Fiber fermentation in pigs and poultry

Transcription

1 Fiber fermentation in pigs and poultry 5 March 2014, Sonja de Vries 1 1 Present: Ingredient Research Centre, Nutreco R&D

2 Improved utilization of complex carbohydrates in pigs and poultry by novel technologies Carbohydrate Competence Center (CCC) Industry partners Wageningen University Food Chemistry Harry Gruppen, Mirjam Kabel, Annemieke Pustjens & Henk Schols Animal Nutrition Walter Gerrits, Wouter Hendriks & Sonja de Vries

3 Project outline Literature study Chemical characterization Processing technologies In vitro degradation In vivo - poultry Enzymes In vivo - pigs

4 Background FIBER IN MONOGASTRIC NUTRITION

5 What is fiber? Many definitions Basically: non-digestible fraction of plant material ~ resistant starch (RS), non-starch polysaccharides (NSP), lignin (Partly) fermented by microbiota in gastrointestinal tract From: McCann and Roberts (1991)

6 Analytical methods to analyze fiber Sugars Oligosaccharides Degree of polymerization >> Polysaccharides Lignin Other Starch NSP 80%- Ethanol soluble Maltodextrins Monosacch Raffinose Disacch Stacchyose Polyols Fructo-oligosacch 40%- Ethanol soluble Amylose Amylopectin Digestible starch RS Pectic polysaccharides Hemicellulose Cellulose Calculated NSP NDF ADF ADL Crude fiber Gravimetric DF Chemical DF Chemical NSP

7 Analytical methods to analyze fiber Sugars Oligosaccharides Degree of polymerization >> Polysaccharides Lignin Other Starch NSP 80%- Ethanol soluble Maltodextrins Monosacch Raffinose Disacch Stacchyose Polyols Fructo-oligosacch 40%- Ethanol soluble Amylose Amylopectin Digestible starch RS Pectic polysaccharides Hemicellulose Cellulose Calculated NSP NDF ADF ADL Variable part of the NSP fraction > depending on feedstuff Crude fiber Gravimetric DF Chemical DF Chemical NSP

8 Fiber degradation Related to solubility Depends on: Chemical composition e.g chain length, linkages b/w sugar molecules Structural arrangements in cell wall Crosslinks b/w polysaccharides, protein and lignin

9 Degradation of NSP Secondary cell wall Insoluble, lignified acidic-(arabino) xylans cellulose s-arabinoxylans β-glucans Parenchymatous tissue Soluble, weakly linked

10 Fiber degradation - pigs Total tract apparent digestibility (TTAD) of cell wall polysaccharides TTAD Pig 1 β - glucans parenchymatous ~100 secondary cell wall ~100 Arabinoxylans parenchymatous secondary cell wall 0-70 Cellulose parenchymatous secondary cell wall From: Bach Knudsen and Hansen, 1991; Bach Knudsen et al., 1993ab; Glitsø et al. 1998; Bach Knudsen and Canibe, 2000

11 Concentration (mg/kg DM) Fiber degradation - poultry Two digestbility markers added to feed: Cr 2 O 3 insoluble Co EDTA - soluble Soluble fraction Chromium Cobalt feed ileum ceca excreta From: Stevens and Hume, 1995

12 Fiber degradation - poultry NSP degradability low: 30-40% barley, wheat, oats I-NSP virtually undegraded Antinutritive effects: e.g. digesta viscosity Starch and protein digestion 14 pp Fat digestion 24 pp

13 Chemical characterization & degradation FIBER-RICH FEED INGREDIENTS

14 Fiber-rich feedstuffs By-products from e.g. biofuel industries Extensively processed Variable quality due to differences in processing conditions Fiber contributes largely to digestive utilization High level of fiber Low degradability

15 DDGS Dried distillers grain with solubles from maize By-product of ethanol production Maize, wheat, barley, sorghum,. mixtures focus on maize! ~ 30% protein ~ 30% NSP > 42% TDF/25% NDF ~ 5 % starch (>RS?) 10% ADF (2% lignin) Fiber composition depends a.o. on ratio of solids ; solubles

16 Fiber structures in corn DDGS Originating from corn Cellulose Hemicellulose Highly substituted glucuronoarabinoxylans (GAX) Single unit side chains (Ara, UA) Oligomeric side chains of (ara, xyl,gal) Ferulic acid, coumaric acid Ester-linked to ara > Cross-links between xylan structures

