Influence of particle size and size distribution of sorghum and field pea on digestibility and growth performance of pigs

Using sorghum and field pea, this project specifically i investigated the influence of milling techniques on particle size characteristics, and how these affected in-vitro digestion of s

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Influence of particle size and size distribution of sorghum and field

pea on digestibility and growth performance of pigs

Giang T Nguyen

Master of Agricultural Sciences

A thesis submitted for the degree of Doctor of Philosophy at

The University of Queensland in 2016

Queensland Alliance for Agriculture and Food Innovation

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Abstract

Cereals have higher starch and lower protein contents than legumes/pulses Field pea, a legume/pulse,

is lower in anti-nutritional factors than other legumes/pulses, and is a desirable protein supplement in cereal-based feeds Grains (cereals and legumes/pulses) are milled to reduce particle size and improve feed efficiency, and while hammer-mills are used more in feed manufacture, disc-mills have different milling mechanisms and are increasing in importance Mill differences manifest in particle size and particle size distribution, and mills can yield narrow, broad and skewed distributions The dependence

of in-vitro and in-vivo digestion on particle size distributions has not been thoroughly studied, and

maximising digestion and energy delivery in pigs, demand defining a grain optimum particle size range Particle size characteristics influence residence time distributions in the gastrointestinal tract (GIT) with nutritional consequences, and understanding the mixing patterns in the GIT provides useful information on this

Using sorghum and field pea, this project specifically (i) investigated the influence of milling

techniques on particle size characteristics, and how these affected in-vitro digestion of starch and

protein; (ii) determined how particle size characteristics affected growth performance of weaner pigs; (iii) modelled residence time distributions in the GIT of grower pigs cannulated at the terminal ileum, and investigated how this modelling revealed mixing in the GIT, which in turn was related to digestion patterns in the pigs; (iv) probed how feed ingredients influenced grain digestion; and (v) examined the status of starch in ileal digesta

Feed mills in Australia and Vietnam were surveyed, revealing differences in particle size characteristics of mash diets within and between grains and mills Also, diets for weaner- and grower-

pigs varied in particle size characteristics In-vitro starch and protein digestions of the milled sorghum

and field pea significantly (p ≤ 0.05) depended on particle size (Dgw), and particle size distribution (Sgw) significantly (p ≤ 0.05) affected the grain water absorption and solubility indices Using a pH drop method for protein digestion, and glucose release for starch digestion, irrespective of the year (2012 - 2014), the rate of protein digestion in the milled grains was much faster than the rate of starch digestion, with implications for nutrient asynchrony Generally, pig feed ingredients significantly (p

≤ 0.05) affected apparent enzyme diffusion coefficients in the milled grains, and the effects on the field pea were generally different from that on the sorghum There were also differences between the effects on starch and protein in both grains

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Growth performance in weaner pigs was investigated with mash diets containing milled sorghum of particle sizes 0.4 - 0.8 mm, and milled field pea of particle sizes 0.6 - 0.8 mm over a 21-day period The weaner-pigs gained weight (average daily gain) during the feeding period, and neither mill type (disc- or hammer-mill) nor grain particle size significantly (p > 0.05) affected the feed conversion ratio (feed intake:weight gain) The particle sizes obtained were inferred to be within the optimum

for the grains for maximum feed efficiency In-vitro digestion parameters reasonably (p ≤ 0.2)

correlated with the growth performance of the weaner pigs

The sorghum and field pea were disc-milled to make mash diets fed to cannulated grower pigs, with titanium dioxide as an indigestible marker Ileal digesta samples, collected through a T-cannula from

0 - 720 min after feeding, generated apparent multi-peak residence time distributions irrespective of the treatments, and gravitational sedimentation mainly in the stomach was proposed to be responsible The distributions were modelled using a compartmental approach, with the stomach and small intestine respectively as mixing and displacement compartments The model revealed pronounced mixing in the stomach, and gastric mean residence time ranged from 3 - 20 hr, while a range of 2 - 4

hr was obtained for the small intestine However, there were no particle size effects on the ileal starch digestibility (95 - 98%), and the particle size characteristics (from light microscopy) of the ileal digesta samples were essentially the same (Dgw-ileal = 0.17 ± 0.03; Sgw-ileal = 0.12 ± 0.03), and independent of the diet particle size characteristics The residence time distribution parameters correlated (r2 > 0.800, p ≤ 0.30) positively with Dgw-diet and negatively with Sgw-diet Confocal microscopy revealed starch granules in, and separated from intact cells, irrespective of the digesta collection time

Using in-vitro and in-vivo approaches, the present study showed how grains, mill types and particle

size characteristics influenced animal performance with insights in apparent enzyme diffusion coefficients, flow profiles in the GIT and the status of starch during passage through the GIT This information is valuable in food and feed processing on grain milling, and in controlling milled grain particle sizes The range (0.4 - 0.9 mm) of particle sizes studied for the field pea and sorghum was within the optimum for the grains, in terms of weaner pig growth performance and grower pig ileal starch digestibility

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Declaration by author

This thesis is composed of my original work, and contains no material previously published or written

by another person except where due reference has been made in the text I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis

I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution I have clearly stated which parts of

my thesis, if any, have been submitted to qualify for another award

I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School

I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis

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Publications during candidature

Peer-reviewed paper

Nguyen, G T., Gidley, M J and Sopade, P A (2015) Dependence of in-vitro starch and protein digestions on particle size of field peas (Pisum sativum L.) LWT-Food Science and Technology, 63,

541-549

Nguyen G T., Bryden, W L., Gidley M J and Sopade P A (2015) Pig feed ingredients affect

enzyme diffusion coefficients Animal Production Science, 55, 1537

Nguyen G.T., Bryden, W L., Gidley M.J and Sopade P.A (2015) Variation in particle sizes of

commercial pig feeds in Vietnam Animal Production Science, 55, 1565

Nguyen G T., Collins, C., Henman, D., Diffey, S., Tredrea, A M., Black, J L., Gidley M J and Sopade P A (2015) Growth performance of weaner pigs fed diets containing grains milled to

different particle sizes I Sorghum Animal Production Science, 55, 1566

different particle sizes II Field pea Animal Production Science, 55, 1567

Conference proceedings

Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) A survey of particle size and particle size variability of milled grains

available for use in Australian pig feeds Manipulating Pig Production XIV - Proceedings of the 14th

Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 55

Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) Design and evaluation of a manual sieving device for monitoring particle

size in feed manufacture Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 56

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Nguyen, G T., Bryden, W L, Gidley, M J., and Sopade, P A (2013) Particle size and particle size

dispersion drive hydration of grains: Field peas (Pisum sativum L.) as a case study (2013) Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian

Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 120

Nguyen, G T., Bryden, W L, Gidley, M J., and Sopade, P A (2013) In-vitro starch and protein digestion in field peas (Pisum sativum L.) reveal particle size dependence Manipulating Pig Production XIV - Proceedings of the 14th Biennial Conference of the Australasian Pig Science

Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 121

Conference abstracts

Nguyen G T., Gidley M J and Sopade P A (2013) Kinetics of protein and starch digestion in field

peas Poster 63rd Annual Conference of the Australian Cereal Chemistry Conference (ACCC), 25 -

28 August 2013, Fremantle, Perth, Australia

Nguyen G T., Zhang, J, Bozec, M., Gidley, M J and Sopade, P A (2014) Can Fourier Transform

Infra-Red spectroscopy contribute to the understanding of protein digestion? Abstract Summer

School 2014 of the Australian Institute of Food Science Technology Food Science (AIFST), 5 - 4 February 2014, The University of Queensland, Brisbane, Australia

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Publications included in this thesis

Incorporated as Chapter 3

Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) A survey of particle size and particle size variability of milled grains

available for use in Australian pig feeds Manipulating Pig Production XIV - Proceedings of the 14th

Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 55

Conducted experiment (40%) Analysed data (80%)

Wrote first draft of paper (100%) Finalised paper (70%)

Reviewed paper (20%)

Conducted experiment (20%) Reviewed paper (10%)

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Finalised paper (30%)

Nguyen G.T., Bryden, W L., Gidley M.J and Sopade P.A (2015) Variation in particle sizes of

commercial pig feeds in Vietnam Animal Production Science, 55, 1565

Analysed data (20%) Reviewed paper (50%) Finalised paper (30%)

Nguyen, G T., Bryden, W L, Gidley, M J., Edwards, A C., Willis, S., Black, J L Wilson, R H and Sopade, P A (2013) Design and evaluation of a manual sieving device for monitoring particle size in feed manufacture Manipulating Pig Production 14th - Proceedings of the Proceedings of the

14th Biennial Conference of the Australasian Pig Science Association (APSA) Edited by Pluske, J and Pulske, J Australasian Pig Science Association (Inc.), Werribee, Victoria, Australia Pg 56

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Reviewed paper (35%) Finalised paper (30%)

Nguyen, G T., Gidley, M J and Sopade, P A (2015) Dependence of in-vitro starch and protein digestions on particle size of field peas (Pisum sativum L.) LWT-Food Science and Technology, 63,

541-549

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different particle sizes II Field pea Animal Production Science, 55, 1567

Analysed data (40%) Reviewed paper (10%)

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Finalised paper (30%)

different particle sizes I Sorghum Animal Production Science, 55, 1566

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Contributions by others to the thesis

Jing Zhang

School of Agriculture and Food Sciences, The

University of Queensland,St Lucia, QLD 4072,

AUSTRALIA

Assisted in various experiements as a research assistant in the funded project, under which the thesis falls

Sylvie van Rijt

HAS Den Bosch, University of Applied

Sciences, s’-Hertogenbosch, NETHERLANDS

Jennifer Waanders

David Appleton

Stephen Appleton

University of Queensland,St Lucia, QLD 4072,

Melbourne School of Land and Environment,

The University of Melbourne, Melbourne, VIC

3010, AUSTRALIA

Sourcing and supervising grower pigs for the cannulation experiment in the funded project, under which the thesis falls

Dagong Zhang

University of Queensland, Gatton, QLD 4343,

AUSTRALIA

Freeze drying of ileal samples in the funded project, under which the thesis falls

Peter Isherwood

University of Queensland, Gatton, QLD 4343,

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Acknowledgements

I am so indebted to Dr Peter Sopade, my principal advisor for everything he has done for me to successfully complete my studies Peter has been a wonderful teacher and his research knowledge has widened my world His patience, understanding and ever encouraging nature helped me complete this challenging task, and I deeply acknowledge his time, effort, guidance, and supervision I would also thank him for teaching me in the professional academic writing styles and for honing my presentation skills There were many times when he sacrificed to guide me through Peter, without you, I would not have come this far, and I sincerely appreciate your efforts and will value these always I thank my co-advisors, Prof Mike Gidley and Prof Wayne Bryden, for all their guidance and supervision during my studies A very special thank-you for your valuable suggestions and comments through the entire research

I am grateful to my sponsor, the Australian Aid for International Development (AusAID) for the scholarship awarded to me to undertake this study Without this scholarship, this study would not have been possible, and I thank AusAID for that The continued support from my employer, An Giang University (AGU), Vietnam is gratefully acknowledged

I am also grateful to Pork CRC (Co-operative Research Centre for High Integrity Australian Pork) for the financial support (Project 4B-112) throughout the experiments, and to attend the Australasian Pig Science Association (APSA) workshops and conferences I acknowledge the technical support received from Rivalea Australia Pty Ltd (Corowa NSW, Australia) and the University of Melbourne for the weaner feeding and cannulation experiments I also thank other Pork CRC associates such as Mrs Sara Willis, Dr Cherie Collins, Prof John Black, Dr David Henman, and Mr Rob Wilson for their technical support

I would like to thank the entire people in the Centre for Nutrition and Food Sciences and School of Agriculture and Food Sciences that supported me in one way or another Special mention to Dr Lesleigh Force, Dr Hotnida Sinaga, Mr David Appleton, Mr Peter Isherwood, and Mr Dagong Zhang for their laboratory support I would also like to thank my Vietnamese friends and colleagues, for their friendship and generous assistance throughout my stay in Australia

Last but not least, I thank my parents, my husband and my children for their continued understanding and encouragement during my study in Australia Dear mom, without your never-ending love, I would not have this achievement

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Keywords: field pea, sorghum, particle size, particle size distribution, in-vitro digestion, weaner pigs,

digestibility, apparent diffusion rate, residence time distribution, growth performance

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code: 070204, Animal Nutrition, 60%

ANZSRC code: 090805, Food Processing, 40%

Fields of Research (FoR) Classification

FoR code: 0702, Animal Production, 60%

FoR code: 0908, Food sciences, 40%

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Table of contents

Statement of parts of the thesis submitted to qualify for the award of another degree xii

2.5 Conclusions from the literature and research thrust 50

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Chapter 4 Dependence of in-vitro starch and protein digestions on particle size of

field peas (Pisum sativum L.)

