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Volatile and non-volatile components of beef marrow bone stocks

Dissertation
Author: Belayet H. Choudhury
Abstract:
Beef bone marrow has been part of the human diet since prehistoric times. Marrow bone stock is important culinary base used by gourmet chefs. It is well known for its distinct savory character in foods. While there has been a great deal published on flavor active components in cooked meats, the flavor composition of bone marrow is still relatively unstudied. For this study, commercial chopped fresh beef marrow bones were simmered in water for seven hours at 90ºC. Three batches of cooked marrow bone mixtures were prepared. First batch was not enzyme treated. The second and third batch was enzyme treated with papain and umamizyme and heated for one hour at 65ºC and 50ºC respectively. All three batches (untreated and enzyme treated) were defatted by microfiltration. Samples from all three batches were heated under pressure at 120ºC or 160ºC for one hour. In another series of experiments, the defatted stock samples of three batches (one untreated and two treated with papain or umamizyme) were heated for one hour with ribose, xylose or methyglyoxal. Head space volatiles of all above samples were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) by Solid Phase Microextraction (SPME). Stock sample prepared at 90ºC without further treatment showed presence of lipid oxidation products including diacetyl, alcohols, aldehydes and ketones. Stock samples both untreated and treated with enzymes and heated at 120ºC for one hour showed additionally Strecker aldehydes, dimethyl sulfides and furans. Stock samples treated with enzymes showed in addition pyrazines. Stock samples both untreated and treated with enzymes and heated at 160ºC for one hour showed all of the compounds identified at 120ºC heating at higher concentration and fatty acids, thiazoles and alkenals. Samples with addition of methylglyoxal showed significantly higher levels of pyrazines and alkenals. The results of our research showed that after heating and especially after treatment with enzymes and addition of ribose, xylose, and methylglyoxal a number of novel flavor alkenals and interesting volatiles are formed that were not previously identified in bone marrow stocks. All three stock samples were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) for non-volatiles mainly amino acids, di- and tri peptides. 20 peptides are identified in the Papain and Umamizyme digested samples. Umamizyme treated stock yielded the most peptides followed by papain treated stock. These non-volatiles act as volatile precursors and generated numerous volatiles when the stocks were treated with ribose, xylose or methylglyoxal.

vii Table of Contents Page ABSTRACT OF THE DISSERTATION ……………………………………………. ii PREFACE .................................................................................................................... iv ACKNOWLEDGEMENTS ........................................................................................... v DEDICATION ............................................................................................................... vi TABLE OF CONTENTS ............................................................................................... vii LIST OF TABLES ......................................................................................................... xi LIST OF FIGURES ....................................................................................................... xiii

1. INTRODUCTION ............................................................................................... 1 2. LITERATURE REVIEW ................................................................................... 4 2.1 Background Information ................................................................................... 4 2.1.1 Beef Bone marrow interest .................................................................... 4 2.1.2 Beef bone marrow stock preparation ..................................................... 4 2.2 Review of Beef Bone Marrow Chemistry and Biology .................................... 6 2.2.1 Beef bone marrow types and composition ............................................ 6 2.2.1.1 Beef bone marrow lipids ................................................................ 7 2.2.1.2 Beef bone marrow proteins and peptides ......................................... 8 2.2.1.3 Beef bone marrow Amino Acids ..................................................... 10 2.2.1.4 Marrow Extracellular Matrix .......................................................... 10 2.2.1.5 Beef bone marrow nucleotides ........................................................ 11 2.2.1.6 Beef bone marrow minerals ............................................................. 11

viii 2.3 Volatile Components Contributing to Aroma ....................................................... 12 2.3.1 Lipid oxidation ................................................................................................ 12 2.3.2 Maillard reaction ............................................................................................. 15 2.3.2.1 Amadori reaction .................................................................................... 15 2.3.2.2 Strecker degradation ............................................................................... 16 2.3.2.3 Mechanisms of pyrazine formation ........................................................ 19 2.3.2.4 Other important volatiles ........................................................................ 21 2.3.2.5 Peptide-specific Maillard compounds .................................................... 22 2.4 Non-volatile components ..................................................................................... 23 2.4.1 Basic tastes .................................................................................................... 24 2.4.1.1 Umami taste ............................................................................................ 24 2.4.1.2 Amadori compounds ............................................................................... 27 2.4.1.3 Maillard Peptides .................................................................................... 27 2.4.1.4 Organic acids .......................................................................................... 28 2.4.1.5 Taste enhancers ...................................................................................... 28 2.5 Enzymatic Hydrolysis .......................................................................................... 29 2.6 Fat Separation ....................................................................................................... 31 2.6.1 Membrane Filtration ...................................................................................... 31 2.7 Flavor analysis ..................................................................................................... 33 2.7.1 Volatile analysis ............................................................................................ 33 2.7.1.1 Solid Phase Micro-Extraction SPME) .................................................... 33 2.7.2 Non-volatile analysis ..................................................................................... 35 2.7.3 Protein identification ..................................................................................... 37