17 Undegraded consituent sugars (% fed) Fermentation of NSP from DDGS Cellulose + GAX

18 Ara:xyl ratio Fermentation of specific fiber structures Arabinose:xylose and uronic acid:xylose ratio Ara:xyl and UA:xyl are indicative for the degree of substitution of arabinoxylans: the higher the ratio, the more arabinose side-chains are present First, linear xylan fragments Ara:xyl UA:xyl Then highly substituted fragments 0 Feed Ileum Colon Total tract

19 Ara:xyl ratio However Arabinose:xylose ratio Real difference microbial degradation?? Experiment 1 Experiment Feed Ileum Total tract

20 Ara:xyl ratio However Uronic acid:xylose ratio Experiment 1 Experiment Feed Ileum Total tract

21 Undegraded phenolic acids (% from fed) Degradation of phenolic acids Less well degraded than NSP > associated with recalcitrant fiber fraction? coumaric acid monoferulic acid diferulic acid ileum colon total tract

22 Rapeseed meal (RSM)= Canola meal Brassicaceae meals By-product of oil production Rapeseed, but several species > Brassica napus, rapa spp. campestris, juncea (mustard seed) focus on Brassica napus! ~ 30% protein ~ % NSP > LMW sugars 18% ADF 24% lignin (10% Acid soluble) Fiber content and composition depends a.o. on species

23 Fiber structures in rapeseed meal Tightly bound pectins and hemicellose linked within cellulose-lignin network Rigid cell wall matrix Pustjens et al. Carbhydr. Polym. 98:

24 Unfermented NSP from RSM - pig Tightly bound in celluloselignin matrix Ester-linkages or H-bondings

25 Unfermented NSP from RSM - pig

26 Unfermented NSP from RSM - broiler

27 Unfermented NSP from RSM - broiler Other Uronic Acid Xylose Arabinose Feed Excreta

28 Literature PROCESSING AND ENZYME TECHNOLOGIES

29 Processing Disruption cell wall matrix depolymerization breaking crosslinks Physicochemical properties particle size solubility viscosity hydration properties Almond cotyledon cell walls

30 Cell wall degrading enzymes Specifically cleave polymers depolymerization remove side-chains Aleurone layer destroyed Cell wall layers detached and disrupted Wheat bran cell walls

31 Analytical methods to analyze fiber Sugars Oligosaccharides Degree of polymerization >> Polysaccharides Lignin Other Starch NSP 80%- Ethanol soluble Maltodextrins Monosacch Raffinose Disacch Stacchyose Polyols Fructo-oligosacch 40%- Ethanol soluble Amylose Amylopectin Digestible starch RS Pectic polysaccharides Hemicellulose Cellulose Calculated NSP NDF ADF ADL Technologies: Shift in fractions! Crude fiber Gravimetric DF Chemical DF Chemical NSP

32 Processing and enzyme technologies Effects of technologies on NSP degradability in pigs and poultry Processing common technologies effective for easy NSP not sufficient for more recalcitrant NSP, e.g. DDSS and RSM Enzymes Contradictory results Mismatching enzyme activities, side-activities, concentration de vries et al. Anim Feed Sci Technol. 178:

33 Combinations of processing + enzymes Processing opens cell wall matrix > better accessibility for enzymes Processing may increase viscosity > enzymes can counteract Enzymes after processing Viscosity: 3-4 fold decrease Fiber digestibility: effects 2-6 x larger in processed diets de Vries et al. Anim Feed Sci Technol. 178:

34 DDGS EFFECTS OF PROCESSING AND ENZYMES

35 Processing and enzyme technologies Wet-Milling Extrusion Autoclaving Acid - Enzymes autoclaving Specifically Hydrothermal Particle Chemical size cleaving treatment reduction polymers Protein denaturation complex (Maillard) Starch gelatinsation retrogradation

36 Treatments Unprocessed Unprocessed+ Wet-milled Wet-milled+ Extruded Extruded+ Autoclaved Autoclaved+ Acid-autoclaved Acid-autoclaved+

37 In Vitro digestion simulation 1. Enzymatic digestion Two step enzymatic incubation (adapted from Boisen and Fernández, 1997) 2. Fermentation Gas production method (Williams et al. 2005)

38 In vitro NSP degradation - DDGS Technologies to modify DDGS cell wall polysaccharides Shear, heat, or mild acid treatments (ph 4.2) not effective No interaction between processing and enzymes More severe hydrothermal acid treatment (ph )

