg) at weaning (age of 3 weeks) 4 piglets per diet for 28 days. Piglets were housed ad libitum in metabolic cages. The LMP, HMP and aSBM, differently affected the digestion processes and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominated community. Pectin supplemented diets led to a higher relative abundance of Bacteroidetes, and reduced abundance of Firmicutes in the colon and feces, but not in the ileum. A LMP related increase in the relative abundance of the genus Dialister was detected in the colonic digesta. LMP was more efficiently fermented in the ileum than HMP, which was mainly fermented in the proximal colon. The protein digestibility was lower for LMP and HMP fed pigs than for CONT fed pigs throughout the ileum and the large intestine. In the large intestine, lowest digestibility of NSP was observed for aSBM-fed pigs, followed by those for LMP- and HMP-fed pigs. Overall, the SCFA patterns in the pig feces were not significantly affected by the diet and intervention time. Effects of pectin on fermentation characteristics, carbohydrate utilisation and microbial community composition in the gastrointestinal tract of weaning pigs (Tian et al. 2017). 73, 104 or 145 g per/kg of DM (Genu pectin type B Rapid Set, CP Kelco) citrus pectin 50 pigs from 10 L weaned at 4 weeks of age (BW 8.6 ± 1.4 kg) and divided into 5 treatment groups. Pigs fed for 9 days, and were then euthanised, entire GIT was removed. Pectin decreased ADFI and ADG, pectin decreased the area of mucin in the crypts of the small intestine. Shorter villi and crypts, unaltered villous height/crypt depth ratio, no dosage effect. Maybe indirect effect of ADFI, but could be a direct effect too. No effect on crypt depth in the colon. Increased luminal viscosity and water binding capacity (J. E. Lindberg, unpublished data), which may have slowed down digesta passage and increased the satiety of the pigs, leading to lower feed intakes. Citrus pectin included in the diets may have had a negative impact on taste, mouth feel of the diets or both. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties (Hedemann et al. 2006). 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp 20 piglets (30 days of age) 2 days after weaning, the animals were of mixed breed and had been given a wheat-based creep feed since 7 days of age. Using low doses, 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp, a low-value industrial by-product, Strube et al. show that high molecular weight RG-I can be solubilised in the stomach of weaning piglets. The release of this fibre is in the order of 22%–38% of the theoretical amount, achieved within 20 min. The catalysis takes place mainly in the stomach of the animal and is then followed by distribution through the small intestines. In situ prebiotics: enzymatic release of galacto-rhamnogalacturonan from potato pulp in vivo in the gastrointestinal tract of the weaning piglet (Strube et al. 2015a) Cornstarch–SBM (Control) or Control with 12% pectin. Intravenously 1.5 mmol/kg BW of L-1- /react-text 13 react-text: 172 C valine (40 mol%) 12 barrows (21 kg mean BW) Dietary pectin inclusion increased plasma levels of glucose, isoleucine and glutamine but had no effect on insulin or urea nitrogen. There were no differences in FSR (Fractional Synthesis Rate) and ASR (Absolute Synthesis Rate) of whole intestinal protein in jejunum and colon. The FSR of mucosal proteins in colon, not in jejunum, was increased with dietary pectin supplementation. Assuming mucosal protein mass is constant, these results imply that the higher protein synthesis in colon mucosa contributes to the reduced THR efficiency observed in pectin-supplemented diet. Effect of feeding fermentable fiber on synthesis of total and mucosal protein in the intestine of the growing pig (Libao-Mercado et al. 2007). Two experiments were carried out using a balanced feed mixture composed of wheat, corn, soyabean meals, casein, and crystalline amino acids (AA) and supplemented with 0, 4, or 8% of apple pectin. Pigs (25–40 kg BW) with the post valve T-caecum (PVTC) cannula to determine AA digestibility and ileal digesta viscosity. Second experiment, the N-balance on pigs 20 and 28 kg BW six animals per diet and DFI and BW measured twice during the 28, 40 day The added pectin decreased standardized ileal AA digestibility (on average up to 5% dig. units) and increased digesta viscosity from about 1 to 88 mPas. The higher pectin level did not affect motility of the duodenum and mid-jejunum, measured as responses to electrical field stimulation and to acetylcholine. However, the response of the duodenum to electrical field stimulation was after feeding the diet with 4% pectin increased compared with other diets. Pectin supplementation did not alter the weight and the length of the small intestine, but induced changes in the intestinal morphology. Muscle layer width increased significantly in the duodenum, the mid-jejunum, and the ileum while villi length increased in the duodenum and ileum. The addition of pectin decreased N retention and increased the F/G ratio but did not affect significantly the ADG of the pigs. The effect of pectin on amino acid digestibility and digesta viscosity, motility and morphology of the small intestine, and on N-balance and performance of young pigs (Buraczewska et al. 2007). Daily infusion of either 60 g of pectin dissolved in 1.8 L of water or 1.8 L of water as a control infusion 2 × 8 barrows growing pigs (BW 30.1 ± 1.3 kg) (German Landrace × Piétrain), the pigs were surgically fitted at the distal ileum with a simple T-cannula In Pectin infusion reduced (P = 0.005) ileal gene copy number of the C. leptum cluster. Ileal bacterial populations and fermentation patterns are susceptible to changes in the intestinal availability of Ca and P as well as to the supply of pectin as a fermentable substrate. Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion (Metzler-Zebeli et al. 2010). Cereal-based diets with 0, 40 or 80 g of Apple pectin (P) or 270 g of rye (R) per kg Four groups (six pigs each), pigs of 15 kg BW Digesta viscosity was increased more by 80 g than by 40 g P or by R. Ileal digestibility of AA and nitrogen retention were negatively affected by P, whereas growth performance was decreased by R. The effect of the supplements on intestinal morphology was variable, except for the increase in myenteron thickness by P and crypt depth by R. The number of goblet cells containing acidic mucins was decreased by 40 g P in crypts in the mid-jejunum and by 40 g P and R in villi in the ileum. Fasting and postprandial plasma levels of free threonine and of threonine dehydrogenase activity in the liver and pancreas were not affected. In conclusion, feeding P or R negatively affects ileal AA digestibility and provokes irregular changes of small intestinal morphology. These effects cannot be attributed to the increase of digesta viscosity as the main factor. The effects of pectin and rye on amino acid ileal digestibility, threonine metabolism, nitrogen retention and morphology of the small intestine in young pigs (Święch et al. 2012). Citrus Pectin (Genu pectin type B Rapid Set, CP Kelco) 150 castrated piglets, five diets with varying contents of citrus pectin (soluble fibre) and barley hulls (insoluble fibre) There was no difference in SCFA caused by dietary treatment, but across treatments there was a correlation between concentration of SCFA and the proportion of butyrate. The correlation was lowest in the caecum and highest in the distal colon. Association between butyrate and short-chain fatty acid concentrations in gut contents and faeces in weaning piglets (Lærke et al. 2007). Mango pectin 30 large Whitemale pigs with an initial weight of ∼19 kg (10 pigs per group) The rheology of ingredients does not necessarily reflect the rheological effect when ingested. Viscous property of pectin hinders amylase activity in pig stomach. Rheological and microstructural properties of porcine gastric digesta and diets containing pectin or mango powder (Wu et al. 2016). Pectin (0%–12% inclusion level) CP Kelco, Wilmington, DE Two groups of eight Yorshire barrows, average BW of 14.3 ± 1.4 kg (Lysine study) and 17.2 ± 1.3 kg (Threonine study) Utilisation of ileal digestible threonine, but not of ileal digestible lysin intake, for PD (protein deposition) decreased linearly with dietary pectin level, and was not influenced by diet cellulose level. Increasing dietary pectin level reduces utilization of digestible threonine intake, but not lysine intake, for body protein deposition in growing pigs (Zhu et al. 2005). 0.5 and 10% pectin Weaned piglets three groups of six castrated male crossbred pigs with an average BW of 8 kg were fitted with T-cannulas at the caecum fed with diets supplemented with 0%, 5% and 10% pectin. Faeces were collected over a period of 3 days. Addition of 5% pectin to the diet lowered the content of dry matter and lactic acid in fecal matter. The pH and the digestibility of pectin, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine. The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs (Zacharias et al. 2004). 4 Citrus pectins with DE (DM) 80, 60%, 46%, 5% Two citrus pectins classified as calcium sensitive (CAS) and calcium insensitive (CAN) and SBP were tested. Piglets (eight per group, n = 32 for control animals) were weaned at 4 weeks of age. Killed 7 weeks of age. Dissimilarity of viscosity between water and GI due to GI juice interaction with pectin structures. DM46 and DM 80 reduced digestibility of organic matter, no difference of digestibility was seen for DM 60, DM5 CAS and CAN compared to the control group. Concluding that the rheological characteristics of pectins in water differ in the GIT. 29 Isolated Pectins: vary in their Functional Properties in the Gut of Piglets (Laerke et al. 2001). Grapefruit pectin 18 female miniature Swine of the Pitman–Moore strain Grapefruit pectin supplementation inhibits hypercholesterolemia and appears to be proportionately protective against atherosclerosis. Grapefruit pectin inhibits hypercholesterolemia and atherosclerosis in miniature swine (Baekey et al. 1988). Control treatment: Corn-SBM basal diet + ZnO (phase 1: 0.05%; phase 2; 0.03%) and four different levels of sugar beet pulp were supplemented in Corn-SBM basal diet (3%, 6%, 9% or 12%). Two phase feeding programs (phase 1: 1–2 weeks; phase 2: 3–5 weeks) were used for 5 week of growth trial. 200 weaning pigs [(Yorkshire × Landrace) × Duroc], averaging 9.01 ± 1.389 kg. Each treatment was composed of four replicates with 10 pigs per pen. No significant differences in growth performance and incidence of diarrhea among treatments. Linear response was observed in Lactobacillus counts as sugar beet pulp supplementation increased (P < 0.05). In addition, IGF-1, IgA and IgG were not affected by dietary treatments. However, the BUN concentration was decreased when pigs were fed the treatments of diets with SBP compared to that of control treatment (P < 0.05). In nutrient digestibility, crude fiber and NDF digestibilities were improved as the sugar beet pulp increased (P < 0.05). However, digestibilities of crude ash, crude fat, crude fiber and nitrogen retention were not affected by dietary sugar beet pulp levels. Effect of Dietary sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal microflora, blood profiles and diarrhea incidence in weaning pigs (Yan et al. 2017). 40 days on one of four diets differing in the level and type of polysaccharide. Control diet with or without 4 or 8% pectin or a diet with rye instead of part of the wheat. 24 male growing pigs with a high lean gain potential (synthetic line 990, Poland), divided into four groups (six pigs each) initial BW of about 20 kg The influence of polysaccharidesonthe epithelial structure was evaluated in the duodenum, mid-jejunum and ileum. Increased crypt depth and mucosa thickness were found after feeding the diet with rye in all measured segments, whereas feeding the diets with 4% and 8% pectin caused an increase in myenteron thickness of the mid-jejunum and ileum, in comparison with other diets. The structure of small intestinal tissue in pigs as influenced by indigestible polysaccharides (Tusnio et al. 2006). Pectin was supplied at levels of 0 and 334 g dry matter day−1 together with a semi-synthetic basal diet to. In addition to Pectin treatments two different levels of thiamine, 0.66 and 2.57 mg day−1 were applied. 