4.2.5 Determination of water absorption and solubility indices 70

4.3.6 Asynchronous relationship between protein and starch digestions 89 4.3.7 Relationship between hydration and digestion properties 91

Chapter 5 Growth performance of weaner pigs fed diets containing field pea milled

to different particle sizes

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5.2.6 Weaner feeding experiment 100

5.3.4 Effect of particle size on growth performance of weaner pigs 115 5.3.5 Relationship between in-vitro digestion and growth performance parameters 116

Chapter 6 Growth performance of weaner pigs fed diets containing sorghum milled

to different particle sizes

6.3.4 Effect of particle size on growth performance of weaner pigs 141 6.3.5 Relationship between in-vitro digestion and growth performance parameters 142

Chapter 7 Residence time distribution and mixing profile in the gastrointestinal tract

of pigs as defined by model protein and carbohydrate sources of different particle sizes

Chapter 8 Particle size effects on ileal starch digestibility in pigs fed carbohydrate

and protein sources

173

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8.3 Results and discussion 177

3.1 Particle size characteristics of milled grains and diets collected from the feed

mills surveyed in Australia

235

3.2 Particle size characteristics of diets for various pig types collected from the

feed mills surveyed in Vietnam

237

4.2 Glucose stock concentration and calibration curve for starch analysis 239

5.1 The diet compositions for weaner feeding experiment with milled field pea 243

6.1 The diet compositions for weaner feeding experiment with milled sorghum 257

7.1 The diet compositions for cannulation experiment with milled field pea 269 7.2 The diet compositions for cannulation experiment with milled sorghum 270 7.3 The ethic approval (1112335.5) of the cannulation experiment 271

7.5 Samples of milled field pea and sorghum grains used in the cannulation

experiment

287

7.6 Relationship between residence time parameters and particle size

characteristics of the milled field pea grain

288 7.7 Relationship between residence time parameters and particle size

characteristics of the milled sorghum grain

289

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List of Figures

Figure 2.1 Top field pea producing countries and Australia from 2009 - 2013 6

Figure 2.3 Diagram of the distribution of the milling techniques obtained from a

feed-mill survey

15

Figure 2.4 Typical disc-, roller- and hammer-mills with the main milling forces 15 Figure 2.5 A manual sieving device to evaluate particle size distribution 20 Figure 2.6 Hypothetical digestograms showing differences between single-point and

time-course measurements

32

Figure 2.8 Predictive ability of assumptions 1 - 3 for non-processed milled sorghum of

average particle size 256 μm

40 Figure 2.9 Steps of cannulation techniques of inserting a simple T-cannula at the distal

and mill types and particle size dispersion shown as error bars

61

Figure 3.3 Average particle size of similar grains and mill types in different mills with

particle size dispersion shown as error bars

62 Figure 3.4 The collection sites in Vietnam with feed mill codes; Average particle size

of different commercial feeds in selected mills

63

Figure 4.2 Typical particle size distributions of the milled field pea 75 Figure 4.3 Hypothetical broad and narrow particle size distributions, showing different

Sgw with the same Dgw

79 Figure 4.4 Typical digestograms of the in-vitro starch digestion of the milled field pea

showing experimental and predicted data

80

Figure 4.5 The relationship between the reciprocal of the rate of in-vitro starch

digestion and the square of the particle size

84 Figure 4.6 Typical digestograms of the in-vitro protein digestion of the milled field

pea showing experimental and predicted data

86

Figure 4.7 The relationship between the reciprocal of the rate of in-vitro protein

digestion and the square of the particle size

88 Figure 4.8 The relationship between the rates of in-vitro digestion of starch and protein

of the milled field pea

90 Figure 4.9 The relationship between the rates of in-vitro digestion of starch and protein

and hydration properties of the milled field pea for the combined mill data

93

Figure 5.2 Typical particle size distributions of the field pea samples 103 Figure 5.3 The relationship between the particle size characteristics of the milled field

pea grains and experimental diets

Figure 5.6 The relationship between the reciprocal of the rate of starch digestion, and

the square of the particle size for the field pea samples

109 Figure 5.7 Digestograms of the in-vitro protein digestion of the milled field pea grains 111 Figure 5.8 Digestograms of the in-vitro protein digestion of the field pea diets 112

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Figure 5.9 The relationship between the reciprocal of the rate of protein digestion, and

the square of the particle size of the field pea samples

114 Figure 5.10 The relationship between the rates of digestion of starch and protein of the

field pea samples

115

Figure 5.11 A simplified relationship between various factors that control voluntary

feed intake in pigs

119

Figure 6.2 Typical particle size distributions of the sorghum samples 129 Figure 6.3 The relationship between the particle size characteristics of the milled

sorghum grain and diet

130

Figure 6.4 Digestograms of the in-vitro starch digestion of the milled sorghum grain 131 Figure 6.5 Digestograms of the in-vitro starch digestion of the sorghum diet 132 Figure 6.6 The relationship between the reciprocal of the rate of starch digestion, and

the square of the particle size for the sorghum samples

135

Figure 6.7 Digestograms of the in-vitro protein digestion of the milled sorghum grain 136 Figure 6.8 Digestograms of the in-vitro protein digestion of the sorghum diet 137 Figure 6.9 The relationship between the reciprocal of the rate of protein digestion, and

the square of the particle size of the sorghum samples

140

Figure 6.10 The relationship between the rates of digestion of starch and protein of the

sorghum samples

141

Figure 7.2 Distribution of marker residence time for an idea reactor consisting of a

Plug Flow and Continuous-stirred tank reactors in series

155 Figure 7.3 Typical particle size distributions of the field pea and sorghum samples 157 Figure 7.4 The relationship between the particle size characteristics of the field pea

and sorghum samples

159 Figure 7.5 Concentration (Ti)-time relationships for the field pea diet 162 Figure 7.6 Concentration (Ti)-time relationships for the sorghum diet 162

Figure 7.8 Cumulative concentration (Ti)-time relationships for the field pea diet 164 Figure 7.9 Cumulative concentration (Ti)-time relationships for the sorghum diet 164

Figure 7.11 Relationship between residence time parameters and particle size

characteristics of the field pea diet

170

Figure 7.12 Relationship between residence time parameters and particle size

characteristics of the sorghum diet

171 Figure 8.1 Digestograms of the in-vitro starch digestions of the milled field pea and

the square of the particle size for the field pea and sorghum samples

181 Figure 8.5 Digestograms of the in-vitro protein digestion of the milled field pea and

the square of the particle size of the field pea and sorghum samples

186 Figure 8.8 The relationship between the rates of digestion of starch and protein of the

field pea and sorghum samples

187

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Figure 8.10 Typical particle size distributions of the ileal digesta collected at 600 min 191 Figure 8.11 Particle size characteristics of the ileal digesta by time 193 Figure 8.12 Ileal starch digestibility by time of the field pea and sorghum samples 195 Figure 8.13 Confocal laser scanning micrographs of digesta samples of field pea 196 Figure 8.14 Confocal laser scanning micrographs of digesta samples of sorghum 197

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pigs as well as feed conversion ratio

