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The evaluation of raspberry pomace as a feedstuff for growing pigs McDougall, N. Ruth 1990

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THE EVALUATION OF RASPBERRY POMACE AS A FEEDSTUFF FOR GROWING PIGS b Y N. RUTH MCDOUGALL B.Sc. (Agric) The University of B r i t i s h Columbia, 1986 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (DEPARTMENT OF ANIMAL SCIENCE) We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1990 (c) N. Ruth McDougall, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Animal Sc ience The University of British Columbia Vancouver, Canada D a t e May 7 r 1QQ0  DE-6 (2/88) A B S T R A C T Raspberry pomace, consisting of seeds, pulp and added r i c e h u l l s , i s the residue from the pressing of raspberries for j u i c e and concentrate production. Through the determination of chemical composition by laboratory analyses and the measurement of feeding value i n animal t r i a l s , the pomace was evaluated as a feedstuff f o r growing pigs. Pomace contains 11.1% crude fat, 10.0% crude protein, 59.5% t o t a l dietary f i b r e , 7.4% soluble carbohydrates, and a gross energy l e v e l of 5220 k c a l * k g - 1 . The acid detergent residue of the pomace contains 11.7% l i g n i n , 6.0% cutin, 2.2% acid detergent ash and 26.0% c e l l u l o s e (by d i f f e r e n c e ) . The d i g e s t i b i l i t y of dry matter, f a t , protein and energy was determined. Pomace dried at 60 C, whole and ground (1mm) was fed to growing male pigs (3 0-3 5 kg) i n a re p l i c a t e d 4X4 Latin Square design with treatments basal (B) , B plus 4 0% unground pomace, B plus 4 0% ground pomace and B plus 40% barley. Grinding of pomace s i g n i f i c a n t l y improved the d i g e s t i b i l i t y of a l l parameters measured, however, the barley-soybean meal basal r a t i o n was consistently better digested than eit h e r of the pomace treatments. The d i g e s t i b i l i t y of whole and ground pomace was respectively: dry matter 10.7% and 20.8% (S.E.M. 1.30), fat 24.1% and 79.7% ( S . E . M . 3.47), pro te in 10.6% and 14.7% ( S . E . M . 4.83) and energy 7.9% and 28.4% ( S . E . M . 1.80). Prote in q u a l i t y of ground (1mm) and f reeze -dr i ed pomace was evaluated with ra t s i n metabolism cages to produce the fo l lowing values: true pro te in d i g e s t i b i l i t y 36.0% ( S . E . M . 0.66), b i o l o g i c a l value 91.0% ( S . E . M . 3.46), and net prote in u t i l i z a t i o n 32.7% ( S . E . M . 1.15). In r a t growth t r i a l s , where pomace replaced barley incremental ly , growth rate was not af fected at replacement l eve l s up to 40%, although feed e f f i c i e n c y dec l ined cons i s t en t ly as the l e v e l of pomace i n the d i e t increased. I t i s suggested that raspberry pomace could replace up to 2 0% of an energy feedstuff i n a r a t i o n for growing swine without s i g n i f i c a n t l y reducing growth rate or feed e f f i c i e n c y . TABLE OF CONTENTS Page ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i i LIST OF FIGURES X ACKNOWLEDGEMENTS x i CHAPTER 1. INTRODUCTION 1 CHAPTER 2. LITERATURE REVIEW 4 1. F r u i t and vegetable processing wastes as live s t o c k feed. 4 1.1. Constraints on the use of processing wastes. 4 1.1.1. N u t r i t i o n a l c h a r a c t e r i s t i c s of processing by-products 5 1.1.2. Dry matter content of by-products. . . 10 1.1.3. Preservation of processing by-products 10 1.1.4. Season of a v a i l a b i l i t y of processing wastes 11 1.1.5. Contamination 11 1.2. U t i l i z a t i o n of processing wastes i n North America 11 1.2.1. United States 11 1.2.2. Canada 12 1.2.3. B r i t i s h Columbia 13 2. N u t r i t i o n a l evaluation of raspberry pomace . . . . 16 2.1. The production of pomace 16 2.2. Chemical composition of raspberry pomace . . 17 2.2.1. Energy, protein and f i b r e content. . . 17 2.2.2. Amino acid composition 18 2.2.3. Mineral composition 19 2.2.4. Composition of raspberry seed o i l . . . 19 2.2.5. Composition of r i c e h u l l s 21 iv 2.3. Short and longterm preservation of pomace. . 22 2.3.1. Chemical preservation 22 2.3.2. E n s i l i n g 23 2.4. In v i t r o (rumen f l u i d ) dry matter d i g e s t i b i l i t y of pomace 23 3. N u t r i t i o n a l evaluation of s i m i l a r by-products. . .25 3.1. Winery pomace 26 3.1.1. Nutrient content of winery pomace. . .26 3.1.2. DMD of winery pomace by growing pigs . 26 3.1.3. Protein d i g e s t i b i l i t y of winery pomace by dairy calves 27 3.2. Tomato pomace 27 3.2.1. Nutrient content of tomato pomace. . . 27 3.2.2. Protein and f a t d i g e s t i b i l i t y of tomato pomace f o r ruminants 28 4. The use of t o t a l dietary f i b r e methodology i n the evaluation of high-fibre feeds for monogastrics 28 4.1. Limitations of the crude f i b r e method. . . . 29 4.2. Limitations of the neutral detergent f i b r e method 3 0 4.3. Total dietary f i b r e method 30 5. The use of rats as models for studies on the n u t r i t i o n of pigs 32 CHAPTER 3. EXPERIMENTAL 3 4 CHAPTER 4. CHEMICAL ANALYSIS OF RASPBERRY POMACE . . . . 36 1. Introduction 3 6 2. Materials and methods 37 2.1. Standard analyses 38 2.2. Total dietary f i b r e method 40 2.3. Analysis of f i b r e components using Van Soest forage f i b r e methods 4 3 3. Results and discussion 44 3.1. Protein and amino acid composition of pomace 4 6 3.2. Fat and gross energy content of pomace . . . 50 3.3. Soluble carbohydrate content 51 3.4. Composition of raspberry pomace f i b r e . . . . 51 3.4.1. Neutral detergent f i b r e 51 3.4.2. Total dietary f i b r e . . 52 3.4.5. Lignin, c u t i n and c e l l u l o s e (by difference) i n raspberry pomace . . . 56 v CHAPTER 5. DIGESTIBILITY OF DRY MATTER, PROTEIN, FAT AND ENERGY OF RASPBERRY POMACE BY GROWING PIGS AND RATS 59 1. Materials and methods 59 1.1. T r i a l 1: Determination of pomace d i g e s t i b i l i t y with growing pigs 59 1.1.1. Analysis of protein i n f e c a l material 63 1.1.2. Analysis of f a t i n f e c a l material. . . 63 1.1.3. Determination of gross energy i n feces 64 1.2. T r i a l 2: Determination of pomace dry matter d i g e s t i b i l i t y with rats using chromic oxide marker method 65 1.2.1. Chromic oxide analysis 68 1.3. T r i a l 3: Determination of pomace protein d i g e s t i b i l i t y by growing rats 7 0 1.3.1. Chemical analysis of urine and f e c a l samples 73 1.4. S t a t i s t i c a l analysis 74 2. Results and discussion 7 5 2.1. Dry matter d i g e s t i b i l i t y of pomace by growing pigs and rats 7 5 2.1.1. Pigs 75 2.1.2. Rats 78 2.1.3. Conclusion 80 2.2. Energy d i g e s t i b i l i t y of raspberry pomace by growing pigs 83 2.3. D i g e s t i b i l i t y of pomace protein by growing pigs and rats 84 2.3.1. Apparent d i g e s t i b i l i t y of pomace protein by pigs 84 2.3.2. TD, BV and NPU of pomace fed to growing rats 85 2.3.3. The e f f e c t of fineness of grind on TD, BV and NPU of pomace fed to growing rats 88 2.3.4. The e f f e c t of heat-treatment on TD, BV and NPU of pomace fed to growing rats 89 2.3.5. BV of freeze-dried, coarsely-ground pomace fed to rats 91 2.3.6. Overall d i g e s t i b i l i t y of pomace protein 92 v i 2.4. D i g e s t i b i l i t y of f a t i n pomace fed to growing pigs 93 2.5. Digestible protein, f a t and energy i n raspberry pomace 95 CHAPTER 6. FEED INTAKE, GROWTH RATE AND FEED EFFICIENCY OF GROWING RATS FED DIETS CONTAINING RASPBERRY POMACE AD LIB 97 1. Materials and Methods 97 1.1. S t a t i s t i c a l analysis 97 2. Results and Discussion 98 2.1. Feed intake 98 2.2. Weight gain 101 2.3. Feed e f f i c i e n c y 105 CHAPTER 7. SUMMARY AND CONCLUSIONS 108 BIBLIOGRAPHY 114 Appendix 1. Total dietary f i b r e method 121 Appendix 2. Acid hydrolysis of f a t i n f e c a l material. . 126 Appendix 3.1. Chemical analyses of raspberry pomace containing r i c e h u l l s 129 Appendix 3.2. Analysis of soluble and insoluble dietary f i b r e components of raspberry pomace with r i c e h u l l s 130 Appendix 3.3. Analysis of f i b r e of raspberry pomace with r i c e h u l l s 131 v i i LIST OF TABLES Page Table 2.1. Use of f r u i t and vegetable processing wastes i n B r i t i s h Columbia i n 1988. (Beames and Taylor, 1988) 14 Table 2.2. Mineral composition of raspberry pomace and r i c e h u l l s 20 Table 2.3. In v i t r o dry matter d i g e s t i b i l i t y (IVDMD) of raspberry pomace subjected to various heating and grinding treatments. (Buckley, 1985) 24 Table 4.1. Composition of raspberry pomace containing r i c e h u l l s (100% D.M.basis) 45 Table 4.2. Amino acid composition of raspberry pomace containing r i c e h u l l s (g a.a. per 100 g pomace D.M.) 47 Table 4.3. Amino acid composition of raspberry pomace containing r i c e h u l l s (g a.a per 100 g CP.) . . . 48 Table 4.4. Insoluble and soluble components of t o t a l dietary f i b r e of raspberry pomace with r i c e h u l l s (100% D.M. basis) 53 Table 4.5. Analysis of f i b r e components of raspberry pomace with r i c e h u l l s (100% D.M. basis) 57 Table 5.1. Composition of basal rat i o n for d i g e s t i b i l i t y t r i a l with growing pigs . . . . . . 61 Table 5.2. Composition of treatment d i e t s f o r d i g e s t i b i l i t y t r i a l with pigs (100% D.M.basis). . 61 Table 5.3. Composition of diets used i n growth t r i a l with rats fed raspberry pomace i n varying amounts (g per lOOg d i e t dry matter) 66 Table 5.4. Composition of trace mineral-vitamin mixture used i n rat growth t r i a l d i e ts (concentration per kg of mix) 67 v i i i Table 5.5. Composition of nitrogen-free mix and mineral supplement added to diets i n nitrogen balance t r i a l with rats 71 Table 5.6. Dry matter, protein, f a t and energy d i g e s t i b i l i t y of whole rations containing 40% of e i t h e r ground or unground raspberry pomace, or barley, by growing male pigs 76 Table 5.7. Dry matter, protein, f a t and energy d i g e s t i b i l i t y (by difference) of barley, and ground and unground raspberry pomace fed to growing male pigs Table 5.8. Dry matter d i g e s t i b i l i t y of a balanced barley-based d i e t containing varying amounts of pomace when fed to growing rats 77 79 Table 5.9. True d i g e s t i b i l i t y , b i o l o g i c a l value and net u t i l i z a t i o n of the protein of raspberry pomace by weanling male rat s . Pomace was dried at two d i f f e r e n t temperatures and ground to two d i f f e r e n t degrees of fineness Table 5.10. Digestible protein, f a t and energy i n ground and unground raspberry pomace for growing pigs 87 96 Table 6.1. Average d a i l y feed intake, weight gain and feed e f f i c i e n c y of growing rats fed varying proportions of raspberry pomace i n a balanced barley-based r a t i o n . 99 Table 6.2. Average d a i l y feed intake (FI), weight gain (ADG) and feed e f f i c i e n c y (F/G) of rats fed varying l e v e l s of raspberry pomace i n a balanced barley-based rat i o n over three weeks of t r i a l (see figures 6.1, 6.2 and 6.3 for weekly data). .101 ix LIST OF FIGURES Page Figure 6.1. Average d a i l y feed intake of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Feed intake was s i g n i f i c a n t l y higher during week two than during weeks one and three . . 102 Figure 6.2. Average d a i l y gain of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Daily gain was highest during week one of the t r i a l , and declined s i g n i f i c a n t l y during each of the subsequent weeks 104 Figure 6.3. Feed/gain of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Feed/gain was s i g n i f i c a n t l y better during week one of the t r i a l than during either of the other weeks 107 x ACKNOWLEDGEMENTS I would l i k e to thank my advisor, Dr. R.M. Beames of the Department of Animal Science, f o r h i s continuing support and encouragement over the past three years, and for his car e f u l attention to d e t a i l during the wri t i n g of t h i s t h e s i s . I am deeply indebted to Jason Mann, fellow graduate student, for h i s endless, s e l f l e s s help during the f i r s t year of my studies. The generosity of Dr. John Hunt of the Agriculture Canada Research Station, Agassiz B.C., who permitted me the use of the Feed M i l l lab f a c i l i t i e s during a part of t h i s project, i s appreciated. At U.B.C., my load of labwork was made l i g h t e r and more pleasant with the help of summer students Tracey Innes and Christa Wallace; a big thank you to both of you as well. F i n a l l y , I would l i k e to acknowledge the B.C. Science Council and East Chilliwack A g r i c u l t u r a l Co-op for t h e i r f i n a n c i a l support of t h i s project. x i INTRODUCTION The Fraser Valley of B r i t i s h Columbia has a combination of temperate climate and l i g h t , well-drained s o i l s which make i t one of the best-suited areas i n the world for the production of raspberries (Dorling, 1967). Because of t h i s , raspberry production i n the Fraser Valley f a r exceeds the demand for fresh berries, with the r e s u l t that there i s an ongoing search for new products containing processed raspberries. During the 1989 production season i n B.C., 900 tonnes of raspberries were sold as fresh product, and 18,200 tonnes were processed into raspberry products (Peters, 1989). Raspberry j u i c e has been one of the most successful new products. Raspberry pomace i s the by-product of the production of j u i c e . I t i s a low dry matter (44%) , highly perishable waste product (Buckley, 1985) which consists of seeds, pulp and trash. I t also contains about 12% r i c e h u l l s which are added as an i n e r t press-aid during j u i c e production. The approximately 500 tonnes of pomace produced annually i n the Fraser Valley (Maclntyre, 1987) creates a considerable disposal problem for the processor i f i t cannot be otherwise u t i l i z e d . Currently, the o i l contained i n the seed of the pomace i s being extracted and sold as a flavouring agent (Maclntyre, 1987). Optionally, the pomace could be used as 1 a feedstuff, which would be made available without cost to a li v e s t o c k producer on the understanding that i t would a l l be removed from the processing plant as i t i s produced. Small amounts of strawberry and blueberry pomace (7 0-100 tonnes annually) are also produced i n the Fraser Valley as a by-product of j u i c e extraction (Maclntyre, 1987). As these are also small seed berry pomaces, i t was considered that data produced i n an inves t i g a t i o n of raspberry pomace might provide an i n d i c a t i o n of the feeding value of these pomaces. By-products of f r u i t and vegetable processing are commonly used as l i v e s t o c k feedstuffs (NRC, 1983), most frequently i n rations for ruminants because of the fibrous nature of the waste products. Raspberry pomace has been previously evaluated as a feedstuff for ruminants, through chemical analysis and i n v i t r o dry matter d i g e s t i b i l i t y studies (Buckley, 1985). In these investigations, the pomace was found to have a very high gross energy content (5400 kcal*k g - 1 ) because of the s i g n i f i c a n t amount of l i p i d material contained i n the seeds, and a protein content s i m i l a r to that of feed grade barley. Such figures indicate that the pomace could be a reasonable source of energy and protein for non-ruminant animals, i n sp i t e of the known high f i b r e content. On the basis of i t s promising chemical 2 composition and the proximity of the processing plant to several swine producers, i t was decided to evaluate the pomace as a feedstuff for growing swine. The study was divided into two parts; determination of the chemical composition of the pomace through laboratory analyses, and measurement of i t s feeding value through animal t r i a l s . Chemical analyses designed to ascertain the amount of r e a d i l y available nutrients, as well as the amount and composition of the unavailable fibrous portion of the pomace, were undertaken. Four animal t r i a l s were designed to determine the feeding value of the pomace for non-ruminants. The dry matter, protein, f a t and energy d i g e s t i b i l i t y of the pomace, and the e f f e c t of heating and fineness of grind on protein d i g e s t i b i l i t y were examined i n three separate t r i a l s . A fourth t r i a l was designed to determine the growth rate and feed e f f i c i e n c y of growing male rats fed varying l e v e l s of raspberry pomace in a balanced ra t i o n . 3 LITERATURE REVIEW 1. FRUIT AND VEGETABLE PROCESSING WASTES AS LIVESTOCK FEED 1.1. Constra ints on the use of process ing wastes The processing of f r u i t and vegetables produces a waste or by-product which consists of skin, pulp, seeds, stalks, rejected product and ef f l u e n t water. Many processing by-products are highly n u t r i t i o u s and thus would be suitable as liv e s t o c k feed. Despite t h i s , t h e i r p o t e n t i a l as feedstuffs has not been widely exploited. Processing plants have found i n the past that the disposal of s o l i d material i n l a n d f i l l s i t e s , and of waste eff l u e n t s into sewage systems or waterways has been the cheapest, simplest method of disposal of f r u i t and vegetable processing wastes. However, the costs of, and r e s t r i c t i o n s on waste disposal are rapidl y increasing. Because l a n d f i l l s i t e s are becoming scarce, s o l i d waste disposal i s no longer free i n many areas, forcing processors to f i n d alternate means of disposal such as the provision to livestock producers who can u t i l i z e t h i s free material i n t h e i r rations. More stringent p o l l u t i o n control measures are forcing processors to concentrate t h e i r l i q u i d wastes, thereby producing a material with a higher dry matter 4 content which i s more economical to transport and thus, as a feedstuff, would have a lower landed cost per unit of nutrient. These disposal problems have stimulated i n t e r e s t i n feeding processing by-products to livestock, and as a consequence, u t i l i z a t i o n has increased r a p i d l y i n recent years (NRC, 1983) . For the most part, l i v e s t o c k producers have not explored the po t e n t i a l of processing wastes because i t has been much less complicated to u t i l i z e t r a d i t i o n a l feedstuffs. As p r o f i t margins from animal production narrow, producers are turning increasingly to alt e r n a t i v e , lower-cost feedstuffs. However, processing by-products have several c h a r a c t e r i s t i c s , discussed i n more d e t a i l below, which may make t h e i r incorporation into l i v e s t o c k feeds complicated and cos t l y . 