12 adult sows weighing 166 ± 17 kg The pectin fermentation rate was determined by three different methods s to account for 94%–96% of pectin intake. The apparent digestibility of nitrogen was reduced by more than 20% at an almost constant true N digestibility. The increased fecal nitrogen excretion with pectin caused a reduction in protein retention to zero, and was composed of 82% bacterial nitrogen. The changes in the proteinaceous water-soluble fraction of fecal nitrogen indicated an increase in the excretion of endogenous proteins. Effects of pectin supplementation on the digestion of different structural carbohydrate fractions and on bacterial nitrogen turnover in the hindgut of adult sows (Roth-Maier et al.1993). Sugar beet fibre Two groups of pigs/ 36 female weanling piglets (14 ± 2 kg) of the Large White breed After 4 week, total serum cholesterol and high-density-lipoprotein cholesterol concentrations were similar on both diets. By contrast, the fasting triacylglycerols were 21% lower (P < 0.05) and apparent feed-conversion efficiency was 47% higher (P < 0.01) on the SBF diet than on the control diet. Accordingly, the effect of SBF did not appear to be mediated by an impairing effect on dietary lipid absorption. The results suggest that the decreasing effect of SBF on triacylglycerols was due to a reduction in very-low-density-lipoprotein synthesis without changes in the size of particles. The low-density-lipoprotein receptor activity of a liver plasma membrane-enriched fraction was not influenced by the dietary treatment; however, a significant negative relationship between cholesterol concentrations and the receptor activity was observed irrespective of the diet. Effects of sugar beet fiber feeding on serum lipids and binding of low-density lipoproteins to liver membranes in growing pigs (Fremont, Gozzelino and Bosseau 1993). Each group of piglets was fed with corn–soybean meal diets containing 5% apple pectin (PEC, purchased from Yuzhong Biotech Corporation, Henan, China) or cornstarch (purchased from Yufeng Cornstarch and Sugar Company, Hebei, China) as the control (CON) Male Duroc × Large White crossbred piglets with an average body weight of 11.05 ± 0.11 kg were randomly divided into 2 groups of 6 animals each and allowed to adapt for 1 week before the start of the 72-day experiment. Growth performance was not affected by the inclusion of 5% pectin in the conventional corn–soybean meal diet over the 72-d trial period n pigs fed with pectin, total cholesterol and low-density lipoprotein cholesterol were lowered but high-density lipoprotein was increased (P < 0.05). Serum triglycerides tended to be lower in the pectin-fed animals (P = 0.093), whereas no change was noted in serum total bile acid. Bile acid pool: In pigs fed with 5% pectin, only cecal ursodeoxycholic acid (P = 0.097) and hyocholic acid (P = 0.088) showed a decreasing tendency. In the ileum, pectin increased in-and-out BA transport on the apical membrane, but it increased the overall BA transport in the cecum. Effects of dietary pectin on the profile and transport of intetinal bile acids in young pigs (Fang et al. 2018). Pectin in feed Animal and or model Observations, parameters under investigation Reference LMP, HMP citrus (Classic CU-L 020/13, Classic CU-L 021/13 Herbstreith & Fox, Neuenburg, Germany) at 3% w/w of basal diet, aSBM soy bean meal, control diet (CONT) Piglet (Dutch Landrace × Large White) (mean 6.1 ± 0.02 kg) at weaning (age of 3 weeks) 4 piglets per diet for 28 days. Piglets were housed ad libitum in metabolic cages. The LMP, HMP and aSBM, differently affected the digestion processes and shaped the colonic microbiota from a Lactobacillus-dominating flora to a Prevotella-dominated community. Pectin supplemented diets led to a higher relative abundance of Bacteroidetes, and reduced abundance of Firmicutes in the colon and feces, but not in the ileum. A LMP related increase in the relative abundance of the genus Dialister was detected in the colonic digesta. LMP was more efficiently fermented in the ileum than HMP, which was mainly fermented in the proximal colon. The protein digestibility was lower for LMP and HMP fed pigs than for CONT fed pigs throughout the ileum and the large intestine. In the large intestine, lowest digestibility of NSP was observed for aSBM-fed pigs, followed by those for LMP- and HMP-fed pigs. Overall, the SCFA patterns in the pig feces were not significantly affected by the diet and intervention time. Effects of pectin on fermentation characteristics, carbohydrate utilisation and microbial community composition in the gastrointestinal tract of weaning pigs (Tian et al. 2017). 73, 104 or 145 g per/kg of DM (Genu pectin type B Rapid Set, CP Kelco) citrus pectin 50 pigs from 10 L weaned at 4 weeks of age (BW 8.6 ± 1.4 kg) and divided into 5 treatment groups. Pigs fed for 9 days, and were then euthanised, entire GIT was removed. Pectin decreased ADFI and ADG, pectin decreased the area of mucin in the crypts of the small intestine. Shorter villi and crypts, unaltered villous height/crypt depth ratio, no dosage effect. Maybe indirect effect of ADFI, but could be a direct effect too. No effect on crypt depth in the colon. Increased luminal viscosity and water binding capacity (J. E. Lindberg, unpublished data), which may have slowed down digesta passage and increased the satiety of the pigs, leading to lower feed intakes. Citrus pectin included in the diets may have had a negative impact on taste, mouth feel of the diets or both. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties (Hedemann et al. 2006). 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp 20 piglets (30 days of age) 2 days after weaning, the animals were of mixed breed and had been given a wheat-based creep feed since 7 days of age. Using low doses, 0.03% enzyme-to-substrate ratio, of the enzymes pectin lyase and polygalacturonase in combination with potato pulp, a low-value industrial by-product, Strube et al. show that high molecular weight RG-I can be solubilised in the stomach of weaning piglets. The release of this fibre is in the order of 22%–38% of the theoretical amount, achieved within 20 min. The catalysis takes place mainly in the stomach of the animal and is then followed by distribution through the small intestines. In situ prebiotics: enzymatic release of galacto-rhamnogalacturonan from potato pulp in vivo in the gastrointestinal tract of the weaning piglet (Strube et al. 2015a) Cornstarch–SBM (Control) or Control with 12% pectin. Intravenously 1.5 mmol/kg BW of L-1- /react-text 13 react-text: 172 C valine (40 mol%) 12 barrows (21 kg mean BW) Dietary pectin inclusion increased plasma levels of glucose, isoleucine and glutamine but had no effect on insulin or urea nitrogen. There were no differences in FSR (Fractional Synthesis Rate) and ASR (Absolute Synthesis Rate) of whole intestinal protein in jejunum and colon. The FSR of mucosal proteins in colon, not in jejunum, was increased with dietary pectin supplementation. Assuming mucosal protein mass is constant, these results imply that the higher protein synthesis in colon mucosa contributes to the reduced THR efficiency observed in pectin-supplemented diet. Effect of feeding fermentable fiber on synthesis of total and mucosal protein in the intestine of the growing pig (Libao-Mercado et al. 2007). Two experiments were carried out using a balanced feed mixture composed of wheat, corn, soyabean meals, casein, and crystalline amino acids (AA) and supplemented with 0, 4, or 8% of apple pectin. Pigs (25–40 kg BW) with the post valve T-caecum (PVTC) cannula to determine AA digestibility and ileal digesta viscosity. Second experiment, the N-balance on pigs 20 and 28 kg BW six animals per diet and DFI and BW measured twice during the 28, 40 day The added pectin decreased standardized ileal AA digestibility (on average up to 5% dig. units) and increased digesta viscosity from about 1 to 88 mPas. The higher pectin level did not affect motility of the duodenum and mid-jejunum, measured as responses to electrical field stimulation and to acetylcholine. However, the response of the duodenum to electrical field stimulation was after feeding the diet with 4% pectin increased compared with other diets. Pectin supplementation did not alter the weight and the length of the small intestine, but induced changes in the intestinal morphology. Muscle layer width increased significantly in the duodenum, the mid-jejunum, and the ileum while villi length increased in the duodenum and ileum. The addition of pectin decreased N retention and increased the F/G ratio but did not affect significantly the ADG of the pigs. The effect of pectin on amino acid digestibility and digesta viscosity, motility and morphology of the small intestine, and on N-balance and performance of young pigs (Buraczewska et al. 2007). Daily infusion of either 60 g of pectin dissolved in 1.8 L of water or 1.8 L of water as a control infusion 2 × 8 barrows growing pigs (BW 30.1 ± 1.3 kg) (German Landrace × Piétrain), the pigs were surgically fitted at the distal ileum with a simple T-cannula In Pectin infusion reduced (P = 0.005) ileal gene copy number of the C. leptum cluster. Ileal bacterial populations and fermentation patterns are susceptible to changes in the intestinal availability of Ca and P as well as to the supply of pectin as a fermentable substrate. Ileal microbiota of growing pigs fed different dietary calcium phosphate levels and phytase content and subjected to ileal pectin infusion (Metzler-Zebeli et al. 2010). Cereal-based diets with 0, 40 or 80 g of Apple pectin (P) or 270 g of rye (R) per kg Four groups (six pigs each), pigs of 15 kg BW Digesta viscosity was increased more by 80 g than by 40 g P or by R. Ileal digestibility of AA and nitrogen retention were negatively affected by P, whereas growth performance was decreased by R. The effect of the supplements on intestinal morphology was variable, except for the increase in myenteron thickness by P and crypt depth by R. The number of goblet cells containing acidic mucins was decreased by 40 g P in crypts in the mid-jejunum and by 40 g P and R in villi in the ileum. Fasting and postprandial plasma levels of free threonine and of threonine dehydrogenase activity in the liver and pancreas were not affected. In conclusion, feeding P or R negatively affects ileal AA digestibility and provokes irregular changes of small intestinal morphology. These effects cannot be attributed to the increase of digesta viscosity as the main factor. The effects of pectin and rye on amino acid ileal digestibility, threonine metabolism, nitrogen retention and morphology of the small intestine in young pigs (Święch et al. 2012). Citrus Pectin (Genu pectin type B Rapid Set, CP Kelco) 150 castrated piglets, five diets with varying contents of citrus pectin (soluble fibre) and barley hulls (insoluble fibre) There was no difference in SCFA caused by dietary treatment, but across treatments there was a correlation between concentration of SCFA and the proportion of butyrate. The correlation was lowest in the caecum and highest in the distal colon. Association between butyrate and short-chain fatty acid concentrations in gut contents and faeces in weaning piglets (Lærke et al. 2007). Mango pectin 30 large Whitemale pigs with an initial weight of ∼19 kg (10 pigs per group) The rheology of ingredients does not necessarily reflect the rheological effect when ingested. Viscous property of pectin hinders amylase activity in pig stomach. Rheological and microstructural properties of porcine gastric digesta and diets containing pectin or mango powder (Wu et al. 2016). Pectin (0%–12% inclusion level) CP Kelco, Wilmington, DE Two groups of eight Yorshire barrows, average BW of 14.3 ± 1.4 kg (Lysine study) and 17.2 ± 1.3 kg (Threonine study) Utilisation of ileal digestible threonine, but not of ileal digestible lysin intake, for PD (protein deposition) decreased linearly with dietary pectin level, and was not influenced by diet cellulose level. Increasing dietary pectin level reduces utilization of digestible threonine intake, but not lysine intake, for body protein deposition in growing pigs (Zhu et al. 2005). 0.5 and 10% pectin Weaned piglets three groups of six castrated male crossbred pigs with an average BW of 8 kg were fitted with T-cannulas at the caecum fed with diets supplemented with 0%, 5% and 10% pectin. Faeces were collected over a period of 3 days. Addition of 5% pectin to the diet lowered the content of dry matter and lactic acid in fecal matter. The pH and the digestibility of pectin, the concentration of total SCFA, acetate, propionate, butyrate, bicarbonate and chloride increased. Dietary pectin of 10% increased the content of total SCFA and acetate further. It was concluded that pectin might be beneficial for the development of fermentative processes in the large intestine. The influence of 5% and 10% dietary apple pectin on parameters of fermentation in faeces and caecal digesta of weaning pigs (Zacharias et al. 2004). 