Conjugated Linoleic Acids and Conjugated Vegetable Oils: Volume 19 (Catalysis Series) - Hardcover

Hardcover

ISBN 10: 1849739005 ISBN 13: 9781849739009

Publisher: Royal Society of Chemistry, 2014

Synopsis

This book presents a comprehensive overview of all different aspects of CLA, and summarises the chemistry of their various applications.

About this title

From the Back Cover

Conjugated linoleic acids (CLA) isomers of linoleic acid - a compound derived from meat and dairy products. Attention was first drawn to their potential anti-carcinogen properties in the 1980's; since then further health benefits have been reported, and applications in the glue and paint industries as a renewable resource have been explored.

This comprehensive book presents an overview of the background and research into CLA and examines each of their applications in the context of the chemistry surrounding them and CLA-enriched oils. The biosynthesis of CLA is presented, with a discussion on how animal husbandry could promote CLA production. Other chapters examine the current strategies for their synthesis using bespoke catalysts and enzymes. Readers from academia and industry will find the layout of the book highly accessible, with sections for each application.

The editors are both active researchers in the field, and have brought together a wealth of expertise from across the globe, presenting a comprehensive guide to this valuable group of compounds and their potential applications.

From the Inside Flap

Conjugated linoleic acids (CLA) isomers of linoleic acid a compound derived from meat and dairy products. Attention was first drawn to their potential anti-carcinogen properties in the 1980 s; since then further health benefits have been reported, and applications in the glue and paint industries as a renewable resource have been explored.

This comprehensive book presents an overview of the background and research into CLA and examines each of their applications in the context of the chemistry surrounding them and CLA-enriched oils.  The biosynthesis of CLA is presented, with a discussion on how animal husbandry could promote CLA production. Other chapters examine the current strategies for their synthesis using bespoke catalysts and enzymes. Readers from academia and industry will find the layout of the book highly accessible, with sections for each application.

The editors are both active researchers in the field, and have brought together a wealth of expertise from across the globe, presenting a comprehensive guide to this valuable group of compounds and their potential applications.

Excerpt. © Reprinted by permission. All rights reserved.

Conjugated Linoleic Acids and Conjugated Vegetable Oils

By Bert Sels

The Royal Society of Chemistry

Copyright © 2014 The Royal Society of Chemistry
All rights reserved.

ISBN: 978-1-84973-900-9

Contents

Chapter 1 Synthesis of Conjugated Linoleic Acid in Ruminants and Humans K. J. Shingfield and R. J. Wallace, 1, 
Chapter 2 Use of CLA in Animal Feed N. Everaert, A. Koppenol and J. Buyse, 66, 
Chapter 3 Health Benefits of Conjugated Fatty Acids Yeonhwa Park and Yan Wu, 94, 
Chapter 4 Commercial CLA and its Chemical Use Rafael Lopes Quirino, 117, 
Chapter 5 Recent Advances in the Production of CLA and Conjugated Vegetable Oils: Microbial and Enzymatic Production of Conjugated Fatty Acids and Related Fatty Acids in Biohydrogenation Metabolism Jun Ogawa, Michiki Takeuchi and Shigenobu Kishino, 131, 
Chapter 6 Recent Advances in the Production of CLA and Conjugated Vegetable Oils: Production of CLA and Conjugated Vegetable Oils via Metal Catalysis K. Belkacemi, N. Chorfa and S. Hamoudi, 151, 
Chapter 7 Analysis of Conjugated and Other Fatty Acids Pierluigi Delmonte, Ali Reza Fardin-Kia, Noelia Aldai, Magdi M. Mossoba and John K. G. Kramer, 172, 
Subject Index 232, 

CHAPTER 1

Synthesis of Conjugated Linoleic Acid in Ruminants and Humans

K. J. SHINGFIELD AND R. J. WALLACE

1.1 Introduction

There is increasing evidence that nutrition plays an important role in the development of human chronic diseases including cancer, cardiovascular disease, insulin resistance and obesity. Developing foods and diets that promote human health is central to public health initiatives for preventing and lowering the economic and social impact of chronic disease. Direct and indirect costs of cardiovascular disease (CVD) have been estimated at $445 billion in the United States and h200 billion within the European Union. Global costs of CVD in 2010 totalled US$863 billion. These costs are projected to increase several-fold by 2030, reaching unsustainable levels due to people living longer and the rapid increase in obesity in developed and developing countries.