1.1.1. N u t r i t i o n a l c h a r a c t e r i s t i c s of processing by-products Energy: Some processing by-products such as grape, tomato and berry pomaces contain a s i g n i f i c a n t amount of l i p i d material which i s found l a r g e l y i n the seeds. This type of by-product has the most po t e n t i a l as a feedstuff for non-ruminants, providing that the seeds are mechanically cracked to make the o i l a v a i l a b l e . The storage l i p i d , which contains f a t t y acids s p e c i f i c to the plant from which i t i s 5 derived, i s highly d i g e s t i b l e by non-ruminants and i s therefore an excellent source of energy (Gurr, 1983). Protein: Most by-products, including berry pomaces, have only a moderate protein content (8% to 14%) , which precludes t h e i r use as protein concentrate replacements. As well, the protein d i g e s t i b i l i t y by non-ruminants frequently i s poor, as a r e s u l t of the high f i b r e and l i g n i n l e v e l s , the presence of tannins, and heat-damage to the protein that might have occurred during processing. In high f i b r e feedstuffs, much of the protein i s bound within the structure of the c e l l wall i n an i n d i g e s t i b l e matrix of c e l l u l o s e , hemicellulose and l i g n i n (Theander and Aman, 1984) . The l i g n i n , which i s co-bonded to both c e l l u l o s e and hemicellulose i n the c e l l wall, prevents microbial digestion of these components and of the c e l l wall protein (Robertson and Van Soest, 1981). Extensive grinding of these high-fibre feedstuffs may be necessary to increase protein d i g e s t i b i l i t y . The presence of tannins i n a feedstuff w i l l s i g n i f i c a n t l y reduce protein d i g e s t i b i l i t y . Tannins have been found to diminish the protein and dry matter d i g e s t i b i l i t y (DMD) of diets fed to rats, hens, swine and c a t t l e (Butler et a l . , 1986). Both condensed and hydrolyzable tannins react with amino acid side chains of 6 proteins i n the gut to produce i n d i g e s t i b l e complexes (Hagerman and Klucher, 1986). They may also bind endogenous enzymes, but t h i s i s not thought to influence d i g e s t i b i l i t y (Butler et a l . , 1986). Tannins react p r e f e r e n t i a l l y with p r o l i n e and possibly with other non-essential amino acids (Hagerman and Klucher, 1986). The actual mechanism of protein binding by tannin has not been f u l l y elucidated. The d i g e s t i b i l i t y of by-product protein can be further impaired by the formation of i n d i g e s t i b l e protein-carbohydrate complexes during processing (Maillard non-enzymic browning products). M a i l l a r d reaction products are the r e s u l t of the reaction of the amino groups of amino acids, peptides and proteins with the " g l y c o s i d i c " hydroxyl group of reducing sugars ( E l l i s , 1959). In the presence of a source of reducing sugar (such as glucose or fructose), the amino acid reacts with the reducing sugar to form a linkage which i s e s s e n t i a l l y non-hydrolyzable by digestive enzymes (Knipfel, 1981) and which passes undigested into the feces. The extent of formation of the M a i l l a r d product i s accelerated i n the presence of heat. Temperatures above 50 C can induce s i g n i f i c a n t heat damage, with the extent of damage increasing as the heating temperature increases (Van Soest, 1965) and as the duration of heating increases (Mauron, 1981). The presence of moisture i s important also. 7 L i t t l e protein damage i s observed with completely dry conditions, or when moisture exceeds 90%; damage i s most severe when the moisture content i s approximately 3 0% (Mauron, 1981) . Because of the use of heat and moisture during processing, i t i s l i k e l y that the protein of most f r u i t and vegetable waste products w i l l have sustained some damage. Ma i l l a r d reactions are numerous. Of i n t e r e s t i n li v e s t o c k n u t r i t i o n are those which occur i n feedstuffs, and which, as a re s u l t , reduce the n u t r i t i o n a l q u a l i t y of the protein. A l l es s e n t i a l amino acids can be damaged by the formation of Ma i l l a r d products. However, ly s i n e i s the amino acid which appears to be p r e f e r e n t i a l l y complexed by the carbohydrate source (Hurrell and Carpenter, 1981), e s p e c i a l l y when heating i s of short duration and at a low temperature (Knipfel, 1981). In feedstuffs i n which lysi n e i s the f i r s t l i m i t i n g amino acid, a severe reduction i n the n u t r i t i o n a l q u a l i t y of the feed can occur even with mild heating. When the temperature of heating i s higher and occurs for a longer period, damage occurs to a l l amino acids, and the o v e r a l l d i g e s t i b i l i t y of the protein i s reduced (Knipfel, 1981). Fibre: Most processing by-products contain a high percentage of f i b r e . The various components of f i b r e , 8 including c e l l u l o s e , hemicellulose, l i g n i n , c u t i n and tannin, are v a r i a b l y available to non-ruminants. Although the microbial populations i n the colon and caecum of growing pigs can degrade s i g n i f i c a n t amounts of the predominant f i b r e components, c e l l u l o s e and hemicellulose, they do t h i s with a lower e f f i c i e n c y than when the nutrient source i s simple carbohydrates. Also, f i b r e digestion i n the pig i s not as e f f i c i e n t as that of a ruminant consuming the same feedstuff ( F a r r e l l and Johnson, 1972; Kennelly et a l . , 1981; Keys and DeBarthe, 1974; Low, 1985). Processing wastes often contain a considerable amount of l i g n i n , an i n d i g e s t i b l e , unfermentable component of the c e l l wall which also prevents the fermentation of the c e l l u l o s e to which i t i s co-bonded within the c e l l wall (Theander and Aman, 1984). Several other components may be found i n substantial amounts in fibrous feedstuffs. Cutin i s a waxy substance composed of complex polymers of hydroxy f a t t y acids that forms a protective water-resistant coating on the outside of leaves and seed h u l l s (Martin and Juniper, 197 0) , and i s also i n d i g e s t i b l e by non-ruminants, as are other waxes found in association with cutin (Theander and Aman, 1984). Tannic material can also reduce the DMD of a feedstuff (Butler et a l . , 1986). 9 1.1.2. Dry matter content of by-products By-products of f r u i t processing have a low dry matter content, ranging from 25-50%, which makes the cost of transportation per unit of nutrient very high (NRC, 1983). Thus, producers who u t i l i z e these by-products can economically haul the waste only a short distance. To compensate for t h i s , the by-product i s normally made ava i l a b l e free of charge although the producer must bear the cost of transportation. 1.1.3. Preservation of processing by-products High-moisture waste by-products are highly perishable and therefore must be fed immediately or preserved i n some way. F r u i t processing wastes i n p a r t i c u l a r are very perishable. For example, mould growth begins on raspberry pomace a f t e r 24 hours at room temperature (Buckley, 1985). Freezing, drying and e n s i l i n g are a v a i l a b l e methods of avoiding spoilage. However, these are expensive processes and, i n general, are uneconomical. By-products are most economically fed "as i s " by the li v e s t o c k producer. Some by-products, because of t h e i r high nutrient content or d i g e s t i b i l i t y , can be processed economically before being sold as feed ingredients. For instance, in some areas of the U.S.A. high qua l i t y by-products such as 10 tomato and apple pomace are oven-dried, ground, and sold as energy ingredients i n prepared dog foods or dairy c a t t l e concentrate rations (Muller et a l . , 1984). In hot, dry areas such as C a l i f o r n i a , tomato pomace i s sundried. 1.1.4. Season of a v a i l a b i l i t y of processing wastes Most processing wastes are av a i l a b l e only during c e r t a i n times of the year, normally l a t e summer and f a l l (NRC, 1983). To u t i l i z e by-products e f f e c t i v e l y , the li v e s t o c k producer must have a f l e x i b l e feeding system which allows the incorporation of the waste product into the rat i o n during the periods when i t i s av a i l a b l e . 1.1.5. Contamination The p o s s i b i l i t y of contamination with pesticides, or 9 with dangerous objects such as metal or glass should be assessed before a producer considers incorporating the by-product into a rat i o n . 1.2. U t i l i z a t i o n of processing wastes i n North America 1.2.1. United States Katsuyama (1973) as quoted by the NRC (1983), calculated that the annual production of waste from 11 vegetable processing i n the U.S. was 3,891,000 tonnes. Of t h i s t o t a l , 2,908,000 tonnes, mostly from corn processing, were used as animal feed while 983,000 tonnes were dumped. Of the t o t a l production of f r u i t processing wastes, 3,243,000 tonnes were u t i l i z e d i n feed, 2,721,000 tonnes of t h i s being c i t r u s waste, while 471,000 tonnes of s o l i d waste were dumped. As c i t r u s processing waste i s the p r i n c i p a l product u t i l i z e d , i t i s c l e a r that much of the waste from other types of f r u i t processing i s not being used. It i s suggested (NRC, 1983) that the degree of u t i l i z a t i o n of by-products has increased s u b s t a n t i a l l y i n recent years, given the continuing r e s t r i c t i o n s on dumping of waste products and the growing i n t e r e s t of liv e s t o c k producers i n the use of these products, although no s t a t i s t i c s were avail a b l e to confirm t h i s . 1.2.2. Canada Very few data are available on the production and u t i l i z a t i o n of Canadian processing wastes. Muller et a l . (1984) estimated that 19,600 to 38,260 tonnes of tomato processing wastes (tomato pomace) were produced i n Ontario and Quebec i n 1980 and that much of t h i s waste pomace was disposed of i n l a n d f i l l s i t e s . However, the authors also stated that many processing plants were arranging for 12 u t i l i z a t i o n of the pomace by feedlot operators, although the actual proportion of the waste being fed was not stated. No d e t a i l e d data are available on the extent of u t i l i z a t i o n of f r u i t processing wastes i n Canada. However, i t i s expected that usage would be less here than i n the U.S., as t h i s country has a smaller processing industry, and longer distances between processors and farms i n some areas. I t i s also expected that, as i n the U.S., l a n d f i l l r e s t r i c t i o n s and environmental control measures w i l l become increasingly stringent, thereby encouraging alternate uses for these by-products . 1.2.3. B r i t i s h Columbia In B.C., r e l a t i v e l y small amounts of f r u i t processing wastes are produced, and t h i s occurs i n two main areas of the province, the Okanagan and the Lower Mainland (Table 2.1). Tree f r u i t and winery pomaces are produced i n the Okanagan Valley as the by-products of j u i c e , cider and wine production. Some of the apple and pear pomace i s used as c a t t l e feed by l o c a l feedlot owners, who incorporate i t fresh into t h e i r rations (Macintyre, 1987) . Most of the winery pomace i s used as f e r t i l i z e r or c a t t l e feed (Beames and Taylor, 1988). In the Fraser Valley, the main f r u i t s grown and processed are raspberries, strawberries and 13 Table 2 .1 . Use of f r u i t and vegetable process ing wastes i n B r i t i s h Columbia i n 1988. (Beames and T a y l o r , 1988) Source Location Quant. End use ( t * y r " 1 ) 1 F r u i t By-products Beaven's Cannery Summerland 40 L a n d f i l l B.C. F r u i t Packers Kelowna 750 L a n d f i l l Jordan/Ste. Michelle C e l l a r s Ltd. Surrey 1500 Fert., feed Monashee Co-op n/a 500 L a n d f i l l Okanagan-S imilkameen Co-op Oliver 3000 L a n d f i l l Pioneer Portion Pak Ltd. Richmond 2500 L a n d f i l l Sun-Dew Foods Ltd. Okan. F a l l s 1500 Catt l e feed Sun-Rype Products Ltd. Kelowna 5000 Cattle feed P a c i f i c F r u i t and Chilliwack 500 Extraction Concentrate Ltd. of raspberry seed o i l Winery Pomace Andres Wines Ltd. Port Moody 200 L a n d f i l l Beaupre Wines Ltd. Langley n/a n/a Brights House of Wine Oliver 1500- Fert., feed 2000 Casabello Wines Ltd. Penticton 2-500 F e r t i l i z e r Calona Wines Ltd. Kelowna 850 Fert., feed Jordan/Ste. Michelle C e l l a r s Ltd. Surrey 450 F e r t i l i z e r A l l Estate Wineries 27 F e r t i l i z e r Metric tonnes per year. 14 Table 2 .1 . con ' t . Use of f r u i t and vegetable processing wastes i n B r i t i s h Columbia i n 1988. (Beames and T a y l o r , 1988) Source Location Quant. End use ( t * y r _ 1 ) Vegetable By-products Al's Salad King Burnaby 1500 L a n d f i l l Armstrong Food and Produce Ltd. Armstrong 50 Pig feed B.C. Coast Vegetable Co-op Assn. Richmond 17000 Catt l e feed Cloverdale Lettuce and Vegetable Co-op Surrey 4000 L a n d f i l l Central Foods Ltd. Vancouver 150 L a n d f i l l Empress Foods Ltd. Clearbrook 400 F e r t i l i z e r Four Seasons Food Ltd. Vancouver 300 L a n d f i l l Fraser Valley Foods Ltd. Chilliwack 2000 F e r t i l i z e r Sardis 5500 Catt l e feed, l a n d f i l l Fraser Valley Mushroom Growers Co-op Assn. Langley 150 L a n d f i l l Nalley's (Canada) Ltd. New West. 100 Pig feed Royal C i t y Foods Ltd. Burnaby n/a Cattle feed, l a n d f i l l Snowcrest Packers Ltd. Abbotsford n/a L a n d f i l l 15 blueberries, and a c e r t a i n amount of pomace from each type of berry i s produced during j u i c e and concentrate production. Raspberry pomace i s currently being sold for o i l extraction, but the 70 to 100 tonnes of strawberry and blueberry pomaces produced annually are not u t i l i z e d . The small amount of other f r u i t processing wastes produced i n the Fraser Valley i s dumped i n l a n d f i l l s . Waste from the processing of fresh vegetables (Table 2.1) i s put back onto f i e l d s as f e r t i l i z e r or hauled to l a n d f i l l s . The exception i s sweet corn processing waste, which i s fed to c a t t l e . C u l l fresh or stored vegetables are almost a l l purchased by l o c a l farmers as c a t t l e feed. 2. NUTRITIONAL EVALUATION OF RASPBERRY POMACE 2.1. The production of pomace Raspberry pomace i s the waste material from the production of raspberry j u i c e . During the raspberry harvest, excess fresh product i s frozen i n large drums and stored at the processing plant. A f t e r the harvest season i s completed, the pomace i s processed into j u i c e and concentrate. In the production of j u i c e , the berries are thawed, poured into a large vat, and brought to an average temperature of between 40 and 50 C, but not exceeding 55 C, 16 for one to two hours. Pectinase i s then added to disrupt the pectin and thus increase the extractable portion of the berry. Rice h u l l s are added as a press-aid, an addition which i s e s p e c i a l l y important for very r i p e , s o f t berries which do not provide enough resistance f o r e f f i c i e n t pressing (Maclntyre, 1987). The r i c e h u l l s , being p r a c t i c a l l y i n e r t chemically, provide l i t t l e but structure to the berry mixture. The proportion of h u l l s i s determined subjectively, with the r e s u l t that s i g n i f i c a n t v a r i a t i o n between batches occurs. The berries are then mechanically compressed. More pectinase i s added to the residue and the mixture i s centrifuged. Depending on the thoroughness of the i n i t i a l extraction, the r e s u l t i n g pomace may be mixed with water and re-extracted. The raspberry pomace, consisting of seeds, pulp and r i c e h u l l s , i s then frozen i n large drums i f i t i s to be sold l a t e r , or dumped in a l a n d f i l l i f a market i s not avai l a b l e . 2.2. Chemical composition of raspberry pomace 2.2.1. Energy, protein and f i b r e content of pomace Buckley (1985) determined the nutrient composition of raspberry pomace. The pomace has a very high gross energy l e v e l (5400 kcal*kg - 1) (D.M. ba s i s ) . This exceeds the gross 17 energy l e v e l of corn (4400 kcal*kg ) which i s considered the highest energy feed grain. The energy l e v e l i s due to the high percentage of o i l i n the seed, averaging 13.9% (D.M. b a s i s ) . The pomace has an average protein l e v e l of 12% (D.M. b a s i s ) . However, i t has been suggested that much of the protein may be unavailable due to binding by l i g n i n , tannins and f i b r e , as i s the case with other seed-containing processing by-products such as tomato pomace (Buckley, 1985) . Acid detergent f i b r e l e v e l of the seeds i s high (55.1%), which would suggest that DMD for non-ruminants would be low (Buckley, 1985). The raspberry pomace used in Buckley's work did not contain r i c e h u l l s . 2.2.2. Amino acid composition The amino acid composition of raspberry pomace has been determined by Papke (1983). The l e v e l s of lysine, methionine and threonine i n the pomace are much higher than the l e v e l s of the same amino acids i n average Canadian feed grade barley, in d i c a t i n g that raspberry pomace i s a p o t e n t i a l l y good source of these l i m i t i n g amino acids (NRC, 1982) . The o v e r a l l amino acid p r o f i l e of raspberry pomace i s better than that of feed grade barley. 18 2.2.3. Mineral composition The mineral composition of raspberry pomace (Buckley, 1985) and of r i c e h u l l s (NRC, 1982) i s shown i n Table 2.2. Because the raspberry pomace analyzed by Buckley did not contain r i c e h u l l s , the mineral composition of r i c e h u l l s i s included for comparison. Raspberry pomace does not meet the requirements of growing and f i n i s h i n g pigs for calcium and phosphorus (NRC, 1988). Rice h u l l s are lower i n calcium and phosphorus than raspberry pomace, so t h e i r addition has a d i l u t i n g e f f e c t on the already low calcium and phosphorus l e v e l s i n pomace. However, a l l cereal grains commonly used in swine d i e t s are d e f i c i e n t i n these minerals, and as a consequence supplementation i s a standard procedure. Raspberry pomace i s very high i n potassium (5.08%) but i t i s not anticipated that t h i s w i l l cause any n u t r i t i o n a l problems i n swine (Underwood, 1981). The presence of r i c e h u l l s i n pomace would d i l u t e the potassium l e v e l to some extent, although even h u l l s contain well above the NRC (1988) recommendation for t h i s mineral. 2.2.4. Composition of raspberry seed o i l Raspberry seed o i l contains 97.5% neutral l i p i d s , which contain mainly the unsaturated l i n o l e i c and l i n o l e n i c acids (Pourrat and Carnat, 1981). T y p i c a l l y , plants which store. 19 T a b l e 2 . 2 . M i n e r a l c o m p o s i t i o n o f r a s p b e r r y pomace A and r i c e h u l l s 2 • m i n e r a l r a s p b e r r y r i c e h u l l s pomace C a 0.17% 0.09% P 0.09% 0.08% K 5.08% 0.34% Mg 0.19% 0.83% Mn 1 5 . 1 ppm 3 3 3 . 3 ppm ' C u n / a 4 ppm Z n n / a 1 8 . 1 ppm B u c k l e y , 1 9 8 5 . Pomace d i d n o t c o n t a i n r i c e h u l l s . 2 N R C , 1 9 8 2 . 20 energy within the seed kernel as l i p i d material do so by producing large amounts of a l i m i t e d range of f a t t y acids (Gurr, 1983). The remaining 2.5% l i p i d material i n the pomace i s unsaponifiable material, and consists of a l i p h a t i c alcohols, carotenoid pigments (mainly beta-carotene), s t e r o l s and xanthophyll pigments. Raspberry seed o i l has antioxidant properties although the compound responsible for t h i s i s not known. O i l l e f t at room temperature or i n sunlight does not become rancid. Although the o i l contains approximately 40 mg*kg - 1 tocopherols (Vitamin E compounds), t h i s amount i s too small to be responsible for the antioxidant properties (Pourrat and Carnat, 1981). If pomace were dried and ground before being fed to livestock, the o i l q u a l i t y would not deteriorate during storage, which i s a problem encountered with other seed o i l s . 2.2.5. Composition of r i c e h u l l s Rice h u l l s are the highly fibrous outermost layer of the r i c e grain. They are added to the l i q u i f i e d raspberries during j u i c e production at about 12% of the dry matter of the mixture as a "press-aid". As r i c e h u l l s contain 72% acid detergent f i b r e and 16% l i g n i n (NRC, 1982), t h e i r addition serves only to reduce the feeding value of the 21 pomace. For both ruminants and non-ruminants, t h e i r d i g e s t i b i l i t y i s p r a c t i c a l l y zero (NRC, 1982). 2.3 . Short and longterm preservat ion of pomace 2.3.1. Chemical preservation Because grey mould growth on raspberry pomace begins within 24 hours of pomace production, some type of preservation i s necessary i f the pomace i s not to be fed immediately. Buckley (1985) investigated methods of preserving pomace for short and long term storage. Ammonium hydroxide and hydrogen peroxide were assessed for t h e i r effectiveness as short term preservatives. Each chemical was added to a separate 200 gram sample of pomace. The sample was then mixed well and stored i n an a i r t i g h t container at 22 C. After s i x days, even the highest l e v e l of i n c l u s i o n of ammonium hydroxide (0.6% of sample wet weight) was i n e f f e c t i v e i n i n h i b i t i n g mould growth. Hydrogen peroxide was a more e f f e c t i v e short term preservative. At a l e v e l of 1600 ppm, i t e f f e c t i v e l y i n h i b i t e d mould growth, although at lower l e v e l s i t was not as e f f e c t i v e . 