23 Table 2.5 Effect of particle size uniformity on nutrient digestibility and growth

performances in grower-finisher pigs

24

Table 2.6 Diets with optimum particle sizes for growth performance of pigs 24 Table 2.7 Assessment of in-vitro and in-vivo digestion techniques 26 Table 2.8 Summary of representative in-vitro starch digestion techniques 28 Table 2.9 Summary of in-vitro protein digestion techniques (single-protease system) 30 Table 2.10 Summary of in-vitro protein digestion techniques (multi-proteases) 31 Table 2.11 Examples of D∞ values above 100% estimated by the LOS approach 38 Table 2.12 Summary of in-vivo digestion techniques in mono-gastric animals 42 Table 2.13 Basal ileal endogenous protein from difference estimations 49

Table 4.2 Physical properties and chemical composition of the field pea 73 Table 4.3 Particle size and particle size distribution of the milled field pea 74

Table 4.5 Regression equations on the hydration properties and particle size

characteristics of the milled field pea

78 Table 4.6 The parameters of the modified first-order kinetic model for the in-vitro

starch digestion of the milled field pea

81 Table 4.7 Regression equations on the in-vitro starch digestion parameters and particle

characteristics of the milled field pea

83

Table 4.8 Regression equations on the rate of in-vitro starch digestion and particle size

of the milled field pea

protein digestion of the milled field pea

87

Table 4.10 Regression equations on the in-vitro protein digestion parameters and

particle characteristics of the milled field pea

88 Table 4.11 Regression equations on the rate of in-vitro protein digestion of the particle

size of milled field pea

89 Table 4.12 Regression equations on the relationship between the hydration and in-vitro

digestion properties of the milled field pea

92

Table 5.1 Mill settings and codes of the disc- and hammer-milled field pea 97 Table 5.2 Composition of the experimental diet (as fed) of the field pea 98 Table 5.3 Particle size characteristics of the milled field pea grain and diet 104 Table 5.4 Parameters of the modified first-order kinetic model for the in-vitro starch

digestion of the milled field pea grains and diets

107 Table 5.5 Linear equation between the square of the particle size and the reciprocal of

the rate of starch digestion of the field pea samples

109

Table 5.6 The parameters of the modified first-order kinetic model for the in-vitro

protein digestion of the milled field pea grain and diet

113 Table 5.7 Linear equations relating the square of the particle size and the reciprocal of

the rate of protein digestion for the field pea samples

114

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Table 5.8 Effect of the field pea diet particle size on the growth performance of

weaner pigs in a 21-day experiment

117 Table 5.9 Pearson correlation analysis between the growth performances and in-vitro

digestion parameters of the field pea samples

118

Table 6.1 Mill settings and codes of the disc- and hammer-milled sorghum 124 Table 6.2 Composition of the experimental diet from the milled sorghum 125 Table 6.3 Particle size characteristics of the milled sorghum grain and diet 130 Table 6.4 The parameters of the modified first-order kinetic model for the in-vitro

starch digestion of the milled sorghum grain and diet

the rate of starch digestion of the sorghum samples

135

Table 6.6 The parameters of the modified first-order kinetic model for the in-vitro

protein digestion of the milled sorghum grain and diet

the rate of protein digestion of the sorghum samples

140 Table 6.8 Effect of the sorghum diet particle size on the growth performance of

weaner pigs in a 21-day experiment

143

Table 6.9 Pearson correlation analysis between the growth performances and in-vitro

digestion parameters of the sorghum samples

144 Table 7.1 Physical properties and chemical composition of field pea and sorghum

digestibility studies

153 Table 7.4 Typical calculation of the titanium concentration in an experimental period

parameters

starch digestion of the field pea and sorghum samples

180

Table 8.2 Linear equation between the square of the particle size and the reciprocal of

the rate of starch digestion of the field pea and sorghum samples

protein digestion of the field pea and sorghum samples

184

Table 8.4 Linear equation between the square of the particle size and the reciprocal of

the rate of protein digestion of the field pea and sorghum samples

186 Table 8.5 Hydration properties of the field pea and sorghum diets 190 Table 8.6 Particle size characteristics of the ileal digesta and starch digestibility of the

diets

192

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List of Abbreviations

ADFI Average daily feed intake

ADG Average daily gain

D0 Digested starch at time t = 0

D∞ Digested starch at infinite time, t  ∞

Dgw Geometric mean diameter or median size of particles by mass

Diff Apparent diffusion coefficient

Dt Digested starch at time t

ΔpH The change in pH from time t = 0 to time t → ∞ (pH0 - pH∞)

ΔpH10 min The change in pH in 10 min from the initial pH of about 8.0

FCR Feed conversion ratio

IVDP Percent in-vitro protein digestibility

K Apparent rate constant; subscript ST isfor starch and PR is for protein

M∞ Maximum cumulative marker concentration

MRT Mean residence time in the mixing and displacement compartments gastrointestinal

tract)

pH0 pH of protein digesta at time t = 0

pH∞ pH of protein digesta at infinite time, t  ∞

pHt pH of protein digesta at time t

r2 Coefficient of determination (r = correlation coefficient)

Sgw Geometric standard deviation of particle diameter by mass

σMRT Standard deviation of the mean residence time

τ First appearance of marker/Residence time of marker due to displacement flow or

compartment (intestines)

Г Residence time of marker in the mixing compartment (stomach)

WAI Water absorption index

WSI Water solubility index

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Chapter 1

Introduction

Livestock production plays an important role in national economies as it provides income and employment for farmers and others (Sundrum, 2001) About nine billion live animals were produced globally in 2013, and due to an increasing demand for meat, global pig production grew from approximately 873 million heads in 2003 to 977 million heads in 2013 (FAOSTAT, 2015) In 2013, over 113 million tonnes of pork were produced globally, and Australia produced 0.4 MT tonnes of pork, which is about 8% of the total national meat production, to rank eighth in the world for pork production (FAOSTAT, 2015) Pork is the most widely consumed source of animal protein,

accounting for about 40% of the world’s meat consumption (Taylor et al., 2006)

Pig production begins on the farm where they are fed mash and/or pellet diets (Petrus et al., 2011)

Similar to humans, pigs have a relatively simple digestive system, and require nutrition for normal body activities including growth Feeds are recommended to supply a balanced diet, which contains not only energy, but also other nutrients such as protein, minerals and vitamins in optimum ratios Numerous ingredients are used in pig diets to supply these nutrients Energy is supplied mostly by carbohydrates, mainly through cereal grains (e.g sorghum, wheat, barley, and triticale), while protein

is commonly sourced from animal (fish meal, meat meal and blood meal) or plant (legume/pulse, and

oil seed meal) products (McDonald et al., 2002) Notable legumes/pulses that are used in pig feeds

include soybean, alfalfa, beans, peanuts, chickpeas, clover, lentils, and field peas