Following the identification of the anti-mutagenic properties of conjugated linoleic acid (CLA) isomers in cooked beef, numerous studies have investigated the biological activity of CLA isomers in cell culture and animal models (http://fri.wisc.edu/cla.php). Much of the research has focused on the effects of cis-9, trans-11 18 : 2 (trivial name rumenic acid) or trans-10, cis-12 18 : 2 due to the cost and availability in a range of mammalian and avian species. In addition to the inhibition of mutagenesis, specific isomers of CLA have been demonstrated to modulate energy metabolism, immunity, inflammation, insulin resistance and bone metabolism in several animal models. However, evidence that the physiological effects described in vitro or in other mammalian species are also replicated in humans remains inconclusive.

The optimal intake of one or more isomers of CLA in humans remains to be established. Direct or exponential extrapolation of data from studies in the rat model of carcinogenesis implicate intakes of cis-9, trans-11 18 : 2 between 95 and 3500 mg per day being required for significant decreases in cancer risk in human populations. Estimates of cis-9, trans-11 18 : 2 consumption in human populations vary between 15 and 1500 mg per day depending on the methodology used to estimate dietary intakes, with marked differences between countries, gender and socioeconomic groups. Isomers of CLA are present in a wide range of foods including milk, beef and lamb, and to a much lesser extent in pork, poultry, fish and eggs, with trace amounts in some vegetable sources. Milk and dairy products are the major source of CLA in the human diet contributing to between 66 and 80% of total intake. Typically concentrations of CLA in pork, chicken and fish are lower than 0.1 g per 100 g lipid, whereas the consumption of lamb, beef and other ruminant meat products account for 15–32% of average daily CLA intakes in developed countries.

The CLA status of humans can be increased using oral supplements or fortification of foods with synthetic sources, which typically contain equal amounts of cis-9, trans-11 18 : 2 and trans-10, cis-12 18 : 2, or from a higher consumption of ruminant-derived foods. In contrast to synthetic sources, meat and milk from ruminants contain numerous positional and geometric isomers of CLA with conjugated double bonds at positions 6,8 through to 13,15, with cis-9, trans-11 18 : 2 being the major isomer, and trans-7, cis-9 18 : 2 or trans-11, cis-13 18 : 2 as the second most abundant.

Producing ruminant-derived foods containing higher amounts of CLA offers the opportunity to increase the consumption of CLA, principally cis-9, trans-11 18 : 2, without requiring major changes in the habitual diet or eating habits. For this reason, a considerable amount of research has been dedicated to understanding the nutritional, physiological and genetic factors influencing CLA concentrations in meat and milk. The present chapter provides a comprehensive review of the most recent evidence on the biochemical, microbial, nutritional and physiological factors influencing the biosynthesis of CLA isomers in ruminants and humans.

1.2 Lipid Metabolism in the Rumen

1.2.1 Substrate Supply

Ruminant diets vary in composition depending on species, physiological state, and the cost and availability of feed ingredients. Diets often contain forage species (grasses, legumes or forage maize) of variable maturity and nutritional value, and differ in composition from those containing forages as the sole feed to combinations of forage, cereals and protein supplements. By-products of the food industry or lipid supplements may also be included. A general characteristic of ruminant diets is the relatively high fibre content (<300 g cell wall constituents per kg dry matter (DM)) and low amounts of lipid (<50 g per kg DM). Lipid in cereal grains, plant oils, marine lipids and by-products are predominantly in the form of triacylglycerol (TAG). Most of the lipid in grasses and legume forages is present as phospholipids (PL) and glycolipids (GL) located within thylakoid membranes of chloroplasts. In forages, GL are the major lipid class, with galactolipids (monoand digalactosyl diacylglyerol) being the most prevalent. These differ from TAG in that one or more carbohydrate molecules are linked to one position of the glycerol backbone. Forages and oilseeds also contain several PL species, (phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine) as structural components of cell membranes. Most PL contain a diacylglycerol covalently bonded to a phosphate group, which is often esterified to a simple organic molecule such as choline. In general, both fatty acid moieties bound to glycerol in glycolipids or PL are unsaturated. Non-esterified fatty acids (NEFA) are minor components of most ruminant feeds, but are the major lipid class in ensiled grasses and forage legumes and in certain proprietary fat supplements. Changes arising during fermentation in silo are characterized by a substantial decrease in the relative abundance of polar membrane lipid and an increase in NEFA, TAG, diacylglycerol (DAG) and monoacylglycerol (MAG) fractions attributable to the activity of plant and microbial lipases.