22 2.3.2. E n s i l i n g Buckley (1985) examined e n s i l i n g as a method of long term storage. Pomace was ensiled alone and with grass i n combination with various additives for 8 weeks. Dried whey, Silage Science (a commercial mixture of organic acids), and propionic acid were a l l i n e f f e c t i v e i n c o n t r o l l i n g mould growth i n both types of s i l a g e . Both pomace and grass-pomace silages were most e f f e c t i v e l y preserved with 0.5% and 1% formic acid (wet weight b a s i s ) . 2.4. In v i t r o (rumen f lu id ) dry matter d i g e s t i b i l i t y (DMD) of pomace Buckley (1985) incubated raspberry pomace in rumen f l u i d to determine the i n v i t r o DMD. P r i o r to incubation the pomace was subjected to drying at eit h e r 50 C or 1 0 6 C, grinding (1mm screen), cooking with added water at 80 C for four hours, and e n s i l i n g . The r e s u l t s are presented i n Table 2.3. The highest i n v i t r o DMD was observed with the ground pomace dried at 50 C, followed c l o s e l y by the whole ensiled pomace samples. The poorest i n v i t r o DMD, which was equal to that of the control, was observed with the ground pomace that was dried at 106 C. Buckley (1985) suggested that of the treatments with the highest i n v i t r o DMD (Table' 2.3), 23 Table 2 .3 . In v i t r o dry matter d i g e s t i b i l i t y (IVDMD) i n rumen f l u i d of raspberry pomace subjected to var ious heating and gr ind ing treatments. (Buckley, 1985) Treatment IVDMD S. D.1 (%) control (whole, dried at 50 C) 16.0 3.8 1 mm ground, dried at 50 C 28.2 4.5 1 mm ground, dried at 106 C 15.7 3.6 whole, cooked at 80 C 20.6 0.7 whole, ensiled 26.4 0.9 Standard deviation. e n s i l i n g would be the most economical way to increase the DMD of raspberry pomace for ruminants as i t also preserves the pomace. When whole and ground raspberry seeds were incubated i n rumen f l u i d , i n v i t r o dry matter d i g e s t i b i l i t i e s were respectively 3.76% and 11.53%. The poor d i g e s t i b i l i t y of the pomace can be p a r t i a l l y a t t r i b u t e d to the low d i g e s t i b i l i t y of the in t a c t seeds. 3. NUTRITIONAL EVALUATION OP SIMILAR BY-PRODUCTS Because so l i t t l e information i s availa b l e on the n u t r i t i o n a l c h a r a c t e r i s t i c s and feeding value of raspberry pomace, a b r i e f examination i s made here of two other by-products of f r u i t and vegetable processing that have s i m i l a r i t i e s to raspberry pomace. Although many by-products of food processing are being u t i l i z e d as live s t o c k feed, very few of these can be compared with raspberry pomace. Two such by-products are grape pomace and tomato pomace. The most important s i m i l a r i t y between these by-products and raspberry pomace i s the high energy value which i s found within the hard-coated seeds. Other c h a r a c t e r i s t i c s common to many processing by-products, including these, are a high f i b r e l e v e l and a moderate crude protein content. 25 3.1. Winery pomace Winery pomace, also known as grape pomace or grape marc, consists of grape skins, seeds and st a l k s , and i s the waste product from j u i c e , wine and alcohol production. Most research with t h i s by-product has concentrated on ruminants, although a small amount of work has been done with non-ruminants . 3.1.1. Nutrient content of winery pomace Winery pomace contains 12.3% crude protein, 8.5% crude fat and 35.4% crude f i b r e (D.M.basis) (Hadjipanayioutou and Louca, 1976) . 3.1.2. DMD of winery pomace by growing pigs Rose and F a r r e l l (1984) formulated d i e t s for growing pigs using dried, hammermilled pomace at l e v e l s of 5, 10, 15 and 20% of basal d i e t dry matter. They found that as the l e v e l of pomace i n the d i e t increased, growth rate and feed e f f i c i e n c y declined. Apparent DMD of the t o t a l d i e t also declined, from 80.4% DMD with 5% pomace i n the d i e t to 65.0% DMD at a l e v e l of 2 0% pomace. The authors suggested that winery pomace could be useful i n growing pig di e t s at le v e l s of 10% to 15%. 26 3.1.3. Protein d i g e s t i b i l i t y of winery pomace by dairy calves Hadjipanayioutou and Louca (1976) found that the d i g e s t i b i l i t y by dairy calves of the protein i n winery pomace was only 19.5%. 3.2. Tomato pomace Tomato pomace i s the residue from the production of tomato j u i c e or paste, and consists of tomato seeds, skins and pulp. Much has been published on t h i s by-product, including extensive chemical analyses, and several d i g e s t i b i l i t y and feeding t r i a l s with sheep, goats and beef animals. No studies u t i l i z i n g the pomace as a feedstuff for non-ruminant animals were found. 3.2.1. Nutrient content of tomato pomace Tomato pomace contains on average 19.8% crude protein (D.M. basis) (Hinman et a l . , 1978), 11.5% crude f a t and 55.9% crude f i b r e (D.M. basis) (Tsatsaronis and Boskou, 1975). 27 3.2.2. Protein and fat d i g e s t i b i l i t y of tomato pomace for ruminants Although d i g e s t i b i l i t y t r i a l s with ruminants give l i t t l e i n d i c a t i o n of the quality of a feedstuff for non-ruminant animals, a b r i e f examination of protein and fat d i g e s t i b i l i t y of tomato pomace by ruminants i s given below. Jayal and Johre (1976) fed sun-dried, ground tomato pomace to h e i f e r s , sheep and goats and found that the protein of the pomace was not as well digested as the protein i n an average concentrate mixture. Hinman et a i . (1978) fed varying proportions of fresh and dried pomace to sheep, with the remainder of the r a t i o n consisting of a l f a l f a hay. They found the d i g e s t i b i l i t y of the protein i n fresh pomace (by difference) to be 71.9%, but that of dried pomace, when provided as the t o t a l ration, to be only 58.5% d i g e s t i b l e . In a t r i a l with sheep, Hinman et a l . (1978) found the d i g e s t i b i l i t y of the fat in tomato pomace to be between 76% and 86%. 4 . THE USE OF TOTAL DIETARY FIBRE METHODOLOGY I N THE EVALUATION OF HIGH-FIBRE FEEDS FOR NON-RUMINANTS Because f i b r e constitutes a sizeable component of most f r u i t and vegetable processing by-products, including 28 raspberry pomace, a comprehensive analysis of the quantity and composition of the f i b r e was considered e s s e n t i a l i n order to accurately describe t h i s by-product. This was f e l t to be e s p e c i a l l y important because the by-product was being examined as a p o t e n t i a l feedstuff for non-ruminants. The most widely used methods for determining f i b r e i n non-ruminant d i e t s (crude f i b r e , neutral detergent fibre) are considered inadequate by many researchers, and for t h i s reason, a more complete method of f i b r e analysis was sought for t h i s research. 4.1. Limitations of the crude f i b r e method For over 100 years, crude f i b r e has been the standard f i b r e determination method used i n non-ruminant n u t r i t i o n . This method measures crude f i b r e as the residue a f t e r sequential digestion of the sample i n d i l u t e acid and d i l u t e a l k a l i solutions. However, a highly variable amount of the f i b r e i s extracted with t h i s procedure. Much of the l i g n i n and hemicellulose, and a l l of the soluble f i b r e components are dissolved, and thus are not included i n the insoluble residue (Robertson and Van Soest, 1981). Therefore, for non-ruminant n u t r i t i o n , t h i s method cannot be considered s u f f i c i e n t l y accurate. 29 4 . 2 . L imi ta t ions of the neutra l detergent f i b r e method The neutral detergent f i b r e method was developed as a replacement for the crude f i b r e method (Goering and Van Soest, 1970). I t recovers a l l of the insoluble f i b r e components, including c e l l u l o s e , hemicellulose, tannin, c u t i n and l i g n i n quantitatively, and i s therefore an improvement over crude f i b r e f or both non-ruminant and ruminant n u t r i t i o n . However, the neutral detergent solution dissolves the soluble f i b r e components, including pectin, gum, beta-glucans and mucilage. These components are p a r t i c u l a r l y important i n non-ruminant n u t r i t i o n because they are fermented almost completely i n the hind gut, producing v o l a t i l e f a t t y acids which contribute to dietary energy ( S p i l l e r and Amen, 1976). By contrast, the insoluble f i b r e components are fermented to a lesser extent, depending on the l i g n i f i c a t i o n of the f i b r e . For t h i s reason, the neutral detergent f i b r e method i s not completely s a t i s f a c t o r y for f i b r e determination i n non-ruminant d i e t s . 4 . 3 . T o t a l d ie tary f i b r e method Because of the d e f i c i e n c i e s i n the e x i s t i n g f i b r e methods, the need has been recognized for some time for a methodology which quanti t a t i v e l y recovers a l l of the components considered to constitute dietary f i b r e . "Total 30 dietary f i b r e " i s a new methodology which more accurately defines and quantifies the f i b r e i n non-ruminant d i e t s . It i s defined as "the sum of l i g n i n and polysaccharides that are not hydrolyzed by the endogenous secretions of the human digestive t r a c t " (Southgate, 1981). Total dietary f i b r e (Prosky et a l . , 1984) recovers a l l of the components of the d i e t defined as f i b r e , including the insoluble f i b r e components, c e l l u l o s e , l i g n i n , tannin, c u t i n and hemicellulose as well as the soluble components v i z . pectin, gum and mucilage. Because of i t s completeness, i t was considered to be the method of choice for t h i s research. Although several d i f f e r e n t t o t a l dietary f i b r e methods have been developed (Asp et a l . , 1983; Prosky et a l . , 1984; Schweitzer and Wursch, 1979; Southgate, 1969; Theander and Aman, 1982), a l l are based on an i n i t i a l enzymatic digestion of the sample which mimics phys i o l o g i c a l digestion by endogenous enzymes. Samples are digested sequentially with amylase, peptidase and amyloglucosidase. Following the procedural step of enzymatic digestion, the methods developed over the past 10 years have diverged into two d i f f e r e n t approaches to the determination of dietary f i b r e . The f i r s t i s a rapid, gravimetric methodology which i s suitable for routine laboratory analysis of f i b r e , and which gives a t o t a l weight of both insoluble and soluble f i b r e 31 components (Prosky et a l . , 1984; Asp et a l . , 1983). These methods are best suited for rapid, quantitative determination of dietary f i b r e . The method of Asp et a l . (1983) contains a modification which allows the separate determination of insoluble and soluble components, i f t h i s i s desired. Because of t h i s , the method of Asp et a l . (1983) was chosen for t h i s thesis work. The second methodology i s more complicated (Southgate, 1969; Schweizer and Wursch, 1979; Theander and Aman, 1982). I t involves enzymatic digestion of the sample as with the method of Prosky (1984), but, following t h i s step, f i b r e components are degraded into t h e i r component sugars and measured qua n t i t a t i v e l y e i t h e r with gas l i q u i d chromatography or c o l o r i m e t r i c a l l y . This methodology i s much more time-consuming and as a r e s u l t would be most appropriate for research which required a detailed determination of each f i b r e component. 5 . THE USE OF RATS AS MODELS FOR STUDIES ON THE NUTRITION OF PIGS Laboratory rats have been used extensively as models in studies on the n u t r i t i o n of pigs. Rats are inexpensive, they have small food requirements which s i m p l i f i e s the preparation of experimental d i e t s , and they grow quickly, 32 allowing the rapid c o l l e c t i o n of experimental data. As well, rats w i l l consume n u t r i t i o n a l l y inadequate d i e t s for a longer period than pigs, allowing the d i r e c t t e s t i n g of feeds of low d i g e s t i b i l i t y . The d i g e s t i b i l i t i e s of protein and energy by pigs are close to values obtained with rats. Just et a l . (1977) (as quoted by Eggum and Beames, 1986) found that the energy d i g e s t i b i l i t y of 57 feedstuffs fed to pigs and rats compared very well, although rats consistently showed 1% to 4% lower d i g e s t i b i l i t y . Eggum (1973) fed 15 di e t s to pigs and rats, and found that protein d i g e s t i b i l i t y was generally s i m i l a r between the two species, but that some values d i f f e r e d s i g n i f i c a n t l y . Other reports have supported t h i s finding (Eggum and Beames, 1986). This suggests that, while d i g e s t i b i l i t y of a protein by rats generally w i l l c l o s e l y predict the d i g e s t i b i l i t y of feedstuffs by pigs, absolute values are sometimes s l i g h t l y at variance. 33 E X P E R I M E N T A L The experimental work of t h i s thesis can be separated into two d i s t i n c t areas of research; determination of the chemical composition of raspberry pomace by laboratory analyses, and measurement of i t s feeding value i n animal t r i a l s . Standard a n a l y t i c a l methods were used for protein, amino acids, f a t , soluble carbohydrates and ash determination. Total dietary f i b r e methodology (Prosky et a l . , 1984), and the Goering and Van Soest (1970) forage f i b r e methods were used to p a r t i t i o n the f i b r e component of the pomace. The res u l t s of chemical analyses are presented in Chapter 4. Four animal t r i a l s were undertaken to estimate the feeding value of the pomace for non-ruminants (Chapters 5 and 6) . In the f i r s t t r i a l , the dry matter, protein, fat and energy d i g e s t i b i l i t i e s of rations containing 4 0% of ground or unground pomace, and of ground and unground pomace (by difference), were determined with growing male pigs. In the second t r i a l , the DMD of rations which contained pomace at 0% to 100% of the d i e t (excluding minerals and vitamins) was determined with growing male rats, using chromic oxide as an in d i g e s t i b l e marker i n the d i e t s . In a t h i r d t r i a l , the true d i g e s t i b i l i t y , b i o l o g i c a l value and net u t i l i z a t i o n 34 of the protein of pomace, and the influence of fineness of grinding and of heating on these parameters, were investigated using male weanling rats as models for growing pigs. F i n a l l y , the growth rate and feed e f f i c i e n c y of growing male rats fed varying l e v e l s of raspberry pomace i n a balanced r a t i o n were measured. 35 CHEMICAL ANALYSIS OF RASPBERRY POMACE 1 . INTRODUCTION In examining raspberry pomace as a p o t e n t i a l feedstuff for non-ruminants, i t was f e l t that i t was inadequate to determine only the feeding q u a l i t y of t h i s processing by-product through animal t r i a l s , but that i t was es s e n t i a l also to undertake a thorough analysis of i t s physical and chemical composition. The methods used i n these analyses, and the r e s u l t s obtained, are presented here. Most by-products of f r u i t and vegetable processing are very fibrous and, as such, are only p a r t i a l l y digested by non-ruminant animals. Thus, when defining a by-product such as raspberry pomace i t i s important not only to quantify those components which generally are of a medium to high a v a i l a b i l i t y to non-ruminant animals, such as protein, fat and simple carbohydrates, but also to characterize the components of the f i b r e f r a c t i o n which are of low and va r i a b l e a v a i l a b i l i t y to these animals, and which influence the a v a i l a b i l i t y of other nutrients. For t h i s reason, many of the analyses i n t h i s thesis were undertaken to more accurately quantify the f i b r e component of the pomace. Two methods of f i b r e analysis were used; t o t a l dietary f i b r e (Prosky et a l . , 1984) and neutral detergent fibre. (Goering 36 and Van Soest, 1970). The f i b r e components - c e l l u l o s e , l i g n i n , tannin, and cutin were also measured using the methods of Goering and Van Soest (197 0). The raspberry pomace samples used for these analyses contained r i c e h u l l s , which are added to the be r r i e s during the j u i c e extraction process as an i n e r t press-aid. Raspberry pomace analysed by other workers (Buckley, 1985; Papke, 198 3) did not contain r i c e h u l l s . P a r t l y because of t h i s , there i s a s i g n i f i c a n t v a r i a t i o n i n a n a l y t i c a l r e s u l t s between e a r l i e r analyses and those presented i n t h i s t h e s i s . 2. M A T E R I A L S AND METHODS To obtain a measure of the v a r i a b i l i t y i n the composition of raspberry pomace, one kilogram samples of pomace were c o l l e c t e d by s t a f f of the P a c i f i c F r u i t and Concentrates processing plant i n Chilliwack, B.C. on 10 days of j u i c e production during December 1986 and February 1987. Samples were frozen immediately a f t e r c o l l e c t i o n and stored at -20 C. A subsample was taken from each of the 10 pomace samples c o l l e c t e d . These were f i n e l y ground (in wet form) i n a ceramic b a l l m i l l . For t h i s grinding, approximately 65 grams of wet pomace, 150 ml of water and 4 0 medium c y l i n d r i c a l ceramic b a l l s (15mm diameter by 15mm length) were put into a 1 L b a l l m i l l j a r which was rotated for 18 37 hours. This ground the pomace to a talcum-powder fineness. The ground subsamples were then s h e l l - f r o z e n and dried to approximately 95% D.M. i n a Labconco freeze-drier and stored at -20 C u n t i l required for analysis. 2 .1 . Standard a n a l y s e s Dry matter determinations were made by drying the sample i n aluminum drying dishes for 24 hours at 100 C. Nitrogen content of pomace was determined by the macro-Kjeldahl method (AOAC, 198 0), and converted to crude protein by multiplying by 6.25. The c a t a l y s t mixture (Kjelpak) contained K 2S0 4, CuS0 4 and pumice. Ammonia was trapped i n a 4% boric acid solution containing as an indicator Methyl red and Bromo Cresol green. This solution was back t i t r a t e d with 0.1 N hydrochloric acid, and t o t a l N was calculated from the volume required to neutralize the solution. Crude f a t was measured using the Goldfisch ether extraction method (AOAC, 1975). Samples were extracted for four hours with anhydrous d i e t h y l ether. Gross energy was determined with a Gallenkamp automatic adiabatic bomb calorimeter following the method outlined in the technical manual. 