1. Fish Nutrition Research Laboratory Dept. of Animal Biosciences
Keratin Waste Products - A Critical Look at the Benefits of Properly Processing this Undervalued Source of Protein into Valuable Feather Meal
Guillaume Pfeuti
&
Dominique P. Bureau
Fish Nutrition Research Laboratory
Dept. of Animal Biosciences
University of Guelph

2. Chicken, Feather, and FeM Production
8.3 billion chickens raised and processed in the USA in 2013
23.2 million tons of live weight
5 to 7% of chicken body weight consist of feathers
~1.4 million tons of feathers co-products generated in the USA
617 thousand tons recycled into feather meal
A fraction of the amount recycled for feather meal is used for textiles and composites
~700 thousand tons of raw feathers incinerated and buried in landfills in the USA only
A great amount of potential dietary protein is disposed of globally

3. Feather Meal 75-85% Crude Protein Rich in: Arginine (5.8%)
Cystine (3.8%)
Isoleucine (4.2%)
Leucine (6.9%)
Phenylalanine (4.1%)
Threonine (3.9%)
Poor in:
Histidine: (0.7%)
Lysine: (1.8%)
Tryptophan: (0.55%)
Cost-effective (~700$/ton)
Slide 3 I would put the cost-effective comment at the end.  i think "cost-effectiveness" depends on price and nutritive value.  You can't state this from the start.
May be worth adding comparison of cost per kg  or tonne of protein to other common ingredients?
High variability in nutritional value!!!

4. Price of Feed Ingredients
Crude
Protein %
Price
$/MT
$/ MT of
Crude Protein
Fishmeal 63
2000
2860
Poultry Meal 65
820
1260
Corn gluten Meal 60
750
1250
Blood Meal 90
900
1000
Soybean Meal 48
450
990
Canola Meal 38
350
920
Meat and Bone Meal 55
475
910
Feather Meal 80
700
875

5. Apparent Digestibility (%)
Variability of Digestibility
Author
Apparent Digestibility (%) DM CP GE
Cho et al. (1982) 75 58 70
Cho and Kaushik (1990) 81 77
Bureau et al. (1999) 79 76 80 82 83 84 87
Cheng et al. (2004)
Gaylord et al. (2008) - 88
Glencross (2011) 59 93 85
Anecdotal evidence among feed industry stakeholders along with an important numbers of studies report that the digestibility of CP and amino acids in FeM are extremely variable, making precise feed formulation of diets containing feather meal to accomplish difficult.

6. Variability of raw materials
This variability is the result of the use of highly diverse raw materials.
Feathers of different origin are often rendered along with hog hair, horn, hooves, and claws.

7. Variability processing equipment
Batch Pressure Cooker
Disc Dryer
Continuous Pressure
Cooker
Ring Dryer
Flash Dryer
This variability is the result of the use of highly diverse commercial processing equipment for cooking and drying the biomass.
The conditions of drying and hydrolysis (temperature, pressure, and time) are highly variable across manufacturing facilities and change according to the type of industrial rendering equipment available.

8. Steam-hydrolysis of keratin
Disulphide
Bond S S
Hydrogen
Bond H O O H
Cysteine level dropping from 7% in raw feathers to 4% in FeM
Raw feathers, hair, hooves, horns, and claws are 90% comprised of the structural protein keratin.
Raw keratins are indigestible in their natural state.
This lack of digestibility is caused be the high degree of disulphide bonds provided by the high level of cystine, which strongly holds together the polypeptide chains into the robust structure.
Using steam hydrolysis, processor break down the chemical bonds present into raw keratin and transform it into digestible FeM.
The cysteine level drops from 7-10% in raw keratin to 4% in FeM due to the cross-linking of amino acids occurring during heat processing
Peptide
Bond

9. Amino Acid Cross-linking
Exposure of keratin biomass to high heat during the cooking and drying steps of FeM processing promotes the β-elimination of cystine and serine, two amino acids found at high level in this material, to give undesired dehydroalanine (DHA).
The double bond of the DHA intermediate reacts actively with cysteine, lysine, histidine, ornithine, and ammonia to form LAN, LAL, HAL, OAL, BAL which have very little nutritional value
Cysteine can be oxidized into nutritionally unavailable cysteic acid
CLAAs in heat and alkali treated proteins were shown to be indicators of amino acid racemization.

