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A Mul&-‐Enzyme NSPase For Enhanced Grain-‐NSP Diges&on and Energy
Recovery
Overview
• Background of Grain Structure, Carbohydrates, and Non-‐Starch Polysaccharides (NSP)
• Primary, Secondary, and Debranching Enzymes • Product Development & InnovaFon • Previous Research in Ruminants • Current Brazilian Research
Composi&on of Component Part, % on a DM basis Component % of Kernel Starch, % Protein, % Oil, % Ash, % Sugars, % Fiber, %
Endosperm 82.9 88.4 8.0 0.8 0.3 0.6 1.9
Germ 11.0 11.9 18.4 29.6 10.5 10.8 18.8
Bran Coat 5.3 7.3 3.7 1.0 0.8 0.3 86.9
Tip Cap 0.8 5.3 9.1 3.8 1.6 1.6 78.6
Whole Kernel 100.0 75.0 8.9 4.0 1.5 1.7 8.9
Typical Composi&on of Yellow Dent Corn
NSP’s
Cellulose
Insoluble in water, alkali or dilute acids
Non-‐cellulosic polymers
Arabinoxylans, mixed -‐linked β-‐Glucans,
mannans, galactans, xylogucan, fructans
Par&ally soluble in water
Pec&c Polysaccharides
Polygalacturonic acids, which may be subs&tuted w/ arabinan, galactan, &
arabinogalactan
Par&ally soluble in water
Comparison of Arabinose/Xylose, Mannose, Galactose & Cellulose Content of Major Grain NSP’s
• Corn ~ 10% (Araxyl 5.2%, Mann 0.2%, Galact 0.6%, Cell 2.0%) • SBM ~ 22% (Araxyl 3.3%, Mann 1.3%, Galact 4.1%, Cell 6.2%) • Wheat ~ 11%(Araxyl 8.1%, Mann (-‐), Galact 0.3%, Cellulose 2.0%)
• NSP structure and content (e.g. soluble vs. insoluble NSP’s) can vary with growth and storage condiFons in many grains further challenging the mixture of degradaFve enzymes required for maximum energy release.
• Enzymes are selecFve for specific substrates and act as catalysts in releasing basic nutriFonal components from complex ingredients in feed.
• Fiber and non-‐starch polysaccharide (NSP) components of feed are composed of complex carbohydrates • The release of simple sugars for improved uFlizaFon by rumen microbes requires a wide spectrum and opFmum combinaFon of degradaFve enzymes.
Enzyme Supplementa&on of Ruminants
Poten&al Mechanisms of Supplemental Enzymes For Enhancing Feedlot Performance
• Supplemental enzymes are complementary and/or augment exisFng microbial enzymes present in rumen and enhance NSP hydrolysis (enzymaFc synergy).
• Exogenous enzymes (e.g. xylanases + cellulases) create addiFonal acachment sites on grain and plant substrates for rumen microbes to acach and enhance fiber breakdown.
• Supplemental enzymes include esterases (e.g. feruloyl esterase) that break lignin-‐NSP and other NSP-‐fiber crosslinking to facilitate digesFon by xylanases and cellulases.
• Supplement includes acid and protease stabile enzymes that contribute to more complete carbohydrate hydrolysis in abomasum and other downstream digesFve compartments.
• AddiFonal enzymes reduce viscosity of digesta in rumen which results in increased feed intake and uFlizaFon.
S.J.Meale, et al. J. Animal Sci. 92: 427-‐442, 2014
Poten&al Variables Influencing The Efficacy of Supplemental Feedlot Enzymes
• Pre-‐exisFng rumen microflora & their secreted carbohydrases – supplemental enzymes must work synergisFcally with rumen enzymes to degrade grain and plant fibers for reproducible energy release and feed efficiency improvement.
• Differences in diet including type of silage or forage fed before start of grain diet can affect enzyme producFon by ruminal microbes.
• Effect of ruminal condiFons such as pH on microbial populaFons. • Diversity of rumen microflora as influenced by cacle breed. • Supplemental enzyme diversity, consistency, mode of delivery and sensiFvity to proteolyFc or anF-‐catalyFc condiFons in the rumen as well as post-‐ruminal compartments.
• DigesFbility of non-‐fibrous grain components (e.g. starch) can be influenced by the rate of fibrolyFc enzyme acFvity (supplemental enzyme Vmax and Kcat properFes).
