novel uses of lignin and hemicellulosic sugars from acidhydrolysed

8/19/2019 Novel Uses of Lignin and Hemicellulosic Sugars From Acidhydrolysed

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NOVEL USES OF LIGNIN AND HEMICELLULOSIC SUGARS FROM ACID-

HYDROLYSED LIGNOCELLULOSIC MATERIALS

by

Mojtaba Zahedifar

(B.Sc., Animal Husbandry - University of Tehran)

(M.Sc., Animal Nutrition - University of Gödölló)

Thesis submitted for the degree of Doctor of Philosophy

in the University of Aberdeen

September 1996


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DECLARATION

I hereby declare that this thesis has been composed by myself and has not been presented

or accepted in any previous application for a degree. The work has been done by myself

and all help given has been acknowledged and sources of information has been specially

acknowledged by means of references.

Mojtaba Zahedifar

September 1996


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DEDICATION

To my wife


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ABBREVIATIONS

ADF acid detergent fibre

BSA bovine serum albumin

CD carpinus duinensis

CrI crystallinity index

DM dry matter

EA ethyl acetate

EAEP ethyl acetate extractable phenolics

ETOH ethanol

FSG functional specific gravity

GLC gas liquid chromatography

H hydrogen

HMF hydroxymethyl furfural

HPLC high performance liquid chromatography

HT heating up timeLM lignocellulosic materials

MW molecular weight

N nitrogen

NDF neutral detergent fibre

PEG polyethylene glycol

PVP polyvinylpolypyrrolidone

RT reaction time

SUG sugar

TEP total extractable phenolics

TT total tannins

VFA volatile fatty acid

WIP water insoluble phenolics

WSE water soluble extract


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ACKNOWLEDGMENT

I am extremely grateful to my supervisor Dr. E.R. Ørskov at The Rowett

Research Institute for his continuous encouragement, comments and advice during my

studies at the Rowett. I should also thank my university supervisor Dr. H. Galbraith for

his valuable comments during the course of this study.

I would like to express my gratitude to Dr. F.B. Castro for his invaluable

comments on my studies and on the manuscript of this thesis.

I am grateful to Dr. R.N.B. Kay for his useful comments on the manuscript of this

thesis.

I would like to thank the Director of The Rowett Research Institute, Professor

W.P.T. James for the provision of the facilities at the Rowett and through him all the

Rowett Research Institute who have helped me in one way or another.

My very appreciation to Drs R. Begbie, G.J. Proven, J.A. Lomax and T.M. Wood

for their valuable comments and discussion during the course of this work.

I am grateful to all the staff from the International Feed Resources Unit with

special reference to Mr D.J. Kyle, Mr W.J. Shand and Mrs A. Marsden, who have

always been helpful and kind. I must also thank Dr X.B. Chen for his valuable

comments throughout this study.

Many thanks to the members of the Analytical Department of The Rowett

Research Institute specially Mr P.J.S. Dewey, Mr D.S. Brown and Miss M.G. Annand

who have helped me in the chemical analysis.


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My great appreciation to the Ministry of Jihad Sazandegi and the Ministry of

Culture and Higher Education of The Islamic Republic of Iran for their financial support

throughout this study.

Finally, I wish to express my sincere gratitude to my wife and my children

Mohsen, Hamed and Sara for their constant encouragement and love.


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SUMMARY

Lignocellulosic materials (LM) are an ever present renewable and available

energy source. The energy stored by photosynthesis in the form of vegetation is about

ten times more than world's annual energy consumption (Zsuffa, 1982). This source is

the only alternative for chemical production after fossil fuels.

Formation of organic acids (mainly acetic acid) from hemicellulose during steam

treatment of LM leads to acid hydrolysis of cell wall components. Solubilization of

hemicellulose and depolymerization of lignin are the most important changes that occur

during the process.

During hydrolysis of LM appreciable amounts of sugar degradation products,

organic acids and phenolics are produced. Inhibitory effects of the compounds on yeast

during alcoholic fermentation have been reported and several methods have been

proposed to overcome the problem. Among the new compounds phenolics derived from

lignin depolymerization have received most attention. Another problem during enzymic

saccharification of cellulose is partial inactivation of cell free enzymes.

The above mentioned constraints were investigated in this study.

Dry matter (DM) loss especially under harsh treatment conditions may be

regarded as a form of nutrient loss. Results of this study showed that greater nutrient loss

could occur through the formation of indigestible soluble and insoluble browning

reaction compounds. In vitro digestion of water soluble extract showed that only

carbohydrates in this fraction were utilized by rumen microbes. The water soluble


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extract from steam-treated LM has been used for some biological purposes due to being

rich in soluble carbohydrates. This fraction can also be used as animal feed for both

ruminants and those monogastric animals which can ferment them in the hind gut to

produce volatile fatty acids (VFA). The highest sugar content (57%) of water soluble

fraction was obtained at 19 bar pressure and 0 min reaction time. At this treatment

condition only about 8% of soluble carbohydrates were in the form of monomeric

pentoses. Increase in harshness (pressure, reaction time and/or exogenous acid) of

treatment conditions negatively affected carbohydrate content of water extract.

Concentration of browning compounds in steam-treated LM is rather high if steam

treatment exceeds optimum conditions. The nitrogen present in steam-treated LM is in

the form of Maillard products. These compounds should be considered as nutrient loss

as they are also indigestible.

The antinutritive materials produced during steam treatment of LM are classified

mainly as lignin-based phenolics and browning reaction compounds.

The study of the inhibitory effect of browning compounds showed that these

compounds were not toxic to rumen microbes. Furfural and hydroxymethyl furfural with

known toxic effect on yeasts, did not affect rumen microbes. Production of these

compounds should be considered only as nutrient loss.

The detailed study of the effect of lignin-based phenolics on rumen microbes un-

der different conditions showed that neither low molecular weight nor high molecular

weight phenolic derived from lignin depolymerization affected ruminal fermentation.

The concentrations of tannin-like compounds in steam-treated wheat straw were too low


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(maximum 0.97% as tannic acid equivalent) to affect rumen microbes. Measurement of

the total monomeric phenolic compounds with a known negative effect on rumen

microbes also showed that their concentrations were far too low to affect microbial

activity. Alkali-extractable lignin did not show any effect on rumen microbes either. In

general it was shown in this study that no material toxic to rumen microbes is produced

during steam treatment of wheat straw.

Steam treatment has been used as an approach to increase enzymic hydrolysis of

cell wall polysaccharides. The advantages of steam treatment in this respect arise from

physical (cell wall swelling and reduction in particle size) and chemical (hemicellulose

and lignin depolymerization) changes which increase the availability of cell wall

polysaccharides to enzymic attack. Reduction in enzymic activity of cell-free enzymes

during saccharification of steam-treated LM has been considered as a constraint as more

enzymes have to be used. Measurement of the enzymic activity of xylanase and cellulase

mixed with lignin extracted from steam-treated wheat straw showed that lignin was

responsible for inactivation of enzymes. Both tannin and lignin were more potent in

precipitating protein (bovine serum albumin - BSA) than in deactivating enzymes.

A study of the lignins extracted from different LM sources (wheat straw, barley

straw, rice straw and sugar cane bagasse) showed that both treatment conditions

(pressure, reaction time and exogenous acid) and LM source affected the protein-

precipitating capacity of lignin. Very harsh treatment conditions (using high pressure

and a long reaction time in presence of exogenous acid) did not improve lignin quality.

Autohydrolysis at 19 bar pressure and 0 min reaction time was found to be more suitable


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for lignin production.

In vitro degradation of protein mixed with lignin showed that lignin, like tannin,

was able to protect the protein from ruminal fermentation. It was also shown that the

protein adsorbed to lignin was released at low pH (≈ 2.2). The similarities between

tannin and lignin suggest the presence of hydrogen bonds and hydrophobic interactions

between lignin and protein since both types of the reactions are responsible for

precipitation of proteins by tannins. The results suggest that, like tannins, lignin could be

used as a protein protective material. In this respect lignin may also be used for the

chemical protection of proteins. An advantage of lignin is that it does not affect normal

ruminal fermentation. However, its low activity compared to tannins is a drawback.

