Fermentation of fibrous material from DDGS and rapeseed meal in pigs. 20 June 2014, Sonja de Vries

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1 Fermentation of fibrous material from DDGS and rapeseed meal in pigs 20 June 2014, Sonja de Vries

2 Fermentation of fibrous material from DDGS and rapeseed meal in pigs I. Fiber fermentation in pigs What is fiber? Fiber in DDGS and rapeseed meal and its degradation in pigs Interaction between fiber and digestive utilization of the diet II. Manipulation of fermentation Processing and enzyme technologies to improve fiber degradation (DDGS)

3 Background FIBER IN MONOGASTRIC NUTRITION

4 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)

5 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 de Vries, S PhD Thesis, chapter 1

6 Fiber degradation 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 Related to solubility

7 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 > recalcitrant fiber

8 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 > 29-46% TDF/20-44% NDF ~ 5 % starch (=RS?) % ADF (2% lignin) Fiber composition depends a.o. on ratio of solids:solubles 1 From : Kim et al. 2008; Pedersen et al 2007; Shurson et al 2012; Stein et al. 2006, 2009; Urriola et al Summarized in: de Vries, S PhD Thesis, chapter 1

9 Fiber structures in maize DDGS Originating from maize 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 Appeldoorn et al. J Agric Food Chem: 58: Huisman et al. Carbohydr Polym 43:

10 Undegraded consituent sugars (% fed) Fermentation of NSP from DDGS Cellulose + GAX de Vries et al. Submitted for publication (chapter 5).

11 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 de Vries et al. Submitted for publication (chapter 5).

12 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 32-35% TDF / 21-35% NDF 16-22% ADF 24% lignin (10-13% Acid soluble) 1 Fiber content and composition depends a.o. on species 1 From : Bach Knudsen 1997; Canola Council of Canada 2001; CVB 2007, Khajali et al Summarized in: de Vries, S PhD Thesis, chapter 1

13 Fiber structures in rapeseed meal Originating from rapeseed Cellulose Hemicellulose Xyloglucan, glucuronoxylan, galactomannan Pectic polysaccharides Homogalacturonan, rhamnogalacturonan, arabinan, arabinogalactan Tightly bound within a lignin-cellulose network > Rigid cell wall matrix Pustjens et al. Carbohydr Polym. 98:

14 Undegraded consituent sugars (% fed) Fermentation of NSP from RSM Partly linked through ester linkages or H-bonds Feed Ileum Cecum Colon Feces Mannose Uronic acid Arabinose Galactose Glucose Xylose Rhamnose Pustjens et al. Indust Crop Prod 58: (chapter 6).

15 DDGS and rapeseed meal INTERACTIONS BETWEEN FIBERS AND OTHER NUTRIENTS

16 Experimental design Effect of additional fibre sources on fermentability of rapeseed meal and DDGS fiber in pigs? Rapeseed meal fiber DDGS fiber β - glucan Resistant starch Fermentable Viscous.. ++.

17 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

18 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

19 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

20 Samples Ileal digesta Pooled samples 48 h > digestibility + viscosity 0, 45, 90, 180, 270, 360, 540, 720 min after feeding > transit time Feces Pooled samples 48 h > digestibility

21 AID starch (%) AID starch a ATTD of starch nearly complete >> Starch not digested in ileum was fermented in large intestine RS was indeed quite resistant b c x y z CM DDGS Control β-glucan RS Effect of fiber source P -value Raw material < 0.01 β-glucan < 0.01 RS < 0.01 Interaction < 0.01 de Vries, S PhD Thesis, chapter 9

22 AID CP (%) AID CP CM β-glucan and RS reduced AID CP DDGS Control β-glucan RS Effect of fiber source P -value Raw material < 0.01 β-glucan < 0.01 RS < 0.01 Interaction 0.06 de Vries, S PhD Thesis, chapter 9

23 ATTD NGP (%) ATTD NGP β-glucan increased ATTD of NGP RS decreased ATTD of NGP 60 Control β-glucan 40 RS 20 0 CM DDGS Effect of fiber source P -value Raw material < 0.01 β-glucan 0.04 RS < 0.01 Interaction 0.13 de Vries, S PhD Thesis, chapter 9

24 Discussion and Conclusion Clear interactions between ingredients Differences between ingredients (β-glucan vs. RS and CM vs. DDGS) Related to physico-chemical properties of digesta, retention time, microbial population etc. β-glucan increased viscosity in ileum. RS? β-glucan and RS decreased MRT of solid digesta in upper GIT

25 MRT of solid digesta (min) Mean retention time upper GIT β-glucan and RS reduced MRT Control β-glucan RS 50 0 CM DDGS Effect of fiber source P -value Raw material 0.36 β-glucan < 0.01 RS 0.04 Interaction 0.20 de Vries, S PhD Thesis, chapter 9

26 Discussion and Conclusion Clear interactions between ingredients Differences between ingredients (β-glucan vs. RS and CM vs. DDGS) Related to physico-chemical properties of digesta, retention time, microbial population etc. β-glucan increased viscosity in ileum. RS? β-glucan and RS decreased MRT of solid digesta in upper GIT Need to reconsider addititvity approach in feed evaluation?

27 Manipulation of fiber fermentation DDGS EFFECTS OF PROCESSING AND ENZYMES

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

29 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

30 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

31 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)

32 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 ) de Vries et al. J Agric Food Chem. 61: (chapter 4)

33 In vitro NSP solubilization (%) In vitro NSP solubilization DDGS Acid-treatment increased 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: (chapter 4)

34 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 de Vries et al. J Agric Food Chem. 61: (chapter 4)

35 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 de Vries et al. J Agric Food Chem. 61: (chapter 4)

36 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

37 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

38 AID (%) Apparent ileal digestibility 100 * ** Acid-treatment tendend to increase NSP degradation but reduced digestibility in small intestine Protein Starch NSP * Control Acid-extrusion * P < 0.10 ** P < 0.05 *** P < 0.01 de Vries et al. Submitted for publication (chapter 5).

39 Hydrothermal acid treatment of DDGS Technologies to modify DDGS cell wall polysaccharides Increased in vitro solubilization of DDGS Improved rate of fermentation in growing pigs but not total extent! Reduced digestibility in small intestine Restrictedly fed, but. Lower feed intake ( P < 0.001) for acid-extruded diet Reduced performance Lower weight gain also reduced feed efficiency (G:F -4 g/g)

40 Technologies to improve degradation of DDGS fiber Sense or nonsense? 35% of fiber undegraded Severe processing required to disrupt cell wall matrix, in vivo degradation not improved (Ester-linked) phenolic acids (coumaric and diferulic acid) seem to be involved in recalcitrance of NSP Alkali-treatments or esterases? Preferred because of specificity

41 Wrap up I. Fermentation of DDGS and RSM fiber in pigs Recalcitrant fiber fractions Degradation hampered by ester-linkages/h-bonds Fiber degradation is affected by other fibers in the diet: Challenges additivity approach in feed formulation II. Technologies to improve degradation of DDGS (and RSM) fiber Focus on undegraded (recalcitrant) fiber structures Technologies might work in vitro, not in vivo (e.g. transit time) Limited scope for processing technologies Enzymes preferred over chemical degradation: Esterases Limitation: enzyme availability!

42 Thank you!