Field peas (Pisum sativum L.) is an annual crop, which has been primarily used for human food but

nowadays all parts of field pea plants are useful for livestock production; its roots enrich soil fertility,

and its grains are used as feed for monogastric animals and forage for ruminants Sorghum (Sorghum bicolor) is a summer crop, and the third most important cereal in Australia, in terms of production behind wheat and barley (Mahasukhonthachat et al., 2010a) Sorghum is essentially used for animal

feed in Australia, particularly in Queensland About 2.2 million MT of sorghum and 0.2 million MT

of field peas were produced in Australia in 2013 (FAOSTAT, 2015)

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In order to optimise digestibility from feed types, producers are concerned not only with the composition and nutrients of the feed, but also how it is processed Animal feeds are supplied in either mash or pellet form, with the latter being the product of heat-moisture treatments including conditioning and pelleting or extrusion It is reported that animal performance increased when pellet diets were fed to pigs, possibly because of the beneficial effects of heat treatment on destructuring

macromolecules and inactivating anti-nutrients, amongst others (Chang et al., 1987; Hansen et al., 1987; Wondra et al., 1995a; Medel et al., 2004; Millet et al., 2012) However, there are cost

implications in the production of pellet diets, and pellet diets are mainly used by large scale commercial piggeries Many small- and medium-scale pig farms in Australia prefer mash diets for ease of production and availability

Irrespective of the diet form, upon grain receival, milling is the first operation in feed production or manufacture, and the main consequence of milling is to reduce particle size Grain particle size has been demonstrated to significantly influence digestion, functional properties, feed efficiency, and

feed cost per kg of weight gain In-vitro and in-vivo studies on particle size of pig feed grains generally

revealed an inverse relationship between particle size, digestibility and animal performance (Choct

et al., 2004; Al-Rabadi et al., 2009; Mahasukhonthachat et al., 2010a; Tinus et al., 2012; I'Anson et al., 2012; Montoya and Leterme, 2011) Some studies have also revealed mill differences For

example, hammer-milled grains produced greater digestibility in finisher pigs fed corn-soybean based diets than the same diets from roller-milling, but the influence on growth rate was minimal (Wondra

et al., 1995a) The influence of mill type would, however, appear to be dependent on grains, because Laurinen et al (2000) did not measure a mill effect on the digestibility of dry matter and gross energy

of barley or wheat based diets There are economic considerations in grinding grains to a small particle size, and some studies showed minimal or reduced changes in digestibility, functional

properties and animal performance below an optimum particle size (Ayles et al., 1996; Lawrence et al., 2003; Morel and Cottam, 2007) Moreover, fine particles can increase wastages, release viscosity-forming components (like β-glucans) from the cell wall to adversely affect gastrointestinal residence time, and may promote erosion of gastrointestinal epithelium layers, which are detrimental to animal

gut health and performance (Ayles et al., 1996; Morel and Cottam, 2007; Sopade and Gidley, 2009; Millet et al., 2012) It is also observed that diets containing small particle sizes (< 400 μm) mostly

led to gastric ulceration that could culminate in pig death (Flatlandsmo and Slagsvol, 1971; Wondra

et al., 1995a;Morel and Cottam, 2007)

Beside the average particle size, the size distribution of milled grains is also important in digestion and animal performance Particle size distributions are influenced by the effective milling force of a

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mill, and/or the nature (roughness, clearance and restriction) of the reaction zone in the mill as well

as grain fracturability (Laurinen et al., 2000; Sopade et al., 2011) Theoretically, it is possible to mill

grains such that they have the same average particle size but different particle size distribution, and therefore associating measured responses to only the average particle size might not be correct Most

previous studies have concentrated on the effects of average particle size (Choct et al., 2004; I'Anson

et al., 2012; Montoya and Leterme, 2011), but there are few reports on how differences in particle

size distribution of test diets affect digestibility, functional properties and animal performance In

their studies on narrow and broad particle size distributions, Wondra et al (1995a) reported the former

to enhance animal performance More studies are, therefore, required to investigate the effects of particle size and particle size distribution on digestibility, functional properties and pig performance Such studies would assist in establishing optimum particle size characteristics for cereals and pulses

in pig diets

In this thesis, the effects on in-vitro and in-vivo digestion of particle size for sorghum and field pea

as examples of pig feed grain and legume components are studied:

Chapter 2 reviews the literature on how particle size and size distribution of these grains influence grain properties and animal performance This Chapter explains the knowledge base, and highlights the knowledge gaps that are addressed in this thesis

Chapter 3 reports on the evaluation of a manual sieving device, and the suitability of the device for particle size measurements and characterisations The Chapter discusses particle size profiles of pig feed mills in Australia and Vietnam, explains the variability and provide recommendations for grain milling for pig feeds

Chapter 4 discusses the dependence of grain hydration properties and in-vitro protein and starch

digestion on particle size parameters of field pea grain milled by disc-, hammer- and roller-mill under laboratory conditions

Chapter 5 reports on in-vitro digestion of field pea that was disc- and hammer-milled under

commercial conditions to replicate practical situations The responses of weaner pigs to diets containing these milled field peas were also discussed in the Chapter, which concludes by highlighting how pig feed ingredients affected apparent enzyme diffusion into grain particles during starch and protein digestions

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Using sorghum as an example of a carbohydrate source, Chapter 6 discusses similar issues as Chapter

5

Chapter 7 reports the relationship between time and concentration of an indigestible marker that was added to milled field pea and sorghum diets Using ileal cannulated grower pigs, this Chapter shows residence time distributions in the gastrointestinal tract (GIT) and model these with a compartmental approach to understand flow profiles and extent of mixing in the GIT The Chapter discusses the dependence of residence time distribution parameters on diet treatments

Chapter 8 explains ileal starch digestibility of the field pea and sorghum diets that were used in Chapter 7 The Chapter discusses changes in ileal starch digestibility on diet treatments Using novel microscopy techniques, the Chapter examines starch status and particle size changes during passage through the GIT up to terminal ileum of cannulated grower pigs

Chapter 9 provides an overview of the overall results obtained with recommendations and conclusions for the body of knowledge in the field of the thesis

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Chapter 2

Literature review

Pig feeds are manufactured from grains and other components to supply the required nutrients for growth Feed components supply carbohydrates and proteins, amongst other nutrients, and generally, while cereals are used as an energy (carbohydrate) source, proteins are usually supplied by the leguminous fractions of the feeds In Australia, field pea is a notable legume and sorghum is an important cereal for pig feeds

This Chapter reviews literature on field pea and sorghum as protein and energy sources in pig feeds, milling as an important unit operation in feed processing/manufacture, the influence of particle size and particle size distribution in pig nutrition, various approaches to assess starch and protein digestion, and different indices of animal performance The Chapter highlights previous research done on some of the issues above, and helps to identify knowledge gaps that could advance the understanding of the relationship between particle size characteristics of grains and optimum animal performance