ix 2.7.3.1 Analysis and separation of sample ions .................................................. 37 2.7.3.2 The peptide sequencing ....................................................................... 38

3. HYPOTHESIS ....................................................................................................... 42 4. OBJECTIVES ........................................................................................................ 43 5. EXPERIMENTAL METHODS............................................................................ 44 5.1 Experimental Design ....................................................................................... 44 5.1.1 Stock preparation ...................................................................................... 44 5.1.2 Sample preparation ................................................................................... 45 5.2 Sample preparation ......................................................................................... 47 5.2.1 Stock preparation ...................................................................................... 48 5.3 Fat separation .................................................................................................. 49 5.3.1 Membrane filtration .................................................................................. 49 5.4 Degree of Hydrolysis ...................................................................................... 50 5.5 Flavor analysis ................................................................................................ 51 5.5.1 Volatile Analysis ....................................................................................... 51 5.5.1.1 Solid Phase Micro Extraction ............................................................ 51 5.5.2 Non-Volatile Analysis .............................................................................. 52 5.5.2.1 Non-volatile mapping ........................................................................ 52 5.5.2.2 Amino acid analysis ........................................................................... 54 5.5.2.3 Peptide fragmentation ........................................................................ 55 5.5.2.4 Mass accuracy determination ............................................................. 56 6. RESULTS AND DISCUSSION ........................................................................... 57

x 6.1 Stock Preparation ............................................................................................ 57 6.2 Volatile analysis ............................................................................................. 58 6.2.1 Votatiles comparison of stock samples S, P and U ................................... 58 6.2.2 Volatiles in stock samples heated at 120ºC and 160ºC ............................. 61 6.2.2.2 Papain treated ..................................................................................... 61 6.2.2.3 Umamizyme treated ........................................................................... 61 6.2.3 The effect of ribose, xylose and methylglyoxal at 160ºC ......................... 66 6.2.3.1 Untreated stock .................................................................................... 66 6.2.3.2 Papain digested stock ........................................................................... 73 6.2.3.3 Umamizyme digested stock ................................................................. 80 6.3 Non-volatile studies ........................................................................................ 102 6.3.1 LC-MS TOF analysis ............................................................................. 102 6.3.2 Amino acids analysis ............................................................................. 104 6.3.3 Peptide analysis ...................................................................................... 108 6.3.3.1 Sample S ........................................................................................ 108 6.3.3.2 Sample P ........................................................................................ 108 6.3.3.3 Sample U ........................................................................................ 111 6.3.4 Interpretation of spectra ......................................................................... 115

7. CONCLUSIONS................................................................................................... 118 8. REFERENCES ................................................................................................... 148 9. VITA .................................................................................................................... 156

xi Lists of tables Page

Table 1. Fatty acids composition of bone marrow of two years old cow ................. 8 Table 2. Beef myelopeptides .................................................................................... 8 Table 3. Peptides isolated from beef bone marrow hemoglobin .............................. 9 Table 4. Amino acids percentages in acid digested cervical vertebre marrow protein ......................................................................... 10 Table 5. Some volatile aldehydes obtained from autoxidation of unsaturated fatty acids ............................................................................ 13 Table 6. Volatile aldehydes from the Strecker degradation .................................... 17 Table 7. Dipeptides and tripeptides reported in literature to evoke umami taste ...................................................................................... 26 Table 8. Immonium and related ion characteristics of amino acids ......................... 41 Table 9. Stock samples ............................................................................................. 46 Table 10. Bone marrow composition ......................................................................... 47 Table 11. pH and protein comparison of undigested, papain and umamizyme digested stock ........................................................................ 50 Table 12. LC-MS parameters ..................................................................................... 53 Table 13. Chromatography parameters ...................................................................... 54 Table 14. LC-MS using hybrid quadropole linear ion trap ........................................ 55 Table 15. Volatiles comparison of stock samples ..................................................... 59 Table 16. Volatile comparison of all three stocks at 120ºC and 160ºC ...................... 62

xii Table 17. Volatile comparisons of samples S-160-R, S-160-X, and S-160-M ........................................................... 67