More research is needed to increase the protein adsorbing capacity of lignin in order to

decrease lignin:protein ratio for agricultural and industrial purposes.


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TABLE OF CONTENTS:

Page No.

CHAPTER 1 - LITERATURE REVIEW

1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Plant cell wall . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3 Cell wall components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3.1 polysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.3.1.1 cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71.3.1.2 hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91.3.1.3 pectic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3.2 proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3.3 phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

1.3.3.1 lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3.3.2 tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.3.3.3 phenolic acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.4 Acidolysis reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181.4.1 cellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191.4.3 hemicellulose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201.4.4 lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

1.5 Protein precipitating capacity of phenolic compounds . . . . . . . . . . . . . . . 27

1.6 The effect of steam treatment-associated toxic compounds onrumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

1.6.1 furfural and 5-hydroxymethyl furfural . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.6.2 phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

CHAPTER 2 - CHARACTERIZATION OF WATER-SOLUBLE EXTRACTFROM STEAM-TREATED WHEAT STRAW


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2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

Page No.

2.2 Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

2.2.2 steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352.2.3 extraction of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382.2.4 statistica analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .382.2.5 total neutral sugar analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372.2.6 dry matter loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

2.2.7 ash content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.2.8 nitrogen (N) content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.2.9 total extractable phenolics analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402.2.10 in vitro gas production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .412.2.11 browning reaction products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

2.3 Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

2.3.1 steam treatment procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 442.3.2 soluble carbohydrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

2.3.3 nutritive value of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . 51

2.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

CHAPTER 3 - IDENTIFICATION OF INHIBITORY COMPOUNDSPRODUCED DURING STEAM TREATMENT OF LIGNOCELLULOSICS

3.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

3.2 Material and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

3.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.2.2 steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.2.3 extraction of water soluble extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 613.2.4 effect of addition of PVP on microbial fermentation . . . . . . . . . . . . . . . . . 633.2.5 extraction of phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

3.2.5.1 ethyl acetate extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

3.2.5.2 extraction of water insoluble phenolics . . . . . . . . . . . . . . . . . . . . . . . . 65


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3.2.6 effect of extraction procedure by ethyl acetate onnutritive value of feed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

3.2.7 HPLC analysis of phenolic compounds of steam-treated wheatstraw samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Page No.

3.2.8 effect of ethyl acetate extractable phenolics and waterinsoluble phenolics on rumen microbes . . . . . . . . . . . . . . . . . . . . . . . . . 66

3.2.9 extraction of phenolic compounds using ethanol solutions . . . . . . . . . . . . 673.2.10 effect of EAEP and WIP at high substrate loading on

microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

3.2.11 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693.2.12 optimum level of substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693.2.13 effect of furfural and HMF on rumen microbes . . . . . . . . . . . . . . . . . . . . . 693.2.14 extractable phenolics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 703.2.15 total tannins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70


3.3.1 effect of PVP on microbial fermentation . . . . . . . . . . . . . . . . . . . . . . . . . 713.3.2 effect of EAEP and WIP on rumen microbes . . . . . . . . . . . . . . . . . . . . . . 74

3.3.3 effect of phenolic compounds from steam-treated wheatstraw on rumen microbes at high substrate loading . . . . . . . . . . . . . . . . . . 81

3.3.4 effect of ethanol extract from steam-treatedwheat straw on rumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . 83

3.3.5 effect of furfural and HMF on ruminal fermentation . . . . . . . . . . . . . . . . . . 84

3.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89

CHAPTER 4 - PROTEIN PRECIPITATING CAPACITY OF LIGNIN

EXTRACTED FROM STEAM TREATED LIGNOCELLULOSICS

4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90


4.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924.2.2 phenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924.2.3 lignin extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924.2.4 enzyme assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94

4.2.4.1 effect of lignin and tannin on xylanase inactivation . . . . . . . . . . . . . . . . . 94


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4.2.4.2 enzymic inhibition by steam-treated lignin usinggas production technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

4.2.5 effect of lignin extracted from steam-treatedwheat starw on rumen microbial activity . . . . . . . . . . . . . . . . . . . . . . . . . 96

4.2.6 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

Page No.

4.2.7 protein precipitating capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 964.2.6 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 974.2.7 reducing sugar analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98


4.3.1 inhibition of enzymes by lignin extracted fromsteam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

4.3.2 protein precipitating capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102

4.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108

CHAPTER 5 - EFFECT OF TREATMENT CONDITIONS AND SOURCE OFLIGNOCELLULOSIC MATERIALS ON PROTEIN PRECIPITATINGCAPACITY OF LIGNIN

5.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109


5.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111 5.2.2steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111

5.2.3 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1115.2.4 effect of hemicellulose on lignin quality . . . . . . . . . . . . . . . . . . . . . . . . . 1125.2.5 lignin extraction and purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125.2.6 protein precipitating capacity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125.2.7 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125.2.8 water soluble fraction of lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135.2.9 total phenolic content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135.2.10 ash content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135.2.11 nitrogen content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1135.2.12 total sugar content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114



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5.3.1 optimization of steam treatment conditions for lignin

production from wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155.3.2 lignin quality from barley straw, rice straw

and sugar cane bagasse . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185.3.3 lignin properties from different agricultural by-products . . . . . . . . . . . . . . . 1205.3.4 effect of hemicellulose removal on lignin quality . . . . . . . . . . . . . . . . . . . 124

Page No.

5.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

CHAPTER 6 - THE USE OF LIGNIN TO PROTECT PROTEIN FROMMICROBIAL DEGRADATION IN THE RUMEN

6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121


6.2.1 substrate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296.2.2 steam treatment conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296.2.3 extraction and purification of lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1296.2.4 In vitro protein and amino acid degradation . . . . . . . . . . . . . . . . . . . . . . . 1296.2.5 NH3 analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1306.2.6 VFA analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1306.2.7 statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1316.2.8 protein analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316.2.9 adsorption of amino acids by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1316.2.10 amino acid analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

6.2.11 release of adsorbed protein frompolyphenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132

6.2.12 specific gravity of lignin-protein complex . . . . . . . . . . . . . . . . . . . . . . . . 133


6.3.1 protection of protein by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1346.3.2 adsorption of amino acids by lignin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1366.3.3 effect of lignin on deamination activity . . . . . . . . . . . . . . . . . . . . . . . . . . . 1366.3.4 postruminal availability of protein adsorbed by lignin . . . . . . . . . . . . . . . . 138

6.4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142


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CHAPTER 7 - GENERAL DISCUSSION AND CONCLUSIONS

7.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .143

7.2 Constraints of steam treatment of lignocellulosic materials . . . . . . . . . . . . 144

7.2.1 nutrient loss . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1447.2.2 identification of compounds toxic to rumen microbes in

steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

Page No.

7.3 Biological activity of lignin extracted from steam-treatedlignocellulosic materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150

REFERENCES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

APPENDIXES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179


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LIST OF FIGURES

Figure No. Title Page No.

1.1.1 Model of arrangement of components in wood cell wall(lignified) (Ker and Goring, 1975) . . . . . . . . . . . . . . . . . . . . . . 2

1.1.2 Relationship between lignin content and digestibility in ryegrass (data from Hartley, 1972) . . . . . . . . . . . . . . . . . . . . . . . . . .2

1.2.1 Illustration of primary and secondary cell walls(Timell, 1964) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

1.3.1 Distribution of cell wall polysaccharides (Worth, 1967) . . . . . . . . .6

1.3.1.1.1 Proposed arrangement of D-glucan chains withincellulose microfibrills of primary cell walls of dicots(Dey andBrinson, 1984) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .8

1.3.1.1.2 Diagrammatic representation of cellulose microfibrillarstructure (Hess et al., 1975) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

1.3.1.2.1 Hypothetical structure of plant xylan(Puls and Poutanen, 1989). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.3.3.1.1 Structure of three precursors of lignin(Sarkanen and Ludwic, 1971) . . . . . . . . . . . . . . . . . . . . . . . . . . 14

1.3.3.1.2 Proposed lignin structure in softwoods (Nimz, 1974) . . . . . . . . . . 14

1.4.1.1 Production of furfural from D-xylose(Tipson and Horton, 1988) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

1.4.1.2 Production of 5-hydroxymethyl-2-furaldehyde fromD-glucose (Tipson and Horton, 1988) . . . . . . . . . . . . . . . . . . . . . 21

1.4.4.1 The susceptible bounds (α-aryl and ß-O-4) in lignin

molecule for cleavage under acidolysis reaction . . . . . . . . . . . . . . 24


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1.4.4.2 Effect of reaction time on depolymerization/repolymerizationof lignin (Wayman and Chua, 1979) . . . . . . . . . . . . . . . . . . . . . .26

Page No.