The amount and composition of constituent fatty acids differs substantially between ingredients in ruminant diets (Table 1.1). For grasses and legume forages fatty acid content is generally lower than 50 g per kg DM with cis-9, cis-12, cis-15 18 : 3 (α-linolenic acid; 18 : 3 n-3) as the major fatty acid (450 g per 100 g fatty acids). However, conservation of grass by drying results in substantial decreases in lipid content, principally due to the disappearance of cis-9, cis-12 18 : 2 (linoleic acid; 18 : 2 n-6) and 18 : 3 n-3 by oxidation and leaf shatter. Oxidation arises from the activity of plant lipoxygenases, which catalyse the incorporation of molecular oxygen in non-esterified 18 : 2 n-6 and 18 : 3 n-3, forming 9and 13-hydroperoxy polyunsaturated fatty acids, respectively, that are highly reactive and rapidly metabolized into a series of oxylipins including volatile leaf aldehydes and alcohols, hydroxyand epoxy-fatty acids and jasmonates. In contrast, 18 : 2 n-6 is the predominant fatty acid in forage maize, whole crop silages and cereal grains. Ruminant diets may also contain up to around 50 g per kg DM of additional lipid in the form of oils or oilseeds. Oils from rapeseeds, high oleic sunflowerseeds, olives and peanuts are a rich source of cis-9 18 : 1 (oleic acid), cottonseeds, safflowerseeds, soyabeans and sunflowerseeds are abundant in 18 : 2 n-6, whereas linseeds and camelina contain relatively high proportions of 18 : 3 n-3 (Table 1.1). In contrast, coconut oil is rich in 12 : 0 (lauric acid), whereas 16 : 0 (palmitic acid) is the major fatty acid in palm oil. Ruminant diets may also be supplemented with fish oil containing cis-5, cis-8, cis-11, cis-14, cis-17 20 : 5 (eicosapentaenoic acid; 20 : 5 n-3) and cis-4, cis-7, cis-10, cis-13, cis-16, cis-19 22 : 6 (docosa-hexaenoic acid; 22 : 6 n-3) or marine algae enriched in 22 : 6 n-3.

1.2.2 Lipolysis

Following ingestion and mastication, the ester linkages of TAG, PL and GL are rapidly hydrolysed in the rumen. Hydrolysis of dietary TAG occurs predominantly as a result of microbial lipases. Forage plant tissues are also rich in endogenous galactoand phospholipases, which remain active once ingested into the rumen for several hours, suggesting that senescence of the plant material itself in the rumen may contribute to ruminal lipolysis in grazing animals. Dawson et al. challenged this idea and concluded that microbial lipases were more important than plant enzymes. These conclusions were drawn using autoclaved grass as a substrate, which the authors noted was not ideal because of the many effects that autoclaving has in addition to enzyme denaturation. This debate has been revisited by Lee et al., who reported increased NEFA and decreased polar lipid abundance after 6 h incubation of fresh ryegrass leaves in buffer, confirming that plant-catalysed lipolysis occurred. Further, Van Ranst et al. reported lipolysis of up to 60% after 8 h incubation of fresh red clover leaves. Both studies suggested the observed lipolysis to be due to active plant lipases that could contribute to overall ruminal lipolysis. However, until plant lipase activity is compared directly with that of ruminal micro-organisms, there will remain an uncertainty about their relative importance. Nonetheless, it may be useful to breed forage plants low in lipase activity in order to increase the amount of polyunsaturated fatty acids escaping the rumen. Plant lipids may also be compartmentalized in the ingested plant material, effectively protecting them from both endogenous and microbial hydrolysis.

Among the various types of ruminal micro-organisms, the bacteria are considered to be most active in lipolysis. The most active bacterial species isolated selectively using TAG as a substrate was Anaerovibrio lipolytica with which most research has been carried out. More recently, Jarvis et al. isolated two bacteria from red deer that hydrolysed TAG and grew on glycerol. The bacteria, which were highly active against tallow, tripalmitin and olive oil, were most closely related to the genus Propionibacterium and clostridial cluster XIVa. Cirne et al. isolated Clostridium lundense sp. nov. from the bovine rumen. Clostridium lundense exhibited lipolytic activity against olive oil, but neither its activity nor its numbers was reported, so it is difficult to assess its likely importance. Thus, a wider range of bacteria than is usually considered may be involved in the lipolysis of TAG in the rumen.

A. lipolyticus lacks the ability to hydrolyse galactolipids and PL and, therefore, other lipolytic species would be expected to predominate in grazing ruminants. The Butyrivibrio spp. appeared to contain all the phospholipase A, phospholipase C, lysophospholipase and phosphodiesterase activities typical of the mixed rumen contents. Their lipase activity against TAG varies between different Butyrivibrio and Pseudobutyrivibrio strains, but not in a manner that corresponds to their position in the phylogenetic tree. Little is known about how other lipases vary across different strains/species, nor whether other recently recognized species may possess such activities in the rumen of grazing ruminants.