38 Ash was measured as the weight of residue following ashing of the samples i n aluminum dishes for 12 hours at 500 C i n a muffle furnace. Water-soluble carbohydrates were determined by the method of the Ministry of Agriculture, F i s h e r i e s and Food, England (1973). The water-extractable components were suspended i n solution by shaking the sample with water, then f i l t e r i n g the solution. Anthrone reagent, which reacts with glucose and other sugars to produce a blue-green colour, was added to the f i l t r a t e , and the samples heated i n a b o i l i n g water bath for 2 0 minutes. To ensure accuracy, standard curves were made with both glucose and fructose, and samples were read with each set of standards. The degree of colour development was read at 620nm absorbance using a Shimadzu colorimeter. Anthrone reagent has been used most frequently to measure the l e v e l of glucose i n solutions. However, i t has been found that fructose and sucrose are also measured accurately with t h i s method (Morris, 1948). There are several problems associated with the use of the anthrone reagent i n the determination of soluble hexose sugars. Acid strength, temperature of the solution during colour development and time of colour development can a l l influence the reading obtained (Johanson, 1954). Pentose sugars, i f present i n s i g n i f i c a n t amounts, can i n t e r f e r e with the 39 determination of hexose sugars under c e r t a i n conditions. In t h i s analysis, care was taken to r e p l i c a t e the ideal experimental conditions as were recommended i n the l i t e r a t u r e i n order to minimize interference. Further, raw raspberries contain no appreciable amount of pentose sugars (Wrolstad and Schallenberger, 1981). Amino acid composition was determined by ion-exchange chromatography with a Beckman 63 00 autoanalyzer following acid hydrolysis of the samples and s t a b i l i z a t i o n of the solu t i o n i n sodium c i t r a t e buffer. Samples were acid-hydrolyzed by suspending the sample i n 3 N HC1 and autoclaving for 17 hours at 121 C and 15 PSI. Hydrolyzed amino acids were then separated i n an ion-exchange column. In t h i s procedure, the column e f f l u e n t i s mixed with ninhydrin colour reagent and the blue colour produced by the amino acid-ninhydrin reaction i s read at 570 nm. The amino acids pr o l i n e and hydroxyproline produce a yellow colour when mixed with ninhydrin, and are read at 44 0 nm (Blackburn, 1968). Neither cystine nor tryptophan were measured. 2 . 2 . Total dietary f i b r e method Total dietary f i b r e was determined using the method of Prosky et a l . (1984) and incorporating the modification of 40 Asp et a l . (1983) to separate t o t a l dietary f i b r e into soluble and insoluble dietary f i b r e components (see Appendix 1 for complete method). Because l i p i d content was greater than 8%, the dried and f i n e l y ground samples of pomace were fat-extracted p r i o r to analysis (using the Soxhlet extraction apparatus). Samples i n t r i p l i c a t e which had been suspended i n a buffer s o l u t i o n were then sequentially digested with a heat-stable alpha-amylase, a protease and f i n a l l y , an amyloglucosidase. The pH of the solution was adjusted before each digestion to ensure maximum enzyme a c t i v i t y . During digestion, samples were agitated at a l l times. To g e l a t i n i z e the starch, the amylase digestion proceeded i n a b o i l i n g water bath, while the other digestions occurred i n a 60 C bath. Immediately following the t h i r d digestion, the hot s o l u t i o n was f i l t e r e d through a dried, weighed f r i t t e d glass c r u c i b l e (medium porosity) containing C e l i t e 545 as a f i l t e r i n g a i d . The c r u c i b l e (containing the insoluble f i b r e component) was dried at 70 C overnight and reweighed. The f i l t r a t e from t h i s f i l t r a t i o n (containing the water-soluble f i b r e components i n solution) was q u a n t i t a t i v e l y c o l l e c t e d and transferred back to the o r i g i n a l beaker. The soluble f i b r e was then p r e c i p i t a t e d out of solution by the addition of four volumes of 95% 41 ethanol which had been pre-heated to 60 C. The p r e c i p i t a t e d soluble f i b r e was f i l t e r e d through a f r i t t e d glass c r u c i b l e as above. This f r a c t i o n was also dried overnight and reweighed the following day. Enzyme a c t i v i t y of the protease was tested using casein, and of the amylase and amyloglucosidase using pure corn starch. The presence of undesirable enzymes, including pectinase and beta-glucanase, was measured by running the analysis using pure pectin and pure beta-glucan. Blanks were run throughout the analysis to determine the residue from enzymes, and f i n a l sample weights were corrected for t h i s amount. A l l samples were corrected for the loss of C e l i t e on ashing. Insoluble and soluble f i b r e were calculated as the weight difference of the cru c i b l e s before and a f t e r f i l t r a t i o n , less the amount of the blanks and other corrections. The insoluble f i b r e f r a c t i o n (from the f i r s t f i l t r a t i o n ) contains c e l l u l o s e , hemicelluloses, l i g n i n , insoluble ash and the protein which i s not digested by the protease enzyme. To determine the fr a c t i o n s of insoluble ash and i n d i g e s t i b l e protein, one of the t r i p l i c a t e samples of insoluble f i b r e was ashed at 500 C for 12 hours, and the remaining two samples were transferred to f i l t e r paper and 42 subjected to N analysis. N i n these two samples was determined using the block digest wet ash method of Wall and Gehrke (1975). Samples were digested i n a soluti o n of concentrated s u l f u r i c acid and hydrogen peroxide, catalyzed by lithiu m s u l f a t e and selenium. N content was read c o l o r i m e t r i c a l l y using a Technicon Auto-analyzer I I . Insoluble protein and ash content were subtracted from the t o t a l weight of insoluble f i b r e . Insoluble f i b r e was also corrected for the fat content of the o r i g i n a l sample, the weight of the blank samples and the loss of C e l i t e when the sample was ashed. The soluble f i b r e f r a c t i o n contains pectin, gums, mucilages and a large component of ash. The ash was determined by ashing as above, and subtracted from the weight of soluble f i b r e . Soluble f i b r e was also corrected for the weight of the blank sample and loss of C e l i t e on ashing. 2.3 . Analys i s of f i b r e components using Van Soest forage f i b r e methods The f i b r e component of the pomace was pa r t i t i o n e d using the methods of Goering and Van Soest (1970). The samples were prepared for further analysis by i n i t i a l treatment with acid detergent f i b r e solution. The residue from t h i s 43 procedure was used for sequential l i g n i n and cutin analysis. Lignin was estimated as the loss i n sample weight following treatment with saturated permanganate solu t i o n . The residue from t h i s procedure (consisting of c e l l u l o s e , c u t i n and ash) was treated with 72% s u l f u r i c acid, and the loss i n weight following treatment determined to be c e l l u l o s e . The residue (cutin and acid-insoluble ash) was then ashed, and the further loss i n weight a f t e r t h i s step determined to be cut i n . Neutral detergent f i b r e residue was measured by ref l u x i n g the sample i n neutral detergent solu t i o n for 1 hour (Goering and Van Soest, 1970). 3. RESULTS AND DISCUSSION The raspberry pomace contained on the average 44.4% dry matter, and consisted of 87.8% raspberry seeds, pulp and trash and 12.2% r i c e h u l l s (D.M. basis) (Table 4.1). The use and composition of r i c e h u l l s has been discussed i n the l i t e r a t u r e review. The percentage of seeds i n the pomace has been reported to range from 57% to 7 6% of the dry matter of pomace without r i c e h u l l s (Buckley, 1985), with the remainder consisting of pulp and trash. Buckley (1985) observed that pomace from the processing of frozen f r u i t contains proportionately less pulp and more seed than pomace 44 Table 4 .1 . Composition of raspberry pomace containing r i c e h u l l s (100% D . M . b a s i s ) 1 . Pomace composition (N=5) Dry matter (N=4) Raspberry seeds, pulp and trash (% of D.M) Rice h u l l s (% of D.M.) Nutr ient composition (N=10) Crude protein Crude f a t 3 Soluble carbohydrates 4 Ash Total dietary f i b r e 5 Neutral detergent f i b r e 6 Gross energy (kcal*kg Average Range S. E.; (%) (%) 44.4 43.3-45.8 0. 52 87 . 8 85.8-89.9 0. 63 12.. 2 10.1-14.2 0. 63 10.0 9.1-12.3 0. 27 11.1 8.6-12.6 0. 37 7.4 5.2- 9.0 0. 39 4.1 3.2- 5.2 0. 21 59.5 51.6-64.6 1. 17 54.1 48.8-56.4 0. 64 5220 5130-5250 33 Appendix 3 contains complete tables of r e s u l t s . Kjeldahl N * 6.25. Goldfisch ether extraction method. Total soluble sugars by water-extraction and colorimetry. Enzymatic digestion method of Asp and Johansson (1981). Method of Goering and Van Soest (1970) . Bomb calorimetry. Standard error of the mean. 45 from fresh f r u i t . The freezing and thawing of the pomace appears to rupture the c e l l wall of the f r u i t , allowing more of the pulp to be extracted i n the j u i c e . This may be a factor i n the large v a r i a b i l i t y reported i n the proportion of seeds i n the pomace. 3.1 . Prote in and amino ac id composition of pomace The mean crude protein l e v e l of pomace used i n these experiments (10.0%) (Table 4.1) i s somewhat lower than the value of 11.9% obtained by Buckley (1985), which was probably due large l y to the d i l u t i o n of protein with the addition of r i c e h u l l s . Rice h u l l s , which contain 3.3% crude protein (NRC, 1982), are included at approximately 12% of the pomace dry matter. The crude protein l e v e l of raspberry pomace compares favourably with that of feed grade barley (NRC, 1982), which i s an energy feedstuff commonly used i n swine rations in western Canada. However, i t i s expected that the a v a i l a b i l i t y of pomace protein w i l l be poor, as i s the case with s i m i l a r processing by-products such as tomato pomace (Jayal and Johre, 1976) and winery pomace (Hadjipanayioutou and Louca, 1976). The amino acid p r o f i l e of raspberry pomace containing r i c e h u l l s i s given i n Tables 4.2 and 4.3 (as g of amino 46 Table 4.2. Amino ac id composition of raspberry pomace containing r i c e h u l l s (g a . a . per 100 g pomace D . M . ) 1 . (N=6) Average Range S.E.M. (g*100g _ 1 (g*100g _ 1) D.M.) D.M.) Lysine 0.57 0.49-0.59 0.014 Methionine 0.13 0.12-0.14 0. 002 Phenylalanine 0.44 0.39-0.45 0. 008 Arginine 0.64 0.61-0.67 0. 009 Leucine 0.77 0.70-0.80 0. 015 Isoleucine 0.47 0.42-0.50 0. 011 Tyrosine 0.26 0.23-0.28 0. 007 Hi s t i d i n e 0.29 0.26-0.32 0. 009 Aspartate 0.99 0.93-1.04 0. 020 Threonine 0. 37 0.34-0.40 0 . 008 Serine 0.48 0.45-0.51 0. 011 Glutamine 1.44 0.98-1.65 0. 087 Proline 0.45 0.41-0.46 0. 007 Glycine 0.53 0.49-0.55 0. 007 Alanine 0. 54 0.50-0.57 0. 010 Valine 0.50 0.43-0.54 0. 015 Measured as amino acid residues. 47 Table 4.3. Amino ac id composition of raspberry pomace containing r i c e h u l l s (g a .a per 100 g C P . ) 1 . (N=6) Average Range S. E.: (g*100g-l (g*100g-l) CP.) CP.) Lysine 5.47 5.26- 6.14 0. 11 Methionine 1.32 1.29- 1.37 0. 01 Phenylalanine 4.39 4.17- 4.68 0. 06 Arginine 6.41 5.97- 6.84 0. 11 Leucine 7.78 7.40- 8.28 0. 11 Isoleucine 4.72 4.42- 4.98 0. 07 Tyrosine 2 . 61 2.44- 2.79 0. 05 H i s t i d i n e 2 . 95 2.54- 3.12 0. 08 Aspartate 9.95 9.09-10.33 0. 17 Threonine 3 .77 3.65- 3.91 0. 04 Serine 4.86 4.37- 5.05 0. 10 Glutamine 14 . 53 9.57-15.98 0. 92 Proline 4.48 4.37- 4.66 0. 04 Glycine 5.32 5.18- 5.58 0. 05 Alanine 5.39 5.07- 5.62 0. 07 Valine 5.08 4.57- 5.42 0 . 12 Measured as amino acid residues. 48 acid per 100 g of pomace dry matter, and as g of amino acid per 100 g of pomace crude protein) . The values for a l l amino acids are considerably lower than those determined by Papke (1983) for raspberry pomace without r i c e h u l l s (see l i t e r a t u r e review). The pomace used herein contained 0.57% l y s i n e and 0.13% methionine. This compares with 1.0% l y s i n e and 0.26% methionine measured by Papke (1983). A s i m i l a r pattern i s observed with the ent i r e amino acid p r o f i l e . Some of the difference i s as a r e s u l t of the lower protein content of the pomace used i n the thesis work because of the protein d i l u t i o n by r i c e h u l l s . However, even considering the d i l u t i o n of protein, i t appears that the protein content of the pomace analysed by Papke (1983) was much higher than that of the pomace used i n t h i s work. This may have been the r e s u l t of changes to the j u i c e production process that had occurred since the previous analysis was done. The berries were extracted twice during the production of j u i c e when the samples for the present work were taken, whereas when the e a r l i e r analyses were done, only a single extraction was performed on the berries (Maclntyre, 1987). Because extraction i s now more complete, the r e s u l t i n g pomace has a much lower content of soluble c e l l components, including protein. 49 Based on the re s u l t s of Papke (1983), raspberry pomace i s a better source of the ess e n t i a l amino acids lysine, methionine and threonine than Canada No.l feed barley (NRC, 1982), although the average protein content of the two feedstuffs i s s i m i l a r . However, the amino acid p r o f i l e of the pomace used i n the present work i s very s i m i l a r to that of feed grade "barley. Further, the high l e v e l of f i b r e i n the pomace would be expected to adversely a f f e c t protein a v a i l a b i l i t y and thus to reduce i t s protein value below that of barley. 3.2. Fat and gross energy content of pomace Raspberry pomace contained on average 11.1% l i p i d (ether-extractable material) (Table 4.1) which i s found almost e n t i r e l y i n the seeds. The gross energy of the pomace, 5220 c a l * g - 1 (Table 4.1), i s very high as a re s u l t of the l i p i d content. I t exceeds the gross energy of corn, considered the highest energy cereal. However, as the l i p i d material i s contained almost exclusively i n the seed of the raspberry, i t would be expected to be unavailable to either non-ruminants or ruminants unless the hard seed coat was cracked p r i o r to i t s i n c l u s i o n i n a feed. Because raspberry pomace also contains a high percentage of f i b r e , which i s lar g e l y unavailable to non-ruminants, i t i s ess e n t i a l that 50 t h i s highly d i g e s t i b l e f a t be released i f t h i s by-product i s to be considered as a feedstuff for non-ruminants. 3 . 3 . Soluble carbohydrate content The amount of water-soluble carbohydrates i n raspberry pomace was, on average, 7.4% of the pomace dry matter (Table 4.1). This water-extractable f r a c t i o n includes non-s t r u c t u r a l sugars and fructosans. I t has been found to be considerably larger and quite variable i n raw raspberries; from 17.3% (Englyst, 1981) to 36% (Wrolstad and Schallenberger, 1981) but, as would be expected, i s large l y removed from the pomace during the mechanical extraction of j u i c e from the ber r i e s . The water-soluble carbohydrate f r a c t i o n i n raw raspberries contains on average 34.6% fructose, 31.3% glucose and 33.6% sucrose (Wrolstad and Schallenberger, 1981). I t i s not known whether raspberry pomace contains sugars in the same proportions. 3 . 4 . Composition of raspberry pomace f i b r e 3.4.1. Neutral detergent f i b r e Raspberry pomace contained on average 54.1% neutral detergent f i b r e (NDF) residue (Table 4.1). This residue consists of those components not soluble i n neutral 51 detergent solution; c e l l u l o s e , hemicelluloses, l i g n i n , c u t i n, tannin, bound c e l l - w a l l protein, heat-damage protein complexes and ash (Robertson and Van Soest, 1981). The average NDF value i s somewhat lower than the average amount of t o t a l dietary f i b r e i n pomace (Table 4.1), l a r g e l y because the NDF residue does not include the soluble dietary f i b r e components while the t o t a l dietary f i b r e determination does (see discussion below). 3.4.2. Total dietary f i b r e Table 4.4 shows the soluble and insoluble dietary f i b r e content of raspberry pomace. Raspberry pomace contained 59.5% t o t a l dietary f i b r e (TDF) (D.M. b a s i s ) , which i s the sum of the insoluble and soluble f i b r e f r a c t i o n s . Components of insoluble dietary f i b r e : The insoluble f i b r e f r a c t i o n of TDF i s composed of three p h y s i o l o g i c a l l y d i s c r e t e f r a c t i o n s ; true insoluble f i b r e , i n d i g e s t i b l e crude protein and insoluble ash. Because the accepted d e f i n i t i o n of dietary f i b r e includes only the i n d i g e s t i b l e polysaccharides i n a feedstuff, or components that modify these polysaccharides (Southgate et a l . , 1978), the i n d i g e s t i b l e protein and ash i n the sample are not included as part of dietary f i b r e and are therefore subtracted from the weight of insoluble f i b r e . 52 Table 4 . 4 . Insoluble and so luble components of t o t a l d i e tary f i b r e of raspberry pomace with r i c e h u l l s (100% D.M. b a s i s ) . 1 (N=10) Average Range S.E.M. (%) (%) Water- insoluble components i n d ie tary f i b r e ana lys i s Insoluble f i b r e 57.2 49.6-62.1 1.20 Indigestible crude protein 5.2 4.4-6.8 0.23 Insoluble ash (residue on ashing) 8.5 4.2-16.4 1.09 Water-soluble components i n d ie tary f i b r e ana lys i s Soluble f i b r e 2.3 1.6-3.4 0.17 Soluble ash (residue on ashing) 1.0 0.5-1.5 0.11 T o t a l d i e tary f i b r e 59.5 51.6-64.6 1.17 (sum of i n s o l . and s o l . fibre) 1 Enzymatic digestion method of Asp and Johansson, 1981 53 The true insoluble f i b r e f r a c t i o n , 57.2% of the pomace dry matter, consists of c e l l u l o s e , hemicelluloses, l i g n i n , c u t i n and tannins (Table 4.4). Of these components, only c e l l u l o s e and hemicelluloses are considered to constitute true c e l l - w a l l f i b r e . Lignin i s included as insoluble f i b r e because i t frequently i s covalently linked to both c e l l u l o s e and hemicelluloses i n the plant c e l l wall, and influences the microbial degradation i n the gut of these polysaccharides (Southgate et a l . , 1978). Prosky et a l . (1984) include cutin i n t h i s f r a c t i o n as well because i t i s very r e s i s t a n t to both digestion and fermentation, and may impair the digestion of other f i b r e components ( S p i l l e r and Amen, 1976), although some researchers dispute t h i s (Southgate et a l . , 1978). The action of cu t i n i n r e l a t i o n to other f i b r e constituents i s less well understood than that of l i g n i n . Tannins are included as a component of insoluble f i b r e because they have been demonstrated to reduce the dry matter d i g e s t i b i l i t y of feedstuffs (Hibberd et a l . , 1982) although they a c t u a l l y have more impact on the a v a i l a b i l i t y of protein (Butler et a l . , 1986) (see l i t e r a t u r e review). The i n d i g e s t i b l e crude protein f r a c t i o n consists of the protein that i s not digested by the peptidase enzyme during the dietary f i b r e analysis. I t i s made up large l y of 54 undigested c e l l - w a l l protein but also includes i n d i g e s t i b l e protein-carbohydrate complexes (Maillard browning products) which may be formed during heating. In raspberry pomace, the i n d i g e s t i b l e protein made up on average 5.2% of the pomace dry matter (Table 4.4). Assuming a mean crude protein content of 10.0% i n raspberry pomace (Table 4.1), 52% of the pomace protein apparently i s i n d i g e s t i b l e by the peptidase enzyme. Asp et a l . (1983) suggest that t h i s i n d i g e s t i b l e protein f r a c t i o n may be a reasonable estimate of i n vivo i n d i g e s t i b l e protein. However, no further research has been found to either confirm or refute t h i s suggestion. Components of soluble dietary f i b r e : Raspberry pomace was found to contain 2.3% soluble f i b r e (Table 4.4) and 1.0% ash residue following ashing of the soluble f i b r e f r a c t i o n . The soluble f i b r e f r a c t i o n contains the water-soluble f i b r e components which have been prec i p i t a t e d out of solution by the addition of ethanol. I t includes pectin, gums, mucilages and beta-glucans. Pectin i s a s t r u c t u r a l component of the c e l l s of a l l plants, although i t i s found in larger amounts in f r u i t s and vegetables (Theander and Aman, 1984) . Gums are generally formed on the outer surfaces of plant leaves and stems i n response to damage ( S p i l l e r and Amen, 1976). Beta-glucans are related to gums 55 and are found p r i n c i p a l l y i n cereals. Mucilages are found in small amounts on the coat of seeds or i n the seed endosperm, and prevent dehydration of the seed during periods of drought ( S p i l l e r and Amen, 1976). Englyst (1981), using a dietary f i b r e method s i m i l a r to that of Asp et a l . (1983), measured 7.5% water-soluble f i b r e i n raw raspberries, a value considerably higher than that which was determined i n t h i s work. However, t h i s i s to be expected as most of the water-soluble f i b r e would be removed with the j u i c e during i t s extraction from the b e r r i e s . As well, the addition of pectinase during j u i c e production would la r g e l y degrade the insoluble pectin to i t s component sugars. 