39 In vitro NSP solubilization (%) In vitro NSP solubilization DDGS Acid-treatment increases NSP solubilization (P<0.001). No effect of enzymes. e e bc b a a d cd unprocessed maleic 2.9 maleic 2.3 sulfuric 2.9 Maleic acid more effective de vries et al. J Agric Food Chem. 61:

40 Ara:xyl ratio Recalcitrant fiber Arabinose:xylose ratio Arabinose more acidlabile No enzyme Enzyme a b e de g f cd c unprocessed maleic 2.9 maleic 2.3 sulfuric 2.9

41 Ara:xyl ratio Recalcitrant fiber Uronic acid:xylose ratio Mainly highly substituted NSP affected No enzyme Enzyme ab ab bcd d abc ab cd d unprocessed maleic 2.9 maleic 2.3 sulfuric 2.9

42 Experimental set-up Effect of acid-extrusion on degradation of DDGS in pigs? Fed as liquid feed # Treatment 1 40% DDGS control 2 40% DDGS Acid-extruded

43 Experimental set-up 48 gilts (Topigs20*Talent, BW 20.8±1.45 kg ) 4 pigs per pen, 12 pens 2 treatments 6 replicates Transition period Experimental feed (ad libitum) (restricted feeding ) Transition period Adaptation period Balance period Dissection Gradually changed from starter feed to 100% experimental feed Feed intake, body weight (BW) (pen level) Fecal samples from rectum 2* per day (pen level) Feed intake, BW (pen level) Chyme samples: ileum, mid colon (pen level) day 1-5 day 6-19 day day 24,25

44 g/pig*day-1 Feed intake Restrictedly fed, but. Lower feed intake ( P < 0.001) for acid-extruded diet Daily feed intake day 6 -end Lower weight gain also reduced feed efficiency (G:F -4 g/g) Unprocessed Acid-extruded

45 AID (%) Apparent ileal digestibility 100 * ** Acid-treatment tends to increase NSP degradation but digestbility in small intestine Protein Starch NSP * Control Acid-extrusion * P < 0.10 ** P < 0.05 *** P < 0.01

46 ATTD (%) Apparent total tract digestibility 100 ** *** Control Acid-extrusion No difference in ATTD of NSP 0 Protein Starch NSP * P < 0.10 ** P < 0.05 *** P < 0.01

47 Hydrothermal acid treatment of DDGS Technologies to modify DDGS cell wall polysaccharides Increased in vitro solubilization of DDGS Improves rate of fermentation in growing pigs but not total extent! Reduces digestibility in small intestine Reduced performance

48 RSM EFFECTS OF PROCESSING AND ENZYMES

49 heat In vitro digestion simulation Enzymes RSM

50 In vitro NSP solubilization (%) In vitro solubilization of rapeseed meal Enzymes increase NSP solubilzation (P<0.001) No enzyme Enzyme c bc bc ab c ab c c c a unprocessed wet-milled extruded autoclaved acid-autoclaved

51 Viscosity (mpa s) Viscosity of supernatant 6 5 Technology x enzyme interaction (P<0.001) 4 3 No enzyme Enzyme cd de a ab d d cd d ab df unprocessed wet-milled extruded autoclaved acid-autoclaved

52 Molecular weight distribution of supernatant

53 In vivo Broilers Effect of processing technologies and enzymes on degradation of rapeseed meal Unprocessed in broilers? Unprocessed+ Wet-milled Wet-milled+ Extruded Extruded+ Acid-extruded Acid-extruded+

54 Experimental set-up Effect of processing technologies and enzymes on degradation of rapeseed meal in broilers? Fed as pellets # Treatment 1 35% RSM control 2 35% RSM control + enzyme 3 35% RSM Wet-milled 4 35% RSM Wet-milled + enzyme 5 35% RSM Extruded 6 35% RSM Extruded + enzyme 7 35% RSM Acid-extruded 8 35% RSM Acid-extruded + enzyme 9 Basal diet 10 Basal diet + enzyme

55 Experimental set-up 750 one-day old female chicks (Ross 308)

56 ATTD of NGP (%) Apparent total tract digestibility of NGP Enzymes increase NGP degradation (P<0.001) No enzyme Enzyme unprocessed wet-milled extruded de Vries et al. Poult Sci. Accepted for publication.