4 Citrus pectins with DE (DM) 80, 60%, 46%, 5% Two citrus pectins classified as calcium sensitive (CAS) and calcium insensitive (CAN) and SBP were tested. Piglets (eight per group, n = 32 for control animals) were weaned at 4 weeks of age. Killed 7 weeks of age. Dissimilarity of viscosity between water and GI due to GI juice interaction with pectin structures. DM46 and DM 80 reduced digestibility of organic matter, no difference of digestibility was seen for DM 60, DM5 CAS and CAN compared to the control group. Concluding that the rheological characteristics of pectins in water differ in the GIT. 29 Isolated Pectins: vary in their Functional Properties in the Gut of Piglets (Laerke et al. 2001). Grapefruit pectin 18 female miniature Swine of the Pitman–Moore strain Grapefruit pectin supplementation inhibits hypercholesterolemia and appears to be proportionately protective against atherosclerosis. Grapefruit pectin inhibits hypercholesterolemia and atherosclerosis in miniature swine (Baekey et al. 1988). Control treatment: Corn-SBM basal diet + ZnO (phase 1: 0.05%; phase 2; 0.03%) and four different levels of sugar beet pulp were supplemented in Corn-SBM basal diet (3%, 6%, 9% or 12%). Two phase feeding programs (phase 1: 1–2 weeks; phase 2: 3–5 weeks) were used for 5 week of growth trial. 200 weaning pigs [(Yorkshire × Landrace) × Duroc], averaging 9.01 ± 1.389 kg. Each treatment was composed of four replicates with 10 pigs per pen. No significant differences in growth performance and incidence of diarrhea among treatments. Linear response was observed in Lactobacillus counts as sugar beet pulp supplementation increased (P < 0.05). In addition, IGF-1, IgA and IgG were not affected by dietary treatments. However, the BUN concentration was decreased when pigs were fed the treatments of diets with SBP compared to that of control treatment (P < 0.05). In nutrient digestibility, crude fiber and NDF digestibilities were improved as the sugar beet pulp increased (P < 0.05). However, digestibilities of crude ash, crude fat, crude fiber and nitrogen retention were not affected by dietary sugar beet pulp levels. Effect of Dietary sugar beet pulp supplementation on growth performance, nutrient digestibility, fecal microflora, blood profiles and diarrhea incidence in weaning pigs (Yan et al. 2017). 40 days on one of four diets differing in the level and type of polysaccharide. Control diet with or without 4 or 8% pectin or a diet with rye instead of part of the wheat. 24 male growing pigs with a high lean gain potential (synthetic line 990, Poland), divided into four groups (six pigs each) initial BW of about 20 kg The influence of polysaccharidesonthe epithelial structure was evaluated in the duodenum, mid-jejunum and ileum. Increased crypt depth and mucosa thickness were found after feeding the diet with rye in all measured segments, whereas feeding the diets with 4% and 8% pectin caused an increase in myenteron thickness of the mid-jejunum and ileum, in comparison with other diets. The structure of small intestinal tissue in pigs as influenced by indigestible polysaccharides (Tusnio et al. 2006). Pectin was supplied at levels of 0 and 334 g dry matter day−1 together with a semi-synthetic basal diet to. In addition to Pectin treatments two different levels of thiamine, 0.66 and 2.57 mg day−1 were applied. 12 adult sows weighing 166 ± 17 kg The pectin fermentation rate was determined by three different methods s to account for 94%–96% of pectin intake. The apparent digestibility of nitrogen was reduced by more than 20% at an almost constant true N digestibility. The increased fecal nitrogen excretion with pectin caused a reduction in protein retention to zero, and was composed of 82% bacterial nitrogen. The changes in the proteinaceous water-soluble fraction of fecal nitrogen indicated an increase in the excretion of endogenous proteins. Effects of pectin supplementation on the digestion of different structural carbohydrate fractions and on bacterial nitrogen turnover in the hindgut of adult sows (Roth-Maier et al.1993). Sugar beet fibre Two groups of pigs/ 36 female weanling piglets (14 ± 2 kg) of the Large White breed After 4 week, total serum cholesterol and high-density-lipoprotein cholesterol concentrations were similar on both diets. By contrast, the fasting triacylglycerols were 21% lower (P < 0.05) and apparent feed-conversion efficiency was 47% higher (P < 0.01) on the SBF diet than on the control diet. Accordingly, the effect of SBF did not appear to be mediated by an impairing effect on dietary lipid absorption. The results suggest that the decreasing effect of SBF on triacylglycerols was due to a reduction in very-low-density-lipoprotein synthesis without changes in the size of particles. The low-density-lipoprotein receptor activity of a liver plasma membrane-enriched fraction was not influenced by the dietary treatment; however, a significant negative relationship between cholesterol concentrations and the receptor activity was observed irrespective of the diet. Effects of sugar beet fiber feeding on serum lipids and binding of low-density lipoproteins to liver membranes in growing pigs (Fremont, Gozzelino and Bosseau 1993). Each group of piglets was fed with corn–soybean meal diets containing 5% apple pectin (PEC, purchased from Yuzhong Biotech Corporation, Henan, China) or cornstarch (purchased from Yufeng Cornstarch and Sugar Company, Hebei, China) as the control (CON) Male Duroc × Large White crossbred piglets with an average body weight of 11.05 ± 0.11 kg were randomly divided into 2 groups of 6 animals each and allowed to adapt for 1 week before the start of the 72-day experiment. Growth performance was not affected by the inclusion of 5% pectin in the conventional corn–soybean meal diet over the 72-d trial period n pigs fed with pectin, total cholesterol and low-density lipoprotein cholesterol were lowered but high-density lipoprotein was increased (P < 0.05). Serum triglycerides tended to be lower in the pectin-fed animals (P = 0.093), whereas no change was noted in serum total bile acid. Bile acid pool: In pigs fed with 5% pectin, only cecal ursodeoxycholic acid (P = 0.097) and hyocholic acid (P = 0.088) showed a decreasing tendency. In the ileum, pectin increased in-and-out BA transport on the apical membrane, but it increased the overall BA transport in the cecum. Effects of dietary pectin on the profile and transport of intetinal bile acids in young pigs (Fang et al. 2018). View Large Various parameters have been studied for the evaluation of pectin in feed as growth and health modulating ingredient, (see Table 1). Parameters include gut development (Hedemann et al. 2006; Buraczewska et al. 2007; Święch et al. 2012), more specifically muscle layer width in the intestine and motility of the duodenum and mid-jejunum (Buraczewska et al. 2007), plasma glucose, mucosal protein synthesis in the intestine (Libao-Mercado et al. 2007), intestinal morphology and enzymatic activity (Hedemann et al. 2006; Wu et al. 2016; Pluschke et al. 2018). Studies on the direct interactions of pectin with the mucosa exist mostly ex vivo (Schmidgall and Hensel 2002; Liu et al. 2005; Thirawong et al. 2007). Investigations of the impact of pectin on the ion exchange and buffering capacity, digestibility of organic matter (Metzler-Zebeli et al. 2010; Tian et al. 2017), dry matter content in feces, amino acid digestibility (Buraczewska et al. 2007; Święch et al. 2012), digesta viscosity (Laerke et al. 2001; Buraczewska et al. 2007; Wu et al. 2016) and mineral absorption (Metzler-Zebeli et al. 2010), as well as effects of pectin on the profile and transport of intestinal bile acids (BA) (Fang et al. 2018) are valuable contributions to the understanding of the interactions of pectin with feed components and the animal's physiology (Table 1). Within this context, information on pectin source and its molecular structure is essential and is often missing even in recent studies. For instance, in the study by Fang et al. 2018, who presented a detailed analysis of the impact of pectin on the profiles and transport of bile acids in young pigs. They reported benefits of pectin supplementation for the cholesterol metabolism, as well as its impact on BA composition and upregulation of intestinal BA transporters and receptors. Unfortunately, the authors did not provide information on the molecular structure and source of the pectin substrate implemented. Furthermore, the analysis of the piglet GM would have been an interesting addition to the study. BAs are known to be metabolised by the GM (Sayin et al. 2013; Ridlon et al. 2014) and the correlation of the changes in the BA pool with potential pectin induced changes in the GM could provide information on such metabolism. Characteristics of pectin such as the DE impact its viscous properties, that can affect daily feed intake (DFI) and nutrient absorption. Bruggeman et al. (2018) recently published an invention, that describes the energy redistribution in animals by pectin with a DE of less than 65%. The findings are explained by a change of the feed intake pattern, characterised by initial latency to feed intake followed by smaller but more frequent meals. The report indicates a role of pectin in the regulation of hunger and satiety (Bruggeman et al. 2018). When included in weaner diets, citrus pectin with a DE of 70% reduced daily feed intake and average daily gain (DG) as reported by Hedemann et al. 2006. Unfortunately, the study did not include investigations of the impact of pectin on the piglet GM and SCFA production (Hedemann et al. 2006). The DG, a measure of animal growth is valuable information. Nevertheless, DG is not only affected by a varying DFI, and the absorption or retention of specific nutrients, it can also be influenced by the level of pectin fibre derived short chain-fatty acids (SCFAs). SCFAs are the end products of fermentation of dietary fibres by the GM and have been shown to exert multiple beneficial effects on mammalian energy metabolism, body weight and mammalian health (den Besten et al. 2013). It is hence essential to understand how different pectin substrates influence the endogenous microbial community, which is the source of SCFA production in vivo. Comprehensive studies with a detailed analysis of pectin associated modulation of the GM and associated metabolites, such as SCFAs in combination with data on DFI and DG for piglets and pigs are still missing. Organic acids can also act as antimicrobials due to their pH lowering properties and are among the candidate replacements for antibiotics. They can increase pancreatic secretion and exhibit trophic effects on the gastrointestinal mucosa as reviewed in Dibner and Buttin 2002 (Dibner and Buttin 2002). These acids have been used in swine diets for decades (Partanen and Mroz 1999; Namkung et al. 2004) and several studies exist on the impact of pectin on SCFA levels (Lærke et al. 2007; Metzler et al. 2009; Metzler-Zebeli et al. 2010; Tian et al. 2017). Zacharias et al. have reported changes in SCFA levels in porcine ceacel and fecal matter after administration of apple pectin (Zacharias, Kerler and Drochner 2004). Metzler-Zebeli et al. found increased ileal d-lactate levels and an increase of the molar proportion of isobutyrate after ileal pectin infusion. In feces, pectin infusion tended to increase total SCFAs and acetate. The study did not include a comprehensive analysis of the whole microbial community, solely selected microbial populations were analysed by quantitative PCR in DNA extracts of ileal digesta, and a reduction in the ileal gene copy number of the Clostridium leptum cluster was reported (Metzler-Zebeli et al. 2010). Recent advances in GM community analysis via 16S rRNA gene sequencing at a reduced cost allow for comprehensive investigations of GM community changes, as demonstrated by Tian et al. who investigated the changes of the whole GM community structure by the supplementation of weaner feed with low methoxyl pectin (LMP) and high methoxyl pectin (HMP). The inclusion of the citrus pectin led to a higher relative abundance of Bacteroidetes, whereas that of Firmicutes was lower in the colon and feces, but not in the ileum. An increased relative abundance of unclassified bacteria within the Lachnospiraceae family was found in the feces of both LMP- and HMP-fed piglets. The increase in relative abundance of unclassified microbes within the family Prevotellaceae also occurred in the colon of both the LMP- and HMP-fed piglets. Whereas, the increase in relative abundance of the Dialister genus in the colonic digesta was LMP specific. The SCFA patterns in the piglet feces were not significantly affected by the diet and intervention time (Tian et al. 2017). In vivo SCFA are absorbed by the host, it would be hence beneficial also to monitor the SCFA levels in the piglet