3.4.5. Lignin, cutin and c e l l u l o s e (by difference) i n raspberry pomace Pomace was treated with acid detergent f i b r e solution as a preparatory step for the determination of l i g n i n and c u t i n . Following t h i s i n i t i a l treatment and subsequent potassium permanganate digestion, raspberry pomace was determined to contain 11.7% l i g n i n (Table 4.5). This value i s higher than the value of 6.8% l i g n i n (D.M. basis) measured i n raspberry seeds alone (Buckley, 1985). I t i s assumed, although i t was not stated by Buckley, that t h i s 56 Table 4.5. Analysis of f i b r e components of raspberry pomace with r i c e h u l l s (100% D.N. b a s i s ) 1 . (N=9) Average Range S.E.M. (%) (%) Acid detergent f i b r e 46.0 43.3-47.2 0.41 (to t a l of l i g n i n , c u t i n A.D. ash and cellulose) Lignin 11.7 9.6-15.4 0.56 Cutin 6.0 3.2-9.1 0.55 Acid detergent ash 2.2 1.4-3.1 0.19 Cellulose (by 26.9 24.5-33.3 0.80 difference) 1 Methods of Goering and Van Soest (1970) 57 value included cutin. The r e s u l t s of the current research and that of Buckley (1985) would suggest that the l i g n i n f r a c t i o n i s found mainly i n the pulp of the pomace and i n the added r i c e h u l l s . The l i g n i n content of r i c e h u l l s ranges from 16 to 21.4% (D.M. basis) (NRC, 1972; 1982). The '"lignin determination also includes tannins (Robertson and Van Soest, 1981) and heat-damage protein (Van Soest, 1965). Cutin, which i s a normal component of seed h u l l s ( S p i l l e r and Amen, 1976), was present at a l e v e l of 6.0% i n the pomace dry matter (Table 4.5). As measured by difference, the pomace contained 26.9% c e l l u l o s e . 58 DIGESTIBILITY OF DRY MATTER, PROTEIN, FAT AND ENERGY OF RASPBERRY POMACE BY GROWING PIGS AND RATS. 1. MATERIALS AND METHODS Pri o r to the s t a r t of the animal t r i a l s , two 200-litre drums of wet, frozen raspberry pomace were purchased from P a c i f i c F r u i t and Concentrate Ltd of Chilliwack. This pomace was thawed, subdivided into smaller bags and refrozen u n t i l the experimental work began. Because pomace i s extremely perishable, i t was thawed i n amounts small enough to be accommodated i n the available drying ovens. Once thawed, the pomace was immediately transferred to drying ovens and dried at 55-60 C for 24 hours or u n t i l the pomace was uniformly dry. With the exception of one t r i a l which used a small amount of freeze-dried pomace, a l l pomace used in the animal experimental work was prepared i n t h i s way. 1.1. T r i a l 1: Determination of pomace d i g e s t i b i l i t y with growing pigs In t h i s t r i a l , d i g e s t i b i l i t y of dry matter, f a t , energy and protein of pomace by pigs was determined by the difference method. Four Yorkshire-Landrace cross weanling barrows (25 to 30 kg i n weight at s t a r t of t r i a l ) were used i n a randomized 59 4X4 Latin Square design. The four treatments were: basal (B) (1.6mm ground), B with 40% barley, B with 40% ground pomace (1.6 mm) and B with 40% unground pomace. The composition of the basal r a t i o n i s shown i n Table 5.1. It provided 17% crude protein (100% D.M. ba s i s ) . The raspberry pomace used i n t h i s experiment was dried at 55-60 C for 24 hours i n a forced-air oven, then ground to 1.6 mm fineness in a Christy and Norris hammermill. F i n a l crude protein, crude f a t and gross energy of the four treatment d i e t s i s shown i n Table 5.2. The barrows were i n d i v i d u a l l y housed i n d i g e s t i b i l i t y crates i n a controlled atmosphere room i n the U.B.C. swine unit. Room temperature was kept r e l a t i v e l y constant at 20 C. Each pig had free access to water from a nipple drinker. Pigs were fed 1.5 kg of a i r dry feed d a i l y i n two equal feedings, at 9:30 a.m. and 4:00 p.m. To minimize wastage, feed was mixed with water i n a one-to-one r a t i o before i t was fed. S p i l l e d feed was co l l e c t e d i n a tray suspended below each feeder and was removed a f t e r each c o l l e c t i o n period, dried and weighed. D i g e s t i b i l i t y c o e f f i c i e n t s were corrected for feed s p i l l a g e . Each period consisted of a f i v e day preliminary period followed by a f i v e day t o t a l f e c a l c o l l e c t i o n period, which was i n turn followed immediately by the s t a r t of the next 60 Table 5 .1 . Composition of basal r a t i o n for d i g e s t i b i l i t y t r i a l with growing pigs Component Percentage of t o t a l diet ( a i r dry basis) Barley Soybean meal Dicalcium phosphate Limestone TM-Vitamin mix Iodized s a l t 80. 3 16.8 0.7 1.2 0.5 0.5 *TM-Vitamin mixture (per kg of mix): Vitamin A 825,000 I.U.; Vitamin D 55,000 I.U.; Vitamin E 2,700 I.U.; Vitamin K 0.4 g; Thiamin 0.1 g; Riboflavin 0.8 g; Niacin 4.0 g; Ca pantothenate (45%) 6.0 g; B12 2.5 mg; Choline 50.0 g; Copper sulphate (5H20) 3 g; Zinc sulphate 25.0 g; Manganese sulphate 7.0 g; Sodium se l e n i t e 15.7 mg. Table 5.2. Composition of treatment diets for d i g e s t i b i l i t y t r i a l with growing pigs (100% D . M .basis). Crude Protein Crude Fat Gross Energy (kcal*kg - 1) (%) (%) Diet 1 (basal B) 17.7 Diet 2 (B + 40% barley) 14.9 Diet 3 (B + 40% ground 1.8 1.9 4250 4330 pomace) 14.8 Diet 4 (B + 40% 6.4 4800 unground pomace) 15.1 6.2 4640 61 preliminary period. Feces were c o l l e c t e d i n large p l a s t i c trays that were suspended beneath the rear section of the d i g e s t i b i l i t y cages. Once d a i l y , trays were removed and the contents d i l u t e d s l i g h t l y with water, mixed into a homogeneous s l u r r y and weighed. Two 200 gram subsamples were taken from the sl u r r y , one for immediate dry matter determination (for c a l c u l a t i o n of t o t a l dry weight of feces per day) and the second to be frozen for subsequent protein, fat and energy analysis. Any fe c a l material which was deposited i n front of the c o l l e c t i o n trays was caught beneath the cages i n large screens which were suspended there. This material was dried, weighed and added to the t o t a l weight of feces from the trays, but was not used i n analyses because of possible contamination with urine. Urine was not c o l l e c t e d . Dry matter, protein, f a t and energy d i g e s t i b i l i t y of the raspberry pomace was determined by difference from d i g e s t i b i l i t y of the basal r a t i o n and of the rations containing pomace. Dry matter d i g e s t i b i l i t y was determined from the net feed consumed and the t o t a l f e c a l production for each treatment r e p l i c a t e . 62 1.1.1. Analysis of protein i n fe c a l material Fecal samples from each animal for the f i v e c o l l e c t i o n days per period were combined for protein analysis. Wet fe c a l samples were weighed onto nitrogen-free f i l t e r paper and transferred to 250-ml Tecator digestion tubes. Samples were then digested i n a solution of s u l f u r i c acid and hydrogen peroxide using the wet ashing method of Wall and Gehrke (1975) . The cat a l y s t mixture contained lithium s u l f a t e and selenium. Total N (as the concentration of the ammonia-salicylate complex) was determined c o l o r i m e t r i c a l l y using the Technicon Autoanalyzer. Protein was calculated as t o t a l N mu l t i p l i e d by 6.25. 1.1.2. Analysis of fat i n fe c a l material Five day composite f e c a l samples were dried at 60 C i n a forced-air drying oven and ground to 0.5 mm fineness i n a Retsch c e n t r i f u g a l m i l l p r i o r to analysis. Because much of the f a t excreted i n feces i s not extracted by a simple ether-extraction procedure (Thorbek and Henckel, 1977), samples were acid-hydrolyzed i n a Buechi acid hydrolysis unit p r i o r to fat extraction i n a Buechi Soxhlet-type extraction apparatus (see Appendix 2 for complete method). Feed samples were analyzed using the same method. This 63 analysis was undertaken at the Agriculture Canada Research Station, Agassiz, B.C.. Hydrolysis: Three gram samples i n duplicate were refluxed i n 4N hydrochloric acid f or 15 minutes following which the solution was quantitatively transferred to scintered glass crucibles (low porosity) containing 10 g washed sea sand and 5 g acid-washed C e l i t e 545, and the f i l t r a t e removed by suction. The hydrolyzed f e c a l samples i n crucibles were dried at high heat i n a microwave oven for 10 minutes then overnight at 70 C i n a forced a i r oven. Fat extraction: The hydrolyzed, completely dry samples in glass crucibles were placed i n the extraction chambers of a Soxhlet-type fat extraction apparatus. Anhydrous di e t h y l ether was added to a weighed glass beaker containing a glass bead (as b o i l i n g chip) and the samples were ether-extracted for three hours. The beakers were dried at 105 C and weighed, and the difference i n beaker weight before and af t e r extraction taken as weight of crude f a t . 1.1.3. Determination of gross energy i n feces Five-day composite f e c a l samples which had been previously dried and ground as above were analyzed for gross energy content by bomb calorimetry using an adiabatic bomb 64 calorimeter. Feed samples were analyzed using the same method. 1.2. T r i a l 2. Determination of pomace dry matter d i g e s t i b i l i t y with growing rats using chromic oxide marker method Th i r t y male Wistar rats weighing between 80 and 90 g were used i n a randomized block design i n which s i x l e v e l s of raspberry pomace i n the di e t were compared. Pomace was included at 0, 19.4, 38.6, 58.0, 77.2 and 96.4% of the d i e t (D.M. basis) with the remainder consisting of barley, minerals and vitamins (Table 5.3). Daily feed intake and weekly weight gain were measured over the three week t r i a l period. Diet dry matter d i g e s t i b i l i t y was determined using chromic oxide as an i n d i g e s t i b l e marker i n each d i e t . The barley used i n the t r i a l was of unknown vari e t y and was ground to 1 mm fineness i n a Christy and Norris hammermill. The pomace was dried for 24 hours at 60 C, and ground as above. The pomace-barley rations (Table 5.3) were supplemented with 0.75% trace mineral-vitamin mix (Table 5.4), 0.5% iodized s a l t and were balanced with additional calcium and phosphorus. Chromic oxide was added at 0.25% ( a i r dry basis) as an i n d i g e s t i b l e marker. Diets were mixed 65 Table 5 . 3 . Composition of diets used i n growth t r i a l with rats fed raspberry pomace i n varying amounts (g per lOOg di e t dry matter). Diet Number 1 2 3 4 5 6 Barley 96.9 77.4 58.0 38.6 19. 3 -Pomace - 19.4 38.6 58.0 77.2 96.4 CaP0 4 0.4 0.7 1.0 1.2 1.4 1.6 CaC0 3 1.2 1.0 0.9 0.7 0.6 0.4 NaCl 0.5 0.5 0.5 0.5 0.5 0.5 T M - V i t . 1 0.75 0.75 0.75 0.75 0.75 0.75 C r 2 0 3 0.25 0.25 0.25 0.25 0.25 0.25 % C P . 2 11.2 10.8 10.6 10.4 10.1 10. 0 See Table 5.4 for composition of trace mineral -v i tamin mixture. Crude pro te in determined by method of Wall and Gehrke, 1975. 66 Table 5 . 4 . Composition of trace mineral-vitamin mixture used i n rat growth t r i a l d iets (concentration per kg of mix) Trace minerals Copper sulphate (5H20) 3.0 g Zinc sulphate 25.0 g Manganese sulphate 7.0 g Sodium se l e n i t e 15.7 mg Vitamins Vitamin A 825,000 I.U Vitamin D 55,000 I.U Vitamin E 2,700 I.U Vitamin K 0.4 g Thiamin 0.1 g Ribo f l a v i n 0.8 g Niacin 4.0 g Calcium pantothenate (45%) 6.0 g Vitamin B12 2.5 mg Choline 50.0 g 67 for f i v e minutes i n a Hobart mixer, and weighed into i n d i v i d u a l a i r - t i g h t j a r s . Rats were housed i n d i v i d u a l l y i n s t a i n l e s s s t e e l s o l i d -sided, wire mesh-bottomed cages approximately 20cmx25cmx25cm high i n a controlled atmosphere room. Feces and urine were c o l l e c t e d beneath the cages i n s t a i n l e s s s t e e l trays l i n e d with paper towelling. This towelling was changed d a i l y , and the trays were washed every two days. Water was f r e e l y a vailable. Rats were fed ad libitum, with feeders being f i l l e d twice d a i l y . The tunnel-shaped, mesh-floored feeders l i m i t e d wastage by making i t awkward for the rats to carry food back to the cage. The waste feed was removed from the feeders every two days, weighed and discarded. The small amount of feed that was found i n the trays beneath the cages was c o l l e c t e d and weighed. Uncontaminated fecal samples were c o l l e c t e d from each rat every two days for l a t e r chromic oxide determination, and were frozen between weekly c o l l e c t i o n s . 1.2.1. Chromic oxide analysis Chromic oxide concentrations i n the feed and feces were determined by the method of Williams et a l . (1962). Feces were dried at 60 C for 18 hours and ground through a 0.5 mm screen i n a Tecator cyclotec m i l l . Feed samples were ground 68 t o 1 mm f i n e n e s s . A l l g l a s s w a r e was a c i d - w a s h e d i n 50% n i t r i c a c i d . S a m p l e s w e r e f i r s t a s h e d a t 500 C , c o o l e d a n d t h e n d i g e s t e d w i t h 3 m l o f p h o s p h o r i c a c i d - m a n g a n e s e s u l f a t e s o l u t i o n a n d 4 m l o f p o t a s s i u m b r o m a t e s o l u t i o n . T h i s s o l u t i o n was t r a n s f e r r e d q u a n t i t a t i v e l y t o a v o l u m e t r i c f l a s k a n d made up t o v o l u m e , t h e n l e t s t a n d o v e r n i g h t t o s e t t l e o u t s i l i c a t e s . D e p e n d i n g o n t h e e x p e c t e d c h r o m i c o x i d e c o n c e n t r a t i o n i n t h e s a m p l e , e i t h e r 10 o r 30 m l o f t h i s s o l u t i o n was p i p e t t e d i n t o a s e c o n d v o l u m e t r i c f l a s k t h e f o l l o w i n g d a y . T e n m l o f t h e 5000 ppm c a l c i u m s o l u t i o n was a d d e d t o f u r t h e r p r e c i p i t a t e s i l i c a t e s , a n d t h e s o l u t i o n was made t o v o l u m e a n d m i x e d . A n a l i q u o t o f t h i s was s t o r e d i n a t i g h t l y - c a p p e d , a c i d - w a s h e d p l a s t i c b o t t l e . F i s h e r c h r o m i u m r e f e r e n c e s o l u t i o n ( S O - C - 1 9 2 ) (1000 ppm c h r o m i u m ) was u s e d t o make t h e p r i m a r y s t a n d a r d . C h r o m i u m -f r e e f e e d a n d f e c a l s a m p l e s w e r e r u n t h r o u g h o u t t h e a n a l y s i s t o d e t e r m i n e t h e b a c k g r o u n d l e v e l o f c h r o m i u m i n t h e d i e t . D i g e s t e d c h r o m i u m - f r e e f e e d a n d f e c e s w e r e u s e d t o make t h e s e c o n d a r y s t a n d a r d s . T h e c o n c e n t r a t i o n o f c h r o m i c o x i d e was d e t e r m i n e d w i t h a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y . T h e 3 5 7 . 9 nm e m i s s i o n was m e a s u r e d u s i n g a c h r o m i u m h o l l o w c a t h o d e l a m p . 69 1.3. T r i a l 3. Determination of pomace pro te in d i g e s t i b i l i t y by growing rats A nitrogen balance t r i a l with rats was undertaken to determine the e f f e c t of heating and fineness of grinding on true d i g e s t i b i l i t y (TD), b i o l o g i c a l value (BV) and net u t i l i z a t i o n (NPU) of raspberry pomace protein. Th i r t y male Wistar weanling rats between 65 and 70 g i n weight were used in a 2X3 f a c t o r i a l design, with s i x treatments, and f i v e rats per treatment. Two degrees of grinding were tested; b a l l - m i l l e d (very fine) and hammermilled through a 1 mm screen. Three heating treatments were included; freeze dried pomace, and previously dried pomace (60 C) heated at either 100 C or 150 C for 2 hours i n a forced a i r oven. The treatment u t i l i z i n g the pomace which had been heat-treated at 150 C was abandoned following the preliminary feeding period because of poor p a l a t a b i l i t y of the ration, thus reducing the experiment to a 2X2 f a c t o r i a l design. T r i a l d i e t s consisted of raspberry pomace balanced with a nitrogen-free mixture (Table 5.5) to provide 1.44 g N per 100 g d i e t D.M. af t e r supplementation with minerals and vitamins (Table 5.5) (because pomace protein l e v e l was too low to balance for the standard 1.5 g N per 100 g diet D.M.). Diets were mixed i n a pharmaceutical mixer for fi v e 70 Table 5 . 5 . Composition of nitrogen-free mix and mineral supplement added to d i e t s i n nitrogen balance t r i a l with rats N-free mixture g*kg 1 Potato starch (autoclaved and ground) 80.67 Cane sugar 8.92 Cellulose powder 5.20 Corn o i l 5.20 Mineral mixture 1 g*kg - 1 Calcium carbonate CaCCu 68.6 Calcium c i t r a t e Ca-j (C 6H 50 7) 2 308.3 Calcium hydrogen phosphate CaHP04.2H20 112.8 Potassium hydrogen phosphate, secondary 218.8 Potassium chloride KC1 124.7 Sodium chloride NaCl 77.1 Magnesium sulphate MgSO4 3 8.3 Magnesium carbonate MgCO3 35.2 Ammonium f e r r i c i t r a t e 15.3 Manganese sulphate MnS04,H20 0.20 Cupric sulphate CuS0 4.5H 20 0.08 Potassium iodide KI 0.04 Sodium f l o u r i d e NaF 0.51 Aluminum ammonium sulphate 0.09 A1 2(S0 4) 3(NH 4) 2.24 H 20 Vitamin mixture 2 10663AIN Vitamin mixture 76 - American I n s t i t u t e of N u t r i t i o n . Mineral mixture added at 20 g per kg experimental d i e t (A.D basis) Vitamin mixture added at 4 g per kg experimental d i e t m(A.D. basis) 71 minutes and stored i n a i r - t i g h t containers at -20 C u n t i l the t r i a l began. The equipment and methods used i n t h i s experiment were those of Eggum (1973). Room temperature ranged between 23 C and 25 C, and humidity varied from 50% to 70% during the t r i a l . Rats were housed i n ind i v i d u a l p l e x i g l a s s cages and had free access to water. They were fed the t r i a l d i e ts for nine days, with a 4-day adaptation period and a 5-day balance period. The rats were weighed when f i r s t put into the cages, again before the s t a r t of the 5-day c o l l e c t i o n period and f i n a l l y at the conclusion of the t r i a l . Total f e c a l and urine c o l l e c t i o n was made for the 5-day balance period. The 4- and 5-day diets for each r a t were weighed out before the t r i a l began and stored i n a i r - t i g h t p l a s t i c containers. The rats were fed once d a i l y . Urine and feces were c o l l e c t e d i n separate vessels by means of a screen situated i n the funnel beneath each cage. This allowed the urine to pass into one vessel containing 50 ml of 5% s u l f u r i c acid, and shunted the feces into a separate vessel. Fecal material was removed d a i l y from the c o l l e c t i o n vessel and stored at -20 C without preservative. The separation screen was sprayed d a i l y with 20% c i t r i c acid to rinse deposited urine into the vessel and to prevent loss of nitrogen. When the c o l l e c t i o n period was finished, the 72 cage bottom, funnel and screen were rinsed with lukewarm water to transfer any residual nitrogen into the urine fl a s k . 1.3.1. Chemical analysis of urine and fe c a l samples Urine: Urine samples were transferred quantitatively from the c o l l e c t i o n vessels to 250 ml volumetric flasks and made up to 250 ml with rinse water. The samples, a f t e r being transferred to p l a s t i c bottles, were re f r i g e r a t e d u n t i l required for analysis. Total N was determined using the wet ash digest method of Wall and Gehrke (197 5) with samples digested i n a Tecator block digestor and read c o l o r i m e t r i c a l l y on a Technicon autoanalyzer, with N calculated from the concentration of the ammonia-salicylate complex i n the digest solution following reaction of the two reagents. F i f t e e n ml of the urine solution were pipetted into a block digestion tube. The digest solution consisted of concentrated s u l f u r i c acid and 30% hydrogen peroxide, catalyzed by a mixture of lithium sulphate and selenium. The block was preheated to 150 C (rather than the standard 380 C) to prevent s p l a t t e r i n g and possible loss of solution during the i n i t i a l stage of the digestion. Digested samples were made up to 250 ml volume, and the f i n a l N determination corrected for the d i l u t i o n factor. 