Table 18. Volatile comparisons of in samples P-160-R, P-160-X, and P-160-M ........................................................... 75

Table 19. Volatile comparison of sample U-160-R, U-160-X, and U-160-M ........................................................ 82

Table 20. Comparison of peptides with mass ranges in undigested, papain digested and umamizyme digested stocks ............. 103

Table 21. Amino acids expressed in gms/100 gms of the stock samples ................... 105

Table 22. Major MS fragments: Beef Marrow Bone Stocks Peptides ...................... 145

xiii List of illustrations Figure Page Figure 1. Strecker degradation of α-amino acids ...................................................... 18 Figure 2. Formation of α-aminoketones and α-aminoaldehydes produced upon Strecker degradation of selected dicarbonyls ................ 19 Figure 3. Pyrazine formed by dimerization or condensation and oxidation of the aminoketones and aminoaaldehydes .......................... 20 Figure 4. Formatiomn of b and y ions during the fragmentation of a three amino acids residue peptide chain .............................................. 40 Figure 5. Experimental design: Fat separation using microfiltration and stock preparation ............................................................................. 44 Figure 6. Experimental design: Flow chart of samples investigated ......................... 45 Figure 7. Comparison of 2,3-butanedione (diacetyl) in stock samples S, P and U .............................................................................................. 60 Figure 8. Comparison of Strecker aldehydes and lipid derived aldehydes in stock samples S, P and U at 120ºC ................................................. 64 Figure 9. Volatiles comparison of samples S, P and U at 120ºC and 160ºC ................................................................................... 65 Figure 10. Comparison of furans in samples S-160-R, S-160-X, and S-160-M .......................................................................................... 70 Figure 11. Volatiles comparison of samples S-160-R, S-160-X, and S-160-M ......................................................................................... 71

xiv Figure 12. Strecker degradation of Phenylalanine to Phenylacetaldehye .................... 72 Figure 13. Comparison of furans in samples P-160-R, P-160-X and P-160-M ......................................................................................... 78 Figure 14. Volatiles comparison of samples P-160-R, P-160-X, and P-160-M ......................................................................................... 79 Figure 15. Comparison of furans and hydrocarbons in samples U-160-R, U-160-X, and U-160-M ......................................................... 85 Figure 16. Volatiles comparison of samples U-160-R, U-160-X, and U-160-M ......................................................................................... 86 Figure 17. Formation mechanisms of methylsulfides from glycine and cysteine ........................................................................................... 89 Figure 18. Formation mechanisms of methylsulfides from methionine ...................... 90 Figure 19. Proposed mechanisms of the formation of 2-Methyl-2-butenal ................. 91 Figure 20. Proposed mechanisms of the formation of 2-Methyl-2-hexenal ................ 92 Figure 21. Proposed mechanisms of the formation of 3(2-Furanyl)-2-Phenyl-2-propenal ........................................................ 93 Figure 22. Proposed mechanisms of the formation of 5-Methyl-2-phenyl-2-hexenal ............................................................... 94 Figure 23. Proposed mechanisms of the formation of 2-Isopropyl-5-methyl-2-hexenal ............................................................ 95 Figure 24. Proposed mechanisms of the formation of 2-Phenyl-2-butenal ................. 96 Figure 25. Proposed mechanisms of the formation of 2,5-Dimethyl- 3-butylpyrazine ..................................................................................... 97

xv Figure 26. Formation mechanism of 2-Acetylthiazole from cysteine and methylglyoxal .................................................................... ..98 Figure 27. Comparison of non-volatile distribution in a 3-D images of LC-MS TOF chromatogram in samples S, P and U ............................. 106 Figure 28. LC-MS-ESI chromatogram of samples S, P and U ................................... 107 Figure 29. LC-MS chromatogram showing amino acids in sample S ........................ 121 Figure 30. LC-MS chromatogram showing amino acids in sample P ........................ 122 Figure 31. LC-MS chromatogram showing amino acids in sample U ....................... 123 Figure 32. Amino acids comparison in stock samples S, P and U .............................. 124 Figure 33. Sample P: LC-MS spectrum of gly-leu ...................................................... 125 Figure 34. Sample P: LC-MS spectrum of val-thr ....................................................... 126 Figure 35. Sample P: LC-MS spectrum of ala-his ....................................................... 127 Figure 36. Sample P: LC-MS spectrum of leu-pro ...................................................... 128 Figure 37. Sample P: LC-MS spectrum of leu-lys ....................................................... 129 Figure 38. Sample P: LC-MS spectrum of phe-arg ..................................................... 130 Figure 39. Sample P: LC-MS spectrum of arg-arg ...................................................... 131 Figure 40. Sample U: LC-MS spectrum of ser-pro ..................................................... 132 Figure 41. Sample U: LC-MS spectrum of val-leu ...................................................... 133 Figure 42. Sample U: LC-MS spectrum of gln-pro ..................................................... 134 Figure 43. Sample U: LC-MS spectrum of phe-leu ..................................................... 135 Figure 44. Sample U: LC-MS spectrum of tyr-leu ...................................................... 136 Figure 45. Sample U: LC-MS spectrum of thr-gly-his ................................................ 137 Figure 46. Sample U: LC-MS spectrum of leu-ser-val ................................................ 138