2.1 Field pea

Field pea (Pisum sativum L.) belongs to the Leguminosae family (Genus: Pisum, subfamily: Faboideae tribe: Fabeae), and has an important ecological advantage as it contributes to the

development of low-input farming systems by fixing symbiotic nitrogen in both natural and

agricultural ecologies (Jing et al., 2012) Ten major field peas producing countries are Canada, China,

Russia, United States of America, India, France,Ethiopia, Ukraine, Australia, and Spain Data from FAOSTAT (2015) shows that from 2009 - 2013, Canada was the biggest producer, with about twice the production of Russia (Figure 2.1) The world production of field pea is rapidly increasing and a

greater quantity of this legume plant is now available for animal feeding (Rodrigues et al., 2012)

About 6 million hectares of agricultural land are used for pea cultivation with an average yield of about 2 MT/ha (FAOSTAT, 2015) Field pea became the first legume/pulse crop to be grown in Australia during the 1980s (Siddique and Sykes, 1997), however in 2013 field pea production made

up only 11% of the total legumes/pulses produced in Australia (Figure 2.2)

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Figure 2.1 Top field pea producing countries and Australia from 2009 - 2013 (Source:

FAOSTAT (2015))

Figure 2.2 Production of legumes/pulses in Australia in 2013 (Source: FAOSTAT (2015))

Field pea is primarily used as a human food, but is also used widely as in animal feed because the seed contains mostly starch and protein It is especially rich in lysine, an essential amino acid, which

is normally deficient in cereal grains (Mihailović et al., 2005)

0 500 1000

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2.1.1 Nutritional value

Field pea (Pisum sativum) is mainly harvested for seed, and has the potential to be a seed for oil

extraction, while it is also consumed in immature form as a vegetable crop (FAO, 1994; Khodapanahi

et al., 2012) Field pea is an excellent source of protein (especially rich in the essential amino acids

tryptophan and lysine), carbohydrates, fibre (soluble and insoluble), and provide many essential vitamins and minerals Its high nutritive value has been associated with a rich source of biologically active components that may bring beneficial health-promoting properties such as managing high

cholesterol and type-2 diabetes, and in cancer prevention (Roy et al., 2010) Peas are one of the

earliest crops to be domesticated, are referred to as dry legume seeds and can be up to two times higher in protein than cereals (D'Mello and Devendra, 1995) The mature seed of pea essentially consists of the coat and the grain/kernel (major component) The kernel is rich in starch (450 g/kg DM) and protein (250 g/kg DM), and has smaller amounts of ash, lipid, fibre, and low molecular

weight carbohydrates that include glucose, fructose, sucrose and oligosaccharides (Castell et al.,

1996)

2.1.1.1 Protein and amino acids

The crude protein content of field pea varies from 21.2 - 27.8%, and it is greatly affected by

environmental conditions, agronomic practices and genotype (Urbano et al., 2005) Like other

legumes, field pea generally has high protein content and satisfactory amino acid composition

(Gueguen, 1983) Bastianelli et al (1998) reported that different varieties of peas provided different

amounts of crude protein; garden peas, coloured peas and wrinkled peas averaging 255, 276 and 281

g/kg DM, respectively According to Zdunczyk et al (1997), the amino acid composition (both

essential and non-essential amino acids) is similar among different field peas, and due to similarities

in the amino acid ratios of legumes, each of the legumes can be used in animal feeding, supplementing and replacing each other Due to its low sulphur amino acids (methionine and cysteine) and marginal tryptophan, field pea shows low digestibility for those three amino acids For that reason, it has been

recommended to add synthetic methionine and tryptophan to high field pea-containing diets (Stein et al., 2004) Compared to common cereal grains, for example sorghum, field pea is rich in dietary

protein and total digestible nutrients (Table 2.1) With respect to amino acid composition, field pea protein contains high amounts of lysine (6.84 g/16gN) and can be used as a protein and lysine supplement in cereal-based diets, which contain sufficient methionine and cysteine but lack lysine

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(Zdunczyk et al., 1997) Therefore, field pea can be mixed with cereals such as sorghum to increase

the feeding value of animal diets

Table 2.1 Chemical composition of field peas in comparison to cereal grains

Source: Stein et al (2006), Vasan et al (2008), Awadalkareem et al (2008), Pozdisek et al (2011), and Etuk et al (2012)

The storage proteins in either cereals or legumes/pulses, include four different fractions according to

the Osborne (Osborne et al., 1914) fractionation: water-soluble albumins, salt-soluble globulins,

alcohol-soluble prolamins and acid/alkaline soluble glutelins The main components of pea proteins are globulins and albumins, while prolamins and glutelins are detected in very small amounts

(Adebowale et al., 2007; Genovese and Lajolo, 1996; Martinez-Villaluenga et al., 2008) When pea

proteins were fractionated based on their solubility, the proportions of albumins, globulins, and insoluble proteins based on total seed protein in different peas varieties ranged from 15 - 23, 48 - 59

and 24 - 29% respectively (Park et al., 2010) The two major components, globulins and albumins

are storage proteins, which are used as nitrogen sources for the new embryos after seed germination

(Tzitzikas et al., 2006) The globulins have been subdivided into two major groups based on their sedimentation coefficients: the 11S legumin and the 7S vicilin (Kimura et al., 2008)

2.1.1.2 Carbohydrates

Starch, which is a major component of pea, generally accounts for up to 50% of seed dry matter,

comparable to lupins, faba beans and soybean meals (Gunawardena et al., 2010; Jezierny et al., 2011)

The total carbohydrate content and starch characteristics of field peas are similar to other legumes/pulses (Table 2.2) The digestibility of starch is generally low compared to that of cereals due to the more robust tissue structure, the presence of various anti-nutritional factors (polyphenols and phytic acid), pronounced starch-protein interaction and a relatively high ratio of amylose/amylopectin (Sandhu and Lim, 2008) The total digestible nutrients of field pea are high due

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to its high soluble carbohydrates and low crude fibre, which is about 6.8 and 1.4% in whole seed and

dehulled seed respectively (Vose et al., 1976)

Table 2.2 Total content and characteristics of carbohydrate of some legume/pulse species

Total carbohydrate (%)

Starch (%)

Amylose (%)

Starch granule Unique

characteristic Beans 54.6 - 76.8 31.8 - 45.3 19.9 - 29.6 Usually oval, but

sometimes elliptical, 10 - 42

µm

High resistance under shear

Chickpeas 52.4 - 70.9 33.1 - 43.9 20.5 - 29.2 Oval shaped,

sized 20 - 35 µm

High swelling content Field pea 56.7 - 74.0 41.6 - 49.0 20.7 - 33.7 Oval or

spherical, large and small, 5 - 30

µm

Develop viscosity slowly, wide variety of viscosities Lentils 61.3 - 67.1 41.5 - 48.5 22.5 - 28.3 Ellipsoid,

10 - 30 µm

High water binding capacity

Source: Hoover and Sosulski (1985), Chavan and Kadam (1989) and Ratnayake et al (2002).