73 Feces: Feces were stored i n weighed p l a s t i c containers at -2 0 C u n t i l required for analysis. Just p r i o r to analysis, samples were thawed, s u f f i c i e n t water was added to the dry feces to make a thick s l u r r y , and the mixture was homogenized using a Polytron blender. The weight of the wet fec a l material and container was recorded. A sample of t h i s s l u r r y , ranging from 5 to 6.5 g wet weight, was transferred into a tared Tecator digestion tube (using a 7 mm diameter glass tube which was lowered into the sl u r r y , and then withdrawn while holding a finger over the upper end to provide a reduced pressure). The fe c a l material was digested using the same method as that of the urine samples. N i n t h i s solution was corrected to the t o t a l weight of wet fec a l material to determine the t o t a l f e c a l N. 1 . 4 . S t a t i s t i c a l analys i s A l l r e s u l t s were subjected to analysis of variance using the SAS s t a t i s t i c a l program (UBC s i t e license) , and means were tested using the Newman-Keuls te s t (Keuls, 1952). 74 2. RESULTS AND DISCUSSION 2.1. Dry matter d i g e s t i b i l i t y of pomace by growing pigs and rats 2.1.1. Pigs The dry matter d i g e s t i b i l i t y (DMD) by pigs of a ration containing 40% ground pomace ( a i r dry basis) was 56.3%, which was s i g n i f i c a n t l y better (P<0.05) than that of the rat i o n with 40% unground pomace (52.2%) (Table 5.6), but s i g n i f i c a n t l y poorer than the DMD of both the basal and basal-barley rations. There was no s i g n i f i c a n t difference between the DMD of the basal and of the basal-barley rations. The DMD by pigs of ground pomace (by difference) was s i g n i f i c a n t l y better than that of unground pomace (P<0.05) (Table 5.7). DMD by pigs of the ground pomace was 20.8% while the unground pomace was only 10.7% digested. Both the basal r a t i o n and barley by difference were s i g n i f i c a n t l y better digested than either the ground or unground pomace. DMD of the basal rat i o n was not d i f f e r e n t from that of barley. 75 Table 5 . 6 . Dry matter, protein, f a t and energy d i g e s t i b i l i t y of whole rations containing 40% of either ground or unground raspberry pomace, or barley, by growing male pigs. T r t . Composition D.M. P r o t e i n Fat Energy of D i e t d i g . d i g . d i g . d i g . (D.M. b a s i s ) (%) (%) (%) (%) A Basal 81.8 a* 78.3 a 28. 7 b 81. l a r a t i o n 1 34. 5 b B Basal + 81. 0 a 77.4 a 80.0 a C 40.2% b a r l e y B a s a l + 56.3 b 57. 7 b 71.2 a 55. 6 b 41.8% ground pomace 2 D Basal + 41.7% unground pomace 52.2 C 55. 7 b 24. 9 b 47. 0 C SEM 0.64 1. 09 3.09 0.99 x Basal r a t i o n c o n s i s t e d of b a r l e y and soybean meal supplemented with m i n e r a l s and v i t a m i n s . 2 Pomace d r i e d a t 60 C and ground t o 1.6mm f i n e n e s s . * Means w i t h i n columns w i t h d i f f e r e n t s u p e r s c r i p t l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05) 76 Table 5 . 7 . Dry matter, protein, f a t and energy d i g e s t i b i l i t y (by difference) of barley, and ground and unground raspberry pomace fed to growing male pigs. Component of D.M. Protein Fat Energy Diet dig. dig. dig. dig. (%) (%) (%) (%) Basal r a t i o n 1 81.8 a Barley 7 9 . 9 ^ Ground pomace2 20. 8 b Unground pomace 10.7 C SEM 1.30 7 8 . 3 A 2 8 . 7 ^ 8 1 . l a 7 5 . 0 A 4 2 . 4 B 7 8 - 6 J 1 4 . 7 B 7 9 . 7 A 2 8 . 5 B 1 0 . 6 b 2 4 . 1 C 7 . 9 C 4 . 8 3 3 . 4 7 1 . 8 0 x Basal r a t i o n consisted of barley and soybean meal supplemented with minerals and vitamins. 2 Pomace dried at 60 C and ground to 1.6mm fineness. * Means within columns with d i f f e r e n t superscript l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). 7 7 2.1.2. Rats Table 5.8 shows the measured dry matter d i g e s t i b i l i t i e s (by chromic oxide marker method) of diets consumed by growing rats fed six l e v e l s of pomace i n a balanced ration. Pomace was included at 0, 19.4, 38.6, 58.0, 77.2 and 96.4% of a complete rati o n (D.M. bas i s ) . With each increment of pomace i n the diet, dry matter d i g e s t i b i l i t y of the complete ra t i o n was reduced s i g n i f i c a n t l y (P<0.05). D i g e s t i b i l i t y of the dry matter of the 96.9% barley r a t i o n was 80.8%, while the d i e t containing 96.4% pomace was 20.9% d i g e s t i b l e . This l a t t e r r e s u l t shows excellent agreement with the d i g e s t i b i l i t y of ground pomace (by difference) by growing pigs (Table 5.7). From the average d i g e s t i b i l i t i e s of the 96.9% barley and 96.4% pomace rations fed to growing rats, the expected d i g e s t i b i l i t i e s of the other four diets have been interpolated (Table 5.8). There i s good agreement between the expected and observed d i g e s t i b i l i t i e s f or a l l diets except d i e t 4, which contained 58.0% pomace. Measured dry matter d i g e s t i b i l i t y of the d i e t containing barley and pomace i n the approximate r a t i o of 40:60 was almost 10% higher than the interpolated d i g e s t i b i l i t y , which was based on the d i g e s t i b i l i t i e s of barley and pomace as sole feedstuffs. Observed d i g e s t i b i l i t y was 54.5% and expected 78 Table 5.8. Dry matter d i g e s t i b i l i t y of a balanced barley-based d i e t containing varying amounts of pomace when fed to growing r a t s . T r t . Level of. pomace Av. D.M. Av. D.M. dig. i n d i e t dig. by i n t e r p o l a t i o n 2 (D.M. basis) (%) (%) (%) 1 0 80.8 a 2 19.4 71.5 b 68.8 3 38.6 5 9 > 3 H 5 6 , 8 4 58.0 54.5 d 44.9 5 77.2 30.7j 32.9 6 96.4 20.9 f S.E.M. 1.136 1 Dry matter d i g e s t i b i l i t y by chromic oxide marker method and atomic absorption spectrophotometry. 2 Interpolated from average d i g e s t i b i l i t i e s of barley and pomace. Means with d i f f e r e n t superscript l e t t e r within columns are s i g n i f i c a n t l y d i f f e r e n t . (P<0.05) 79 d i g e s t i b i l i t y was determined to be 44.9%. This suggests that d i g e s t i b i l i t y may have been p o s i t i v e l y stimulated by the associative e f f e c t s of the two feeds i n t h i s r a t i o (Schneider and F l a t t , 1975), although the factor responsible for t h i s i s not known. This e f f e c t was not observed with diets 2, 3 or 5. 2.1.3. Conclusions The poor DMD of both ground and unground raspberry pomace i s l a r g e l y the r e s u l t of the high proportion of i n d i g e s t i b l e components i n the pomace. Raspberry pomace contains on average 59.5% t o t a l dietary f i b r e (Table 4.4), which i s completely i n d i g e s t i b l e by the endogenous enzymes of the non-ruminant animal (Prosky et a l . , 1984). The i n d i g e s t i b l e f r a c t i o n consists of on average 26.9% c e l l u l o s e (by difference) (Table 4.5), 11.7% l i g n i n (which includes tannin and a r t i f a c t l i g n i n ) , 6% cutin and an undetermined amount of hemicellulose. The microbial population i n the colon, and to a lesser extent, the cecum ( F a r r e l l and Johnson, 1972) of growing pigs can degrade a s i g n i f i c a n t amount of c e l l u l o s e and hemicellulose, using the resultant v o l a t i l e f a t t y acids for energy (Kennelly et a l . , 1981; Keys and DeBarthe, 1974; Low, 1985). Keys and DeBarthe (1974) determined that f i n i s h i n g 80 pigs consuming diets which contained several d i f f e r e n t sources of f i b r e providing a standard amount of c e l l u l o s e and hemicellulose, degraded approximately 30% of the d i e t dry matter i n the colon. Kennelly et a l . (1981) determined that v o l a t i l e f a t t y acids produced by growing pigs on a barley-soybean meal di e t containing 27% a l f a l f a provided 15.5% of the metabolizable energy requirements. However, the extent of degradation of these polysaccharides by microbes w i l l depend to a large extent on the degree of l i g n i f i c a t i o n of the feedstuff (Robertson and Van Soest, 1981). Lignin w i l l protect twice to three times i t s weight of plant c e l l wall polysaccharide from degradation by microbes (Van Soest, 1985). The presence of a r e l a t i v e l y large amount of l i g n i n i n pomace suggests that fermentation of the c e l l u l o s e and hemicellulose i n the gut of growing pigs w i l l be minimal. The pomace contains several other i n d i g e s t i b l e constituents which could reduce i t s dry matter d i g e s t i b i l i t y . Cutin appears to i n h i b i t d i g e s t i b i l i t y of c e l l wall material i n a way that i s s i m i l a r to the action of l i g n i n ( S p i l l e r and Amen, 1976). Complexes of tannic material and heat-damage protein (both included i n the l i g n i n determination) reduce the amount of protein available for absorption. Tannin also reduces the dry matter 81 d i g e s t i b i l i t y of feedstuffs, although the mechanism for t h i s i s not known (Butler et a l . , 1986). I t has been shown that high tannin diets are less well digested than are those low in tannin (Cousins et a l . , 1981). These factors are discussed i n more d e t a i l i n the l i t e r a t u r e review and i n the discussion of dietary f i b r e i n Chapter 4. The nutrients i n the pomace which are p o t e n t i a l l y d i g e s t i b l e by endogenous enzymes consist of 11.1% l i p i d material (D.M. basis), 10.0% crude protein (of which 52% may be unavailable), 7.4% soluble carbohydrates (Table 4.1) and 2.3% water-soluble f i b r e (Table 4.4). The water-soluble f i b r e components of pomace, including pectin, gums and mucilage, are highly fermentable i n non-ruminant systems ( S p i l l e r and Amen, 1976). There are several reasons for the improvement i n d i g e s t i b i l i t y of ground pomace over unground (Table 5.7). Of most sig n i f i c a n c e , grinding breaks the seed coat which makes the l i p i d contained therein available for digestion. Grinding also disrupts the i n d i g e s t i b l e c e l l wall material and makes the d i g e s t i b l e components within the c e l l a v a i l a b l e to digestive enzymes (Theander and Aman, 1984). I t reduces the rate of passage and thus increases the time during which feed p a r t i c l e s can be digested by enzymic action (Just, 1982). 82 2 . 2 . Energy d i g e s t i b i l i t y of raspberry pomace by growing pigs Rations containing 4 0% ground pomace had a s i g n i f i c a n t l y higher energy d i g e s t i b i l i t y by pigs than those containing 40% unground pomace (P<0.05) (Table 5.6). Grinding also had a s i g n i f i c a n t e f f e c t (P<0.05) on the energy d i g e s t i b i l i t y of pomace as the enti r e ration (calculated by difference) (Table 5.7). Ground pomace had an energy d i g e s t i b i l i t y of 28.5% compared with 7.9% for unground pomace. The basal and basal-barley rations had consistently higher energy d i g e s t i b i l i t i e s than e i t h e r of the pomace-containing rations. This trend was also observed with the ratio n components (by di f f e r e n c e ) ; the basal ration and barley were both s i g n i f i c a n t l y better digested than eithe r ground or unground pomace (P<0.05). The energy d i g e s t i b i l i t y of a l l four rations mirrors DMD c l o s e l y (Table 5.6). The energy d i g e s t i b i l i t y of the rat i o n components (Table 5.7) i s less consistent with DMD; that of ground pomace i s higher than DMD (28.5% vs. 20.8%) while that of unground pomace i s somewhat lower than DMD (7.9% vs. 10.7%). The high d i g e s t i b i l i t y of the l i p i d material i n the ground pomace may be responsible for t h i s discrepancy. S i m i l a r l y , the l i p i d material i n the unground pomace i s poorly digested, which may account for the lower 83 energy d i g e s t i b i l i t y observed. Overall, however, the factors that influence the DMD of the pomace (see discussion of DMD) also contribute to the poor energy d i g e s t i b i l i t y of both the ground and unground pomace. 2.3. D i g e s t i b i l i t y of pomace protein by growing pigs and rats 2.3.1. Apparent d i g e s t i b i l i t y of pomace protein by pigs The apparent d i g e s t i b i l i t y of the protein i n basal rations containing 40% of ground or unground raspberry pomace, and of pomace as the sole r a t i o n ingredient (by difference) when fed to growing pigs i s presented i n Tables 5.6 and 5.7. There was no s i g n i f i c a n t e f f e c t of grinding on the protein d i g e s t i b i l i t y of rations containing 40% pomace. However, the protein i n both pomace-containing rations was s i g n i f i c a n t l y less well digested than that of either the basal r a t i o n or the basal-barley r a t i o n (P<0.05). Protein d i g e s t i b i l i t y of ground pomace determined by difference was 14.7% while unground pomace was only 10.6% digested. Both the basal r a t i o n and barley alone (by difference) had s i g n i f i c a n t l y higher protein d i g e s t i b i l i t i e s than eit h e r of the pomace treatments (P<0.05) (Table 5.7). It i s in t e r e s t i n g that grinding of the raspberry pomace 84 (1.6mm screen) did not increase protein d i g e s t i b i l i t y over that of unground pomace. However, i n t h i s experiment, the calculated values for d i g e s t i b i l i t y by difference of protein within the two pomace treatments were highly v a r i a b l e . This fact may have masked any r e a l difference between treatments. It has been suggested that protein d i g e s t i b i l i t y by difference cannot be calculated accurately because of the influence on protein digestion of associative e f f e c t s which are created by mixtures of feedstuffs (Schneider and F l a t t , 1975). These e f f e c t s are unpredictable and can produce large v a r i a b i l i t y between observations, and i n t h i s experiment may have contributed to the large v a r i a b i l i t y observed. 2.3.2. TD, BV and NPU of pomace fed to rats True d i g e s t i b i l i t y (TD), b i o l o g i c a l value (BV) and net u t i l i z a t i o n (NPU) by growing rats of protein i n raspberry pomace were measured i n a standard d i g e s t i b i l i t y experiment. The e f f e c t s of two degrees of grinding and two heat-treatments of the pomace on the protein parameters were also measured. TD i s the amount of N digested and absorbed from a known amount consumed, corrected for metabolic f e c a l N (Eggum, 1973) . BV i s the percentage of digested and 85 absorbed N that i s retained and u t i l i z e d by the body, corrected for endogenous f e c a l and urinary losses of N. It i n d i r e c t l y evaluates how well a protein source supplies e s s e n t i a l amino acids to the animal (Lloyd et a l . , 1978). NPU i s calculated as TD*BV, and estimates the e f f i c i e n c y of growth (as accretion of N) of the animal consuming the test protein (Church, 1984). The average TD of the protein of raspberry pomace consumed by growing rats measured i n t h i s t r i a l was 33.3% (Table 5.9). The average BV of the protein was determined to be 79.3%, but was as high as 91.0% i n the pomace that was both freeze-dried and coarsely ground (1mm). This i s considerably better than the average BV of cereal grains (50% to 65%) (Church, 1984), which are the energy feedstuffs that the pomace might p a r t i a l l y replace i n a ra t i o n for growing swine. Even though the pomace protein i s poorly digested and absorbed, that which i s digested apparently contains a good amino acid p r o f i l e as i t i s well u t i l i z e d by the animals. The o v e r a l l NPU of raspberry pomace i s poor (26.6%) because of the low true d i g e s t i b i l i t y of protein. This indicates that the o v e r a l l e f f i c i e n c y of growth of non-ruminant animals consuming a di e t where pomace i s a major component would be poor (Church, 1984). 86 Table 5 . 9 . True d i g e s t i b i l i t y , b i o l o g i c a l value and net u t i l i z a t i o n of the protein of raspberry pomace by weanling male ra t s . Pomace was dried at two di f f e r e n t temperatures and ground to two d i f f e r e n t degrees of fineness. Treatments True digest. (TD) B i o l o g i c a l value (BV) Net protein u t i l i z a t i o n (NPU) (%) (%) (%) Average true d i g e s t i b i l i t y S.E.M. 33.3 0.33 79.3 1.73 26.6 0. 57 Ef f e c t of fineness of grind Coarse (1mm) Fine (ball-milled) S.E.M. 35.7 30.9* 0.47 a* 82.0 a 7 6. 5 a 2.44 29.3' 23. 8* 0.81 E f f e c t of drying temperature Low (freeze-dried) High (100 C) S.E.M. 33 .8 a 32.3 a 0.47 83. l a 75.6 a 2.44 28.2' 25.01 0.81 Interaction between treatments 1. Frz dry x 1mm 2. Frz dry x B.M. 3. 100 C x 1mm 4. 100 C x B.M. 5. E.M. 36.0b 31.5a 35.4b 30. 2a 0. 66 91. 0b 75.2a 73.1a 78. l a 3.46 32.7b 23 .7a 25. 9a 24.0a 1.15 Means grouped within columns with d i f f e r e n t superscripts are s i g n i f i c a n t l y d i f f e r e n t (P< 0.05) 87 Considerable v a r i a b i l i t y was observed within treatments i n t h i s experiment which made i t d i f f i c u l t to dis t i n g u i s h differences between treatments. This appears to have been caused by the contamination of the feces and urine with some of the feed. Because o v e r a l l p a l a t a b i l i t y was poor and the volume of pomace to be consumed large (because of i t s low density), the rats ate t h e i r r a t i o n i n several feedings which gave more opportunity for portions of feed to be car r i e d out of the feeding tunnel on t h e i r feet. 2.3.3 The e f f e c t of fineness of grind on TD, BV and NPU of pomace fed to growing rats A l l protein u t i l i z a t i o n parameters measured i n rats consuming raspberry pomace indicated that the protein of the coarsely-ground pomace (1mm) was more available to the rats than that i n the finely-ground treatments (ball-milled) (Table 5.9). The TD of the protein i n the coarsely ground pomace was 35.7%, which was s i g n i f i c a n t l y higher (P<0.05) than the d i g e s t i b i l i t y of that of the f i n e l y ground pomace (30.9%). The BV of the coarsely ground pomace was higher (82.0%) than the b a l l - m i l l e d pomace (76.5%), although t h i s e f f e c t was not s i g n i f i c a n t . The NPU of the coarsely ground pomace was s i g n i f i c a n t l y better than of the f i n e l y ground treatments, because of the additive e f f e c t of the 88 combination of the other two parameters (NPU=TD*BV) (P<0.05). It was expected that the protein i n the more f i n e l y -ground treatments would have been more avail a b l e to the growing rat s . The observation of the opposite e f f e c t may in fact be because of the extremely fine grind achieved with b a l l - m i l l i n g . Tannins, which are bound i n the outer s h e l l of the raspberry seed, may have been released by the f i n e -grinding of the pomace and could have formed i n d i g e s t i b l e complexes with proteins and amino acids i n the g a s t r o i n t e s t i n a l t r a c t (Hagerman and Klucher, 1986). I t i s u n l i k e l y that heat-damage to the pomace protein during the b a l l - m i l l i n g process caused the reduced a v a i l a b i l i t y because of the small increase i n temperature associated with t h i s process. 