10. Racemization of Amino Acids

11. It is all about finding the sweet spot Optimal Bioavailability
Optimal Processing of FeM
It is all about finding the sweet spot
Under processing
→ High level of keratin
→ High level of disulphide bonds
→ High level of D-amino acids
Over Processing
High level of thiols ←
High level of cross-linked AA ←
High level of D-amino acids ←
Optimal processing =
Optimal Bioavailability

12. Current Quality Assessment Tool
According to AFFCO (Association of America Feed Control Officials)
HCl-Pepsin digestibility 0.2% Pepsin
0.075 M HCL (pH 2)
45°C
16 hours
→ A pepsin digestibility > 75% is considered the “standard” to indicate proper hydrolysis
Official test adopted by the Association of Official Analytical Chemists
(AOAC) in 1960.
No relationship between in vitro and in vivo digestibility of CP in FeM
(Papadopoulous et al., 1987)
The Industry use the HCl-Pepsin assay to evaluate the FeM quality as prescribed by AAFCoà
The HCl-Pepsin Assay is a quality assessment tool used by the industry to assess the quality of feather meal
The Association of American Feed Control Officials (AAFCO) stipulates that no less than 75% of the protein content in FeM must be digestible in 0.2% pepsin solution to assure adequate processing of the ingredient.
The official test adopted by the Association of Official Analytical Chemists (AOAC) in 1960.

13. HCl-Pepsin Digestibility Test
Native, undamaged protein
Damaged protein
Absorbable/soluble cross-links without nutritive value
Easily hydrolysable soluble peptides
Water-soluble peptides, measured as “digestible”
Indigestible cross-linked protein

14. Feather Meal: Effectiveness of a Simple Chemical Pre-Treatment
Pre-treatment of 2 commercial feather meals (FeM)
2% sodium sulfite (%FeM w/w)
0.05% Protease (%FeM w/w)
200% water (%FeM w/w)
24h incubation
sulfonate

15. Pre-Treatment of Steam-hydrolyzed Feather Meals
2- Proteolysis using a commercial protease
1- Sulfitolysis using sodium sulfite (Na2SO3)
sulfonate
Cystine
+ Sulfite
Bunte Salt
+ Cysteine

16. Results: Digestibility of Ingredients
FeM1
PTFeM1
FeM2
PTFeM2
Proximate composition (a)
Dry matter (%)
78.3b
87.7ab
86.9ab
93.2a
Crude protein (%)
85.4b
94.7a
81.9b
95.5a
Gross energy (kJ g-1)1
87.2ab
86.0ab
94.4a
Essential amino acids (%)
Arginine
86.3b
95.6a
84.9b
95.3a
Histidine
53.6b
102.5a
72.8ab
114.8a
Isoleucine
86.0b
94.2a
87.9b
96.5a
Leucine
82.3b
96.1a
99.4a
Lysine
74.1b
96.9ab
87.5ab
105.1a
Methionine
73.3b
87.0ab
88.1a
Phenylalanine
83.0b
96.4a
85.1b
99.0a
Threonine
80.1b
91.0a
79.2b
91.9a
Valine
84.3b
96.2a
Non-essential amino acids and lanthionine (%)
Alanine
81.3b
96.8a
84.0b
99.9a
Aspartic acid
80.4c
92.9ab
84.7bc
97.9a
Cyst(e)ine
78.8b
86.5a
75.4b
84.8a
Glutamic acid
82.8b
93.0a
84.8b
Glycine
96.6a
88.1b
96.0a
Proline
85.8bc
83.0c
90.4ab
Serine
86.9b
95.0a
94.1a
Lanthionine
79.8b
84.6a
66.6c
76.8b