• Responses to enzyme supplementaFon have previously been inconsistent due to various factors. • Single strain, substrate-‐specific, rumen ecology, mode of delivery • AmylolyFc vs. fibrolyFc vs. NSPase vs. combinaFon products • Absence of debranching enzymes
• Observed improvements in diges&bility may not translate to improved growth performance and feed efficiency, the true economic drivers of implementa&on
• The complexity of fiber and non-‐starch components in feedstuffs guided JBS United scienFsts in developing both the formulaFon and producFon environment for JBSU3677 that ensures opFmal performance on a variety of substrates commonly fed
Enzyme Supplementa&on of Ruminants
Principal NSPase Ac&vi&es In JBSU3677 • A blend of culture extracts from two fungal organisms
– Aspergillus niger: high in acid cellulase and β-‐glucanase – Trichoderma reesei: high in xylanase, α-‐galactosidase
• Typical major enzyme acFviFes in JBSU3677, units/g1
• Also contains lower levels of amylase. • AddiFonal debranching enzymes, however, are truly opFmal
for maximum efficacy
Xylanase Acid Cellulase β-‐Glucanase Mannanase α-‐galactosidase
3100 2900 2200 378 10
1 DNS assay: xylanase tested at pH 4.5 & 40°C; cellulase tested at pH 4.8 & 50°C; β-‐glucanase and mannanase tested at pH 5 & 40°C; α-‐galactosidase tested at pH 5.5 & 37° C.
11
Corn Arabinoxylan Hydrolyzate: Proposed Structure
Proposed schemaFc of the sugar moieFes of corn arabinoxylan hydrolysate obtained at 40-‐60% fracFonaFon with ethanol. Araf = Arabifuranose, Xylp = Xylopyranose, D-‐Galp = Galactopyranose, D-‐GLCp = Glucuronic acid, Fer = Ferulic acid.
Structure is highly branched and integrated.
Debranching must occur to expose xylose backbone.
Enzymes Required for Arabinoxylan Degrada&on Arabinoxylan
Endo 1,4-‐ β-‐xylanase
Feruloyl esterase
α-‐Arabinofuranosidase
Ferulate
β-‐xylosidase
Xylose
Smaller Polysaccharide
Arabinose
α-‐Arabinofuranosidase
Arabinoxylan Debranching Enzyme Ac&vi&es Present In JBSU3677 (U/g)
Debranching Enzyme Units/gram
ß-‐xylosidase 1,569 α-‐L-‐arabinase 193 Acetyl esterase 3,405 Feruloyl esterase 951
1. Fungal Diversity
2. Controlled Fermenta&on of JBSU3677
Single bacterial or fungal strain
Narrow spectrum coverage
Complicated: MulFple products for mulFple substrates
Other Fiber and NSP Enzymes Two fungal strains selected for their ability to more effecFvely degrade fiber and non-‐starch
components
Simple: One product for mulFple substrates
Broad spectrum coverage
Dis&nc&ve Features Of JBSU3677 vs. Single Enzyme or Fungal Organism-‐Produced NSPase Products
Fungal Diversity • Dual fungal enzyme product from Aspergillus and Trichoderma species – Very diverse and prolific enzyme producers, mulFple enzyme acFviFes contributed by each
• Provides mulFple xylanase acFviFes with both endo-‐ and exo-‐ properFes – More effecFve breakdown of complex arabinoxylan chains into smaller xylose units that
are more digesFble to the animal.
• Fungal debranching enzymes assist xylanases in digesFng arabinoxylans in corn endocarp, enhancing kernel breakdown, and starch release.
• MulF-‐carbohydrase enzymes work in a more concerted acFon to becer degrade complex substrates than single source or enzyme products.
• Provides a greater enzyme diversity over a broader spectrum of substrate specificiFes and affiniFes – Presence and diversity of criFcal enzymes that may be produced in limited quanFFes when using only a single fungal or bacterial strain
– This diversity is paramount when the composiFon of substrates varies in the raFon
Controlled Fermenta&on • Harnessing the power of two fungal strains to produce a parFcular repertoire of enzymes also depends upon the fermentaFon condiFons used to grow the fungi • DirecFng the enzyme diversity via custom-‐designed growth condiFons
– Substrate, temperature, pH changes alter the enzyme profile produced
• Providing improved enzyme diversity and a broader range of acFvity
Single bacterial or fungal strain
Narrow spectrum coverage
Complicated: MulFple products for mulFple substrates
Other Fiber and NSP Enzymes
Two fungal strains selected for their ability to more effecFvely degrade fiber and non-‐starch components
Simple: One product for mulFple substrates
Broad spectrum coverage
Using JBSU3677 to Improve Fiber Diges&on • AddiFon of JBSU3677 improved in vitro dry macer digesFbility (IVDMD) of ! High moisture corn ! Wet disFllers grains plus solubles ! Corn bran ! Corn husks
• Increased gas producFon of ! Corn leaves ! Corn bran
• Conclusion: Trea&ng feeds with JBSU3677 improved in vitro diges&bility of feeds high in hemicellulose.