xvi Figure 47. Sample U: LC-MS spectrum of leu-leu-pro ............................................... 139 Figure 48. Sample U: LC-MS spectrum of met-val-pro .............................................. 140 Figure 49. Sample U: LC-MS spectrum of phe-trp ..................................................... 141 Figure 50. Sample U: LC-MS spectrum of glu-gly-tyr ............................................... 142 Figure 51. Sample U: LC-MS spectrum of pro-phe-met ............................................. 143 Figure 52. Sample U: LC-MS spectrum of leu-asp-phe .............................................. 144

1    1. INTRODUCTION

Beef bone marrow is part of the human and animal diet since prehistoric time. It is part of the culinary practices and has been used in gourmet cooking, soup, broth and stock preparation. Meat industry today uses marrow bones as an industrial by-product and salvages as much as possible. Fat is extracted and prepared as tallow while the protein is salvaged as meat extracts. Thus bone marrow is part of our food chain prepared from by products of mechanically deboned meat (Field et al., 1981; Field et al., 1999). Bone marrow stock is well known for its unique savory flavor. There are many published literature on the immunological role of beef bone marrow. However, the knowledge of the flavor active compounds in beef bone marrow is very limited. Volatile and non-volatile flavor active compounds in bone marrow flavor have not been studied.

The volatiles and non-volatiles in beef meat have been extensively studied. As the flavor chemistry of beef marrow bones is little understood, it is assumed that beef marrow bone proteins will behave similar to meat proteins. The lipid-derived compounds are the most abundant compounds in meat products rich in fat. On the other hand, Maillard reaction, which occurs between amino compounds and reducing sugars, is one of the most important routes of flavor compounds in meat products. The Maillard reaction involving peptides, amino acids and reducing sugars generates volatile compounds that contribute the final cooked meat flavor. A cascade of Maillard reactions follows to generate further volatiles.

2    Meat flavor is generated through the complex interactions among amino acids, peptides, sugars, thiamine, nucleotides, lipids and products of lipid oxidation (Imafidon and Spanier, 1994). In the past, it has been proven that the water soluble non-volatile components mainly amino acids, peptides, organic acids contribute to a distinct savory broth character in meat products. Most of the flavor precursors of meat are amino acids and peptides. These precursors react with lipids or lipid degradation products during cooking. During heating and enzyme hydrolysis, the proteins are degraded to yield peptides of various length and amino acids. The size of the peptides, their amino acid contents and sequences play an important role in meat flavor development. The short chain low molecular weight peptides participate as flavor precursors and contribute to the taste. The major components of the water soluble components of bone marrow are peptides, amino acids, nucleotides, organic acids, minerals, vitamins, breakdown products of lipids, collagen, and glycogen. Therefore, bone marrow stock is an ideal candidate for Maillard reaction generating volatile components.

Beef marrow stock is cooked in water at simmering temperature. The beef marrow bones contain very high amount of fat and small amount of protein. The investigation of protein and its break down products are a major focus of the studies. It is a challenging task to separate the protein from the fat. Enzyme treatment is needed as heating alone may not effectively hydrolyze the peptide bonds that link the protein molecule. Heating followed by enzyme treatment and fat separation using microfiltration increases the protein yield of the stock and increases the palatability. The degree of hydrolysis of protein increases with enzyme treatment. The protein

3    molecules are broken further into smaller peptides and they are available in the aqueous phase. Without the enzyme treatment, bulky protein molecules are lost in the filtrate. Besides, the compounds from the Maillard reaction can also react with other volatiles such as aldehydes and other carbonyls formed during lipid oxidation, which react readily with Maillard intermediates. Such interaction contributes to the generation of characteristic flavor of marrow bones stock. The stocks possess volatiles mainly of lipid derived aldehydes. When treated with various temperatures they participate in Maillard reaction and generate numerous volatiles besides lipid derived aldehydes. The stocks in reaction with ribose, xylose and methylglyoxal generate further more volatiles. The volatiles yield will depend upon the degree of hydrolysis. A profound knowledge of these volatiles will better understand the flavor chemistry of beef marrow bone. Therefore, the main objectives of this research are the characterization of the flavor compounds of beef marrow bones stock.