2.1.2.3 Anti-nutritional factors

Anti-nutritional factors, which limit utilisation of field pea as a feed, decrease its nutritive value and

can cause health problems (when taken in large amounts) in both humans and animals (Mikić et al.,

2009) Most legume grains contain considerable amounts of these anti-nutritional factors (protease inhibitors, lectins, polyphenols, flatulence factors, saponins, antihistamines, and allergens), but the

main ones are protease inhibitors (Gupta et al., 1958; Kakade et al., 1974; Adamidou et al., 2011)

Protease inhibitors belong to two major classes, the Kunitz trypsin inhibitors, which are mainly present in soybeans, and the Bowman-Birk trypsin/chymotrypsin inhibitors, which occur widely in

grain legumes (Jain et al., 2009) Protease inhibitors strongly inhibit the activity of trypsin and

chymotrypsin, and consequently reduce the digestive efficiency of the enzymes This leads to the protein in the feed not being effectively digested, which reduces the amount of amino acids absorbed (Pisulewska and Pisulewski, 2000) Trypsin inhibitor activity increases as seed maturation progresses However, most protease inhibitors in legumes can be destroyed by heat (boiling, microwave, autoclave, and roasting) and other physical treatments like soaking, fermentation and micronisation (Khattab and Arntfield, 2009) Trypsin inhibitor is quantified according to the trypsin inhibitor activity (TIA), which is expressed by trypsin inhibitor unit (TIU) per dry matter On this basis, pea

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varieties are classified into four groups; very low activity (2 - 4 TIU/mg DM), low activity (4 - 7 TIU/mg DM), medium activity (7 - 10 TIU/mg DM) and fairly high activity (10 - 13 TIU/mg DM) However, agronomic studies have produced new field pea varieties, with low or very low trypsin inhibitor activity to respond to the demands of animal husbandry Consequently, pigs can tolerate these varieties well, and the palatability of the resulting diets is comparable to diets from corn and

soybean meals (Stein et al., 2006)

2.1.2 Field peas in pig feeds

Field pea has been used in food and feed for centuries, and can meet the nutrient requirements of the

pig industry (Leguen et al., 1995; Grosjean et al., 1998; Stein et al., 2006) For feeding to pigs, only peas harvested at maturity are used (Stein et al., 2004) In comparison with soybeans, field peas

contain less anti-nutritional factors so they can be fed directly to pigs instead of needing to be treated

prior to feeding (Stein et al., 2010) However, feed processing techniques such as pelleting, extrusion,

flaking, and expansion, are heat-moisture treatments, and they further reduce trypsin inhibitors in peas-containing diets

The chemical composition and protein profile of field pea are intermediate between corn and soybean meals The digestibility of most nutrients in field pea is similar to that in soybean meals, and the concentration of digestible energy in field pea is similar to that in corn Thus, field pea can replace 100% of the soybean meals in rations fed to grower and finisher pigs without any negative effects on

the growth performance, carcass composition, carcass quality, or pork palatability (Stein et al., 2006)

Lower carcass drip losses and a more desirable colour of the longissimus muscle have been reported for pigs fed diets containing field pea, with no substantial effects on other carcass characteristics Likewise, the palatability of pork chops and ground pork patties is not changed by the inclusion of

field pea in the diets (Stein et al., 2006) While field peas can be a viable source of both energy and

protein, cereal grains are cheaper and therefore the major energy source in both pig and poultry diets Cereal grains are an excellent source of carbohydrates (starch), palatable, and can be highly digestible

(Pozdisek et al., 2011) A cereal of interest in this thesis is sorghum

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2.2 Sorghum

Sorghum (Sorghum bicolor L.) is the fifth most important cereal in the world, following maize, rice,

wheat, and barley (FAOSTAT, 2015) In 2013, Australia produced 2.2 million MT of sorghum, which was mainly grown in Queensland and Northern New South Wales, ranking third after wheat and barley Sorghum tolerates heat and drought better than other cereals This character makes sorghum

a rotation crop of preference in the tropical and subtropical areas which receive marginal rainfall

(Nyachoti et al., 1997) There are many varieties of sorghum with different physical and chemical

compositions The colour of the seed coat of grains also varies widely with white, cream, brown, and red being the most common (Boren and Waniska, 1992) Besides being a staple food, sorghum is also

used in animal feeds as a good source of carbohydrates (Wedad et al., 2008)

2.2.1 Nutritional value

Sorghum kernels have three parts, the pericarp layer, the germ or embryo, and the endosperm or storage tissue In general, these represent 5, 8 and 87% of the whole kernel weight respectively (Haikerwal and Mathieso, 1971) Some varieties have a thin layer underneath the pericarp called the testa This layer may contain tannins, phenolic compounds similar to those in fruits and red wine The grain colour is determined by the colour and thickness of the pericarp, the endosperm, and the

presence or absence of testa (Sedghi et al., 2012) Sedghi et al., (2012) reported that the sorghum

crop may be divided into three main varieties, depending on genotypes and tannin contents Type I sorghum does not have a pigmented testa and is tannin free, type II sorghum has a pigmented testa that contains condensed tannins and type III sorghum contains tannins both in the testa and pericarp

The nutrient composition of sorghum is summarised in Table 2.1, and its in-vitro digestibility is

estimated to be 65 - 75% for protein and 70 - 84% for starch while it is 66 - 86% digestible by animals

(Wong et al., 2009; Mariscal-Landín et al., 2010; Selle et al., 2010; Etuk et al., 2012; Giuberti et al.,

2012a; 2012b)

2.2.1.1 Protein and amino acids

Most sorghum varieties contain a higher level of protein than corn (9 - 15%) with similar essential

amino acids (Douglas et al., 1990) The major storage proteins in sorghum are kafirins, which are aqueous alcohol-soluble prolamins (Belton et al., 2006) Using different extraction procedures, Hamaker et al (1995) reported that kafirins accounted for 68 - 73% of whole sorghum protein, and

h77 - 82% of the endosperm proteins Kafirins have been further classified into alpha (α)-, beta (β)-,

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and gamma (γ)-kafirins based on molecular weight, extractability and structure (Liu et al., 2013; Shull

et al., 1991) Alpha-kafirin reportedly constitutes about 80% of the total kafirins, is located in the

interior of the protein bodies and considered the principal storage protein of sorghum, followed by β-

and γ-kafirins, which comprise about 5% and 15% respectively (Shull et al., 1992; Mazhar and Chandrashekar, 1993; Belton et al., 2006) Kafirins have relatively poor digestibility, probably due

to their low solubility and location in the protein bodies (Oria et al., 1995; Shull et al., 1992) Beta-

and γ-kafirin are found mostly on the outside of the protein bodies, and are reported to be the first

proteins to be digested, followed by α-kafirin (Shull et al., 1992; Mazhar and Chandrashekar, 1993)