There have been few recent studies that investigate protozoal lipolysis. Wright suggested Epidinium spp. to be responsible for 30–40% of the lipolytic activity in the rumen. Epidinium ecaudatum was reported to liberate galactose from galactolipids, suggesting galactosidase activity, although lipase activity per se was not demonstrated. Another protozoal species, Entodinium caudatum, was shown to have phospholipase activity, but it is possible that this activity was more relevant to the intracellular metabolism of the protozoa than to the digestion of dietary lipids. The earlier studies to determine the contribution of protozoa to the lipolytic activity in the rumen were conducted using fractionated rumen fluid, with the possibility that lipolytic activity in protozoal fractions was more due to the activity of bacteria that the protozoa had ingested than that of the protozoa themselves. Once again, given that protozoa comprise up to half the microbial biomass present in the rumen, their lipolytic properties warrants further investigation.

Much is known about bacterial lipases in general. They comprise eight well-documented families and their modes of action are reasonably well characterized. The lipase activity of A. lipolyticus was investigated in some detail by methods available at the time, some four decades ago. Perhaps surprisingly, no cloning and sequencing studies seem to have been done when the technology became widely available, and the most detailed recent study was derived from genomic analysis. Three enzymes from A. lipolyticus were identified from genomic analysis of A. lipolyticus. The alipA, alipB and alipC encoded 492-, 438and 248-amino acid peptides, respectively. Phylogenetic analysis indicated that alipA and alipB clustered with the GDSL/ SGNH family II, and alipC clustered with lipolytic enzymes from family V. Subsequent expression and purification of the enzymes showed that they had esterase activities with substrate specificities favouring the hydrolysis of caprylate-, laurateand myristate-containing substrates. Genomes are available for several species in the Butyrivibrio group, but to date no similar analysis appears to have been carried out for their lipase activities.

Metagenomic methods will be invaluable in order to understand the full complement of lipolytic enzymes that are present in the rumen. Expression libraries may be useful. Liu et al. screened a metagenomic library from the rumen of grazing Holstein cows for lipase activity, using trioleoylglycerol as substrate. Out of 15 360 bacterial artificial chromosome (BAC) clones, only two were found to have high lipase activity, which seems surprisingly small. The likely origin of the genes was investigated, based on the other open reading frames (ORFs) present in the BAC clones. It was impossible to decipher the host for the first gene, Rlip1, but the second, Rlip2, gave most similar homologues from Thermosinus carboxydivorans, which has not previously been associated with the ruminal ecosystem. Nonetheless, by BLASTing their deposited lipase sequences it was discovered that the genes most likely encoded carboxyl esterases. Thus the prevalence of these lipases in the overall community is far from certain. Clearly many more lipase sequences must be analysed from the metagenome, and assignment to lipases assured, in order to understand the true nature of lipolytic enzymes active in the rumen.

Lipolysis is considered rate limiting for the biohydrogenation of dietary unsaturated fatty acids in the rumen. During ruminal digestion of grasses, mono- and digalactosyl diacylglycerides are released following the rupture of chloroplasts. Early studies involving incubations of C-labelled substrate with strained rumen fluid indicated that lipolysis conforms to first-order kinetics, occurring at a high rate with a short lag time. These observations led Hawke and Silcock to conclude that hydrolysis of dietary acyl lipids in the rumen would be sufficiently rapid not to impede biohydrogenation. However, such conclusions were not supported by observations that the profile of products formed during intra-ruminal infusion of esterified or non-esterified 18 : 2 n-6 in sheep differed.

Much of what is known about the factors influencing ruminal lipolysis is based on incubations of various substrates with rumen fluid. Laboratory-scale experiments have obvious drawbacks with respect to mimicking conditions in vivo. Accepting these limitations and the assumptions therein, a number of factors have been determined to influence the rate and extent of lipolysis in a simulated rumen environment (Figure 1.1). Increases in dietary nitrogen content were found to increase the rate at which triolein was hydrolysed, whereas the rate of lipolysis was decreased by replacing fibre with starch or when more mature forages were incubated. As could be expected, lipolysis increases with incubation time, but is often decreased at low rumen pH. The extent of lipolysis is also decreased for lipids with a higher melting point containing relatively high proportions of saturated fatty acids. Increasing the amount of substrate incubated also lowers progressively the rate and extent of lipolysis in vitro. After 24 h incubations with buffered rumen fluid, between 73.7% and 89.5% of TAG in fish oil, linseed oil or sunflower oil was found to be hydrolysed. Similarly, lipolysis of lipid in cocksfoot and red clover after 24 h incubations were reported to vary between 65.0% and 82.0%.

(Continues...)

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Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2014
• Language: English
• ISBN 10: 1849739005
• ISBN 13: 9781849739009
• Binding: Hardcover
• Number of pages: 263
• Editor: Bert Sels