plasma. Understanding the microstructure related mode of action of pectin and its impact on the GM and its metabolites along the GIT is critical for the successful tailoring of pectin formulations for in feed applications. Fig. 1 depicts an overview of direct and indirect interactions of pectin with the GM as well as with the host- associated gut barrier and immune system. Figure 1. View largeDownload slide Overview of direct and indirect interactions of pectin within the gut. Interactions are indicated with arrows. The host and gut barrier are displayed as coloured background.. Figure 1. View largeDownload slide Overview of direct and indirect interactions of pectin within the gut. Interactions are indicated with arrows. The host and gut barrier are displayed as coloured background.. Feeding the animal and its microbiome: gut microbiota related challenges in pig production Neonatal piglet diarrhea caused by pathogens or viral infections is leading to considerable economic losses in the pig industry (Wittum et al. 1995; Wieler et al. 2001; Jacobson et al. 2003). Diarrhea impairs the welfare of the animals in the short term and may disrupt the normal bacterial succession in the GIT and hence affect animal health in the long run as well. There is increasing pressure to replace the antibiotics and zinc oxides, that are implemented to counteract diarrhea in pig production. This pressure is leading to increased efforts to explore other avenues to promote gut health, such as the implementation of pro- and prebiotics (Brüssow 2017; Li 2017), which are thought to strengthen and stabilise gut health via a balanced gut microbiome. Bacteria colonise the GIT from birth onwards (Kenworthy and Crabb 1963). This colonisation impacts the maturation of the GIT, the immune system and its capacity to defend against pathogens (Bezirtzoglou 1997; Ley et al. 2008). Mammalian milk plays a vital role in the nurturing of a healthy GM. The milk oligosaccharides (MOs) are a group of diverse and complex glycans (≈200 structures) and an abundant component of the mammalian milk. MOs evolved to nurture the mammalian GM, nursing beneficial microbes and inhibiting pathogens. These glycans thus play an essential role in the establishment of health during early life (Sela and Mills 2010). They are known to lower the risk for viral, bacterial, protozoan, parasite infections and necrotising enterocolitis (Jantscher-Krenn et al. 2011). Porcine milk oligosaccharides (PMOs) are the representatives of these oligosaccharides in pigs (Poroyko et al. 2010). In earlier farm practice piglets were weaned spontaneously after 10–12 weeks. In current days piglets are weaned at the age of 3–5 weeks, a time when the sows produce the maximum amount of milk. The sudden weaning results in various adverse reactions such as diarrhea (Nabuurs 1998). The absence of PMOs contributes to these effects, through the weakening of the endogenous GM and reduced pathogen defence. It is, therefore, necessary to use prebiotic ingredients as components in milk replacers and weaner feed formulations. Examples of current alternatives for PMOs are galacto-oligosaccharides (GOS) and fructo-oligosaccharides (FOS). Mannan oligosaccharides have also been shown to promote health in piglets (Castillo et al. 2008; Che et al. 2011). Whereas, Chen et al. demonstrated the improvement of growth performance and immunity after administration of dietary pectic oligosaccharides (POS) in weaned pigs infected by rotavirus (Chen et al. 2017). Unfortunately, the study did not include an analysis of the impact of POS on the whole piglet microbiome. The inclusion of specific POS in weaner formulations might aid to mimic PMOs and promote GIT health during animal production at an early age. POS with varying degrees of polymerisation can be obtained via the hydrolysis of α and β-glucosidic links of the principal chains of HG, RG-I and RG-II and their side chains (Gullón et al. 2011). POS have been suggested as a new class of prebiotics (Hotchkiss Jr et al. 2003) and this subject has been recently reviewed in detail in ‘Pectic oligosaccharides and other emerging prebiotics’ by Míguez et al. (2016). Various in vitro studies have shown evidence for the prebiotic effects of POS when fermented by human fecal inoculum (Olano‐Martin, Gibson and Rastall 2002; Suzuki et al. 2004; Al‐Tamimi et al. 2006; Holck et al. 2011; Onumpai et al. 2011; Chen et al. 2013; Gómez et al. 2014; Moon et al. 2015; Gómez et al. 2016; Li et al. 2016). A recent study examined fermentability of POS from sugar beet pulp with human and pig fecal inoculum (from multiparous non-lactating sows, Dutch Landrace) in vitro. The study demonstrated lowered degradation of acetylated RG oligosaccharides by pig fecal microbiota (Leijdekkers et al. 2014). Few studies exist to date on POS in animal feed applications (Wang et al. 2016; Chen et al. 2017), also due to current limitations in the production of POS. These are hence just the beginnings of the exploration of POS for in feed applications. In vitro models such as the CoMiniGut model (Wiese et al. 2018) operating at a low fermentation volume, requiring only small amounts of substrate are ideal for the initial investigation of novel pectic substrates just available in small quantities. Diet is a principal factor in shaping the GM composition and functionality (Scott et al. 2013). Depending on the age of the animal target group and the associated maturity and carbohydrate degradation capacity of the target group's GM, different pectin substrates can be matched for in feed applications. In a grown mammal the matured GM has an expanded capacity to degrade more complex carbohydrates and continues to have a profound influence on its nutrition, organ, tissue and immune system (O'Hara and Shanahan 2006). The GM provides a large set of metabolic functions for the host, that go beyond the production of SCFA, it also plays a role in the synthesis of K- and B-vitamins, the protection from pathogenic bacteria through competitive exclusion, stimulation of enterocyte turnover (Sekirov et al. 2010; LeBlanc et al. 2013) and assistance in development and maturation of the GIT and the mucosal system (Sommer and Bäckhed 2013). Emerging studies such as the study by Ramayo-Caldas et al. 2016 provide an integrated overview of the porcine GM and can identify microbial enterotypes associated with specific traits such as growth. An enterotype is a robust microbial cluster, driven by species composition including a functional understanding of the overall community (Arumugam et al. 2011). An enterotype-like group with a significant over-abundance of genera such as Prevotella and Streptococcus was reported to be correlated to increased body weight and average daily weight gain during the post-weaning period (Ramayo-Caldas et al. 2016). The study would have benefited from additional measurements and correlations of SCFA levels in colon content, feces and plasma with the identified enterotypes and body weights. In vitroandex vivo studies on the effects of pectin on the gut microenvironment Several ex vivo studies investigated the mucoadhesive effect of pectin using porcine mucosa tissue or material, from different segments of the GIT, such as the buccal, stomach, small intestine and the large intestine. Mucoadhesion facilitates contact with the gut epithelium and may aid in the modulation of the immune response (Furness et al. 2013). It may also be used for drug delivery solutions (McConnell, Fadda and Basit 2008; Wong, Colombo and Sonvico 2011), as well as for the topical coating of the damaged intestinal wall in inflammatory diseases. The mucoadhesive properties of pectin have been shown to differ between different GIT sites and also vary based on molecular properties such as the net charge, molecular weight and DE (Schmidgall and Hensel 2002; Liu et al. 2005; Thirawong et al. 2007). An overview of studies is displayed in Table 2. Table 2. In vitro (and ex vivo) studies investigating the effect of pectin on the gut microenvironment with focus on pigs. In Vitro Model Pectin/substrate Observations Reference In vitro:pig colonic digesta (C) or faeces (F) Batch: 24 or 48 hr, no pH control Apple pectin from ZPOW Pektow in Sp. z o.o. (Jasło, Poland) Potato protein and casein were fermented each with cellulose, pectin or raw potato (resistant) starch. The total SCFA concentration was greater after C than F fermentation, regardless of the substrates. The total amines concentration after C and F fermentation of potato protein with all fibres was the same, while after C fermentation of casein it was affected by the type of fibre (P = 0.001 and P = 0.000 after 24 and 48 hr, respectively). It was very high with cellulose, lower with starch and the lowest with pectin. Concluding that in vitro bacterial proteolysis is greatly affected by the interaction of type of protein and fibre. The effects of type of protein and fibre fermented in vitro with different pig inocula on short-chain fatty acids and amines concentrations (Taciak et al. 2015). In vitro: pig and human fecal inocula: Pig Fecal Inoculum. Three multiparous non-lactating sows (Dutch Landrace, aged 4 years) Pooled fecal inoculum Sugar beet pectic oligosaccharides SBPOS consisted mainly of partially acetylated RGOS and partially methyl- esterified/acetylated HGOS. Some SBPOS contained an unsaturated galacturonic acid residue at their non-reducing end. SBPOS could be completely fermented by human and pig fecal microbiota, thereby producing butyrate yet mainly acetate and propionate as metabolites. The degradation of SBPOS by pig fecal microbiota was different and much slower compared to human fecal microbiota. In general, RGOS were degraded slower than HGOS. Acetylation of RGOS lowered the degradation rate by pig fecal microbiota but not by human fecal microbiota. No classic bifidogenic effect was shown for SBPOS using human fecal inoculum. However, several other potentially interesting modifications in the microbiota composition that can be associated with host health were observed. In Vitro Fermentability of Sugar Beet Pulp Derived Oligosaccharides Using Human and Pig Fecal Inocula (Leijdekkers et al. 2014). In vitro: single strain fermentations: 18 bacterial strains most strains were of human origin; two strains originated from porcine feces: Lactobacillus acidophilus L. fermentum swine Soy arabinogalactan (AGPS), sugar beet arabinan (AOS), wheat flour arabinoxylan, polygalacturonan, rhamnogalacturonan from apple, FOS Lactobacillus acidophilus L. fermentum (Porcine origin) degraded FOS and AGPS partially. Overall: AOS of DP 2–6 were fermented by Bi. longum and C. clostridiiforme. E. coli and B. adolescentis fermented only arabinotriose while Clostridium beijerinckii and Clostridium sartagoformum only degraded the arabinotriose and arabinotetraose to some extent. Clostridium butyricum degraded only arabinotetraose were unable to degrade the oligosaccharides. Rhamnogalacturonan-Enriched Polysaccharide Fraction (RGAPS) and (Rhamno)galacturonooligosaccharides ((R)GAOS). Fermentation of plant cell-wall-derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria (Van Laere et al. 2000). In vitro: pig fecal inoculum from feces of three multiparous sows (Dutch Landrace). BW 264–368 kg. fed twice daily with a high-fat (18.3% (w/w)), low-fibre (7.1% (w/ w)) diet, the diet contained wheat (20% (w/w)) and barley (20% (w/w)) as fibre sources. Human inoculum faeces of three healthy donors High methyl esterified (HM) citrus pectin (C74) from CP Kelco (Lille Skensved, Denmark) other fibres were: Guar gum, alginatye, tapioca starch, glucomannan. Maize starch, oat b-glucan, Inulin, FOS, soy pectin (Soyafibe-S-DA- 100), xanthan gum. Pig inoculum produced less SCFA than human inoculum for most of the fibres. Both inocula produced the highest amount of total SCFA from soy pectin (9.06–10.59 mmol/g). Fibres containing uronic acids induced the production of acetate, whereas fibres containing neutral sugars induced the production of propionate or butyrate. Except pectin and xanthan gum, the soluble fibres in this experiment were well fermented by both inocula with less than 5% (w/w) of the fibres remaining after fermentation. For xanthan gum, the analysis of sugar composition of the remaining soluble sugars showed that human inoculum did not ferment 13% of all glucose, 16% of all mannose and 14% of all glucuronic acid. For pig inoculum, it was shown that 30% of all glucose, 41% of all mannose and 54% of all glucuronic acid were not fermented. In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans (Jonathan et al. 2012). In vitro: incubated 6, 12, 24, 48 hr with ruminal fluid from cattle or feces from dogs, pigs, horses and human Cellulose (Solka Floc), beet pulp (Michigan sugar), citrus pulp (Freeman Industries) and citrus pectin (HM rapid; TIC Gums Belcamp, MD) When data were pooled across all species and substrate disappearance and SCFA production ranked from the least to greatest in the following order: cellulose< beet pulp< citrus pulp< citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microbiota from various species seem to be dependant not only on the fermentative activity of the microbial population but other factors as well, perhaps lag time and rate of digesta passage. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using faecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle (Sunvold et al. 1995). In vitro: faeces from the adult pigs from four castrated finisher pigs (Dutch Landrace × Great Yorkshire. The unweaned piglet faeces were collected from 19 unweaned piglets (male and female) which were 27 to 30 days old Pectin Comparison of in vitro microbial activity was made between inocula from faeces of adult and unweaned pigs, using a range of carbohydrate-rich substrates. The substrates tested were classified into groups (fibre-rich, grains, gums, pectin, saccharides, storage carbohydrates and miscellaneous). There were significant differences in the fermentation patterns both due to source of substrate and inoculum. It would appear that the metabolic activity of the microflora does differ significantly between adult and weanling pigs, though this varied for the different carbohydrates tested. Microbial activities of faeces from unweaned and adult pigs, in relation to selected fermentable carbohydrates (Bauer et al. 2001). In vitro:gut contents of piglet terminal ileum Potato RGI The fibers showed high fermentability, with lactate in particular being increased. A significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms was observed. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fibre being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galactorhamnogalacturonan (Strube et al. 2015b). Ex vivo: porcine GI mucosa, i.e. buccal, stomach, small intestine and large intestine, texture analyser with mucoadhesive platform. Buccal mucosa with simulated saliva fluid pH 6.75 (SSF), gastric mucosa with simulated gastric fluid USP without pepsin (SGF) or citric-phosphate buffer, pH 4.8 (representing the fasted or fed state, respectively), small intestinal mucosa (duodenum part) and large intestinal mucosa with simulated intestinal fluid pH 6.8 (SIF) Four pectins with different DEs Herbstreith & Fox KG (Germany) HMP (CU201 70% De, MW 200 kDa, 0% DA), CU501 56% De 0% DA, Mw 180 kDa), LMP (CU701 (38% De, Mw 80 kDA, 0% DA CU020 29% De, 20% DA, Mw 150 kDA). Two parameters derived from texture analysis, namely maximum detachment force (Fmax) and work of adhesion (Wad) were used as parameters for comparison of mucoadhesive performance. Pectins showed a stronger mucoadhesion on large intestinal mucosa than on small intestinal mucosa. The mucoadhesive properties of pectins on gastric mucosa depended on pH of the medium, a higher Fmax and W ad in a pH 4.8 medium than a pH 1.2 medium was revealed. The results also demonstrated that the mucoadhesive performance of pectins largely depended on their characteristics, i.e. higher degree of esterification and molecular weight gave a stronger mucoadhesion. The rank order of mucoadhesive performance of examined pectins on to the GI mucosa appeared to be similar to the rank order of their degree of esterification and molecular weight (i.e. CU201 > CU501 > CU020 > CU701). Mucoadhesive properties of various pectins on gastrointestinal mucosa: An in vitro evaluation using texture analyzer (Thirawong et al. 2007). Ex Vivo:In an ex vivo system based on porcine colonic tissue Rhamnogalacturonan (RG) derived from pectin.(apple pectin, citrus fruit pectin and pectin acid from Roth (Karlsruhe, Germany) and sugar beet pectins Sûdzucker AG (Obrigheim, Germany). Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast, highly esterified pectins and neutral polysaccharides were ineffective. Within a structure activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Bioadhesive properties of polygalacturonides against colonic epithelial membranes (Schmidgall and Hensel, 2002). The ex vivo studies on the mucosal adsorption of pectins and incorporated proteins were conducted on the surfaces of porcine colonic mucus membranes Citrus pectins with the DE of 25% (P-25) or 94% (P-94), Pectin derivatives carrying side chain primary amine groups (P-N) were prepared from P-25. Pectins with higher net charges were more mucoadhesive than the other pectins. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergise with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P- 94, also synergised with the mucosal glycoproteins to form a gel structure via coil entanglements. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. The mucin–gel complex formed between P-N or P-94 and mucus tissue constructs a surface barrier, which effectively prohibited the penetration of Triton X-100. P-25 gel did not inhibit Triton X-100 irritation of the mucus tissue as effectively as the gel formed by P-N/mucus complex Interaction of various pectin formulations with porcine colonic tissues (Liu et al. 2005). In vitroassay Six pectin types from three manufacturers (Pectin classic, Genu Pectin, Grinsted Pectin) The factors which varied were; DM (three levels; high, low and medium), degree of amidation (DA) (two levels; ami-dated or nonamidated). Due to the manufacturing process, the molecular weight was correlated with the DM. Mucoadhesive properties were measured using a texture analyzer. It was found that an intermediate degree of methoxylation (35% and 36%) improved the specific mucin interaction. Amidation did not increase mucin interaction. Samples from different manufacturers did not alter these conclusions. This study indicates that the general classification of pectin as a poor mucoadhesive, without differentiating between the amount and type of substituents, probably is an oversimplification. In Vitro measurements of mucoadhesive properties of six types of pectin (Hagesaether and Sande 2007) In Vitro Model Pectin/substrate Observations Reference In vitro:pig colonic digesta (C) or faeces (F) Batch: 24 or 48 hr, no pH control Apple pectin from ZPOW Pektow in Sp. z o.o. (Jasło, Poland) Potato protein and casein were fermented each with cellulose, pectin or raw potato (resistant) starch. The total SCFA concentration was greater after C than F fermentation, regardless of the substrates. The total amines concentration after C and F fermentation of potato protein with all fibres was the same, while after C fermentation of casein it was affected by the type of fibre (P = 0.001 and P = 0.000 after 24 and 48 hr, respectively). It was very high with cellulose, lower with starch and the lowest with pectin. Concluding that in vitro bacterial proteolysis is greatly affected by the interaction of type of protein and fibre. The effects of type of protein and fibre fermented in vitro with different pig inocula on short-chain fatty acids and amines concentrations (Taciak et al. 2015). In vitro: pig and human fecal inocula: Pig Fecal Inoculum. Three multiparous non-lactating sows (Dutch Landrace, aged 4 years) Pooled fecal inoculum Sugar beet pectic oligosaccharides SBPOS consisted mainly of partially acetylated RGOS and partially methyl- esterified/acetylated HGOS. Some SBPOS contained an unsaturated galacturonic acid residue at their non-reducing end. SBPOS could be completely fermented by human and pig fecal microbiota, thereby producing butyrate yet mainly acetate and propionate as metabolites. The degradation of SBPOS by pig fecal microbiota was different and much slower compared to human fecal microbiota. In general, RGOS were degraded slower than HGOS. Acetylation of RGOS lowered the degradation rate by pig fecal microbiota but not by human fecal microbiota. No classic bifidogenic effect was shown for SBPOS using human fecal inoculum. However, several other potentially interesting modifications in the microbiota composition that can be associated with host health were observed. In Vitro Fermentability of Sugar Beet Pulp Derived Oligosaccharides Using Human and Pig Fecal Inocula (Leijdekkers et al. 2014). In vitro: single strain fermentations: 18 bacterial strains most strains were of human origin; two strains originated from porcine feces: Lactobacillus acidophilus L. fermentum swine Soy arabinogalactan (AGPS), sugar beet arabinan (AOS), wheat flour arabinoxylan, polygalacturonan, rhamnogalacturonan from apple, FOS Lactobacillus acidophilus L. fermentum (Porcine origin) degraded FOS and AGPS partially. Overall: AOS of DP 2–6 were fermented by Bi. longum and C. clostridiiforme. E. coli and B. adolescentis fermented only arabinotriose while Clostridium beijerinckii and Clostridium sartagoformum only degraded the arabinotriose and arabinotetraose to some extent. Clostridium butyricum degraded only arabinotetraose were unable to degrade the oligosaccharides. Rhamnogalacturonan-Enriched Polysaccharide Fraction (RGAPS) and (Rhamno)galacturonooligosaccharides ((R)GAOS). Fermentation of plant cell-wall-derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria (Van Laere et al. 2000). In vitro: pig fecal inoculum from feces of three multiparous sows (Dutch Landrace). BW 264–368 kg. fed twice daily with a high-fat (18.3% (w/w)), low-fibre (7.1% (w/ w)) diet, the diet contained wheat (20% (w/w)) and barley (20% (w/w)) as fibre sources. Human inoculum faeces of three healthy donors High methyl esterified (HM) citrus pectin (C74) from CP Kelco (Lille Skensved, Denmark) other fibres were: Guar gum, alginatye, tapioca starch, glucomannan. Maize starch, oat b-glucan, Inulin, FOS, soy pectin (Soyafibe-S-DA- 100), xanthan gum. Pig inoculum produced less SCFA than human inoculum for most of the fibres. Both inocula produced the highest amount of total SCFA from soy pectin (9.06–10.59 mmol/g). Fibres containing uronic acids induced the production of acetate, whereas fibres containing neutral sugars induced the production of propionate or butyrate. Except pectin and xanthan gum, the soluble fibres in this experiment were well fermented by both inocula with less than 5% (w/w) of the fibres remaining after fermentation. For xanthan gum, the analysis of sugar composition of the remaining soluble sugars showed that human inoculum did not ferment 13% of all glucose, 16% of all mannose and 14% of all glucuronic acid. For pig inoculum, it was shown that 30% of all glucose, 41% of all mannose and 54% of all glucuronic acid were not fermented. In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans (Jonathan et al. 2012). In vitro: incubated 6, 12, 24, 48 hr with ruminal fluid from cattle or feces from dogs, pigs, horses and human Cellulose (Solka Floc), beet pulp (Michigan sugar), citrus pulp (Freeman Industries) and citrus pectin (HM rapid; TIC Gums Belcamp, MD) When data were pooled across all species and substrate disappearance and SCFA production ranked from the least to greatest in the following order: cellulose< beet pulp< citrus pulp< citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microbiota from various species seem to be dependant not only on the fermentative activity of the microbial population but other factors as well, perhaps lag time and rate of digesta passage. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using faecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle (Sunvold et al. 1995). In vitro: faeces from the adult pigs from four castrated finisher pigs (Dutch Landrace × Great Yorkshire. The unweaned piglet faeces were collected from 19 unweaned piglets (male and female) which were 27 to 30 days old Pectin Comparison of in vitro microbial activity was made between inocula from faeces of adult and unweaned pigs, using a range of carbohydrate-rich substrates. The substrates tested were classified into groups (fibre-rich, grains, gums, pectin, saccharides, storage carbohydrates and miscellaneous). There were significant differences in the fermentation patterns both due to source of substrate and inoculum. It would appear that the metabolic activity of the microflora does differ significantly between adult and weanling pigs, though this varied for the different carbohydrates tested. Microbial activities of faeces from unweaned and adult pigs, in relation to selected fermentable carbohydrates (Bauer et al. 2001). In vitro:gut contents of piglet terminal ileum Potato RGI The fibers showed high fermentability, with lactate in particular being increased. A significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms was observed. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fibre being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galactorhamnogalacturonan (Strube et al. 2015b). Ex vivo: porcine GI mucosa, i.e. buccal, stomach, small intestine and large intestine, texture analyser with mucoadhesive platform. Buccal mucosa with simulated saliva fluid pH 6.75 (SSF), gastric mucosa with simulated gastric fluid USP without pepsin (SGF) or citric-phosphate buffer, pH 4.8 (representing the fasted or fed state, respectively), small intestinal mucosa (duodenum part) and large intestinal mucosa with simulated intestinal fluid pH 6.8 (SIF) Four pectins with different DEs Herbstreith & Fox KG (Germany) HMP (CU201 70% De, MW 200 kDa, 0% DA), CU501 56% De 0% DA, Mw 180 kDa), LMP (CU701 (38% De, Mw 80 kDA, 0% DA CU020 29% De, 20% DA, Mw 150 kDA). Two parameters derived from texture analysis, namely maximum detachment force (Fmax) and work of adhesion (Wad) were used as parameters for comparison of mucoadhesive performance. Pectins showed a stronger mucoadhesion on large intestinal mucosa than on small intestinal mucosa. The mucoadhesive properties of pectins on gastric mucosa depended on pH of the medium, a higher Fmax and W ad in a pH 4.8 medium than a pH 1.2 medium was revealed. The results also demonstrated that the mucoadhesive performance of pectins largely depended on their characteristics, i.e. higher degree of esterification and molecular weight gave a stronger mucoadhesion. The rank order of mucoadhesive performance of examined pectins on to the GI mucosa appeared to be similar to the rank order of their degree of esterification and molecular weight (i.e. CU201 > CU501 > CU020 > CU701). Mucoadhesive properties of various pectins on gastrointestinal mucosa: An in vitro evaluation using texture analyzer (Thirawong et al. 2007). Ex Vivo:In an ex vivo system based on porcine colonic tissue Rhamnogalacturonan (RG) derived from pectin.(apple pectin, citrus fruit pectin and pectin acid from Roth (Karlsruhe, Germany) and sugar beet pectins Sûdzucker AG (Obrigheim, Germany). Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast, highly esterified pectins and neutral polysaccharides were ineffective. Within a structure activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Bioadhesive properties of polygalacturonides against colonic epithelial membranes (Schmidgall and Hensel, 2002). The ex vivo studies on the mucosal adsorption of pectins and incorporated proteins were conducted on the surfaces of porcine colonic mucus membranes Citrus pectins with the DE of 25% (P-25) or 94% (P-94), Pectin derivatives carrying side chain primary amine groups (P-N) were prepared from P-25. Pectins with higher net charges were more mucoadhesive than the other pectins. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergise with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P- 94, also synergised with the mucosal glycoproteins to form a gel structure via coil entanglements. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. The mucin–gel complex formed between P-N or P-94 and mucus tissue constructs a surface barrier, which effectively prohibited the penetration of Triton X-100. P-25 gel did not inhibit Triton X-100 irritation of the mucus tissue as effectively as the gel formed by P-N/mucus complex Interaction of various pectin formulations with porcine colonic tissues (Liu et al. 2005). In vitroassay Six pectin types from three manufacturers (Pectin classic, Genu Pectin, Grinsted Pectin) The factors which varied were; DM (three levels; high, low and medium), degree of amidation (DA) (two levels; ami-dated or nonamidated). Due to the manufacturing process, the molecular weight was correlated with the DM. Mucoadhesive properties were measured using a texture analyzer. It was found that an intermediate degree of methoxylation (35% and 36%) improved the specific mucin interaction. Amidation did not increase mucin interaction. Samples from different manufacturers did not alter these conclusions. This study indicates that the general classification of pectin as a poor mucoadhesive, without differentiating between the amount and type of substituents, probably is an oversimplification. In Vitro measurements of mucoadhesive properties of six types of pectin (Hagesaether and Sande 2007) View Large Table 2. In vitro (and ex vivo) studies investigating the effect of pectin on the gut microenvironment with focus on pigs. In Vitro Model Pectin/substrate Observations Reference In vitro:pig colonic digesta (C) or faeces (F) Batch: 24 or 48 hr, no pH control Apple pectin from ZPOW Pektow in Sp. z o.o. (Jasło, Poland) Potato protein and casein were fermented each with cellulose, pectin or raw potato (resistant) starch. The total SCFA concentration was greater after C than F fermentation, regardless of the substrates. The total amines concentration after C and F fermentation of potato protein with all fibres was the same, while after C fermentation of casein it was affected by the type of fibre (P = 0.001 and P = 0.000 after 24 and 48 hr, respectively). It was very high with cellulose, lower with starch and the lowest with pectin. Concluding that in vitro bacterial proteolysis is greatly affected by the interaction of type of protein and fibre. The effects of type of protein and fibre fermented in vitro with different pig inocula on short-chain fatty acids and amines concentrations (Taciak et al. 2015). In vitro: pig and human fecal inocula: Pig Fecal Inoculum. Three multiparous non-lactating sows (Dutch Landrace, aged 4 years) Pooled fecal inoculum Sugar beet pectic oligosaccharides SBPOS consisted mainly of partially acetylated RGOS and partially methyl- esterified/acetylated HGOS. Some SBPOS contained an unsaturated galacturonic acid residue at their non-reducing end. SBPOS could be completely fermented by human and pig fecal microbiota, thereby producing butyrate yet mainly acetate and propionate as metabolites. The degradation of SBPOS by pig fecal microbiota was different and much slower compared to human fecal microbiota. In general, RGOS were degraded slower than HGOS. Acetylation of RGOS lowered the degradation rate by pig fecal microbiota but not by human fecal microbiota. No classic bifidogenic effect was shown for SBPOS using human fecal inoculum. However, several other potentially interesting modifications in the microbiota composition that can be associated with host health were observed. In Vitro Fermentability of Sugar Beet Pulp Derived Oligosaccharides Using Human and Pig Fecal Inocula (Leijdekkers et al. 2014). In vitro: single strain fermentations: 18 bacterial strains most strains were of human origin; two strains originated from porcine feces: Lactobacillus acidophilus L. fermentum swine Soy arabinogalactan (AGPS), sugar beet arabinan (AOS), wheat flour arabinoxylan, polygalacturonan, rhamnogalacturonan from apple, FOS Lactobacillus acidophilus L. fermentum (Porcine origin) degraded FOS and AGPS partially. Overall: AOS of DP 2–6 were fermented by Bi. longum and C. clostridiiforme. E. coli and B. adolescentis fermented only arabinotriose while Clostridium beijerinckii and Clostridium sartagoformum only degraded the arabinotriose and arabinotetraose to some extent. Clostridium butyricum degraded only arabinotetraose were unable to degrade the oligosaccharides. Rhamnogalacturonan-Enriched Polysaccharide Fraction (RGAPS) and (Rhamno)galacturonooligosaccharides ((R)GAOS). Fermentation of plant cell-wall-derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria (Van Laere et al. 2000). In vitro: pig fecal inoculum from feces of three multiparous sows (Dutch Landrace). BW 264–368 kg. fed twice daily with a high-fat (18.3% (w/w)), low-fibre (7.1% (w/ w)) diet, the diet contained wheat (20% (w/w)) and barley (20% (w/w)) as fibre sources. Human inoculum faeces of three healthy donors High methyl esterified (HM) citrus pectin (C74) from CP Kelco (Lille Skensved, Denmark) other fibres were: Guar gum, alginatye, tapioca starch, glucomannan. Maize starch, oat b-glucan, Inulin, FOS, soy pectin (Soyafibe-S-DA- 100), xanthan gum. Pig inoculum produced less SCFA than human inoculum for most of the fibres. Both inocula produced the highest amount of total SCFA from soy pectin (9.06–10.59 mmol/g). Fibres containing uronic acids induced the production of acetate, whereas fibres containing neutral sugars induced the production of propionate or butyrate. Except pectin and xanthan gum, the soluble fibres in this experiment were well fermented by both inocula with less than 5% (w/w) of the fibres remaining after fermentation. For xanthan gum, the analysis of sugar composition of the remaining soluble sugars showed that human inoculum did not ferment 13% of all glucose, 16% of all mannose and 14% of all glucuronic acid. For pig inoculum, it was shown that 30% of all glucose, 41% of all mannose and 54% of all glucuronic acid were not fermented. In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans (Jonathan et al. 2012). In vitro: incubated 6, 12, 24, 48 hr with ruminal fluid from cattle or feces from dogs, pigs, horses and human Cellulose (Solka Floc), beet pulp (Michigan sugar), citrus pulp (Freeman Industries) and citrus pectin (HM rapid; TIC Gums Belcamp, MD) When data were pooled across all species and substrate disappearance and SCFA production ranked from the least to greatest in the following order: cellulose< beet pulp< citrus pulp< citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microbiota from various species seem to be dependant not only on the fermentative activity of the microbial population but other factors as well, perhaps lag time and rate of digesta passage. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using faecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle (Sunvold et al. 1995). In vitro: faeces from the adult pigs from four castrated finisher pigs (Dutch Landrace × Great Yorkshire. The unweaned piglet faeces were collected from 19 unweaned piglets (male and female) which were 27 to 30 days old Pectin Comparison of in vitro microbial activity was made between inocula from faeces of adult and unweaned pigs, using a range of carbohydrate-rich substrates. The substrates tested were classified into groups (fibre-rich, grains, gums, pectin, saccharides, storage carbohydrates and miscellaneous). There were significant differences in the fermentation patterns both due to source of substrate and inoculum. It would appear that the metabolic activity of the microflora does differ significantly between adult and weanling pigs, though this varied for the different carbohydrates tested. Microbial activities of faeces from unweaned and adult pigs, in relation to selected fermentable carbohydrates (Bauer et al. 2001). In vitro:gut contents of piglet terminal ileum Potato RGI The fibers showed high fermentability, with lactate in particular being increased. A significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms was observed. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fibre being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galactorhamnogalacturonan (Strube et al. 2015b). Ex vivo: porcine GI mucosa, i.e. buccal, stomach, small intestine and large intestine, texture analyser with mucoadhesive platform. Buccal mucosa with simulated saliva fluid pH 6.75 (SSF), gastric mucosa with simulated gastric fluid USP without pepsin (SGF) or citric-phosphate buffer, pH 4.8 (representing the fasted or fed state, respectively), small intestinal mucosa (duodenum part) and large intestinal mucosa with simulated intestinal fluid pH 6.8 (SIF) Four pectins with different DEs Herbstreith & Fox KG (Germany) HMP (CU201 70% De, MW 200 kDa, 0% DA), CU501 56% De 0% DA, Mw 180 kDa), LMP (CU701 (38% De, Mw 80 kDA, 0% DA CU020 29% De, 20% DA, Mw 150 kDA). Two parameters derived from texture analysis, namely maximum detachment force (Fmax) and work of adhesion (Wad) were used as parameters for comparison of mucoadhesive performance. Pectins showed a stronger mucoadhesion on large intestinal mucosa than on small intestinal mucosa. The mucoadhesive properties of pectins on gastric mucosa depended on pH of the medium, a higher Fmax and W ad in a pH 4.8 medium than a pH 1.2 medium was revealed. The results also demonstrated that the mucoadhesive performance of pectins largely depended on their characteristics, i.e. higher degree of esterification and molecular weight gave a stronger mucoadhesion. The rank order of mucoadhesive performance of examined pectins on to the GI mucosa appeared to be similar to the rank order of their degree of esterification and molecular weight (i.e. CU201 > CU501 > CU020 > CU701). Mucoadhesive properties of various pectins on gastrointestinal mucosa: An in vitro evaluation using texture analyzer (Thirawong et al. 2007). Ex Vivo:In an ex vivo system based on porcine colonic tissue Rhamnogalacturonan (RG) derived from pectin.