As a cereal, sorghum protein is limited in lysine, but rich in methionine and cysteine amino acids

(Awadalkareem et al., 2008) Although sorghum grain provides dietary carbohydrates and protein,

diet containing a high percentage of sorghum is often marginal or low in protein content and of limited

biological protein quality (Duodu et al., 2003; Awadalkareem et al., 2008) Protein digestibility in sorghum is poor (Duodu et al., 2003) due to exogenous factors (e.g grain structure, polyphenols,

phytate, starch, and starch polysaccharides) and endogenous factors (e.g disulphide and disulphide crosslinking, and kafirin hydrophobicity) Heat treatments that are essentially low

non-moisture improve sorghum (protein) digestibility (Mahasukhonthachat et al., 2010a) However, high moisture heat treatments as in cooking are detrimental to sorghum protein digestibility (Duodu et al., 2002; Correia et al., 2010) As a cereal, sorghum is more important for its carbohydrate content

et al., 2000)

Giuberti et al (2012a; 2012b) investigated in-vitro starch digestibility of various cereals grains, and

found that sorghum had the highest resistant starch (275 g/kg DM), lowest potential digestibility of starch (70.4 g/100 g dry starch), and slowest rate of starch digestion (0.018/min.), which is the same

as high amylose maize (0.017/min.), and the lowest predicted glycaemic index This is associated

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with sorghum starch granules being enveloped in a thick layer of protein matrix (Black, 1999) Furthermore, the protein matrix surrounding the starch granules contains a high level of γ-kafirin with many disulphide bonds which is resistant to enzymatic digestion However, the most common difficulty of feeding sorghum is the presence of anti-nutritional factors in the grains

2.2.1.3 Anti-nutritional factors

The most important anti-nutritional factor in sorghum grain is tannins, a water-soluble polyphenolic

compound located in the grain with a pigmented testa (Duodu et al., 2003; Dykes and Rooney, 2005; Etuk et al., 2012) Although tannins are known to protect the grain against insects, birds and fungi, this agronomic benefit goes with nutritional disadvantages and reduces the grain quality (Duodu et al., 2003) Boren and Waniska (1992) reported that seed coat colour could relate to tannin content

and, hence, the nutritional quality of the grain Sorghum that is light in seed coat colour could be low

in tannins, and in turn high in nutritional value than those that are dark in colour Tannins affect the feed value of sorghum, and adversely affect its metabolisable energy and protein utilisation (Boren

and Waniska, 1992; Nyachoti et al., 1997; Black et al., 2005) Butler et al (1984) reported that

sorghum tannins (2 - 4%) can bind and precipitate at least 12 times its own weight of protein, and it was thought that the tannin-protein complex in sorghum involves hydrogen bonding and non-polar hydrophobic associations Sorghum proteins differ in their affinity for tannins, as electrophoresis shows that proteins which bind strongly to sorghum tannins are relatively large, of loose characteristics and rich in prolines such as albumins, globulins and prolamins It has also been suggested that tannins in sorghum reduce α-amylase activity, and adversely affect starch digestion

(Duodu et al., 2003; Etuk et al., 2012)

Sorghum also contains relatively high concentrations of phytate or phytic acid, another nutritional factor, which is naturally present in feedstuffs of plant origin as phytate (0.3 - 1%) (Elkhalil

anti-et al., 2001; Osman, 2004; Badigannavar et al., 2015) Phytic acid can bind positively charged

molecules in the diet and in endogenous gastrointestinal tract secretions such as digestive enzymes and mucins to form insoluble complexes leading to the reduction of nutrient digestibility and increasing endogenous secretions (Woyengo and Nyachoti, 2013)

However, various treatments have been reported to reduce anti-nutritional factors in sorghum and increase its nutritional value of sorghum For example, ammoniation was found to completely remove tannins, and soaking in alkalis and salt solutions was also found to be effective (Mulimani and

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Supriya, 1994) Schons et al (2012) found that enzymes tannase and phytase could be added to

sorghum diets for pigs to reduce tannins and phytic acid

2.2.2 Sorghum in pig feeds

About 48% of global sorghum grain production is used for animal feeding (Dowling et al., 2000) Paulka et al (2015) fed pellet diets from roller-milled sorghum of different particle sizes (724, 573

and 319 μm) to grower-finisher pigs, and found pig growth performance or carcass characteristics to

be comparable to pigs fed pellet diets from corn As a by-product of ethanol production, sorghum distiller dried grains with solubles (DDGS) is an ingredient that is commonly used in pig diets to

reduce feed cost (Cerisuelo et al., 2012; Sotak et al., 2014; Sotak et al., 2015) Depending on the

source, DDGS consists of 89 - 90% dry matter, 29 - 30% protein, 7 - 9% fat, and 4 - 5% fibre

(Cerisuelo et al., 2012; Sotak et al., 2015) While feeding150 g/kg sorghum DDGS in diet caused a reduction in voluntary feed intake of nursery pigs, a level of 300 g/kg or 350 g/kg of sorghum DDGS

in grower-finisher pig diets resulted in no negative influence on growth performance and carcass

yield, but increased back fat thickness (Cerisuelo et al., 2012) However, in weaner pigs, ileal

apparent digestibility of protein and amino acids was observed to be lower than in grower-finisher

pigs and linearly decreased as the amount of sorghum in the diet increased (Mariscal-Landín et al.,

2010) As with other feed grains and ingredients, sorghum is physically processed by milling to

change or improve its feeding value (Kim et al., 2000)

2.3 Particle size in pig feeds

Milling is a unit operation in which particle size is reduced, and because of differences in fracturability of a material, for example grains, a particle size distribution is normally obtained after

milling (Tinus et al., 2012) According to Sopade et al (2011), there are many types of mills, which

include hammer-, roller-, disc-, ball-, pin, and cryo- or freezer-mills Among these mills, disc-, hammer- and roller-mills are the most commonly used in feed processing or manufacturing, as

revealed in a survey (Sopade et al., 2011) of feed mills in Queensland, Australia (Figure 2.3)

In reducing particle size of materials, the internal components are exposed and the surface area for

enzyme-substrate interactions, amongst other effects, is increased (Sopade et al., 2011) The effective

milling forces of these mills are different depending on impact, compressive and shearing or attrition force (Figure 2.4) Also, because of process economics and issues (e.g cost, throughput, mode of

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operation, and automation), some mills are better suited for commercial operations, where tonnes of

grains are milled daily (Sopade et al., 2011)

Figure 2.3 Diagram of the distribution of the milling techniques obtained from a feed-mill

survey (Source: Sopade et al (2011))

Figure 2.4 Typical disc-, roller- and hammer-mills with the main milling forces (Source:

Sopade et al (2011))

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