4    2. LITERATURE REVIEW

2.1 Background Information 2.1.1 Bone Marrow Interest

Beef bone marrows have been of culinary interest for centuries. Beef marrow bones are considered a food industrial by-product. They have been used to make soup, broth and stock. The bone marrow stock may be defined as a clear, thin liquid flavored by soluble substances extracted from the bones. This stock can be utilized in meat dishes, soups, vegetable dishes, sauces or gravies (Ockerman, 2000).

Stock made from the bones of animals, has been consumed as a source of nourishment for humankind throughout the ages. Muscle based food products derived from advanced meat recovery system may contain bone marrow. Lipid oxidation is a major issue with bone marrow generating flavor and off flavors (Miller, 1982; Mancini, 2004).

2.1.2 Beef Bone Marrow Stock Preparation

Beef stock also known as broth or bouillon. The gradual heating of the liquid is of the highest importance for the clarity as well as for the flavorfulness of the stock. Famous French chef Marie-Antoine Careme proposed an explanation in L’Art de la cuisine francaise au XIXe siecle in 1833 that the stock must come to a boil very

5    slowly, otherwise the albumin coagulates and hardens, the water do not penetrate the protein. Justus von Liebig is universally known for his pioneering studies in organic chemistry and for broths and meat extracts. Using vacuum to evaporate the stock produced by cooking minced meat in cold water, Liebig obtained a “beef extract” that he sold throughout the world (Harve This, 2006). This concentrated stock is known as broth. A patent procedure illustrated beef bone marrow broth preparation at simmering temperature (Vollmer and Riney, 1979).

6    2.2

Review of Beef Bone Marrow Chemistry and Biology

Although bone marrow non-volatiles influences the thermal aroma generation and contribute to taste, no research has assessed the role of bone marrow non-volatiles mainly peptides and amino acids in taste and flavor generation. There is an abundance of peer-reviewed literature focused on the mechanism of meat flavor generation from muscle meat. However, there is a lack of published research evaluating flavor generation by bone marrow peptides or lipids. Because the amount of bone marrow research in published literature is limited, the literature review will focus on bone marrow from a biological standpoint. Bone marrow components that may have a role in flavor generation will be reviewed.

2.2.1 Bone Marrow Types and Composition

Bone marrow is housed in a hollow central cavity. Beef bone marrow is found in vertebrae, sternum, ribs and long bones. Long bones such femur bones are composed of “fatty marrow” while he rests are abundant in “red marrow” that is composed of hematopoietic cells (Agar, 1983). It is estimated that marrow is very high in fat, low in protein and carbohydrates. Average bone marrow composition of femur bone was determined to be: protein 2.6%, fat 79.5%, moisture 17.9% (Vollmer, 1979).

7    2.2.1.1 Bone Marrow Lipids

The lipid content of beef bone marrow tends to change with animal age (Field, 1980). The fat content of marrow from young calves is relatively low compared to steers. The nonpolar lipids are increased with age while polar lipids are decreased. Within the polar lipid fraction of bone marrow, palmitic and oleic acid are increased whereas stearic and linoleic acids are decreased as the cattle is matured. Anatomical location affects the lipid content. The femur bones are composed of fatty white marrow, whereas ribs and vertebrae are abundant in red marrow. Beef femur bones contain 85% lipid compared with 26-56% in vertebrae (Kunsman, 1981). Beef bone marrow lipids contain small amount of phospholipids. Table 1 represents the bone marrow fatty acids composition of a two years old cow.