2.3.4. The e f f e c t of heat-treatment on TD, BV artd NPU of pomace fed to growing rats Heating of the pomace (previously dried at 60 C) at 100 C for two hours reduced TD, BV and NPU of the protein below that of the freeze-dried control, although the decrease was s i g n i f i c a n t only for NPU (P<0.05) (Table 5.9). This was apparently an additive e f f e c t , as both TD and BV were reduced (non-significantly) with heating. Heating of the 89 pomace at t h i s temperature does not appear to cause a large degree of damage to pomace protein. However, the high v a r i a b i l i t y observed within treatments may have masked s i g n i f i c a n t differences between treatments. P a l a t a b i l i t y problems with heated pomace may be more of a concern than any reduction of d i g e s t i b i l i t y of the protein. A t h i r d l e v e l of heating, at 150 C for 2 hours, was not tested because the pomace was rejected almost completely by the animals during the preliminary period. P r e - t r i a l consumption problems were encountered with the pomace heated at 100 C as well, although a f t e r the rats had become accustomed to the l a t t e r d i e t for three days, they began to f i n i s h t h e i r entire d a i l y r a t i o n . This problem was not observed to such an extent with the freeze-dried treatments. At the end of the four-day p r e - t r i a l period, the rats consuming the di e t s containing pomace which had been heated at 100 C had feed refusals of an average of 28% compared with 9% of the freeze-dried pomace. During the d i g e s t i b i l i t y t r i a l , there were no problems with consumption of any of the diets containing freeze-dried or 100 C dried pomace. 90 2.3.5. BV by rats of freeze-dried, coarsely-ground pomace The BV of the pomace which was both freeze-dried and coarsely (1mm) ground (Treatment 1) was s i g n i f i c a n t l y higher (P<0.05) than that of the other three treatment combinations (Table 5.9, Interaction). This e f f e c t was also observed with the measurement of NPU, but not with TD. The heat treatment that was used i n t h i s experiment (100 C for two hours) had an adverse e f f e c t on the BV and NPU of the coarsely ground pomace but did not a f f e c t the b a l l - m i l l e d pomace. The observed reduction of BV, but not of TD, of heat-treated j coarsely-ground pomace indicates that the heat-treatment of t h i s pomace caused heat-damage which reduced the q u a l i t y of the protein for the rats . These re s u l t s suggest that to avoid heat-damage to protein and to maximize protein BV, raspberry pomace should be coarsely ground (1mm) and dried at a low temperature (less than 60 C). The reduction i n BV was not observed when the b a l l -m i l l e d pomace was heated for two hours. Perhaps protein damage occurred during the i n i t i a l b a l l - m i l l i n g which resulted i n the observed s i g n i f i c a n t l y lower TD of these treatments, and t h i s masked any further damage that was caused by the subsequent heating. 91 2.3.6. Overall d i g e s t i b i l i t y of pomace protein The o v e r a l l poor d i g e s t i b i l i t y of pomace protein observed i n both the rat and pig d i g e s t i b i l i t y t r i a l s (Tables 5.7 and 5.9) indicates that the amount of soluble and e a s i l y d i g e s t i b l e protein i n the pomace i s very small, as i s suggested by the t o t a l dietary f i b r e analysis (Table 4.4) and by an examination of the process of juic e production (see l i t e r a t u r e review). I t has been suggested that the protein that i s not enzymatically digested during the determination of t o t a l dietary f i b r e ( i n d i g e s t i b l e crude protein - Table 4.4) represents the proportion that i s p h y s i o l o g i c a l l y unavailable to the animal (Asp et a l . , 1983). I f t h i s i s accurate, more than h a l f of the protein in raspberry pomace may be unavailable to non-ruminant animals. Low d i g e s t i b i l i t y of the protein of f r u i t and vegetable processing by-products has been observed by other workers, and i s t y p i c a l of t h i s type of by-product. While no research has been found i n which protein d i g e s t i b i l i t y by non-ruminant animals of a s i m i l a r by-product protein has been determined, d i g e s t i b i l i t y by ruminants of the protein of grape pomace was determined to be 19.5% (Hadjipanayiotou and Louca, 1976) and of tomato pomace protein to be 62% (Jayal and J o h r i , 1976). The poor protein d i g e s t i b i l i t y of 92 raspberry pomace may be the r e s u l t of several factors. These include: the large amount of f i b r e i n the pomace, and the possible presence of tannins and heat-damage protein-carbohydrate complexes i n the by-product. Each of these factors i s discussed more f u l l y i n the l i t e r a t u r e review. Growing rats consuming 100% raspberry pomace digested a higher percentage of the protein i n the di e t (Table 5.9) than pigs consuming the same rati o n (calculated by difference) (Table 5.7). The average apparent protein d i g e s t i b i l i t y (by difference) of pigs fed ground pomace which had been dried at 60 C and ground through a 1.6mm screen was 14.7%, while rats fed s i m i l a r l y prepared pomace as the entire ration (freeze-dried and 1mm ground) digested 21.8% of the protein (apparent d i g e s t i b i l i t y ) . This discrepancy could be as a r e s u l t of the d i f f e r e n t proportions of pomace fed i n the two t r i a l s , or as a re s u l t of differences between the two species i n t h e i r a b i l i t y to digest the protein i n pomace. 2 .4 . D i g e s t i b i l i t y o f f a t i n pomace f e d to growing pigs The l i p i d material i n the rati o n containing 40% ground raspberry pomace was 71.2% digested, which was s i g n i f i c a n t l y better (P<0.05) than the d i g e s t i b i l i t y of the l i p i d i n the rations containing 40% unground pomace (24.9%), 40% barley 93 (34.5%) or of the basal r a t i o n (28.7%) (Table 5.6). D i g e s t i b i l i t y of the fat i n ground pomace (by difference) was 79.7% while unground pomace was s i g n i f i c a n t l y less well digested at only 24.1% (P<0.05) (Table 5.7). The f a t i n the ground pomace was also s i g n i f i c a n t l y better digested than the f a t i n the basal rat i o n and i n barley as the entire rat i o n (by difference), while the f a t i n barley tended to be more d i g e s t i b l e than that of the basal r a t i o n (P<0.05). These re s u l t s demonstrate that the l i p i d contained in the raspberry seed i s well digested by growing pigs and that i t w i l l provide a valuable source of energy. The high d i g e s t i b i l i t y values obtained for the ground pomace are due to the unsaturated f a t t y acids which make up most of the l i p i d material i n raspberry seed o i l (Pourrat and Carnat, 1981), and which generally are well digested and absorbed by non-ruminant animals (Gurr, 1983). However, i t i s cle a r from the observed poor f a t d i g e s t i b i l i t y of unground pomace that grinding of the pomace i s necessary to wholly release t h i s source of nutrients from the seed kernel. The d i g e s t i b i l i t y of the fat i n the ground pomace may have been i n h i b i t e d by the high l e v e l of f i b r e i n the pomace. Experimentally, i t has been determined that apparent f a t d i g e s t i b i l i t y decreases by 1.3-1.5% for each additional 1% crude f i b r e i n the d i e t beyond a base l e v e l 94 (Just, 1982). Large amounts of f i b r e i n a d i e t depress the absorption of nutrients i n the small i n t e s t i n e and stimulate fermentation i n the colon, which appears to reduce fat d i g e s t i b i l i t y (Stahly, 1983). It i s in t e r e s t i n g that 24.1% of the fat i n the unground pomace apparently was digested by the pigs. Because the fat i s found larg e l y i n the seeds which, without grinding, are e s s e n t i a l l y i n d i g e s t i b l e , i t was expected that only a small amount of fat would be digested. I t therefore seems l i k e l y that a sizeable proportion of the seeds are cracked during mastication. The d i g e s t i b i l i t y of the fat i n the basal ration, and of barley by difference was low and quite variable (Table 5.7). However, the low l e v e l of fat i n the feedstuffs themselves would have made i t d i f f i c u l t to determine d i g e s t i b i l i t y accurately, p a r t l y because of the unknown amount of metabolic f a t i n the feces (Schneider and F l a t t , 1975). The ether-extractable f r a c t i o n of these feedstuffs may also contain considerable amounts of i n d i g e s t i b l e l i p i d s such as plant s t e r o l s and pigments. 2.5 . D i g e s t i b l e p r o t e i n , fa t and energy i n raspberry pomace Table 5.10 shows the calculated d i g e s t i b l e protein, fat and energy values for the pomace. The l e v e l of d i g e s t i b l e 95 protein i n both ground and unground pomace i s poor; only 11% to 15% of the average amount of 10.0% crude protein (Table 4.1) i n the pomace i s available to non-ruminants. Of an average of 11.1% crude f a t , 8.9% i s available from ground pomace while only 2.7% i s digested from unground pomace. The d i g e s t i b l e energy l e v e l of ground pomace i s 1490 k c a l * k g - 1 while that of unground pomace i s much lower. This compares with an average gross energy l e v e l i n raspberry pomace of 5220 k c a l * k g - 1 . Table 5.10. Digestible protein, f a t and energy i n ground and unground raspberry pomace for growing pigs (100% D.M. b a s i s ) . 1 Component of d i e t Protein (%) Fat (%) Energy (kcal* kg" 1) Ground pomace Unground pomace 1.5 1.1 8.9 2.7 1490 410 Calculated from average crude protein, crude f a t and gross energy values of pomace (Table 4.1), and from d i g e s t i b i l i t y values determined (by difference) with growing pigs (Table 5.7). 96 FEED INTAKE, GROWTH RATE AND FEED EFFICIENCY OF GROWING RATS FED DIETS CONTAINING RASPBERRY POMACE AD LIB 1. MATERIALS AND METHODS This experiment, designed and executed as described in Chapter 5, section 1.2, measured the feed intake and growth rate of rats fed diets containing increasing amounts of raspberry pomace. Daily feed intake for each rat ( a i r dry basis) was determined by weighing the feed j a r and feed at the s t a r t of the t r i a l and once d a i l y thereafter, and c a l c u l a t i n g the feed intake as the d a i l y loss of weight of j a r and feed. Rats were weighed at the s t a r t and end of the t r i a l , and once weekly during the t r i a l . 1.1. S t a t i s t i c a l analysis A l l r e s u l t s were subjected to analysis of variance using the SAS s t a t i s t i c a l program (UBC s i t e license) , and means were tested using the Newman-Keuls t e s t (Keuls, 1952). 97 2. RESULTS AND DISCUSSION 2.1 . Feed intake Up to and including 77.2% pomace in the d i e t (D.M. basis) (treatments 1 to 5), feed intake of rats over the three week t r i a l period was not s i g n i f i c a n t l y d i f f e r e n t (Table 6.1).- Only those rats consuming a d i e t of 96.4% raspberry pomace (treatment 6) showed a s i g n i f i c a n t reduction i n average d a i l y feed intake over the other treatments (P<0.05). The dry matter feed intake of rats consuming di e t s containing between 0 and 77.2% pomace ranged from 19.2 to 21.6 g per day over the t r i a l period, while rats consuming a d i e t of 96.4% pomace ate on average 16.5 g of feed. The intake on a l l treatments was within the range of expected feed intakes for rats of t h i s age (NRC, 1978). These r e s u l t s suggest that when included as a sizeable component of a d i e t (up to approximately 80%), raspberry pomace does not i n h i b i t feed intake, but as a complete ratio n , p a l a t a b i l i t y problems occur. Because the pomace i s considerably less dense than a barley-based d i e t , a large volume of pomace must be consumed by the animal to ingest the equivalent weight of the basal d i e t , and t h i s may act as a deterrent to feed intake at the 100% l e v e l . The inclusion 98 Table 6.1. Average d a i l y feed intake, weight gain and feed e f f i c i e n c y of growing rats fed varying proportions of raspberry pomace i n a balanced barley-based d i e t . T r t . Percentage DAILY TREATMENT AVERAGES of pomace in d i e t Feed Weight Feed Intake Gain E f f i c i e n c y (D.M. basis) (g dry (g) (g feed*g feed) g a i n - 1 ) 1 0 19.2 a* 4.7 a 4.3 a 2 19.4 19.8 a 4.5 a 4.8 a 3 38.6 21.6 a 3.9 b 6.2a 4 58.0 21.2 a 3.2J 7.4a 5 77.2 19.9 a 2.3 d 9.6a 6 96.4 16.5 b 1.0 e 2 6 . l b SEM 0.65 0.14 1.19 * Means within columns with d i f f e r e n t superscript l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). 99 of raspberry pomace at l e v e l s higher than 80% of a die t i s therefore not recommended for p a l a t a b i l i t y reasons. Over a l l treatments, feed intake between weeks varied s i g n i f i c a n t l y (P<0.05) (Table 6.2 and Figure 6.1). Intake for a l l t r i a l treatments was highest during the second week of the t r i a l . 2.2. Weight gain When pomace was included at up to 19.4% (D.M. basis) of a balanced diet, average d a i l y weight gain of t r i a l rats was not s i g n i f i c a n t l y reduced (Table 6.1). However, average d a i l y gain dropped s i g n i f i c a n t l y with each incremental increase i n the l e v e l of pomace i n the diet, from an average of 4.6 g * d - 1 on the 96.9% barley d i e t and the die t containing only 19.4% pomace, to 1.0 g * d - 1 on the d i e t of 96.4% pomace (P<0.05). These re s u l t s suggest that raspberry pomace could be included at up to approximately 20% of an ad l i b - f e d complete rati o n for growing swine without a s i g n i f i c a n t reduction i n weight gain by the animals. Feed intake was reduced s i g n i f i c a n t l y only at the highest l e v e l of inclusion of pomace, thus i t had l i t t l e influence on weight gain i n the other four pomace-containing treatments. This suggests that poor u t i l i z a t i o n of the pomace caused the s i g n i f i c a n t l y lower weight gains observed 100 Table 6.2. Average d a i l y feed intake (FI), weight gain (ADG) and feed e f f i c i e n c y (F/G) of rats fed varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n over three weeks of t r i a l (see figures 6.1, 6.2 and 6.3 for weekly data). Week FI ADG F/G 1 19.0 b* 4.5 a 5.2 b 2 22.5 a 3.0 b 10.2 a 3 17.5c 2.2c 13.8a * Means within columns with d i f f e r e n t superscript l e t t e r s are s i g n i f i c a n t l y d i f f e r e n t (P<0.05). 101 Figure 6.1. Average d a i l y feed intake of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based ra t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Feed intake was s i g n i f i c a n t l y higher during week two than during weeks one and three. I wk 1 wk 3 0 19.4 38.6 58.0 77.2 96.4 % pomace in diet 102 with treatments 3, 4 and 5. As i s cle a r from the chemical analyses performed, raspberry pomace contains a high l e v e l of fibrous c e l l - w a l l material and other i n d i g e s t i b l e constituents (Tables 4.4 and 4.5) which has been shown to r e s u l t i n poor d i g e s t i b i l i t y by non-ruminants. For a l l treatments, the average d a i l y weight gain was s i g n i f i c a n t l y higher during week one of the t r i a l than during the other two weeks (P<0.05) (Figure 6.2 and Table 6.2). With the exception of the rats consuming 96.4% pomace, the average d a i l y weight gain of a l l animals declined through weeks two and three of the t r i a l . The s i g n i f i c a n t l y higher weight gains o v e r a l l during week one may have been a r e s u l t of compensatory gains by the t r i a l animals. P r i o r to t h i s t r i a l , the rats had been consuming a di e t of 100% raspberry pomace for 10 days and were receiving about h a l f as much feed as they consumed when fed ad l i b during t h i s t r i a l . Once they were given unlimited access to feed which contained a higher percentage of d i g e s t i b l e nutrients, they could make rapid weight gains, compensating for the r e s t r i c t e d growth rate during the previous t r i a l . Although d i e t 6 did not actually contain a higher l e v e l of d i g e s t i b l e nutrients than the diets fed i n the e a r l i e r t r i a l , the increased feed consumption of rats fed t h i s diet 103 Figure 6.2. Average d a i l y gain of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Daily gain was highest during week one of the t r i a l , and declined s i g n i f i c a n t l y during each of the subsequent weeks. I 111 0 19.4 38.6 58.0 77.2 96.4 % pomace in diet 1 0 4 could account for t h e i r observed higher weight gains during the f i r s t week of t h i s t r i a l . Growth data for two st r a i n s of laboratory rat (Poily, 1972) (neither of which were used i n t h i s t r i a l ) indicate that for the age of the rats at the s t a r t and f i n i s h of t h i s t r i a l (from 35 to 56 days of age) , d a i l y weight gain would normally have been increasing during t h i s period. This i s the opposite of the growth pattern observed during the t r i a l and further suggests that compensatory gain may have been occurring. It i s i n t e r e s t i n g to note that the week during which the highest average d a i l y weight gains occurred (week one) was not the week during which feed intake was highest (week two) . 2.3. Feed e f f i c i e n c y As a r e s u l t of the consistent decline i n average d a i l y weight gain with each increment of pomace in the diet, feed/gain increased steadily, although not s i g n i f i c a n t l y , up to the i n c l u s i o n rate of 77.2% pomace i n the d i e t (Table 6.1). Feed/gain for the d i e t with the highest l e v e l of pomace (96.4%) was s i g n i f i c a n t l y higher than for the other f i v e diets (P<0.05). Feed e f f i c i e n c y increased from an average of 4.3 g of feed consumed per g of gain (D.M.basis) 105 with the barley-based ration, to 2 6.1 g feed per g gain for rats fed 96.4% pomace. V a r i a b i l i t y within the treatment means was high which may have prevented the i d e n t i f i c a t i o n of s i g n i f i c a n t differences between treatment means. Over a l l treatments, feed/gain for a l l animals was best during week one, and increased consistently during weeks two and three, with the exception of treatment 6 (Figure 6.3 and Table 6.2). 106 Figure 6 .3 . Feed/gain of growing rats over three weeks of t r i a l consuming varying l e v e l s of raspberry pomace i n a balanced barley-based r a t i o n . Pomace was included at 0 ( t r t 1), 20, 40, 60, 80 and 100% ( t r t 6) of rations. Feed/gain was s i g n i f i c a n t l y better during week one of the t r i a l than during either of the other weeks 0 19.4 38.6 58.0 77.2 96.4 % pomace in diet 107 SUMMARY AND CONCLUSIONS Raspberry pomace has a dry matter content of 44.4%, and consists of approximately 87.8% seeds, pulp and trash and 12.2% added r i c e h u l l s . I t contains on the average 10.0% crude protein, 11.1% crude f a t , 7.4% soluble carbohydrates (sugars) and 4.1% ash (D.M. ba s i s ) . The crude protein and ess e n t i a l amino acid content of raspberry pomace are s i m i l a r to that of Canada No. 1 feed grade barley. The pomace has a gross energy content of 5220 k c a l * k g - 1 , l a r g e l y due to the l i p i d material contained i n the seed. I t contains on average 54.1% neutral detergent f i b r e residue (an estimation of c e l l wall material), 57.2% insoluble dietary f i b r e and 2.3% soluble dietary f i b r e . The acid detergent f i b r e residue consists of 11.7% l i g n i n , 6.0% cutin, 2.2% A. D. ash and 26.9% c e l l u l o s e (by difference from acid detergent value). The l i g n i n determination includes true l i g n i n , tannin material and heat-damage protein-carbohydrate complexes. Dry matter d i g e s t i b i l i t y (DMD) of raspberry pomace was determined by feeding the pomace to growing pigs and rats. Ground (1.6mm) and unground pomace were included at 40% (air dry basis) of a balanced d i e t fed to growing pigs. Grinding of the pomace s i g n i f i c a n t l y improved the DMD of the pomace-containing diets and of the pomace (by difference) . 