2.2.2.1a The High Pressure Steam Treatment Vessel in operation . . . . . . .36

2.2.2.1b Components of the High Pressure Vessel . . . . . . . . . . . . . . . . . .36

2.2.2.1c Putting the sample in the beaker (c) and transferringinto the vessel (d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

2.3.2.1 Effect of pressure and reaction time on solublecarbohydrate (sCHO) content of the water solubleextract from steam-treated wheat straw . . . . . . . . . . . . . . . . . . . 49

2.3.3.1 Nutritive value of the water soluble extract fromsteam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

3.2.5.1 Extraction procedure of phenolic compounds (EAEP and WIP)from steam-treated wheat straw and Carpinus duinensis

by ethyl acetate and acetone solution. . . . . . . . . . . . . . . . . . . . . 64

3.2.9.1 Extraction procedure by ethanol solution fromacid-hydrolysed (19 bar pressure and 10 min reaction time)wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .68

3.3.2.1 Efficiency of extraction by ethyl acetate of phenoliccompounds from water soluble extract of steam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

3.3.2.2 Effect of ethyl acetate extractable phenolics andand water insoluble phenolics extracted from C.duinensis on rumen microbial activity . . . . . . . . . . . . . . . . . . . .78

3.3.3.1 Effect of substrate (rye grass) loading on pH . . . . . . . . . . . . . . . 82

4.2.3 Extraction procedure of lignin from steam-treated(19 bar pressure and 0 min reaction time) wheat straw . . . . . . . . .93

4.3.1.1 lignin effect on cellulase activity . . . . . . . . . . . . . . . . . . . . . . . 101

4.3.2.1Effect of lignin on the Bradford method for determining


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protein adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .103

4.3.2.3 Protein (BSA) adsorbing capacity of 1 g of the threephenolic compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105

Page No.

5.3.1.1 Effect of steam treatment conditions on proteinprecipitating capacity of lignin extracted fromauto-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . 116

5.3.1.2 Effect of steam treatment conditions on proteinprecipitating capacity of lignin extracted fromacid-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . 116

6.3.1.1 Effect of levels of lignin extracted from steam-treatedwheat straw on degradation of casein by rumen microbesas indicated by production of NH3. . . . . . . . . . . . . . . . . . . . . . .135

6.3.3.1 Effect of lignin extracted from steam-treated

(19 bar pressure and 5 min reaction time) wheat strawon deamination activity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .139

6.3.4.1 Effect of pH on releasing of protein fromtannin (tannic acid) and lignin . . . . . . . . . . . . . . . . . . . . . . . . . 140

7.2.1 Physical and chemical changes of LM during steam treatment . . .145

7.3.1 Method for reducing protein degradation in the rumen . . . . . . . . .155


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LIST OF TABLES

Table No. Title Page No.

2.2.7.1 Composition of macro-mineral, micro-mineral and buffersolutions used for preparing artificial saliva . . . . . . . . . . . . . . . . 42

2.3.1.1 Effect of sample DM content on chemical composition of steam-treated wheat straw at 19 bar and 0 min reaction time . . . . 45

2.3.1.2 Sugar, phenolics contents and DM loss of wheat straw bysteam treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

2.3.2.1 Neutral sugar composition of total and monomericcarbohydrates in water soluble extract from steam-treatedwheat straw at different conditions . . . . . . . . . . . . . . . . . . . . . . .48

2.3.3.1 Chemical analysis of water soluble extract fromsteam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52

3.2.2.1 Steam treatment conditions applied to wheat straw . . . . . . . . . . . .62

3.3.1.1 Effect of steam treatment and addition of PVP onthe in vitro gas production after 12 and 24 h incubation . . . . . . . . 72

3.3.1.2 Total extractable phenolic and total tannin content fromauto-hydrolysed and acid-hydrolysed wheat straw . . . . . . . . . . . . .73

3.3.2.1Effect of ethyl acetate extraction procedure onsugar fermentation by rumen microbes . . . . . . . . . . . . . . . . . . . . 75

3.3.2.2 Extractable phenolic, tannin and soluble carbohydratesdata of the ethyl acetate extractable phenolics and waterinsoluble phenolics from steam-treated wheat straw andCarpinus duinensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76

3.3.2.3 Effect of ethyl acetate extractable phenolics and waterinsoluble phenolics from steam-treated wheat straw and

Carpinus duinensis on in vitro gas production . . . . . . . . . . . . . . . 79


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3.3.2.4 Concentration of monomeric phenolic compounds insteam-treated wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

Page No.

3.3.3.1 Effect of ethyl acetate extractable phenolics at highsubstrate loading on rumen microbes . . . . . . . . . . . . . . . . . . . . . . . 82

3.3.4.1 Effect of supernatants and precipitates obtained by ethanol

solutions on in vitro gas production from rye grass . . . . . . . . . . . . .85

3.3.4.2. Amount of dry matter extracted (supernatants and precipitates)from 380 g acid-hydrolysed wheat straw by ethanol extraction . . . . .85

3.3.5.1 Production of furfural and hydroxymethyl furfural fromsteam-treated wheat straw under different treatment conditions . . . . . 86

3.3.5.2 Effects of furfural and hydroxymethyl furfural ongas production in vitro from rye grass . . . . . . . . . . . . . . . . . . . . . .87

4.3.1.1 Effect of lignin and tannin on xylanase activity . . . . . . . . . . . . . . .100

4.3.1.2 Effect of different levels of lignin on rumenmicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .101

5.3.2.1 Protein precipitating capacity of lignin extracted fromauto-hydrolysed wheat straw, barley straw, rice straw andsugar canebagasse at different treatment conditions . . . . . . . . . . . . . 119

5.3.3.1 Characteristics of lignin extracted from auto-hydrolysedand acid-hydrolysed wheat straw . . . . . . . . . . . . . . . . . . . . . . . . . 121

5.3.3.2 Characteristics of lignin samples from barley straw,rice straw and sugar cane bagasse . . . . . . . . . . . . . . . . . . . . . . . . . 122

4.3.4.1 Protein precipitating capacity of lignin and quantity of lignin extraction from acid detergent fibre of wheat straw . . . . . . . . 125

6.3.1.1 Effect of levels of lignin extracted from steam-treated

wheat straw on iso-acid production. . . . . . . . . . . . . . . . . . . . . . . . 135


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6.3.1.2 Functional specific gravity of lignin-protein complexes . . . . . . . . . . 137

7.3.1 Lignosulphonate sale in Europe, 1986(Wayman and Parekh, 1992) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152


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1

CHAPTER 1

LITERATURE REVIEW

1.1 Introduction

Cellulose, hemicellulose and lignin are the main organic compounds which make

up the biomass of trees and agricultural by-products (lignocellulosic materials - LM).

LM are the most important renewable resources of the terrestrial ecosystem and have

been used for many biological and industrial purposes. From the nutritional point of

view, agricultural by-products such as cereal straws can provide only a fraction of the

daily requirement of energy by ruminant animals. The main constraint for using LM as

an animal food is its low nutritive value. Cell wall barriers observed in such materials

negatively affect the bio-degradation of both cellulose and hemicellulose.