8    Table 1: Fatty acid composition of bone marrow of two years old cow (Mello et al., 1976)

Fatty Acids % %

C14:0 Myristic acid 1.2 C15:0 Pentadecanoic acid 0.6 C16:0 Palmitic acid 19.2 C16:1 Palmitoleic acid 3.6 C17:0 Heptadecanoic acid 1.7 C18:0 Stearic acid 18.9 C18:1 Oleic acid 47.2 C18:2 Linoleic acid 3.6 C18:3 Linolenic acid 0.2 C20:4 Arachidonic acid 1.2 C22:0 Docosanic acid 0.1

2.2.1.2 Bone Marrow Protein and Peptides

Bone marrow peptides were unknown up until the middle of 1970’s. They were analyzed by Russian scientists from porcine bone marrow and named myelopeptides (MPs) (Petrov et al., 1997; Akhmedov et al., 2004; Karelin et al., 1998). Table 2 represents these myelopeptides.

Table 2: Myelopeptides

Phe-Leu-Gly-Phe-Pro-Thr (MP-1) Leu-Val-Val-Tyr-Pro-Trp (MP-2) Leu-Val-Cys-Tyr-Pro-Gln (MP-3) Phe-Arg-Pro-Arg-Ile-Met-Thr-Pro (MP-4) Val-Val-Tyr-Pro-Asp (MP-5) Val-Asp-Pro-Pro (MP-6)

9    Table 3: Peptides isolated from beef bone marrow hemoglobin (Ivanov et al., 1991)

Segments of the α-chain of the hemoglobin:

1 VLSAADKGNVKAAWGK 16 (val-leu-ser-ala-ala-asp-lys-gly-asn-val-lys-ala- ala-trp-gly-lys)

16 KVGGHAAEYGAEA 28 (lys-val-gly-gly-his-ala-ala-glu-tyr-gly-ala-glu-ala)

27 AEALERM 32 (ala-glu-ala-leu-glu-arg-met)

76 LPGALSELS 84 (leu-pro-gly-ala-leu-ser-glu-leu-ser)

109 LASHLPSDFTPAV 121 (leu-ala-ser-his-leu-pro-ser-asp-phe-thr-pro-ala-val)

Segments of the β-chain of the hemoglobin:

1 MLTAEEKAAVT 11 (met-leu-thr-ala-glu-glu-lys-ala-ala-val-thr) 15 GKVKVDEVGGEALGRL 30 (gly-lys-val-lys-val-asp-glu-val-gly-gly-glu-ala- leu-gly-arg-leu) 71 SNGMKGLDDLKG 82 (ser-asn-gly-met-lys-gly-leu-asp-asp-leu-lys-gly) 94 KLHVDPE 100 (lys-leu-his-val-asp-pro-glu) ARNFGKFF (ala-arg-asn-phe-gly-lys-phe-phe) NFGKFFTPV (asn-phe-gly-lys-phe-phe-thr-pro-val)

These above endogenous peptides (Table 3) were isolated from beef bone marrow hemoglobin. These peptides have shown biological and immunological activity. However, no flavor activity has been reported on these peptides.

10    2.2.1.3 Beef Bone Marrow Amino Acids

Beef marrow contains most of the amino acids. The predominant amino acids are illustrated in the Table 4 below.

Table 4: Amino acids percentages in acid digested cervical vertebrae marrow protein (Field et al., 1978)

Amino Acids % Leucine 13.2 Lysine 9.4 Glutamic acid 9.2 Aspartic acid 8.6 Alanine 7.3 Valine 6.5 Arginine 5.8 Glycine 5.4 Serine 5.3 Threonine 4.9 Phenylalanine 4.7 Histidine 4.6 Proline 4.0 Cysteine 2.8 Methionine 2.7 Tyrosine 2.6 Isoleucine 2.5

2.2.1.4 Marrow Extracellular Matrix

Extracellular matrix consists of fibronectin, laminin, hyaluronic acid, chondroitin sulfate, keratin sulfate and collagen (Lee et al., 1999). Attached to a core protein are long strands of glycosaminoglycans (GAGs) also called mucopolysaccharides. These structures are naturally jellylike and upon heating forms gel similar to collagen. Hyaluronic acid is a linear polysaccharide consisting of linked disaccharide units of glucuronic acid and N-acetylglucosamine. Chondroitin sulfate is the most abundant

11    mucopolysaccharide comprised of alternating units of β-1, 4 linked glucuronic acid and β-1,3-N-acetyl galactosamine and is sulfated on either the 4, or 6 position of galactosamine residue.