(apple pectin, citrus fruit pectin and pectin acid from Roth (Karlsruhe, Germany) and sugar beet pectins Sûdzucker AG (Obrigheim, Germany). Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast, highly esterified pectins and neutral polysaccharides were ineffective. Within a structure activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Bioadhesive properties of polygalacturonides against colonic epithelial membranes (Schmidgall and Hensel, 2002). The ex vivo studies on the mucosal adsorption of pectins and incorporated proteins were conducted on the surfaces of porcine colonic mucus membranes Citrus pectins with the DE of 25% (P-25) or 94% (P-94), Pectin derivatives carrying side chain primary amine groups (P-N) were prepared from P-25. Pectins with higher net charges were more mucoadhesive than the other pectins. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergise with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P- 94, also synergised with the mucosal glycoproteins to form a gel structure via coil entanglements. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. The mucin–gel complex formed between P-N or P-94 and mucus tissue constructs a surface barrier, which effectively prohibited the penetration of Triton X-100. P-25 gel did not inhibit Triton X-100 irritation of the mucus tissue as effectively as the gel formed by P-N/mucus complex Interaction of various pectin formulations with porcine colonic tissues (Liu et al. 2005). In vitroassay Six pectin types from three manufacturers (Pectin classic, Genu Pectin, Grinsted Pectin) The factors which varied were; DM (three levels; high, low and medium), degree of amidation (DA) (two levels; ami-dated or nonamidated). Due to the manufacturing process, the molecular weight was correlated with the DM. Mucoadhesive properties were measured using a texture analyzer. It was found that an intermediate degree of methoxylation (35% and 36%) improved the specific mucin interaction. Amidation did not increase mucin interaction. Samples from different manufacturers did not alter these conclusions. This study indicates that the general classification of pectin as a poor mucoadhesive, without differentiating between the amount and type of substituents, probably is an oversimplification. In Vitro measurements of mucoadhesive properties of six types of pectin (Hagesaether and Sande 2007) In Vitro Model Pectin/substrate Observations Reference In vitro:pig colonic digesta (C) or faeces (F) Batch: 24 or 48 hr, no pH control Apple pectin from ZPOW Pektow in Sp. z o.o. (Jasło, Poland) Potato protein and casein were fermented each with cellulose, pectin or raw potato (resistant) starch. The total SCFA concentration was greater after C than F fermentation, regardless of the substrates. The total amines concentration after C and F fermentation of potato protein with all fibres was the same, while after C fermentation of casein it was affected by the type of fibre (P = 0.001 and P = 0.000 after 24 and 48 hr, respectively). It was very high with cellulose, lower with starch and the lowest with pectin. Concluding that in vitro bacterial proteolysis is greatly affected by the interaction of type of protein and fibre. The effects of type of protein and fibre fermented in vitro with different pig inocula on short-chain fatty acids and amines concentrations (Taciak et al. 2015). In vitro: pig and human fecal inocula: Pig Fecal Inoculum. Three multiparous non-lactating sows (Dutch Landrace, aged 4 years) Pooled fecal inoculum Sugar beet pectic oligosaccharides SBPOS consisted mainly of partially acetylated RGOS and partially methyl- esterified/acetylated HGOS. Some SBPOS contained an unsaturated galacturonic acid residue at their non-reducing end. SBPOS could be completely fermented by human and pig fecal microbiota, thereby producing butyrate yet mainly acetate and propionate as metabolites. The degradation of SBPOS by pig fecal microbiota was different and much slower compared to human fecal microbiota. In general, RGOS were degraded slower than HGOS. Acetylation of RGOS lowered the degradation rate by pig fecal microbiota but not by human fecal microbiota. No classic bifidogenic effect was shown for SBPOS using human fecal inoculum. However, several other potentially interesting modifications in the microbiota composition that can be associated with host health were observed. In Vitro Fermentability of Sugar Beet Pulp Derived Oligosaccharides Using Human and Pig Fecal Inocula (Leijdekkers et al. 2014). In vitro: single strain fermentations: 18 bacterial strains most strains were of human origin; two strains originated from porcine feces: Lactobacillus acidophilus L. fermentum swine Soy arabinogalactan (AGPS), sugar beet arabinan (AOS), wheat flour arabinoxylan, polygalacturonan, rhamnogalacturonan from apple, FOS Lactobacillus acidophilus L. fermentum (Porcine origin) degraded FOS and AGPS partially. Overall: AOS of DP 2–6 were fermented by Bi. longum and C. clostridiiforme. E. coli and B. adolescentis fermented only arabinotriose while Clostridium beijerinckii and Clostridium sartagoformum only degraded the arabinotriose and arabinotetraose to some extent. Clostridium butyricum degraded only arabinotetraose were unable to degrade the oligosaccharides. Rhamnogalacturonan-Enriched Polysaccharide Fraction (RGAPS) and (Rhamno)galacturonooligosaccharides ((R)GAOS). Fermentation of plant cell-wall-derived polysaccharides and their corresponding oligosaccharides by intestinal bacteria (Van Laere et al. 2000). In vitro: pig fecal inoculum from feces of three multiparous sows (Dutch Landrace). BW 264–368 kg. fed twice daily with a high-fat (18.3% (w/w)), low-fibre (7.1% (w/ w)) diet, the diet contained wheat (20% (w/w)) and barley (20% (w/w)) as fibre sources. Human inoculum faeces of three healthy donors High methyl esterified (HM) citrus pectin (C74) from CP Kelco (Lille Skensved, Denmark) other fibres were: Guar gum, alginatye, tapioca starch, glucomannan. Maize starch, oat b-glucan, Inulin, FOS, soy pectin (Soyafibe-S-DA- 100), xanthan gum. Pig inoculum produced less SCFA than human inoculum for most of the fibres. Both inocula produced the highest amount of total SCFA from soy pectin (9.06–10.59 mmol/g). Fibres containing uronic acids induced the production of acetate, whereas fibres containing neutral sugars induced the production of propionate or butyrate. Except pectin and xanthan gum, the soluble fibres in this experiment were well fermented by both inocula with less than 5% (w/w) of the fibres remaining after fermentation. For xanthan gum, the analysis of sugar composition of the remaining soluble sugars showed that human inoculum did not ferment 13% of all glucose, 16% of all mannose and 14% of all glucuronic acid. For pig inoculum, it was shown that 30% of all glucose, 41% of all mannose and 54% of all glucuronic acid were not fermented. In vitro fermentation of 12 dietary fibres by faecal inoculum from pigs and humans (Jonathan et al. 2012). In vitro: incubated 6, 12, 24, 48 hr with ruminal fluid from cattle or feces from dogs, pigs, horses and human Cellulose (Solka Floc), beet pulp (Michigan sugar), citrus pulp (Freeman Industries) and citrus pectin (HM rapid; TIC Gums Belcamp, MD) When data were pooled across all species and substrate disappearance and SCFA production ranked from the least to greatest in the following order: cellulose< beet pulp< citrus pulp< citrus pectin. The fermentability of different fibrous substrates by fecal or ruminal microbiota from various species seem to be dependant not only on the fermentative activity of the microbial population but other factors as well, perhaps lag time and rate of digesta passage. In vitro fermentation of cellulose, beet pulp, citrus pulp, and citrus pectin using faecal inoculum from cats, dogs, horses, humans, and pigs and ruminal fluid from cattle (Sunvold et al. 1995). In vitro: faeces from the adult pigs from four castrated finisher pigs (Dutch Landrace × Great Yorkshire. The unweaned piglet faeces were collected from 19 unweaned piglets (male and female) which were 27 to 30 days old Pectin Comparison of in vitro microbial activity was made between inocula from faeces of adult and unweaned pigs, using a range of carbohydrate-rich substrates. The substrates tested were classified into groups (fibre-rich, grains, gums, pectin, saccharides, storage carbohydrates and miscellaneous). There were significant differences in the fermentation patterns both due to source of substrate and inoculum. It would appear that the metabolic activity of the microflora does differ significantly between adult and weanling pigs, though this varied for the different carbohydrates tested. Microbial activities of faeces from unweaned and adult pigs, in relation to selected fermentable carbohydrates (Bauer et al. 2001). In vitro:gut contents of piglet terminal ileum Potato RGI The fibers showed high fermentability, with lactate in particular being increased. A significant increase in the numbers of Lactobacillus and Veillonella organisms and an insignificant increase in the numbers of Clostridium organisms as well as a decrease in the numbers of Streptococcus organisms was observed. Multivariate analysis showed clustering of the treatment groups, with the group treated with purified potato fibre being clearly separated from the other groups, as the microbiota composition was 60% Lactobacillus and almost free of Clostridium. In situ prebiotics for weaning piglets: in vitro production and fermentation of potato galactorhamnogalacturonan (Strube et al. 2015b). Ex vivo: porcine GI mucosa, i.e. buccal, stomach, small intestine and large intestine, texture analyser with mucoadhesive platform. Buccal mucosa with simulated saliva fluid pH 6.75 (SSF), gastric mucosa with simulated gastric fluid USP without pepsin (SGF) or citric-phosphate buffer, pH 4.8 (representing the fasted or fed state, respectively), small intestinal mucosa (duodenum part) and large intestinal mucosa with simulated intestinal fluid pH 6.8 (SIF) Four pectins with different DEs Herbstreith & Fox KG (Germany) HMP (CU201 70% De, MW 200 kDa, 0% DA), CU501 56% De 0% DA, Mw 180 kDa), LMP (CU701 (38% De, Mw 80 kDA, 0% DA CU020 29% De, 20% DA, Mw 150 kDA). Two parameters derived from texture analysis, namely maximum detachment force (Fmax) and work of adhesion (Wad) were used as parameters for comparison of mucoadhesive performance. Pectins showed a stronger mucoadhesion on large intestinal mucosa than on small intestinal mucosa. The mucoadhesive properties of pectins on gastric mucosa depended on pH of the medium, a higher Fmax and W ad in a pH 4.8 medium than a pH 1.2 medium was revealed. The results also demonstrated that the mucoadhesive performance of pectins largely depended on their characteristics, i.e. higher degree of esterification and molecular weight gave a stronger mucoadhesion. The rank order of mucoadhesive performance of examined pectins on to the GI mucosa appeared to be similar to the rank order of their degree of esterification and molecular weight (i.e. CU201 > CU501 > CU020 > CU701). Mucoadhesive properties of various pectins on gastrointestinal mucosa: An in vitro evaluation using texture analyzer (Thirawong et al. 2007). Ex Vivo:In an ex vivo system based on porcine colonic tissue Rhamnogalacturonan (RG) derived from pectin.