In the mature plant cell wall polysaccharides are encrusted with lignin (Fig 1.1.1)

thereby limiting the access of enzymes (Baker, 1973) and rumen microbes (Hartley,

1972) (Fig 1.1.2) into the cell wall matrix and limiting its degradation. Another

important factor is the presence of lignin-carbohydrate complexes in the cell wall which

inhibit enzymic activity (Morrison, 1979; Neilson and Richards, 1982). Unavailability of

carbohydrates in lignin-carbohydrate complexes to rumen microorganisms has also been

reported (Wallace et al., 1991).

A possible solution to overcome such barriers is to subject the LM to specific


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treatments, e.g. chemical (Chandra and Jackson, 1971), physical (Morrison, 1983) or


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3

Fig 1.1.1 Model of arrangement of components in wood cell wall (lignified) (Ker andGoring, 1975).

Fig 1.1.2 Relationship between lignin content and digestibility in rye grass (data fromHartley, 1972).


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biological (Agosin et al., 1986; Akin et al., 1993).

Amongst the treatments that have been studied over the years steam treatment has

shown promise method for upgrading LM bio-availability. The need for specific

equipments and the high cost of setting up the processing unit have been important

constraints for its application on a farm scale. Nevertheless, steam treatment has been

widely used in sugar refineries in Brazil (Wayman and Parekh, 1990) as a method to

upgrade sugar cane bagasse for animal feed.

Steam treatment affects cell wall structure as a result of an acid-hydrolysis type of

reaction (Baugh and McCarty, 1988). Production of organic acids from acetyl and

formyl groups present in hemicellulose leads to acidolysis of cell wall components.

Solubilization of hemicellulose and depolymerization of lignin are the main results of

this reaction. The water soluble fraction which contains easily fermentable carbohydrates

(hemicellulosic sugars) could be used for many biological purposes such as alcohol and

single cell protein production or as an animal feed. Production of chemicals such as

furfural and 5-hydroxymethyl furfural is an alternative use for steam treatment (Sproul et

al., 1985). The solid fraction, rich in cellulose, can be used as a raw material for the pulp

industry, for alcohol production or as a ruminant feed.

Although steam treatment is an effective method for upgrading LM, production

of inhibitory compounds are still considered to be an important constraint. Such

compounds restrict enzymic hydrolysis as well as fermentative batch processes (Mes-

Hartree and Saddler, 1983; Sutcliffe and Saddler, 1986). Inhibition of rumen microbial


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activity has also been reported in steam-treated lignocellulosics (Britton, 1978; Castro et

al., 1995). There is no detailed report of the inhibitory compounds formed during steam

treatment of LM.

Depression of enzymic activity during hydrolysis of steam-treated LM with cell

free enzymes have also been reported (Sinitsyn et al., 1982). One possible way in which

free enzymes are inactivated is through binding to phenolic compounds. It should be

mentioned that extractable phenolic compounds detected in steam-treated LM are

formed during lignin depolymerization. It is worth noting that natural phenolic

compounds, e.g. tannins are capable of complexing with proteins and protecting them

from rumen microbial degradation.

All the effects related to inhibitory compounds formed during steam treatment

have been described as negative from the point of view of enzymic hydrolysis and

fermentation processes. Such properties can however be used to control certain

biological processes; e.g., extent of rumen protein degradation. To do so, it is essential to

identify such compounds and measure their activities in specific processes.

In the present study two insight were obtained concerning the effects of steam

treatment upon the properties of both lignin and hemicellulosic sugars. First a negative

effect which normally is detrimental to the utilization of food and second a beneficial

effect in which such products of steam treatment can be used to manipulate fermentation

and protein degradation.


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1.2 Plant cell wall

Plant cell walls consist of primary and secondary cell walls (Fig 1.2.1). Primary

walls are deposited at early stages of growth while the cells are expanding (Fry, 1987).

The middle lamella which forms a common boundary layer between adjacent cells

occupies the site of the cell plate (Timell, 1964; Fry, 1987). Contiguous cells are bound

together by deposition of lignin in the middle lamella (Ornston et al., 1987).

Secondary cell walls are formed at later stages either lignified or unlignified.

Unlignified walls have a high tensile strength and stiffness mainly due to the mechanical

properties of the cellulose microfibrils (Northcote, 1972). Lignification results in a rigid

cell wall with high compressive strength and low porosity. The highest concentration of

lignin is observed in the middle lamella and the primary wall (Saka and Goering, 1985).

The secondary wall contains most of the cell lignin as it forms the greatest volume of

cell wall (Harris, 1990).

1.3 Cell wall components

Plant cell wall is a layer of structural material involving the protoplast which can

be 0.1-10 µm thick and composed of polysaccharides (cellulose, hemicellulose and

pectin) and lignin (Fry, 1987). The presence of polysaccharides in different parts of the

cell wall is illustrated in Fig 1.3.1. Cell wall contains a small amount of glycoproteins

(Cassab and Varner, 1988).


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Fig 1.2.1 Illustration of primary and secondary cell walls (Timell, 1964)


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8

Fig 1.3.1 Distribution of cell wall polysaccharides (Worth, 1967)


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1.3.1 polysaccharides

Classically, cell wall polysaccharides have been grouped into three fractions. a)

Cellulose: the most resistant to chemical disruption. b) Hemicellulose: extracted by

relatively strong alkali solution or mild acid hydrolysis; and c) Pectic polysaccharides:

extracted by hot water, ammonium oxalate solution, weak acids or chelating agents:

(Dey and Brinson, 1984; Chesson and Forsberg, 1988).

1.3.1.1 cellulose. Cellulose is the most abundant cell wall polysaccharide in nature and

consists of long chains of ß-1,4 linked glucose residues. The chains are held together by

hydrogen bonds between oxygen of alternating glycosidic bond in one glucan chain and

the primary hydroxyl groups at position 6 of glycosyl residues in another chain

(Wolfgang et al., 1973) to form thin, flattened, rod-like structures that are referred to as

microfibrils (Fig 1.3.1.1.1) (Dey and Brinson, 1984).

The cellulose microfibrils are bound to each other and to hemicellulose polymers

by hydrogen bonding (Valnet and Albersheim, 1974) but there is no evidence of

covalent linkage between cellulose and other cell wall constituents (Morrison, 1979).

Cellulose microfibrils contains regions with highly oriented molecules or less

oriented microfibrils called crystalline and amorphous regions respectively (Cowling and

Kirk, 1976) (Fig 1.3.1.1.2). The crystallinity index of cellulose, i.e. degree of

microfibrils orientation, is highly variable and depends on the source and age of the

tissue (Harris, 1990). Because of its structure, cellulose is insoluble in most


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Fig 1.3.1.1.1 Proposed arrangement of D-glucan chains within cellulose microfibrills of

primary cell walls of dicots (Dey and Brinson, 1984).


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Fig 1.3.1.1.2 Diagrammatic representation of cellulose microfibrillar structure (Hess et al., 1975).


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solvents and has a low accessibility to aqueous acids and cell-free enzymes (Tipson and

Horton, 1988).

Several properties of cellulose can influence its susceptibility to enzymic

degradation (Cowling and Kirk, 1976), i.e capillary structure in relation to the size of

cellulases, crystallinity index, dimension of crystalline portions of the microfibrils and

the nature of substances (lignin) with which the cellulose is associated (Kirk 1983,

Shambe and Kennedy, 1984). Among different characteristics of cellulose, crystallinity

index has been reported to be the most important factor affecting cellulose digestibility

(Fan et al., 1981; Gharpuray et al., 1983).

1.3.1.2 hemicellulose. The term hemicellulose is applied to cell wall polysaccharides

which occur in close association with cellulose, especially in lignified tissues. It is often

restricted to substances extracted with alkaline reagents (Aspinal, 1959).

Hemicelluloses are built up of a relatively limited number of sugar residues (100-

200 sugar units), eg. D-xylose, D-mannose, D-glucose, D-galactose, L-arabinose, 4-O-

methyl-D-glucuronic acid, D-galacturonic acid, D-glucuronic acid and to a lesser extent,

L-rhamnose, L-fucose and various O-methylated neutral sugars (Timell, 1964).