57 NGP:Co ratio ATTD of NGP related to NGP in ceca 25 NGP:Co in ceca correlated (r =0.53, P<0.001) to ATTD of NGP No enzyme Enzyme 5 GMD: 818 µm GMD: 220 µm GMD: 288µm 0 d ab bc a cd ab unprocessed wet-milled extruded

58 Undegraded constituent sugars (%) Unfermented NSP structures Arabinose Xylose Uronic Acid Other Enzymes mainly affect branched water-soluble arabinan unprocessed wet-milled extruded

59 Conclusions Technologies to modify RSM cell wall polysaccharides Pectolytic enzymes increase TTAD of NGP, 9 20 % units coinciding with increased concentration of NGP in ceca Particle size reduction (wet-milling, extrusion) facilitates NGP solubilization > increased concentration of NGP in ceca Without help of additional pectolytic enzymes still not degraded No interaction between processing and enzymes

60 Synthesis and implementation PROCESSING AND ENZYME TECHNOLOGIES

61 DDGS Technologies to improve degradation of DDGS and RSM fiber 35% of fiber undegraded Acid treatment effective to increase rate, but not extend of fermentation Ileal digestbility of other nutrients and performance reduced (Ester-linked) phenolic acids (coumaric and diferulic acid) seem to be involved in recalcitrance of NSP Alkali-treatments or esterases?

62 RSM Technologies to improve degradation of DDGS and RSM fiber 30% of fiber undegraded Enzyme treatment effective to increase NSP degradation in broilers Rigid matrix of ester-linked or H-bound pectins linked to cellulose/lignin Alkali-treatments or esterases?

63 Preliminary experiment Hydrothermal alkali treatment to improve degradation of RSM fiber Chemical additive Moisture (%) Product temperature ( C) Pressure (psi) Extrusion maleic acid (2%, wt/wt DM) Acidextrusion Alkaliextrusion NaOH (2%, wt/wt DM)

64 In vitro disappearance (%) In vitro degradation of rapeseed meal No effect of alkali-extrusion Dry matter Protein Fiber Control Extruded Acid extruded Alkali extruded

65 Conclusions Technologies to improve degradation of DDGS and RSM fiber Focus on degradation of recalcitrant fiber structures Enzymes preferred over chemical degradation limitation: enzyme availability!

66 Methodology SEPARATION OF DIGESTA FRACTIONS

67 Experimental set-up Two digestbility markers added to feed: Cr 2 O 3 insoluble Co EDTA - soluble # Diets 1 Basal diet > Low fiber (7 % NSP) 2 35% unprocessed RSM diet > High fiber (18% NSP) 3 35% unprocessed RSM + enzyme

68 ATTD Total collection vs. marker Low -fiber RSM RSM + Total collection CP NGP P = Cr 2 O 3 CP NGP P = Co-EDTA Recovery: % Recovery: % P = CP NGP NS

69 cr:co (g/g) Marker methods *** Low-fiber diet RSM diet RSM+ diet *** ** feed 0 ileum ceca excreta Separation of digesta fractions

70 Apparent ileal digestbility Low -fiber RSM RSM + Cr 2 O 3 CP NGP Really? Co-EDTA Opposite to ATTD CP NGP Overestimation of digestbility (exceeding ATTD by 16-42% units) Effects due to enzyme treatment misjudged

71 Another example SEPARATION OF DIGESTA FRACTIONS?

72 Experiment at University of Alberta, Canada

73 Experimental design Effect of additional fibre sources on fermentability of rapeseed meal and DDGS fiber in pigs? β-glucan Pearled barley flour (U of A) ~30% pure Rapeseed meal fiber Resistant starch DDGS β - glucan fiber Resistant starch Fermentable (27% starch, 11% protein) Viscous Extract viscosity 390 mpa s -1 Retrograded tapioca starch (Actistar, Cargill) ~50% resistant

74 Experimental diets Diet RSM DDGS Corn starch Β-glucan % Resistant starch 1 RSM Control 50 - ~ DDGS Control - 50 ~ RSM viscous 50 - ~20 20 (~6) - 4 DDGS viscous - 50 ~20 20 (~6) - 5 RSM fermentable ~40 6 DDGS fermentable ~40 TiO 2 added as digestbility marker

75 Animals Ileal cannulated pigs (grower/finisher) Initial BW 35.8 ± 1.29 kg Final BW 96.5 ± 4.05 kg 6 x 6 Latin square each diet fed to every animal (36 observations) Incomplete 46 observations in 10 animals

76 To be continued!