Collagen is a major connective tissue protein. Fibers of collagen run throughout the bone matrix, which are created by stringing together amino acids, the building blocks of protein. About one third of collagen is composed of glycine, the smallest amino acid while another one third of collagen is composed of proline and hydroxyproline (Kaufman, 2001). Collagen is a source of hydroxyl amino acids and sugars. Collagen is denatured and the peptide bond breakage occurs during prolonged heating of meat above 70ºC. However, collagen fragments were confirmed after cooking which indicates the heat stability of these peptides possibly contributed by high proportion of proline resdues (Bauchart et al, 2006). 2.2.1.5 Bone Marrow Nucleotides:

Marrow from steers contained 15.7 and 4.8 times more DNA and RNA than muscle respectively (Arasu, 1981) and is influenced by vertebrae locations. Cervical marrow contained greater amounts of DNA and RNA than lumbar marrow. 2.2.1.6 Beef Bone Marrow Minerals Bone and cartilage are both classified as connective tissue and contains mineral deposited in an organic matrix. The minerals include calcium, phosphorus, sodium, magnesium, potassium, fluorides, and chlorides.

12    2.3 Volatile Components Contributing to Aroma Lipid oxidation and the Maillard reaction are possibly the two most important reactions in food chemistry. Both reactions include a network of reactions yielding extraordinary complex mixture of compounds and follow parallel reaction pathways, producing a number of reactive intermediates which are responsible for the later formation of highly colored polymers in both reactions by aldol condensations and/or carbonyl-amine polymerization (Hodge, 1953; Hidalgo and Zamora, 2004).

2.3.1 Lipid Oxidation

Lipids upon autoxidation produce carbonyl compounds to form brown, high molecular weight products. “Lipid-derived” volatile compounds dominate the flavor profile of pork cooked at temperatures below 100ºC (Chen and Ho, 1998). Fats play an important role in the development of flavors and off-flavors through autoxidation and produce aldehydes and ketones. At certain parts per million (ppm) level these carbonyl compounds are desirable while in excess may cause off-taste. The off taste may include waxy, fatty, painty undesirable notes in food and food products. Alkane dienals such as 2,4 decadienals possess roasty, nutty notes. Lipid oxidation and Maillard reaction are interrelated. Strecker-type degradation of amino acids occur at as low as 37ºC by some lipid oxidation products (Hidalgo et al., 2004).

13    Table 5: Some volatile aldehydes obtained from autoxidation of unsaturated fatty acids. (Ho and Chen, 1994)

Fatty Acids Hydroperoxides Aldehydes

Oleic acid 8-OOH 2-Undecenal Decanal 9-OOH 2-Decenal Nonanal 10-OOH Nonanal 11-OOH Octanal

Linoleic Acid 9-OOH 2,4-Decadienal 3-Nonenal 13-OOH Hexanal

Linolenic Acid 9-OOH 2,4,7-Decatrienal 3,6-Nonadienal 12-OOH 2,4 Heptadienal 3-Hexenal 13-OOH 3-Hexenal 16-OOH Propanal

Arachidonic Acid 8-OOH 2,4,7-Tridecatrienal 3,6-Dodecadienal 9-OOH 3,6-Dodecadienal 11-OOH 2,4-Decadienal 3-Nonenal 12-OOH 3-Nonenal 15-OOH Hexanal

Lipid oxidation gives a wide range of aliphatic products, including both saturated and unsaturated hydrocarbons, alcohols, aldehydes (Table 5), ketones, acids, and esters as well as cyclic compounds (such as furans, lactones and cyclic ketones). Many of

14    these contribute intense aroma and taste in foods (Farmer, 1994). Some long chain alkyl substituted heterocyclic compounds have been identified in meat flavors. These may originate from the reaction of aldehydes from lipid degradation with heterocyclic compounds formed from the Maillard reaction. Long chain aliphatic aldehydes possess fatty notes.

Phospholipids are constituents of bone marrow fat and have been shown to contribute to the animal specific meaty character. They contribute through lipid- derived volatiles generated by thermally induced lipid oxidation and interaction of lipid intermediates with the Maillard reaction, both of which modify the overall aroma of the cooked meat. Hexanal, nonanal, 2-octenal, 2-decenal, 1-octen-3-ol, 2-pentylfuran are the major odor-active volatiles degradation product of heated phospholipids as this can be generated from various fatty acids, C18:2 and C20:4 (Lin and Blank, 2003).