Xylans are quantitatively the most important hemicellulose of graminaceous cell

walls (Fig 1.3.1.2.1) (Dey and Brinson, 1984). Xylan is composed of chains of 1-4

linked ß-D-xylopyranose residues. Different plants may contain the same basic xylan

structure but different arrangements with other sugar residues, especially L-arabinose,


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D-glucuronic acid and 4-methyl ether, may occur (Wilkie, 1979).


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14


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Fig 1.3.1.2.1 Hypothetical structure of plant xylan (Puls and Poutanen, 1989).


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Xylans from cereals and grasses are generally characterized by the presence of L-

arabinofuranose residues linked to the backbone as single-unit side-chains, usually to

position 3 of xylose. Similarly, D-glucuronic acid and/or 4-O-methyl-D-glucoronic acid

residues are also present in a similar proportion. The hemicellulose in wheat straw is

characterized by the xylan backbone, carrying through position 3 of D-xylose residues,

side-chains terminated by non reducing L-arabinofuranosyl-(1-3)-O-ß-D-xylopyranosyl-

(1-4)-D-xylopyranose (Aspinal, 1959).

There is evidence of the presence of xyloglucans in primary cell walls of mono

and dicotyledonous plants which account for approximately 2 and 19% of the cell wall,

respectively (Dey and Brinson, 1984). In monocotyledons, xyloglucan polymers are

hydrogen bonded to cellulose fibrils forming a non-covalent link in the network of

polymers which cross-link the cellulose fibres (Burke et al., 1974; Valnet and

Albersheim, 1974).

All xylans contain backbones composed of ß-(1-4) linked xylosyl residues. There

is a wide variety in the nature of the side chains attached to this xylan backbone. The

most common side chains encountered are single L-arabinofuranosyl groups attached to

O-3 of some of the backbone xylosyl residues or single D-glucosyluronic or 4-O-methyl-

D-glucosyluronic groups attached to O-2 of some of the backbone xylose units.

However, oligomeric side chains containing other glycosyl residues are also found

(Wilkie, 1979).

The composition of hemicellulose in softwoods and hardwoods is different from


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that in grasses. Galactoglucomannans are the major constituents (20%) of softwood

hemicelluloses. Hardwood hemicellulose contains glucoxylan (15-30%) polymer

backbone similar to that of the softwoods (Timell, 1965).

Hemicellulose is linked to lignin through D-galactose, L-arabinose and D-xylose

by glycosidic linkages (Sarkanen and Ludwic, 1971). Isolation of lignin-carbohydrate

complexes from rumen liquor of animals fed roughage confirms the presence of bonds

between lignin and carbohydrates (Jung, 1988; Wallace et al., 1991). According to

Chesson et al. (1983), ester linkage by the O-5 position of arabinose to core lignin seems

to be a major bond between lignin and hemicellulose in forages.

1.3.1.3 pectic compounds. Pectic polysaccharides make up approximately 35% of the

primary cell walls of dicotyledonous plants, the main components being galactosyluronic

residues (Worth, 1967). The middle lamella is particularly rich in pectic polysaccharides.

Other major polysaccharide component found in pectic polysaccharides are rhamnose,

arabinose, and galactose. Monocotyledons appear to contain minor proportion of such

polysaccharides (Dey and Brinson, 1984). Pectic substances are hydrophillic and

therefore have certain adhesive properties which may also be a means of translocation of

water (Worth, 1967).

1.3.2 proteins

Proteins are a minor component of the plant cell wall which may be covalently


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cross-linked with lignin and polysaccharides (Lamport, 1965). Extensins

(hydroxyproline-rich glycoprotein) are the most abundant protein in the plant cell wall

(Cassab and Varner, 1988). Primary cell walls of dicotyledons contain between 5 and

10% extensin which is rich (20%) in hydroxy-L-proline (Dey and Brinson, 1984).

1.3.3 phenolic compounds

1.3.3.1 lignin. Lignin is the most abundant natural non-carbohydrate organic compound

in fibrous materials. The importance of lignin in plants should be considered from

different aspects, i.e. its role in plant development, contribution to mechanical strength

and protection from degradation (Walker, 1975). From the nutritional point of view,

lignin has always been blamed as an important barrier to polysaccharide utilization. (Van

Soest, 1994).

Lignin is made up of three primary precursors, ie. trans-coniferyl, trans-sinapyl

and trans- p-coumaryl alcohols (Fig 1.3.3.1.1 and Fig 1.3.3.1.2) (Sarkanen and Ludwic,

1971). Lack of enzymic control during lignin polymerization (formation) results in an

almost random series of bonding and a very complex structure (Jung and Fahey, 1983).

The existence of strong carbon-carbon (C-C) and ether (C-O-C) linkages in the lignin

affects its susceptibility to chemical disruption (Harkin, 1973).

Lignins are always associated with hemicellulose, not only in intimate physical

mixture, but also anchored to the latter by actual covalent bonds (Sarkanen and Ludwic,


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1971). Soluble lignin-carbohydrate complexes have been isolated from LM (Morrison,

1974a and b; Nordkvist et al., 1989). Most lignins contain some aromatic carboxylic

acids (P-coumaric and ferulic acids) in ester bonds (Hartley, 1972).


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P-Coumaryl alcohol Coniferyl alcohol Sinapyl alcohol

Fig 1.3.3.1.1 Structure of three precursors of lignin (Sarkanen and Ludwic, 1971).


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Fig 1.3.3.1.2 Proposed lignin structure in softwoods (Nimz, 1974).


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Lignins differ mainly in the proportion of the three alcohol units. Softwood

lignins are made up of approximately 80% coniferyl, 14% p-coumaryl and 6% sinapyl

alcohols. In contrast, hardwood lignins are composed of 56% coniferyl, 4% p-coumaryl

and 40% sinapyl alcohols. Grass lignins are rich in p-coumaryl units (Jung and Fahey,

1983).

Sarkanen and Ludwic (1971) classified lignins into two groups, namely guaiacyl1

and guaiacyl-syringyl lignin. Most of the gymnosperm lignins are typical guaiacyl

lignins, although they contain small amounts (


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hydrolysis of plant cell wall by sulphuric acid produces a residue containing lignin,

cutin, polyphenols, carbohydrate degradation products and nitrogenous material

(Theander et al., 1977). During H2SO4 hydrolysis some lignin may be lost due to partial

conversion to soluble phenolics (Hartley, 1981).

Goering and Van Soest (1970) proposed a method based on treatment with acid

detergent solution to render a residue free of hemicellulose and soluble compounds. The

cellulose-lignin residue is then hydrolysed with 72% sulphuric acid solution to remove

cellulose.

Determination of lignin by the permanganate method has also been described

(Van Soest and Wine, 1968). Important differences between the permanganate and the

acid detergent - sulphuric acid hydrolysis method arise from the fact that cutin is largely

retained in the lignin by the latter whereas it is removed in the former. Polyphenolic and

other unsaturated substances such as tannins, pigments and proteins that may not be

completely removed in the acid detergent fibre may react with permanganate and appear

as lignin.

The acetyl bromide method (Morrison, 1972) is another option for lignin

determination. It is based on the treatment of fibre in acetyl bromide solution to break

down the cross-linkages between structural units of lignin. Soluble phenolics are

measured by UV spectrophotometry. The limitations of this method are the lack of any

entirely satisfactory standard and the presence of non-lignin compounds, e.g. proteins,

phenolic acids and tannins, which interfere with the reading.


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1.3.3.2 tannins

Tannins are known to occur in the vacuoles of plant cells (Forsyth, 1964; Jung

and Fahey, 1983). All tannins are polyphenolic compounds with a high molecular weight

(MW 500-3,000) (Haslam, 1981). Their presence in trees and woody shrubs produces a

bitter taste and astringency which may affect palatability and voluntary intake (Arnold et

al., 1980). Tannins can be divided into condensed and hydrolysable tannins. Condensed

tannins (proanthocyanidins) are the most widely distributed in vascular plants and are

made up by condensation of hydroxyflavans, leucoanthocyanidin (flavan-3,4-diol) and

catechin (flavan-3-ol) (Cope and Burns, 1974). Hydrolysable tannins are restricted to

angiosperm dicotyledons and usually contain glucose as a central core (Swain, 1979).