108 However, the barley-soybean meal basal ration was consistently better digested than either of the pomace treatments. DMD of the whole rati o n containing 40% ground pomace was 56.3%, which was s i g n i f i c a n t l y better than that of the ra t i o n containing 40% unground pomace (52.2%). DMD of ground pomace (by difference) was found to be 20.8% while that of unground pomace was only 10.7% . DMD (by chromic oxide marker method) of barley-raspberry pomace diets was determined with growing rats. Ground pomace (1mm screen) was included at approximately 0, 20, 40, 60, 80 and 100% of the ration, excluding minerals and vitamins. The 100% barley d i e t was 80.8% digested. With each increment of pomace i n the die t , DMD was s i g n i f i c a n t l y reduced. Raspberry pomace as the sole d i e t ingredient was 2 0.9% digested by the rats. The poor DMD of pomace by non-ruminants i s believed to be as a r e s u l t of the high proportion of i n d i g e s t i b l e components. Although c e l l u l o s e and hemicelluloses can be extensively degraded i n the colon and cecum of non-ruminants, the high l e v e l of l i g n i n and other i n d i g e s t i b l e components i n the pOmace probably w i l l l i m i t t h i s microbial degradation (Robertson and Van Soest, 1981). The protein d i g e s t i b i l i t y of pomace was determined by the difference method with pigs, and d i r e c t l y with rats i n a 109 nitrogen balance t r i a l . Because of the d i f f i c u l t y of measuring protein d i g e s t i b i l i t y by difference, i t i s suggested that the res u l t s obtained d i r e c t l y with rats may be more accurate. The apparent d i g e s t i b i l i t y of the protein (by difference) of ground raspberry pomace fed to growing pigs was 14.7% which was higher than that of unground pomace (10.6%), although t h i s e f f e c t was not s i g n i f i c a n t . The protein of the barley-soybean basal r a t i o n was s i g n i f i c a n t l y better digested than that of the pomace. True protein d i g e s t i b i l i t y (TD) by growing male rats of raspberry pomace as the sole protein source was 33.3%. The b i o l o g i c a l value (BV) of the absorbed protein was, on average, 79.3% and the net protein u t i l i z a t i o n (NPU) was 26.6%. Although the o v e r a l l protein d i g e s t i b i l i t y , and thus the NPU, were poor, the protein which was digested and absorbed (BV) was well u t i l i z e d , suggesting that i t contained a good p r o f i l e of ess e n t i a l amino acids. The protein of b a l l - m i l l e d pomace (<0.5mm fineness) was less well u t i l i z e d by rats than that of more coarsely ground pomace (1mm screen). Heating of previously-dried pomace for two hours at 100 C did not s i g n i f i c a n t l y influence protein d i g e s t i b i l i t y . However, i t appears that pomace which was both ground to 1mm fineness and freeze-dried had a higher BV 110 for rats than pomace that was heat-treated at 100 C and either b a l l - m i l l e d or 1mm ground. These r e s u l t s suggest that the u t i l i z a t i o n of pomace protein w i l l be maximized with low-temperature drying ( i f drying i s necessary) and with grinding not f i n e r than 1mm. The poor protein d i g e s t i b i l i t y can be attributed to several factors. Much of the protein i n a fibrous material such as raspberry pomace i s embedded i n the c e l l wall and i s unavailable to non-ruminants because of t h e i r limited a b i l i t y to degrade c e l l wall material (Theander and Aman, 1984) . The presence of tannin i n the pomace would promote the formation of i n d i g e s t i b l e tannin-protein complexes (Robertson and Van Soest, 1981) . The heat of processing could have created protein-carbohydrate complexes (Maillard browning products) which are also i n d i g e s t i b l e (Knipfel, 1981). Grinding s i g n i f i c a n t l y increased the apparent d i g e s t i b i l i t y of the l i p i d material i n pomace. Pigs consuming rations containing 4 0% ground pomace digested 71.2% of the fat i n the rati o n versus only 24.9% i n the rat i o n containing the same amount of unground pomace. Si m i l a r l y , when f a t d i g e s t i b i l i t y of ground and unground pomace was determined by difference, i t was found that 79.7% 111 of the fat i n ground pomace was digested as compared with only 24.1% of the unground pomace l i p i d . Growing rats were fed a supplemented barley-based diet containing ground pomace i n the approximate l e v e l s of 0, 20, 40, 60, 80 and 100% (excluding minerals and vitamins). With up to 2 0% pomace i n the die t , growth rate was not s i g n i f i c a n t l y reduced, but with each increment of pomace beyond 20% of the die t , rate of gain declined s i g n i f i c a n t l y . Feed intake was reduced s i g n i f i c a n t l y only at the highest dietary l e v e l of pomace. Raspberry pomace has a high gross energy value because of the seed l i p i d , and a crude protein and amino acid content s i m i l a r to that of Canada No.l feed grade barley, both of which suggest that i t may be a good substitute for a portion of the barley i n diets for growing swine. The pomace also contains a high proportion of i n d i g e s t i b l e components. D i g e s t i b i l i t y t r i a l s have shown that grinding of the pomace improves the d i g e s t i b i l i t y of dry matter, energy, protein and fat by growing pigs and ra t s . However, even when the pomace i s ground, the d i g e s t i b i l i t y of dry matter, energy and protein i s poor compared with that of other energy feedstuffs commonly used i n swine rations, although the d i g e s t i b i l i t y of the l i p i d i s high. A growth t r i a l showed that the average d a i l y gain of rats was not 112 s i g n i f i c a n t l y reduced when pomace was included at up to 20% of an ad - l i b fed, supplemented rati o n and that feed intake was not in h i b i t e d with up to 80% pomace i n the d i e t . These r e s u l t s suggest that raspberry pomace could be included at up to 20% of the die t of growing swine without markedly influencing growth rate or feed e f f i c i e n c y . 113 BIBLIOGRAPHY AOAC. Association of O f f i c i a l A n a l y t i c a l Chemists. 1975. O f f i c i a l methods of analysis, 12th Ed. Association of O f f i c i a l A n a l y t i c a l Chemists, Washington, D.C. AOAC. Association of O f f i c i a l A n a l y t i c a l Chemists. 1980. O f f i c i a l methods of analysis, 13th Ed. Association of O f f i c i a l A n a l y t i c a l Chemists, Washington, D.C. Asp, N., C-G. Johansson, H. Hallmer. and M. Siljestrom. 1983. Rapid Enzymatic Assay of Insoluble and Soluble Dietary Fibre. 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Free sugars and s o r b i t o l i n f r u i t s - A compilation from the l i t e r a t u r e . J . Assoc. O f f i c . Anal. Chem. 64:91-103. 120 APPENDIX 1. TOTAL DIETARY FIBRE ANALYSIS The method described here i s that of Prosky et a l . (1984), but i t incorporates the modification of Asp et a l . (1983) which allows.the separation of soluble and insoluble dietary f i b r e . This method also incorporates some suggestions by the author which may make the procedure less troublesome. A more complete explanation of the theory behind the method i s found i n the above references. Reagents: 1. Ethanol - 95%, technical grade. 2. Ethanol - 74%. In 1L volumetric flask, add 250mls d i s t i l l e d water and make to volume with 95% ethanol. Mix, and d i l u t e to volume again with ethanol i f required. 3. Phosphate buffer pH 6.0 - Dissolve 1.5g sodium phosphate dibasic and 10.Og sodium phosphate monobasic monohydrate i n ca. 700ml d i s t i l l e d water. Dilute to 1L with water and adjust pH to 6.0 by adding drops of d i l u t e monobasic or dibasic sodium phosphate, i f necessary. 4. NaOH solution - 0.285N. Dissolve 11.4g NaOH i n ca. 700 ml water i n 1L volumetric f l a s k . Dilute to volume with water. 5. Phosphoric acid solution. - 0.329M. Dissolve 37.9g phosphoric acid (85%) i n water i n 1L volumetric flask. Dilute to volume with water. 6. C e l i t e 545. - Fisher S c i e n t i f i c Co., F a i r Lawn, NJ. 121 7. Termamyl (heat-stable alpha-amylase) s o l u t i o n . . - 120L (Novo Laboratories Inc., Wilton, CT.) Store a l l enzyme solutions i n r e f r i g e r a t o r a f t e r each use. 8. Protease P-5380. - Subtilopeptidase A, Type VIII (Sigma Chemical Co., St. Louis, MO 63178). 9. Amylogulcosidase A-9268. - E.C.3.2.1.3 (Sigma Chemical Co.) 10. To measure the a c t i v i t y of the above enzymes and to check for the presence of undesirable enzymes, Sigma Chemical TDF assay control k i t (TDF-C10 ki t ) i s available, containing v i a l s of casein, beta-glucan, pectin, corn and wheat starch, and arabinogalactan, or these can be purchased separately. Determination of t o t a l d ie tary f i b r e (TDF): Note: two clean, dry f r i t t e d c r ucibles of porosity 2 w i l l be required for each sample. Add ca 0.5g C e l i t e to clean crucibles and dry at 130 C for 1 hr to obtain constant weight. Record weight of c r u c i b l e and C e l i t e . F i l t e r i n g problems may be encountered i f crucibles are clogged with mineral matter. I t i s recommended that they be demineralized before use. See Goering and Van Soest (1970) for suitable cleaning solution. 1. I f fat content of samples i s greater than 5%, fat extract before analyzing, recording the weight loss of fat so that f i n a l weight of TDF can be corrected for t h i s l o s s . 2. Run blanks through entire procedure along with samples to measure any contribution from reagents to residue. 3. Weigh, i n t r i p l i c a t e , l g sample accurate to O.lmg (sample weights should not d i f f e r by more than 20mg) into 600ml t a l l - f o r m beakers. Add 50ml pH 6.0 phosphate buffer to each beaker. Because timing of digestion and f i l t r a t i o n i s important, i t i s recommended that only 6 samples be digested at once ( i . e . 2 samples i n t r i p l i c a t e ) . 122 4. Add 0.1ml Termamyi solution. 5. Cover beakers with aluminum f o i l and place i n b o i l i n g water bath. Shake gently every 5 min. Continue for 15 minutes a f t e r inner temperature of solution has reached 95 C. 6. Cool solutions to room temperature. Adjust pH to 7.5 (+/- 0-1) by adding ca 10ml 0.285N NaOH solution. Check with pH meter and adjust accordingly. 7. Add 5rag protease. (Just p r i o r to use, make a 50mg/ml solution with protease powder and phosphate buffer, and pipet 0.1ml into each beaker). 8. Cover beakers with aluminum f o i l and place i n a 60 C constant temperature water bath with constant a g i t a t i o n . Incubate for 30min a f t e r beaker i n t e r n a l temperature has reached 55 C. 9. Cool to room temperature. Add ca 10ml 0.329M phosphoric acid solution to adjust pH to 4.5 (+/- 0.2). Check with pH meter and correct i f necessary. 10. Add 0.3ml amyloglucosidase solution. 11. Same as 8 above. Separating soluble and insoluble f i b r e : 12. Remove from water bath and immediately f i l t e r solution through one of the two dry, weighed crucibles containing C e l i t e (as f i l t e r a i d ) . Save f i l t r a t e q u a n t i t a t i v e l y as i t contains the soluble f i b r e i n soluti o n . Rinse beaker with 2 X 10ml d i s t i l l e d water to wash out water soluble f i b r e , and save t h i s rinse water also. Pour f i l t r a t e back into o r i g i n a l beaker and set aside for subseqent p r e c i p i t a t i o n of soluble f i b r e . Insoluble f i b r e residue i n 1st cr u c i b l e : 13. Wash f i b r e mat with 2 X 10ml 95% ethanol and 2 X 10ml acetone. 123 14. Dry cru c i b l e at 105 C to constant weight (overnight). Weigh a f t e r cooling i n dessicator. Difference i n weight of crucible with and without f i b r e i s weight of crude insoluble f i b r e . 15. Determine N i n two of the t r i p l i c a t e s by Kjeldahl or wet ash method and subtract average weight of i n d i g e s t i b l e protein from crude insoluble f i b r e value. (To analyze, scrape f i b r e and C e l i t e quantitatively out of crucible onto N-free f i l t e r paper and place i n Kjeldahl f l a s k ) . 16. Ash the t h i r d t r i p l i c a t e (in crucible) at 475 C for at leas t 5 hours. Weigh a f t e r cooling i n dessicator. Difference between o r i g i n a l c r u c i b l e weight and t h i s weight i s insoluble ash. Ash a blank c r u c i b l e containing C e l i t e to measure loss of C e l i t e on ashing. F i l t r a t e containing soluble f i b r e : 17. Adjust the volume of combined f i l t r a t e and washing waters to 100ml (ignore t h i s i f the volume i s more than 100ml). 18. Add 400ml warm (60 C) 95% ethanol (or 4X volume of f i l t r a t e , i f t h i s i s l a r g e r ) . Allow to p r e c i p i t a t e overnight (or for a minimum of 60min.). P r e c i p i t a t i o n time should be the same for a l l samples and blanks. 19. F i l t e r solution through the second dry, weighed crucible containing C e l i t e . 20. Rinse beaker and f i b r e with 2 X 10ml portions of 78% ethanol, 2 X 10ml 95% ethanol and 2 X 10ml acetone. 21. Dry at 105 C overnight. Weigh, a f t e r cooling i n dessicator. Increase i n weight i s weight of crude soluble f i b r e . Repeat steps 15 and 16 above for determination of i n d i g e s t i b l e protein and ash i n soluble f i b r e . 124 Calculation: F i n a l weights of soluble and insoluble f i b r e are obtained a f t e r correcting crude values for: 1. o r i g i n a l sample dry weight 2. loss of f a t during fat-extraction ( i f samples contained more than 5% fat) 3. weight of residues from reagents (blank value), 4. i n d i g e s t i b l e crude protein (by Kjeldahl or s i m i l a r method) 5. ash (by incineration of sample) 6. loss of C e l i t e during inci n e r a t i o n 125 APPENDIX 2. ACID HYDROLYSIS OF FAT IN FECAL MATERIAL The acid hydrolysis was done with a Buechi hydrolysis unit, B428, and the subsequent f a t extraction was performed i n a Buechi Soxhlet-type extraction unit. A l l glassware was custom-designed for use with t h i s equipment. Acid hydolysis and subsequent fat extractions performed as part of t h i s thesis were undertaken at the Agriculture Canada Research Station, Agassiz, B.C. This method could be modified to be undertaken without the Buechi hydrolysis and extraction equipment i f t h i s was necessary, although i t would be more awkward. Hydrolys i s procedure: 1. Weigh out and add approximately 5 g of C e l i t e 545 to a digestion tube. 2. Accurately weigh 2-3 g of sample onto weigh paper, record weight and add to digestion tube (Note: much larger sample sizes can e a s i l y be accomodated i n the glassware). 3. Add 100 ml 4N HCl to tube - pour around sides of tube to wash down any sample c l i n g i n g to the sides (4N HCl: 400ml concentrated HCl d i l u t e d with 600ml d i s t i l l e d water). 4. Swirl the mixture u n t i l homogeneous and no sample i s s t i c k i n g to sides or bottom of fl a s k . 5. Weigh out and add approximately lOg of sea sand (cleaned and annealed) to a sintered glass hydrolysis c r u c i b l e . 126 6. Weigh out and add approximately 5g C e l i t e 545 to the cru c i b l e i n a uniform layer on top of sand (these are both for improved f i l t r a t i o n ) . 7. F i t the cru c i b l e into the extraction tube by loosening the caps on the suction tube, i n s e r t i n g the cru c i b l e extension through the hole i n the cap and tightening the cap down. Position the raising/lowering handle i n the uppermost position. 8. Place the digestion tube containing the sample and C e l i t e into the digestor (which has been preheated at control setting 6 for 15 minutes). 9. Set the heat control knob at 3 for the hydrolysis. 10. I n s t a l l the sample aspiration tubes by i n s e r t i n g them through the hole i n the caps on top of the digestion tubes, snapping them into the grooved pieces and connecting them to the glass c r u c i b l e with a U-clamp. Screw down the U-clamp t o ensure good vaccum. Ensure that the raising/lowering arm i s i n the upright p o s i t i o n or sample w i l l be aspirated into the crucibles before hydrolysis i s complete. 11. Turn on water supply to provide suction to remove the HC1 fumes during hydrolysis. 12. After the solution i n the digestion tubes has begun to b o i l , hydrolyze for 15 minutes. 13. Lower the extraction system into the digestion tubes by unscrewing and lowering the arm. Aspirate the sample into the scintered glass c r u c i b l e . The hydrolyzed sample w i l l remain i n the cru c i b l e and the acid solution w i l l be removed. 14. Once the sample has been aspirated from the tube, rinse each tube with 50ml warm d i s t i l l e d water (50-60 C) and aspirate out to remove any residual sample. Repeat for 5 washes; 250ml water per sample. 15. Remove the cru c i b l e from the unit and dry cru c i b l e and sample i n preparation for fat-extraction. A suitable method of drying for samples that are heat-sensitive i s : microwave for 10-15 minutes on high then dry overnight at 70 C. For those samples not sensi t i v e to temperature, dry overnight at 130 C. 127 Fat extraction procedure: 1. Insert a large plug of g lass wool into each c r u c i b l e before extract ion to ensure that no sample f l oa t s out during the ex trac t ion . 2. Put the c r u c i b l e s into the Buechi Soxhlet-type fat ex trac t ion u n i t , ensuring that there are springs i n the extractors to hold the c r u c i b l e s . 3. Extract with petroleum ether or anhydrous d i e t h y l ether for 3 hours fol lowing the standard Soxhlet method. Adjust the d r i p rate of ether so that i t i s as rapid as poss ib le without f i l l i n g the c r u c i b l e more qu ick ly than the ether can be absorbed by the sample. This i s done by a l t e r i n g the temperature of the hotplates beneath the beakers. 4. The weight of fa t i s ca lcu la ted as the weight of the beaker before and a f ter ex trac t ion . 128 APPENDIX 3 . 1 CHEMICAL ANALYSES OF RASPBERRY POMACE CONTAINING RICE HULLS (100% D . M . B A S I S ) . Sample # Crude P r o t e i n 1 (%) Crude F a t 2 (%) Soluble CHO3 (%) Ash (%) Total Dietary Fibre'* (%) G.E. 5 (kcal* kg" 1) 1 10 . 3 10.7 6.8 61.8 2 9.5 12.2 7.0 3 . 3 57.5 5340 3 9.3 12 .1 8.5 4.1 62.9 -4 9.8 10.7 8.3 4.3 61. 1 5206 5 10.1 10.7 9.0 3.2 59.8 -6 12.3 8.6 9.0 5.2 54 .8 -7 9.4 12.0 7.3 4.2 59.4 -8 10.1 11.2 5.2 4.1 64.6 5161 9 10. 3 12 . 6 6.2 3.4 61.4 5254 10 9.1 10.0 6.5 4.8 51.6 5127 Average 10. 0 11.1 7.4 4.1 59.5 5217.6 Crude protein by Macro-kjeldahl method (AOAC, 1980) Crude f a t by Goldfisch ether extract method (anhydrous di e t h y l ether). Soluble CHO i s water-soluble carbohydrates including glucose and other sugars. Method of Asp et a l . (1983) Measured by bomb calorimetry 129 APPENDIX 3.2 ANALYSIS OF SOLUBLE AND INSOLUBLE DIETARY FIBRE COMPONENTS OF RASPBERRY POMACE WITH RICE HULLS (100% D.M. BASIS). TOTAL INSOLUBLE FIBRE SOLUBLE FIBRE Sample Total Insol. Indig . Insol Sol. Sol. dietary f i b r e C P . ash f i b r e ash f i b r e (%) (%) (%) (%) (%) (%) 1 61.8 59.8 5.8 6.6 1.9 1.0 2 57.5 55.6 5.9 10.2 2.0 1.1 3 62.9 60.9 5.0 4.2 2.0 0.9 4 61.1 59.1 5.1 6.6 2.0 1.0 5 59.8 58.2 5.0 8.5 1.6 1.1 6 54.8 •51.4 6.8 12.2 3.4 1.4 7 59.4 56.9 4.5 8.4 2.5 0.5 8 64 . 6 62 .1 4.4 5.2 2.5 0.5 9 61.4 58.4 4.4 6.4 3 .1 0.5 10 51.6 49.6 5.4 16.4 2 . 0 1.5 Average 59.5 57.2 5.2 8.5 2.3 1.0 Note: Total dietary f i b r e i s the sum of insoluble and soluble dietary f i b r e and does not include i n d i g e s t i b l e crude protein or insoluble and soluble ash. 130 APPENDIX 3.3 ANALYSIS OF FIBRE OF RASPBERRY POMACE WITH RICE HULLS (100% D.M. BASIS). 1 Sample N.D.F. A.D.F. Lignin Cutin Acid C e l l . 2 Detergent Ash (%) (%) (%) (%) (%) (%) 1 55.0 n/a n/a n/a n/a n/a 2 53 . 3 44.9 9.8 9.1 1.5 24.5 3 54.3 46.7 10.9 7.8 .2.3 25.6 4 54.8 46.0 12.0 5.9 2.3 25.8 5 56.4 45.2 12.2 6.0 1.4 25.6 6 48.8 43.3 9.7 4.2 3.1 26.3 7 54.5 47.2 15.4 3.2 2.2 33.3 8 56.3 47 . 0 12.4 5.8 2 . 2 26.6 9 53.4 46.6 12.6 5.5 1.8 26.8 10 54 . 3 47.2 10.3 6.6 2.9 27.4 Av. 54. 1 46.0 11.7 6.0 2.2 26.9 Methods of Goering and Van Soest (1970) Cellulose calculated as ADF-(lignin+cutin+ash) 131 

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