The volatiles in meat and bone meal by-products was hexanal, heptanal, octanal, 3-octene-2-one, nonanal, pentanal, 3,5-octadien-2-one, 1-octen-3-ol, 2-pentyl furan, trans-2-alkenals, trans, trans-2,4-alkadienals, and several pyrazines. These compounds can be formed by autoxidative degradation of fatty acids such as arachidonic acid, linoleic or linolenic acids. 3,5-octadien-2-one and 3,5-undecadien-2-one may be derived from linolenic and arachidonic acids, respectively, via enzymatic oxidative reactions. 2-pentyl furan has been identified to be responsible for reversion flavor in soybean oil (Smouse and Chang, 1967). It was postulated that 2-pentyl furan originates from linoleic acid (Greenberg, 1981). The high fat content usually greater than 14% can accelerate autoxidation.

15    2.3.2 The Maillard reaction

The raw bone marrow has little aroma with bloody metallic taste while cooked bone marrow has a unique umami and delicious taste. Like in all meat components, a complex series of thermally induced reactions occur between nonvolatile components of fatty tissues, protein and degraded polysaccharides. The major source of volatile compounds in heated foods is the Maillard reaction between amino acids and reducing sugar, and thermal degradation of lipids (Gerard, 2006).

A number of factors such as pH, water content, temperatures are very important in generating volatile carbonyl compounds (Mottram, 1995). In heated foods, the main source of the aldehydes is Strecker degradation of amino acids, while in high fat containing foods is lipid oxidation. Acidic pH favors furan and furan derivatives. Pyrazines is increased with the increasing reaction pH. Both pH 6.0 and 8.0 are favorable conditions for sulfur-containing compound formation (Tai and Ho, 1998).

2.3.2.1 Amadori reaction

Maillard reaction undergoes in three stages: initial, intermediate and final. At the initial stage, sugar-amine condensation, instable Schiff base formation, isomerization occurs. Glycosylamine as an intermediate occurs with sugar-amine condensation which kicks off a series of chain reactions forming an unstable Schiff base. This triggers isomerization, forms sugar dehydration and fragmentation, flavor compounds,

16    precursors and pigments. In the final stage, products of the intermediate stage further reacts with amino components or amino acid degradation, compounds produces heterocyclic compounds. Glycosylamine further rearranged into aldosamine (Heynes compounds) and ketosamine (Amadori compounds). Maillard reaction enolization is pH mediated. At an acidic pH, the Amadori compound produces 3-deoxyhexosone via 1,2-enolization and at basic pH, the Amadori compound produces to 1-deoxyhexosone via 2,3- enolization.

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Abstract: Beef bone marrow has been part of the human diet since prehistoric times. Marrow bone stock is important culinary base used by gourmet chefs. It is well known for its distinct savory character in foods. While there has been a great deal published on flavor active components in cooked meats, the flavor composition of bone marrow is still relatively unstudied. For this study, commercial chopped fresh beef marrow bones were simmered in water for seven hours at 90ºC. Three batches of cooked marrow bone mixtures were prepared. First batch was not enzyme treated. The second and third batch was enzyme treated with papain and umamizyme and heated for one hour at 65ºC and 50ºC respectively. All three batches (untreated and enzyme treated) were defatted by microfiltration. Samples from all three batches were heated under pressure at 120ºC or 160ºC for one hour. In another series of experiments, the defatted stock samples of three batches (one untreated and two treated with papain or umamizyme) were heated for one hour with ribose, xylose or methyglyoxal. Head space volatiles of all above samples were analyzed using Gas Chromatography-Mass Spectrometry (GC-MS) by Solid Phase Microextraction (SPME). Stock sample prepared at 90ºC without further treatment showed presence of lipid oxidation products including diacetyl, alcohols, aldehydes and ketones. Stock samples both untreated and treated with enzymes and heated at 120ºC for one hour showed additionally Strecker aldehydes, dimethyl sulfides and furans. Stock samples treated with enzymes showed in addition pyrazines. Stock samples both untreated and treated with enzymes and heated at 160ºC for one hour showed all of the compounds identified at 120ºC heating at higher concentration and fatty acids, thiazoles and alkenals. Samples with addition of methylglyoxal showed significantly higher levels of pyrazines and alkenals. The results of our research showed that after heating and especially after treatment with enzymes and addition of ribose, xylose, and methylglyoxal a number of novel flavor alkenals and interesting volatiles are formed that were not previously identified in bone marrow stocks. All three stock samples were analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS) for non-volatiles mainly amino acids, di- and tri peptides. 20 peptides are identified in the Papain and Umamizyme digested samples. Umamizyme treated stock yielded the most peptides followed by papain treated stock. These non-volatiles act as volatile precursors and generated numerous volatiles when the stocks were treated with ribose, xylose or methylglyoxal.