Tannins are known to affect the grazing behaviour and consequently depress the

food intake in sheep (Cope and Burns, 1974). Feed utilization appears to be negatively

correlated with tannin content due to depression in rumen microbial activity (Donnely

and Anthony, 1970; McLeod, 1974; Walker, 1975; Terrill et al., 1989).

1.3.3.3 phenolic acids

Phenolic acids are structural components of the lignin core in plant cell wall

(Shimada, et al., 1971). Their presence in food products has been associated with


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astringency (Durkee and Thivierge, 1977), discoloration (Dorrell, 1976) and antioxidant

properties (Senter et al., 1980). The presence of carboxyl and phenolic groups in

phenolic acids enable such compounds to link to lignin and carbohydrates by ether

(Scalbert et al., 1985) or ester (Scalbert et al., 1986) bonds.

The concentration of phenolics in grass cell walls varies from 8 to 28 mg/g and is

less than 3 mg/g in legumes (Eraso and Hartley, 1990). Ester bonds are labile to alkali

treatment and as phenolic acids are ester-linked to both lignin and hemicellulose they

can be released by alkali treatment (Hartley and Jones, 1978; Hartley et al., 1985;

Scalbert et al., 1985).

The effect of phenolic acids on rumen microbial fermentation has been

extensively studied. Chesson et al. (1982) reported that p-coumaric and ferulic acids

were the most toxic phenolic acids to rumen cellulolytic bacteria. p-Coumaric acid has

an inhibitory effect on colonization of fibres by fungi (Akin and Rigsby, 1985) and

cellulolytic bacteria (Borneman et al., 1986; Varel and Jung, 1986; Akin et al., 1988).

1.4 Acidolysis reactions

Although steam treatment has been classified as a physical treatment, in fact it is

a chemical (thermo-chemical) treatment of acid hydrolysis nature. At high temperature

under acidic conditions hemicellulose is partly disrupted thereby releasing acetyl and

formyl groups of hemicellulose and pectin, e.g. 1-2% dry weight of grasses (Bacon et

al., 1975; Bacon and Gordon, 1980). The release of endogenous organic acids, e.g.


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acetic and formic acids, will help to catalyze further reaction (Baugh and McCarty,

1988) as described below.

1.4.2 cellulose

Acid hydrolysis (saccharification) of cellulose produces a random cleavage of

glucosidic linkages containing hemiacetal and hydroxyl terminal groups (Harris, 1949).

Cellulose acid hydrolysis is dependent upon H+ concentration. A high cellulose

hydrolysis rate is observed even below 100oC when acid concentration is high. However,

under less acidic conditions higher temperature and/or longer reaction time are required

for achieving similar cellulose hydrolysis (Brownell et al., 1986).

The crystallinity index (CrI) of native cellulose has been suggested as a limiting

factor for enzymic hydrolysis (Fan et al., 1981). Interestingly, cellulose CrI is increased

during acid hydrolysis (Carrasco et al., 1994) whilst greater cellulose bio-availability is

achieved (Dekker and Wallis, 1983; Saddler et al., 1982; Wong et al., 1988; Toussaint et

al., 1991; Sawada et al., 1995). The reason for such an increase in cellulose availability

is the significant change observed in the chemical structure of lignin as well as in the

degree of polymerization of cellulose and accessible surface area (fibre swelling).

The degree of polymerization of cellulose can be significantly decreased during

acid hydrolysis (Knappert et al., 1980; Puri, 1984). The reduction of the degree of

polymerization of cellulose during steam treatment is also considered to be an important


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factor affecting cellulose availability to enzymes (Cowling and Kirk, 1976; Wei and

Cheng 1985).

Fibre swelling also plays an important role in increasing cellulose bio-availability

from acid-hydrolysed lignocellulosics (Puri 1984; Morjanoff and Gray, 1987; Wong et

al., 1988).

1.4.3 hemicellulose

Hemicellulose is significantly more susceptible to hydrolysis than cellulose

(Kuznetsov et al., 1990; McDonald and Clark, 1992). Hemicellulose is very susceptible

to depolymerization at high temperature and under acidic conditions (Carrasco et al.,

1994) and the hemicellulosic sugars, e.g. xylose, arabinose, glucose, mannose and

galactose, released by hydrolysis of hemicellulose undergo the specific reactions

mentioned below.

At high temperatures (>100oC) and acidic condition all aldopentoses form 2-

furaldehyde (furfural) (Fig 1.4.1.1) in large quantity. Among the pentoses, D-xylose is

the most effective pentose in production of furfural (Tipson and Horton, 1988). Several

low molecular weight aldehydes (formaldehyde, acetaldehyde, and 2-butenal) have been

isolated from D-xylose after acid treatment.

Under acidic conditions hexoses produce mainly 5-(hydroxymethyl)-2-

furaldehyde (HMF) as a major end-product (Fig 1.4.1.2). The rate of formation of HMF

varies considerably among the hexoses. For instance, in sulphuric acid solution (2 M at


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100oC) the order of reactivity is D-mannose > D-galactose > D-glucose (Tipson and

Horton 1988). Treatment of D-glucose, D-fructose, D-glucuronic acid and


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Fig 1.4.1.1 Production of furfural from D-xylose (Tipson and Horton, 1988).

Fig 1.4.1.2 Production of 5-hydroxymethyl-2-furaldehyde from D-glucose (Tipson andHorton, 1988).


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D-galacturonic acid in acetate buffer at pH 3.5-4.0 at 96oC yields phenolic compounds at

a rate of 0.3% for hexoses and 7% for glucuronic acid (Popoff and Theander, 1976).

Similarly pectins can yield furfural under acid hydrolysis in aqueous solution at

high temperature as do pentoses.

Upgrading LM using steam treatment is normally associated with nutrient loss

via volatilization (Castro, 1994) and formation of undegradable browning reaction

products (Van Soest and Mason, 1991). Castro et al., (1993) reported an increase in acid

detergent fibre (ADF) after steam treatment of LM. The increase in ADF content was

attributed to formation of protein-carbohydrate complexes and polymerization of

hemicellulose breakdown products.

Three types of browning reactions have been described (Hodge, 1953). The

Maillard reaction (carbonylamino reaction) is the most common one and it includes the

reaction of aldehydes, ketones and reducing sugars with amines, amino acids, peptides

and proteins (Tipson and Horton, 1988). Heating of LM generates undegradable

Maillard products which are detected by an increase in the nitrogen content of the cell

wall. Maillard polymer is considered to be a lignin-like compound and poorly

degradable (Van Soest and Mason, 1991). There are many factors affecting the

formation of Maillard reaction products, e.g. temperature, reaction time, moisture

content, H+ concentration and type of substrate (Tipson and Horton, 1988).

A caramelization reaction is another type of browning reaction. It occurs when

polyhydroxycarbonyl compounds, e.g. sugar and polyhydroxycarboxylic acids are heated


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to high temperature in the absence of amino compounds (Hodge, 1953).

A third type is known as oxidative reactions. Conversion of ascorbic acid and

polyphenols into di- or polycarbonyl compounds are examples of oxidative reaction

(Hodge, 1953).

1.4.4 lignin

Most research on chemical structure, metabolic pathways and decomposition

reactions of lignin have been carried out on woody materials (Karina et al., 1992;

Hishiyama and Sudo, 1992). Grass lignins are comparatively much less studied than

wood lignins.

Lignin structure has been discussed previously (Section 1.3.3.1). In order to

elucidate lignin structure and reactivity several studies on lignin acidolysis have been

completed (Lundquist and Hedlund, 1967; Lundquist and Lundgren, 1972; Wayman and

Obiaga, 1974; Hishiyama and Sudo, 1992; Karina et al., 1992).

Lignin disruption under acidolysis is essentially attributed to the cleavage of

various ether bonds, the most important being of the arylglycerol-ß-aryl ether bond

(Lundquist and Hedlund, 1967; Lundquist and Lundgren, 1972) (Fig 1.4.4.1).