(apple pectin, citrus fruit pectin and pectin acid from Roth (Karlsruhe, Germany) and sugar beet pectins Sûdzucker AG (Obrigheim, Germany). Rhamnogalacturonans with a low degree of esterification and linear oligogalacturonids derived from pectin showed significant bioadhesion against colonic mucous membranes. In contrast, highly esterified pectins and neutral polysaccharides were ineffective. Within a structure activity relationship linear, strongly acidic homogalacturonides were shown to be most adhesive agents. Esterification, branching or non-linear backbone structures will reduce the adhesive properties. The bioadhesive effects were concentration-dependent. Bioadhesive properties of polygalacturonides against colonic epithelial membranes (Schmidgall and Hensel, 2002). The ex vivo studies on the mucosal adsorption of pectins and incorporated proteins were conducted on the surfaces of porcine colonic mucus membranes Citrus pectins with the DE of 25% (P-25) or 94% (P-94), Pectin derivatives carrying side chain primary amine groups (P-N) were prepared from P-25. Pectins with higher net charges were more mucoadhesive than the other pectins. Both the negatively charged pectin formulation, P-25, and the positively charged formulation, P-N, were able to synergise with the mucus to produce rheologically strengthened gels. The highly esterified pectin, P- 94, also synergised with the mucosal glycoproteins to form a gel structure via coil entanglements. When incubated with porcine intestinal mucus membrane, P-94 gels were found generally bound to the lumen area, P-25 gels were able to penetrate deeply near the wall area, P-N gels interacted with mucins via electrostatic bonding and dispersed into the whole area from the lumen to the wall. The mucin–gel complex formed between P-N or P-94 and mucus tissue constructs a surface barrier, which effectively prohibited the penetration of Triton X-100. P-25 gel did not inhibit Triton X-100 irritation of the mucus tissue as effectively as the gel formed by P-N/mucus complex Interaction of various pectin formulations with porcine colonic tissues (Liu et al. 2005). In vitroassay Six pectin types from three manufacturers (Pectin classic, Genu Pectin, Grinsted Pectin) The factors which varied were; DM (three levels; high, low and medium), degree of amidation (DA) (two levels; ami-dated or nonamidated). Due to the manufacturing process, the molecular weight was correlated with the DM. Mucoadhesive properties were measured using a texture analyzer. It was found that an intermediate degree of methoxylation (35% and 36%) improved the specific mucin interaction. Amidation did not increase mucin interaction. Samples from different manufacturers did not alter these conclusions. This study indicates that the general classification of pectin as a poor mucoadhesive, without differentiating between the amount and type of substituents, probably is an oversimplification. In Vitro measurements of mucoadhesive properties of six types of pectin (Hagesaether and Sande 2007) View Large The mucosal glycome can differ based on genetic factors as well as the developmental stage of the animal (Hesselager et al. 2016; Priori et al. 2016). In the future, knowledge should be also acquired on the mucoadhesive properties of pectin in the GIT of animals of varying age. There is increasing acknowledgement and development of advanced in vitro screening tools for the investigation of digestive processes and gut microbial fermentation dynamics in vitro reviewed in Macfarlene & Macfarlane and Payne et al. (Macfarlane and Macfarlane 2007; Payne et al. 2012). Several studies have investigated the effect of pectin on pig associated gut microbial communities in vitro (Sunvold et al. 1995; Van Laere et al. 2000; Bauer et al. 2001; Jonathan et al. 2012; Leijdekkers et al. 2014; Taciak et al. 2015). Examples are given in Table 2. In vitro studies such as by Bauer et al. who tested the microbial activities of feces from unweaned and adult pigs, reported significant differences in the fermentation patterns of carbohydrates both due to the source of substrate, as well as the inoculum source and age (Bauer et al. 2001). There is hence a potential to implement in vitro tools for a pre-selection of pectin substrates based on animal target group specific GM activity. Initiatives, such as the one by the european infogest community, (www.cost-infogest.eu), to harmonise and validate in vitro simulation protocols (Egger et al. 2016), with in vivo data in pigs (Egger et al. 2017; Portmann et al. 2017), are currently setting the stage for an increased application of in vitro models in research on gut microbial processes, avoiding the high costs and ethical constraints of in vivo studies. The lack of ethical constraints, for instance, facilitates the possibility to study the potential efficacy of carbohydrates to reduce pathogen load (Martín-Peláez et al. 2010). The reduced cost of in vitro screening tools allows the investigation of the bioavailability of pectin within different feed matrices. The model-driven experimentation using a combination of in vitro gut fermentation and in vitro cell, tissue or mucoadhesion models (for examples see Table 2) can lead to an advanced strategy to identify structure-related modes of action of pectin on the gut microenvironment before in vivo studies. The novel in vitro methods should be implemented to expand knowledge on pectin compounds and their potential to improve animal growth and health via the modulation of the GM and its metabolome. THE POTENTIAL OF PECTIN TO CONTROL AND ELIMINATE PATHOGENS The gut microbiota plays a pivotal role in the inhibition of pathogen growth in the gut lumen right from the beginning of life. The phenomenon is referred to as colonization resistance and efficiently blocks infections by Clostridium difficile, Salmonella spp. and many other pathogenic bacteria. Addition of fermentable fibre such as pectin to pig diets may stimulate growth and metabolic activity of beneficial bacterial species in the GIT (Van der Waaij, Berghuis-de Vries and Lekkerkerk-Van der Wees 1971; Berends et al. 1996; Schley and Field 2002; Knudsen, Lærke and Hedemann 2008). Furthermore, various studies have demonstrated the antiadhesive properties of pectin. For example, it has been shown that POS purified from the root of Panax ginseng displays selective inhibitory effects against the adhesion of Helicobacter pylori to gastric epithelial cells (Lee et al. 2006). Rhoades et al. 2008 have reported orange peel derived POS – mediated inhibition of the adhesion of pathogenic Escherichia coli strains to gut epithelial cells in vitro (Rhoades et al. 2008). The use of antiadhesives can aid in pathogen control and represents an alternative to antibiotic therapy. Decoy oligosaccharides bind to the microbe's carbohydrate-binding proteins, and pathogens are cleared by the bulk fluid movement in the GIT (Zopf and Roth 1996; Sharon and Ofek 2000). Reports also exist on the capacity of pectin substrates to inhibit pathogen-derived toxins (Olano-Martin et al. 2003). The potential of pectin to limit bacterial infections via the stabilisation of a healthy microbiome as well as via decoy mechanisms should be further explored. ANTIVIRAL PROPERTIES OF PECTIN Rotavirus infection is one of the main causes of gastroenteritis and diarrhea in young animals (Chung et al. 2017; Yodmeeklin et al. 2017). A few studies have reported the antiviral properties of pectin. As early as 1947 Green et al. investigated a number of simple and complex carbohydrates containing large amounts of galacturonic acid for their ability to inhibit agglutination of chicken red blood cells by influenza A virus. Apple pectin was found (Green and Woolley 1947) to be able to inhibit virus hemagglutination and virus multiplication in embryonated eggs. Whereas, the hemagglutination-inhibiting citrus pectin, did not reduce the multiplication of the virus (Green and Woolley 1947; Woolley 1949). The application of pre and probiotics against rotaviral infections have been recently reviewed by Gonzalez-Ochoa et al. (Gonzalez-Ochoa et al. 2017). Fibres with prebiotic potential may improve the generalised antiviral response and may reduce the rotaviral infectivity leading to a shorter duration and severity of rotavirus-associated diarrhea (Gonzalez-Ochoa et al. 2017). Recent reports suggest antiviral properties of POS. Rigo-Adrover et al. 2017 have demonstrated, that a mixture of prebiotics scGOS, lcFOS, POS and heat-treated probiotics in fermented milk components, has led to reduced clinical signs in suckling rats with rotavirus (RV)-induced diarrhea (Rigo-Adrover et al. 2017). Decreased viral shedding may be attributed to the interaction of prebiotics with the viral particles, avoiding the entry into enterocytes and therefore reducing its replication. Other potential mechanisms are reviewed in Gonzalez-Ochoa et al. (Gonzalez-Ochoa et al. 2017). Chen et al. recently reported that the dietary POS supplementation could improve the growth performance and immunity in weaned pigs infected by porcine rotavirus (PRV), possibly because POS administration improved the immune function and the utilisation of nutrients in the PRV-infected piglets (Chen et al. 2013). MODULATION OF THE IMMUNE RESPONSE Complex interactions among diet, external pathogens and local immunological and non-immunological processes are constantly integrated in the GIT. The consumption of fibre can affect the immune system directly and via changes in the gut microbial community composition. The GM can mediate immune changes through the contact of bacteria with immune cells, the production of SCFA or by changes in the synthesis of mucin. Fermentable dietary fibre can modulate various properties of the immune system, including those of the gut-associated lymphoid tissues, secondary lymphoid tissues and peripheral circulation, effects of dietary fibre on the immune-system have been reviewed by Schley and Field (Schley and Field 2002). The structural features of the pectin macromolecule determine its effect on the immune system. Popov and Ovodov 2013 have reviewed the poly-potency of pectin substrates as immunomodulatory agents, that can both stimulate and suppress the immune response (Popov and Ovodov 2013). Recent reports demonstrated the potential of citrus pectin to attenuate an endotoxin shock via the suppression of a toll-like receptor signalling in payer's patch myeloid cells (Ishisono, Yabe and Kitaguchi 2017). Furthermore, Tang et al. 2017 reported a unique phenotype of macrophages treated with RG-I from apple pectin (Tang et al. 2017). Such publications illustrate the expansion of knowledge of the potential of pectin substrates to be implemented for the modulation of the mammalian immune response. These studies are providing additional guidance for the tailoring of pectin formulations for in feed applications. STRUCTURE AND FUNCTION: EXAMPLE RG I Aside from the above-mentioned studies by Tang et al. 2017, further studies exist on the direct effects of RG-I on the immune system via immunomodulation of the activity on Peyer's patches and macrophages (Grønhaug et al. 2011), stimulation of macrophages (Ho et al. 2015), inhibition of colon cancer cell proliferation and cell cycle progression (Cheng et al. 2013), prevention of tumor cell migration and cell-to-cell and cell-to-substratum adhesion (Platt 2009). A recent study examined the digestion of RG-I from potato pulp simulating the upper piglet GIT in vitro. The potato RG-I was highly fermentable by terminal ileal piglet gut content in vitro, reflected by a dose-dependent decrease in pH and an increase in the organic acid content, in particular lactate. Analysis of the GM in fermentates revealed a significant increase in the numbers of Lactobacillus, Veillonella and Clostridium, as well as a decrease in the numbers of Streptococcus (Strube et al. 2015b). Strube et al. also demonstrated an enzymatic release of RG-I from potato pulp in vivo. Despite the demonstration of the enzymatic release of potato RG-I in vivo, the data presented by Strube et al. 2015a did not justify the claim of the production of an in situ prebiotic as stated in the title ‘In situ prebiotics: enzymatic release of galacto-RG from potato pulp in vivo in the GIT of the weaning piglet’, as the authors did not investigate potential prebiotic effects of the released RG-I in the different colon regions in vivo (Strube et al. 2015a). Several studies have investigated the fermentability of RG-I by human fecal microbiota. Interestingly, Khodaei et al. 2016 produced galactose-rich oligosaccharides (oligo-RG-I) from potato RG-I and examined their fermentability with human fecal microbiota in vitro. They have found oligo-RG-I to be a more selective prebiotic and lead to higher absolute amounts of SCFAs than RG-I fermentations (Khodaei et al. 2016). The production of oligo-RG-I substrates might also be an interesting avenue to be explored for weaner feed or other in feed applications. CONCLUDING REMARKS Pectin should be further explored for a wide range of applications including prebiotics, milk replacers and as an antimicrobial and antiviral agent. The challenge lies in the unraveling of structure-to-function relationships of different pectin substrates in the GIT and the appropriate matching of specific pectin structures and formulations with the animal target group and application. The current state-of-art in molecular biology and in vitro tools can aid to unravel the modes of action and aid to develop a systematic approach for the design of in vivo studies. ACKNOWLEDGEMENTS I want to thank Todd Hutcherson for cross-checking the grammar. Conflicts of interest. None declared. FUNDING This work was supported by the Innovation Foundation Denmark, Grant Number 5190-00025B. REFERENCES Al‐Tamimi M , Palframan R , Cooper J et al. . 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TI - The potential of pectin to impact pig nutrition and health: feeding the animal and its microbiome
JF - FEMS Microbiology Letters
DO - 10.1093/femsle/fnz029
DA - 2019-02-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-potential-of-pectin-to-impact-pig-nutrition-and-health-feeding-the-llxSMsEHyg
VL - 366
IS - 4
DP - DeepDyve
ER -