(https://jkosary.wordpress.com/) (Kosary.Judit@etk.szie.hu)Dr. Kosáry Judit egyetemi tanárBiochemistry of the storage of food raw materials

Food raw materials are themselves derived from living organisms, so their biochemical processes determine their changes during storage. Therefore, their proper storage and subsequent processing in the food industry require very thorough and specialized biochemical knowledge. The basic features of this knowledge are dealt with in general biochemistry, which was previously a curriculum focused on primary metabolic processes. An appropriate level of knowledge is essential to master the biochemical processes of storage. In the biochemical part of our material we first briefly review the primary metabolic processes, then in the biochemical part of the food we first deal with protein-based foods and then with plant-based foods.

1. An overview of the most important processes of primary metabolism

1.1. The energetic background of the functioning of living organisms

The metabolism of living organisms is a series of continuous metabolic changes. The degradation (catabolic) and biosynthetic (anabolic) processes do not work in the same way and with the same set of enzymes, allowing the organism, which is not in simple chemical equilibrium with the environment, to constantly maintain its organizational stability. The living organism is an open system in terms of equilibrium; its stability is actually a steady state, so it is in constant exchange of energy and matter with the environment. The energy absorbed covers the energy needs of the living organism to survive.

In terms of energy absorption, organisms can be divided into two groups. Autotrophic organisms, using solar energy (in some cases chemical energy), make up the body's own biomolecules (including photosynthetic plants) from water and carbon dioxide (with some nitrogen and mineral salts) taken from the environment. The uptake of heterotrophic organisms also requires the uptake of organic molecules, called essential molecules. Heterotrophic organisms provide their survival energy needs with energy from nutrient degradation.

The rules of reaction kinetics also apply to biochemical processes. Effective collision of reaction partners and activation energy are also essential for the processes occurring in the living organism to take place. In organisms, due to the high activation energy required at body temperature, most organic chemical reactions are practically zero-speed, and can only occur with the aid of catalysts. Catalysts (biocatalysts) in living organisms are enzymes. Enzymes by complexing with substrates (forming enzyme-substrate, shortly E–S complex) open a reaction pathway for the chemical transformation that requires significantly less activation energy than the original pathway. Namely, the formation of these complexes requires needs low activation energy because the mostly secondary bonds inside. Like catalysts, enzymes do not alter the free enthalpy, reaction endothermic or exothermic character of the reaction they catalyze. During enzymatic catalysis, the enzyme temporarily participates in the reaction, forming a complex (enzyme-substrate complex, ES) loosely coupled to the starting material, substrate (usually the reaction partner requires a similar reaction), and the transition is taking place between partners in

favorable spatial positions. After the conversion, the product (product) comes off and the enzyme molecule ultimately remaining unchanged.

Reaction diagram without and with enzyme

Some energy-demanding syntheses occur by hydrolyzing one or more macroerg bonds in parallel with the reaction, and the energy released covers the energy required for synthesis. In this case, the substrate and the macroerg bond molecule (usually nucleoside triphosphate, in short NTP, often adenosine triphosphate, in short ATP in short) form a common macroerg bonded intermediate, not only the product but also the molecules derived from the hydrolysis of NTP can be appear.

Reaction diagram without and with enzyme

Metabolic processes, including enzymes, are based on various interactions and interactions between biomolecules. The interactions of biomolecules respond to the molecular rules of life.

According to the rule of bioaffinity, each biomolecule can form an association, complex with at least one other biomolecule, usually reversibly, by secondary bonds, in which the ratio fit, three dimensional arrangement, and association-dissociation balance of the components can be accurately determined. Accordingly, the ES complex is formed in a similar way.

The rule of biocatalysis deals with enzymes. Enzymes are also biomolecules, their function is reaction-specific (catalyzing only a given reaction) and substrate-specific (they can convert only one biomolecule, or some of its analogues), their kind in the cell is practically as many types of chemical processes in the cell. Really, the cell's enzyme set regulates all the properties of the cell.