Chua and Wayman (1979) reported that lignin-lignin and lignin-carbohydrate

bonds are more sensitive to temperature than acidity. Therefore, under auto-hydrolysis

conditions (t>160oC in absence of exogenous catalyst) one could expect greater lignin

depolymerization as compared to mild temperature acid hydrolysis (100-140oC) with


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exogenous acid).


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Fig 1.4.4.1 The susceptible bonds (α-aryl and ß-O-4) in lignin molecule for cleavageunder acidolysis reaction.


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Nimz (1974) isolated and identified a number of low-molecular weight

degradation products of the wood lignin under acid-hydrolysis conditions. During acid

hydrolysis of lignin considerable amounts of monomeric, dimeric and oligomeric

phenols are formed, the former being found in much smaller proportion. This is due to

the presence of C-C linkages between the monomers which prevent their degradation to

lower molecular weight phenolic compounds (Lundquist and Hedlund, 1967; Lundquist

and Lundgren, 1972). The rate at which phenolic monomers are released during

acidolysis depends upon the lignin structure. For example, acidolysis of birch wood

yields more monomers than spruce wood. This is due to the fact that phenylpropane

units in birch lignin are syringyl in type in which the 3 as well as the 5-position is

occupied by methoxy groups able to link to adjacent units by ether linkages (Lundquist

and Lundgren, 1972) (Fig 1.4.4.1).

Chua and Wayman (1978) reported the effect of auto-hydrolysis treatment on

aspen lignin. When aspen wood was subjected to auto-hydrolysis at 195oC it was

observed that treatment reaction time significantly affected the degree of lignin

depolymerization/repolymerization (Fig 1.4.4.2). Lignin depolymerization was

quantitatively more important than repolymerization reactions with treatment up to 30

min. Longer treatment shifted this to a greater rate of repolymerization reaction

compared to depolymerization. It has been postulated that hemicellulose degradation

products, e.g. furfural and its precursors, can react with lignin during auto-hydrolysis.

This has accounted for the apparent increase in lignin content (Chua and Wayman,


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1978). The nature of such lignin is different from native lignin as it is more condensed


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Fig 1.4.4.2 Effect of reaction time on depolymerization/repolymerization of lignin(Wayman and Chua, 1979)


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compared to original lignin (Wayman and Chua, 1979b). Auto-hydrolysis causes an

increase in the proportion of C to O/H and a decrease in content of methoxy group

(Chua and Wayman, 1978).

1.5 Protein precipitating capacity of phenolic compounds

Phenolic acids are known to inactivate enzymes (Martin and Akin, 1988) through

decreasing enzyme solubility (Sharma, 1985) and/or formation of a soluble and inactive

enzyme inhibitor complex at very low concentration (Zanobini et al., 1967). The extent

of enzymic inhibition by phenolic acids depends on the type of enzyme (Vohra et al.,

1980) and the phenolic compound involved (Sharma et al., 1985).

The ability of tannins to bind and precipitate proteins has been well documented

(Makkar et al., 1987; Makkar, 1989; Makkar et al., 1993b). This precipitating property

is explained by the ability of phenolic hydroxyl groups to form cross-links with proteins

(Feeny, 1969; Swain, 1979). Although tannins are considered to be toxic to rumen

microbial activity (Waghorn et al., 1990) their precipitating capacity may have a positive

effect by preventing bloat in animals feeding on legume pastures and protecting proteins

from microbial degradation (Driedger and Hatfield, 1972).

It is reported that proteins bound to tannins are more likely to escape rumen

degradation than unbound proteins (Driedger and Hatfield, 1972). The protein-tannin

complex is dissociated at abomasal pH (Oh and Hoff, 1987; Waghorn et al., 1990), thus

releasing protein to be digested in the lower tract. This property has been a matter of


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research on protein protection for ruminants for several years (Nishimuta and Boling,

1973).

Beltrame et al. (1992) observed a significant increase in the extent of enzymic

hydrolysis of cellulose after removal of the lignin fraction from steam-treated wheat

straw.

Kawamoto et al. (1992) reported a protein-precipitating capacity in lignin

extracted from steam-exploded LM. Such protein-precipitating capacity of phenolics

was attributed to the formation of hydrogen bonds between lignin hydroxyl groups and

protein carboxyl groups.

1.6 The effect of steam treatment-associated toxic compounds on rumen microbial

activity

1.6.1 furfural and 5-hydroxymethyl furfural

As mentioned previously in this chapter, formation of toxic end-products during

steam treatment may be a major negative effect of treatment. These products can inhibit

yeast growth (Fireoved and Mutharasan, 1986) and methane production (Baugh and

McCarty, 1988). In these two reports such a toxic effect was attributed mainly to

furfural.

Furfural is the most widely distributed simple furan in nature (Dean, 1963). It is

formed during production and storage of fruit juices (Robertson and Samaniego, 1986)


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and wines (Simpson, 1980), and is thus very important in the food industry. Furfural is

also a major degradation by-product of the steam treatment of LM (Sproull et al., 1985).

Sanchez and Bautista (1988) observed that both furfural and HMF were toxic to

yeast. Furfural has shown a greater toxicity compared to HMF. It has been reported that

such aldehydes are first converted into furoic acid and further reutilized by the yeast. In

another study, Van Tran and Chambers (1985) reported that furfural inhibited respiration

of Pichia stipitis at a concentration of 2g/l and the metabolic by-product furfuryl alcohol

reduced its growth rate.

The effects of furfural on animal performance has been also investigated. The

effect of furfural on food intake has been associated with its antimicrobial action and

mucosal irritation (Kyuma et al., 1991). Furfural depressed dry matter consumption rate

and dry matter intake by goats up to 4 h after feeding. However, there was no effect on

the total daily feed intake and digestibility. Cellulose digestibility in vitro using rumen

liquor as inoculum was depressed when furfural addition was greater than 200 ppm.

Such a depression was more marked between 4 and 12 h incubation. Contrary to Kyuma

et al. (1991), Castro et al. (1995) observed that furfural was not toxic to rumen microbial

activity in vitro up to a concentration of 1,300 ppm. It was also demonstrated that rumen

microbes can almost completely degrade both furfural and HMF within 6 h fermentation

in vitro.


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1.6.2 phenolic compounds

Britton (1978) reported a positive effect of removing inhibitory materials from

steam-treated sawdust by ethanol solutions on microbial activity in vitro. Unfortunately

the nature of such inhibitory materials was not investigated in that study. Castro et al.

(1995) reported inhibition of methane production in vitro from acid-hydrolysed wheat

straw. Such an effect was attributed to lignin degradation products.

According to Clark and Mackie (1984) phenolic compounds derived from lignin

depolymerization inhibited yeast activity. These authors reported a greater inhibitory

effect from low molecular weight lignin-based phenolics than from carbohydrate

degradation products.

Production of materials inhibitory to microbes and cell-free enzymes during

steam treatment of fibrous materials limits the value of this process for improving the

fermentability of these materials by microbes or enzymes. This problem has received

surprisingly little attention.

Regarding the advantages of steam treatment which were discussed before,

different aspects of this type of treatment, specially negative ones, need to be studied.

The ultimate goal would be to find a suitable method to reduce such negative effects or

to try to make good use of them such as using the inhibitory compounds to manipulate

ruminal fermentation.

In the present study the biological effects of such compounds were examined


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with emphasis on ruminant nutrition. The inhibitory capacity of such compounds were

studied in two biological systems, i.e. rumen microbial fermentation and cell-free

enzymic hydrolysis. The ability of phenolics extracted from steam-treated

lignocellulosics to protect proteins was also studied.


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CHAPTER 2

CHARACTERIZATION OF WATER-SOLUBLE EXTRACT FROM

STEAM-TREATED WHEAT STRAW

2.1 Introduction

Hemicellulose solubilization (Carrasco et al., 1994; Eklund et al., 1995) and

lignin depolymerization (Wayman and Chua, 1979a) are two important changes that

occur during steam treatment. The extent of hydrolysis of cell wall polysaccharides

depends on treatment conditions (Forsberg et al., 1986). A significant increase in water

soluble extract (WSE) from steam-treated LM occurs due to depolymerization of

hemicellulose and lignin into soluble compounds (Matsuzaki et al., 1994). Treatment

conditions affect both degree of polymerization of soluble carbohydrates (Matsuzaki et

al., 1994) and sugar composition (Grohmann, et al., 1985).