2014 Nebraska Beef Ca:le Report
Harding, J.L., G.E. Erickson, and J.C. MacDonald. 2014. Using Enspira to Improve Fiber DigesFon. Nebraska Beef Cacle Report.
• Conducted using commercial herd ! Mid-‐lactaFon cows (n=60) ! Average LactaFon= 3, DIM=148 ! Cows were normalized on similar TMR for 1-‐week ! NSP3677 was fed to provide 5 g/hd/day ! Cows fed JBSU3677 for a 28-‐d experimental period ! Analyzed both daily and weekly Energy-‐Corrected Milk (ECM) yields
• Results • Cows fed NSP3677 produced + 3.56 lbs. more milk than those fed control diets with no enzyme supplementaFon.
Effects of JBSU3677 on Milk Produc&on and Persistency of Lacta&ng Dairy Caile Pond Hill Dairy Research Farm, WI
C.M. Peter and M. Cooney (2014): JBS United Internal Research Report 14-‐R066
• 36 yearling Angus x Charolais x Nelore crossbred bulls (IniFal BW = 391 kg) • Completely randomized design, 3 treatments, 6 replicates, 2 head/pen • 94-‐d trial with 10-‐d adaptaFon period followed by four 21-‐d periods • Diets formulated to meet or exceed nutrient recommendaFons
• 85% whole corn; 15% protein, vitamins, minerals • Fed one of three treatments
1. Control 2. Control + 10 g/hd/d EXP3066 (amylase-‐based) 3. Control + 10 g/hd/d JBSU3677 (NSPase-‐based)
• DigesFbility and its effects on growth performance, carcass quality, and other metrics were evaluated.
Evalua&on of enzyme effec&veness in high energy density diet f feedlot finishing yearling bulls. UNICENTRO-‐ Central-‐West State University
Nuemann et al. (2015)
Effects of JBU3677 on Diges&bility and Fecal Grain Content
! DigesFbility improvements, however, do not always translate to improved growth performance.
Neumann et al. (2015). Means without a common superscripts are differ (P < 0.05).
[VALUE]b
[VALUE]a [VALUE]a
82,00
83,00
84,00
85,00
86,00
87,00
88,00
Apparent Diet Diges&bility, %
Control EXP 3066 JBSU 3677
[VALUE]a
[VALUE]b [VALUE]ab
0,00
10,00
20,00
30,00
40,00
50,00
60,00
Fecal Grain Content, g/kg
Control EXP 3066 JBSU 3677
Effects of JBSU3677 on Growth Performance and Feed Efficiency
[VALUE]0b
[VALUE]b
[VALUE]a
1,35
1,40
1,45
1,50
1,55
1,60
1,65 ADG, kg/d
+ 11%
Control EXP 3066 JBSU 3677
[VALUE]a
[VALUE]ab
[VALUE]b
5,00
5,20
5,40
5,60
5,80
6,00
6,20 Feed Efficiency, kg/kg
+ 11%
Control EXP 3066 JBSU 3677
! No differences in DMI (kg/d) were observed among treatments.
Neumann et al. (2015). Means without a common superscripts are differ (P < 0.05).
Effects of JBSU3677 on Carcass Daily Gain and Weight
! No differences in carcass yield or quality measures were observed.
Neumann et al. (2015). Means without a common superscripts are differ (P < 0.05).
[VALUE]b
[VALUE]a [VALUE]a
286,00
288,00
290,00
292,00
294,00
296,00
298,00
Hot Carcass Weight, kg
[VALUE]b
[VALUE]a [VALUE]a
1,04
1,06
1,08
1,10
1,12
1,14
1,16
1,18
Carcass Daily Gain, kg/d
Control EXP 3066 JBSU 3677 Control EXP 3066 JBSU 3677
Conclusions
• SupplemenFng both EXP3066 and JBSU3677 significantly improved diet digesFbility and reduced fecal grain content.
• The digesFbility improvements for amylase-‐based JBSU3066 did not translate into significant improvements in performance and feed efficiency.
• DigesFbility improvements observed when supplemenFng NSPase-‐based JBSU3677, however, did directly result in significant improvements in growth performance (+11%), feed efficiency (+11%), and final end product.
Research Driven • Based on fungal diversity • Produced via precision-‐designed fermentaFon • Concentrated broad spectrum enzyme efficacy
RelaFonship Focused • CompeFFve cost structure • Excellent ROI for producer
Results Oriented • Both in vitro and in vivo improvements observed • Univ. of Nebraska: ↑ in vitro digesFbility of feedstuffs high in
hemicellulose • Pond Hill Dairy Research: ↑ ECM by 3.56 lb/hd/d; • AddiFonal Field Research: ↑ milk yield by 2 lb/hd/d in milk yield
observed; • 11% improvement in both ADG and feed efficiency in feedlot cacle
JBSU3677 Benifits