17. Results: Digestibility of Ingredients
FeM1
PTFeM1
FeM2
PTFeM2
Proximate composition (a)
Dry matter (%)
78.3b
87.7ab
86.9ab
93.2a
Crude protein (%)
85.4b
94.7a
81.9b
95.5a
Gross energy (kJ g-1)1
87.2ab
86.0ab
94.4a
Essential amino acids (%)
Arginine
86.3b
95.6a
84.9b
95.3a
Histidine
53.6b
102.5a
72.8ab
114.8a
Isoleucine
86.0b
94.2a
87.9b
96.5a
Leucine
82.3b
96.1a
99.4a
Lysine
74.1b
96.9ab
87.5ab
105.1a
Methionine
73.3b
87.0ab
88.1a
Phenylalanine
83.0b
96.4a
85.1b
99.0a
Threonine
80.1b
91.0a
79.2b
91.9a
Valine
84.3b
96.2a
Non-essential amino acids and lanthionine (%)
Alanine
81.3b
96.8a
84.0b
99.9a
Aspartic acid
80.4c
92.9ab
84.7bc
97.9a
Cyst(e)ine
78.8b
86.5a
75.4b
84.8a
Glutamic acid
82.8b
93.0a
84.8b
Glycine
96.6a
88.1b
96.0a
Proline
85.8bc
83.0c
90.4ab
Serine
86.9b
95.0a
94.1a
Lanthionine
79.8b
84.6a
66.6c
76.8b

18. Slope-Ratio assay to assess the bioavailability of pre-treated feather meals
We attempted to assess the effect of the pre-treatment on the bioavailability of arginine in FeM by comparing the slope of the responses of fish fed diets containing increasing levels of arginine either provided by crystalline L-arginine, FeM or PTFeM.

19. Objective 2 : Arginine Bio-Availability trial
Ingredients 1 2 3 4 5 6 7 8 9 10 11
Arginine Level
1.20
1.35
1.50
L- Arginine
0.15
0.30
Feather meal 1– Original 20
Feather meal 1– Treated
Feather meal 2– Original
Feather meal 2– Treated

20. Growth Trial: Results Arg Dig. = 95% Arg Dig. = 86% Arg Dig. = 96%
Slide 25 is a bit busy with two parameters. Not sure audience will know what is TGC.  I guess the flow of information will make sense when you present.
Arg Dig. = 96%
Arg Dig. = 86%

21. Cross-Linked Amino Acids Levels May be Inversely Correlated with Amino Acid Bioavailability
Ingredients
FeM1
PTFeM1
FeM2
PTFeM2
Proximate composition (as is)
Dry matter (%)
93.4
93.3
86.6
93.1
Crude protein (%)
81.9
80.3
76.3
81.7
Lipid (%)
8.3
7.9
6.5
Total carbohydrates (%)1
1.3
1.5
0.6
Ash (%)
1.9
3.8
2.3
4.3
Gross energy (kJ g-1)1
22.6
22.1
20.7
21.8
Essential amino acids (% as is)
Arginine
5.9
5.7
6.1
Histidine
0.7
0.8
Isoleucine
4.0
3.9
3.5
Leucine
6.7
6.2
6.6
Lysine
1.8
2.2
Methionine
0.5
Phenylalanine
3.4
3.6
Threonine
Valine
6.0
5.8
5.1
5.6
Non-essential amino acids (% as is)
Alanine
3.7
Asparatic acid
5.5
Cyst(e)ine
4.1
Glutamic acid
9.2
9.0
9.7
10.1
Glycine
6.3
Proline
7.8
6.8
7.3
Serine
9.3
8.8
8.1
8.4
Cross-linked amino acids (% as is)
Lanthionine
3.18
3.17
2.55
2.80
DL-Lysinoalanine
0.16
0.15
0.06
0.07
Β-aminoalanine
0.14
0.13
0.05

22. Take-home Message
The pre-treatment significantly improves the digestibility of amino acids and the bioavailability of arginine in FeMs.
Digestibility of CP and amino acids in feed ingredients is influenced by disulphide bond levels.
Disulphide bonds and cross-linked amino acids may be indicators of digestibility of CP and amino acids in feed ingredients.
Cross-linked amino acids may be indicators of bioavailability of amino acids in feed ingredients.

23. Acknowledgments
Sanimax
Anna-Kate Shoveller,
James Longstaffe
Elijah Kiarie
Leonid S. Brown