Due to its high content of readily fermentable compounds, WSE can be used for

some biological purposes, e.g. production of alcohol (Van Tran and Chambers, 1985;

Delgenes et al., 1990), single cell protein (Qu et al., 1992) and animal feed. Both

chemical composition and degree of polymerization of soluble carbohydrates are

important aspects when considering nutritive value of WSE (efficiency and site of

digestion).

With respect to monogastric feeding, the presence of non-monomeric pentoses


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may be a limiting factor as such compounds cannot be digested and absorbed in the

small intestine and may have negative effects on an animal (Longstaff et al., 1988), e.g.

vomiting and diarrhoea, at high levels of feeding (Yule and Fuller, 1992). Oligomers due

to presence of ß 1-4 linkages between sugar residues are not hydrolysed by the enzymic

system in the intestine. However, they can be degraded by microbes in the lower gut into

volatile fatty acids (VFA) which can be absorbed and metabolised by the animal.

Although microbial degradation is less efficient as compared to the enzymic process in

the small intestine (McDonald et al., 1988), the use of WSE containing large quantities

of oligomeric forms of sugar may have a potential market for animal feeding.

Ruminants are more tolerant to some of the constraints described above as they

can degrade oligomeric forms of sugars in the rumen.

Another important constraint for using the WSE as an animal feed is the presence

of material inhibitory to microbial activity (Clark and Mackie, 1984). Also relevant is

the presence of a significant amount of undegradable compounds (Maillard reaction

compounds) which make no contribution to the energy value of this fraction (Van Soest

and Mason, 1991).

Optimum treatment conditions (temperature, reaction time, moisture content and

presence of exogenous catalyst) are achieved when cell wall structure is disrupted in

such a way so as maximize bio-utilization of cell wall polysaccharides while producing

minimum quantities of toxic materials. As mentioned in Section 1.4.3, furans are end-

products of browning reaction of sugars under acidic conditions. Caramelization is


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another type of reaction (Section 1.4.3) which also affects soluble carbohydrate (Hodge

,1953). The nutritive value and toxicity of the products of such reactions have not been

reported in steam-treated LM.

The aim of the work described in this chapter was to investigate the nature and

biological value of water-soluble extract from steam-treated wheat straw produced under

different treatment conditions. Various components from this fraction were tested by

both chemical and biological approaches and possible implications on animal digestive

efficiency are discussed.


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2.2 Materials and methods

2.2.1 substrate

Wheat straw variety Riband was used in this experiment. An air dried sample was

hammer milled through a 1 mm screen and stored at room temperature.

2.2.2 steam treatment conditions

A laboratory scale 1.5 litre high pressure vessel designed at the Rowett Research

Institute was used. To standardize treatment conditions nine batches were first carried

out at 19 bar. Two heating up times (time required to reach 19 bar starting from

atmospheric pressure) (14 and 19 min) and three dry matter (DM) contents (40, 50 and

60%) were tested. The material was then held at this pressure for various reaction times,

including 0 min reaction time which indicates immediate termination of the pressure

treatment.

The vessel (Fig 2.2.2.1) was kept at 250OC prior to treatment. Water was added

to wheat straw to achieve the appropriate moisture content. A 500 ml aluminium beaker

containing boiling water was placed in the vessel and a second similar beaker containing

wet sample (≈ 30 g DM basis) was put on top. The system was closed by the top lid.

Rise of pressure was manually controlled by adjusting the ball valve. Treatment

reproducibility was assessed by producing a series of samples (n=4) treated at 15 and 19

bar.


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Fig 2.2.2.1a The High Pressure Steam Treatment Vessel in operation.


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Fig 2.2.2.1b Components of the High Pressure Vessel.


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(c)

(d)


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After validation another experiment was designed to determine the effect of

pressure (15, 17 and 19 bar), reaction time (RT) and heating up time (HT) (14 and 19

min) upon the properties of the WSE as described below.

2.2.3 extraction of water soluble extract

A 1 g sample was soaked in 30 ml water and kept at 40oC for 2 h. The aqueous

mixture was filtered through a No. 2 sintered glass filter and was centrifuged (Sorvall

RC-SB) at 10,000 x g and 4oC for 10 min. The supernatant was freeze dried and kept in

dark at room temperature for further analyses.

2.2.4 statistical analysis

Effect of DM content on soluble carbohydrate and total extractable phenolic

contents of steam-treated wheat straw was evaluated by analysis of variance (ANOVA).

Differences in nutritive value of water soluble extract from steam-treated wheat straw as

compared to control sample was tested by Bonferroni T test p


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incubated at 25OC in water bath for 30 min with occasional vortex mixing. Five ml water

was added to the tube, which was then vortex mixed, incubated at 100OC for 2 h, cooled

and centrifuged at 3,000 x g for 5 min. One ml of allose/inositol standard (1 mg/ml) was

added to a 3 ml aliquot of the supernatant hydrolysate. The mixture was neutralized by

adding 0.75 ml concentrated ammonia solution (12 M). A 0.4 ml NaBH4 solution (100

mg/ml in 0.05 M NH4OH) and 1 drop of octan-2ol (antifoaming agent) were added to

the neutralized aliquot which was incubated at 40oC for 60 min. Four hundred µl acetic

acid was added to acidify and destroy excess NaBH4. Two ml of acetic anhydride and

0.3 ml 1-methyl imidazole were added to 200 µl of reduced hydrolysate. The reaction

mixture was mixed and allowed to react for 10 min. Five ml of water was added and the

mixture allowed to stand for 10 min to destroy any excess of acetic anhydride. The

alditol acetates were extracted by adding 2 ml of dichloromethane and vortex mixing for

15 seconds. After centrifuging (3,000 x g for 10 min) the organic layer was transferred

to a vial. The dichloromethane was evaporated off under a stream of N2 at 40OC. The

alditol acetates were dissolved in 150 µl ethyl acetate and 1 µl sample was injected into

the GC fitted with a flame ionization detector (FID) and a wide bore capillary column

(30m, 0.75 mm I.D., Supelco SP-2330). The following temperatures were used: injector

(t=250oC), column oven (t=180oC, temperature increased at a rate of 10oC/min until

220oC and time of 3 min and FID (t=275

oC). Helium gas was used as carrier with a flow

rate of 7 ml/min.

Monomeric sugars in water extracts were measured by dissolving a 20 mg sample


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in 5 ml water. A 0.4 ml inositol standard (internal standard) was added to a 2 ml aliquot.

The remaining steps for measuring of monomeric sugars (derivatization to alditol acetate

derivatives, addition of internal standard, extraction of alditol acetates by

dichloromethane from aqueous solution and GLC conditions) were the same as

mentioned above.

2.2.6 DM loss

DM loss during steam treatment was directly measured by weight difference

before and after treatment.

2.2.7 ash content

Ash content was determined by incinerating 1 g sample in a muffle furnace at

550oC for 6 h.

2.2.8 nitrogen (N) content

N content was measured using a Macro N Analyzer (Foss Electric, UK). The

method is based on the oxidative combustion of a given sample (200 mg) at >1,000oC

converting the nitrogen in the sample to N2, which is detected by thermal conductivity.

2.2.9 total extractable phenolic analysis

The phenolic content of water extracts was measured by the Folin and Ciocalteu


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reagent (Julkunen-Tiitto, 1985). Five ml 70% acetone solution (v/v) was added to a test

tube containing 100 mg freeze dried sample (Muller-Harvey and Dhanoa, 1991) to

extract phenolic compounds. Nine hundred µl water, 0.5 ml 1 M Folin and Ciocauteu

phenol reagent and 2.5 ml 20% Na2CO3 were added to 0.1 ml extract.

novel uses of lignin and hemicellulosic sugars from acidhydrolysed

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