The rule of bioregulation deals with the coordination of enzyme-catalyzed processes. As a result of the interaction between enzymes and certain molecules (regulatory molecules), the activity of the enzyme may be increased (activator) or decreased (inhibitor). Regulated enzymes also effect the metabolism of molecules that carry out bioregulation. This sophisticated regulatory system coordinates metabolic processes and allows the organism to adapt to ever-changing environmental influences. In higher organisms, hormones coordinate the function of each organ.

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1.2. Brief summary of the fundamental metabolic processes

During metabolic processes, the living molecules' biomolecules and nutrient molecules in the body are usually degraded on an oxidative way by the oxygen in the air (catabolism). On the one hand, it releases the energy needed to maintain the living organisms (including, in particular, heterotrophic organisms as energetically open systems). On the other hand, it produces the intermediates that are needed to build up the body's materials; these are the biosynthetic processes (anabolism). At the end of the degradation processes, the biomolecules eventually oxidize ("burn") to carbon dioxide and water.

Scheme of the biosynthesis of biomolecules

In plant autotrophic organisms, the energy required to maintain the body is provided by light energy (h) through glucose photosynthesis. The building-up processes take place through photosynthesis (CO2 and H2O incorporation) and ammonia incorporation. The outline of the metabolic processes cannot detect the

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multi-layered regulatory network that coordinates the metabolic processes, thus allowing the harmonious functioning of the living organism.

6 H2O + 6 CO2 + 12 hν C6H12O6 + 6 O2

Except for simple lipids, nutrients and nucleic acids that enter the body are complex molecules (glycosides, acid amides and esters) which, upon hydrolysis, release the simple biomolecules (sugars, amino acids, fatty acids, etc.) that are the starting materials for oxidative degradation processes. The degradation of these biopolymers is accomplished by hydrolase enzymes (with the help of which the substrate molecule is cleaved by water so that the water constituents, the proton and the hydroxide anion are incorporated as hydrogen and hydroxyl groups at the end of the two moieties), usually in the cytosol.

Biomolecule Enzyme IntermediatePolysaccharides Glycosidases/

PhosphorylasesSugars/Sugar phosphates

Proteins Proteases/Peptidases Amino acidsNeutral triglycerides(triacylglycerols)

Lipases Fatty acids and glycerol

Nucleic acids Nucleases NucleotidesThe first phase of the degradation of biomolecules

The enzymes that hydrolyze the glycosidic bonds of polysaccharides are called glycosidases. Exogenous glycosidases generally begin cleavage from the non-reducing end of the chain. Starch glycosidases: -amylase endoglycosidase (cleaves inside the polysaccharide chain), -amylase exo-glycosidase (cleaved at the end of the chain), cleaves maltose units from the non-reducing end of the chain. Maltose is hydrolyzed by maltase into two glucose. Cellulase-degrading cellulases are produced by only a few microorganisms. Sucrose is hydrolyzed by invertase into glucose and fructose. Certain polysaccharide-degrading enzymes, phosphorylase from certain polysaccharides, such as glycogen, cleave glucose-1-phosphate with one molecule of inorganic phosphate.

Proteins that enter the body are hydrolyzed to amino acids by proteases (peptidases). Among the exo-peptidases is the amino-peptidase. Hydrolysis begins at the N-terminus and carboxypeptidases cleave the C-terminus. Endo-peptidases cleave peptide bonds between specific amino acids (e.g., pepsin and trypsin).

Lipase enzymes hydrolyze ester groups of complex lipids. Neutral fats produce decomposition products of lipases from fatty acids and glycerol, and phospholipids from phospholipases produce fatty acids, glycerol, phosphoric acid and another esterification of phosphoric acid.

Nucleic acids are broken down by nucleases into nucleotide units. DNA is cleaved by DNases and RNA is cleaved by RNases. Exo and endo nucleases are also distinguished between nucleases. Endonucleases also include relaxedriction enzymes, which have a major role in genetic engineering.

Biomolecules formed by hydrolysis of biopolymers are further degraded by different oxidative ways. The decomposition processes of biomolecules can be divided into two large, successive parts. Each type of biomolecule is first degraded by unique pathways, first in the cytosol and then in the mitochondria to a common intermediate, the acetyl Coenzyme A molecule.

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The hydrolysis of polysaccharides liberates simple sugars. The most common simple sugar produced is glucose. Other simple sugars are capable of converting directly or in the form of their derivatives into glucose or its derivatives. Glucose is a key component of carbohydrate metabolism in the body, so we only deal with its oxidative degradation.

Glucose is degraded to pyruvate by glycolysis and then converted either anaerobically (lactic acid and alcoholic fermentation) or in an oxidative process, with loss of carbon dioxide, to acetyl Coenzyme A.

Material balance of glycolysis

Material balance of lactic acid and alcoholic fermentation of glucose

Material balance of oxidative process catalyzed by pyruvate dehydrogenase

Amino acids lose their amino group in a process known as transamination to form -keto-carboxylic acids. Transamination of alanine produces pyruvate, and other amino acids to form -keto-carboxylic acids, which are intermediates in one of the steps of the oxidative degradation of acetyl-Coenzyme-A formed during the decomposition of biomolecules. The α-keto-carboxylic acids formed by transamination residues other than the above are usually converted to the acetyl Coenzyme A molecule or possibly an intermediate of the citrate cycle after longer or shorter oxidative degradation. The amino group of amino acids is incorporated into glutamic acid, which, after a prolonged process, is excreted in the form of a nitrogen-containing derivative such as urea: H2N-CO-NH2 in mammals.

Of the hydrolysis products of complex lipids, glycerol is involved in glycolysis in a modified form. Only after activation, fatty acids are able to degrade to acetyl-Coenzyme A molecules by -oxidation. During activation, the fatty acid is converted to a mixed acid anhydride (acyl-AMP) containing an ATP molecule by a

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macroerg bond, the energy released by hydrolysis of the pyrophosphate (PP i) derivative of the ATP molecule to cover the reaction's energy requirement. Acyl-AMP reacts with a molecule of Coenzyme A to form an acyl-Coenzyme A molecule containing a macroerg thiol ester bond, which is the starting material for -oxidation. Simple lipids are also converted to acetyl Coenzyme A by oxidative degradation.

Acetyl-Coenzyme-A molecules from various nutrients are converted into carbon dioxide and water ("burn") in oxidative degradation processes in the mitochondria, with a significant amount of energy generated, some of which is met by the conversion of ADP-ATP to macroerg bonds stored. This involves two processes, the citrate cycle or citric acid cycle and the terminal oxidation, commonly referred to respiration that is a concept in physiology.

The stage of the common degradation process in the mitochondria, where carbon from the nutrient source (and oxygen content) carbon dioxide is initially formed and the nutrient-derived hydrogens are further transported in the form of reduced coenzymes, is called the citrate cycle.

Material balance of citrate cycle

The process in which the reduced coenzymes are reoxidized, that is, regenerated, the nutrient-derived hydrogens form water with the oxygen in the air, that is, the oxygen is reduced (electron transport), while ADP and inorganic phosphate (Pi) produce ATP (oxidative phosphorylation) is called terminal oxidation.

In electron transport, the enzyme complexes in the inner membrane of the mitochondria transfer hydrogens (together electrons and protons at the beginning of the process, later in separated form) from the more negative standard redox potential to the more positive standard redox potential.

For transitions with a large difference in standard redox potential, electron transfer results in a large negative change in free enthalpy, ATP synthesis occurs. During oxidative phosphorylation, the energy released in electron transport is built into macroerg bonds, a process described by Mitchell's chemiosmotic coupling theory. Mitchell hypothesizes that the energy released at the appropriate sites of electron transport will pump protons from the space of the mitochondrial matrix into the space through the FO unit of FOF1-ATPase. This proton pump, which produces a proton concentration gradient (difference) between the two sides of the inner membrane, is the osmotic energy that drives ATP synthesis.

Material balance of terminal oxidation

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Taking into account all stages of the degradation of glucose, as well as the mitochondria (redox shuttle, also known as extra mitochondrial NADH) of reduced coenzymes in the cytosol, the total mass of aerobic degradation of glucose is:

C6H12O6 + 6 O2 6 CO2 + 6 H2O + 36 (38) macroerg bond (ADPATP)

This corresponds to the experience that 12 photons are required for the synthesis of a glucose molecule, which corresponds to the energy of 36 macroerg bonds.

Biomolecules in plants are synthesized from glucose produced by photosynthesis, and intermediates formed during its formation and degradation, in reductive biosynthetic processes. In heterotrophic organisms, the starting materials of the construction processes can only be intermediates in the degradation processes of food biomolecules. There are biomolecules or their intermediate molecules which heterotrophic living organisms cannot be synthesized. These molecules are essential molecules (e.g. vitamins and ten of the twenty protein-forming amino acids). Heterotrophic organisms are also capable of synthesizing glucose. The biosynthesis of glucose from pyruvate is called gluconeogenesis.

Amino acids are mostly synthesized from -keto-carboxylic acids by transamination reactions in the opposite direction. Simple lipids and fatty acids are synthesized from acetyl-Coenzyme A units in reductive processes.

Biosynthesis of different biopolymers is a process that requires a significant amount of energy. In any case, biomolecules must be activated before attachment.

In order to form polysaccharides and to form O-glycosidic bonds, sugar, most often glucose molecules must be activated. During activation, glucose-6-phosphate produced by glycolysis can be converted to glucose-1-phosphate by the enzyme glucose-phosphate mutase (transferase) with the help of the glucose-1,6-bisphosphate cofactor. The cofactor is required because the phosphate group cannot jump from position 6 to position 1, and an isomerization step is replaced by two transfer processes. Glucose 1,6-bisphosphate contains the appropriate substituent at both sites. From the 6th position of the cofactor, the phosphate group moves to the substrate so that the cofactor becomes a product, the substrate becomes a cofactor, and finally all the substrate molecules are converted to the product through the cofactor state, except one which becomes a cofactor. Similar mutase-catalyzed conversion occurs in glycolysis.

Glucose-1-phosphate generates NDP-glucose containing a macroerg bond with a nucleoside triphosphate (UTP for sucrose synthesis, ATP for starch formation, GTP for cellulose formation), and the energy released by the hydrolysis of the residual pyrophosphate is covered by the reaction energy. This active intermediate, which contains the macroerg phosphoric anhydride, is already capable of reacting with the alcohol or glycosidic hydroxyl group of the other sugar moiety to form the O-glycoside in the appropriate position.

Aminoacyl-tRNA molecules are required for protein biosynthesis and translation. The formation of aminoacyl-tRNA molecules from the corresponding amino acid and ATP molecule forms a mixed amino acid acyl-AMP containing a macroerg bond, and the energy released during hydrolysis of pyrophosphate (PPi), which also contains a macroerg bond, covers the energy requirement for the formation of aminoacyl-AMP. Amino-acyl-AMP reacts with the 3'-hydroxy group of the tRNA, and the breakdown of its macroerg bond covers the energy requirement for the formation of the amino-acyl-tRNA.

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The biosynthesis of the ester bond of the composite lipids requires the use of an acyl-Coenzyme A or a nucleotide diphosphate-activated alcoholic hydroxyl group containing a macroerg bond.

The metabolic processes involved in the formation and degradation of living molecules (biopolymers) that form the living organisms described above are referred to as primary metabolism. Intermediates and end products from primary metabolism produce a large number of compounds that play a role not only in cellular structure but also in its function or in the coordination of cellular functions or other systemic functions. These substances are called secondary metabolites and their metabolic processes are referred to as secondary metabolism.

2. The role of amino acids in the storage of food raw materials

During storage, the metabolism of amino acids results in the production of many compounds that can influence the quality of food raw materials and foods. Already during the hydrolysis of proteins, amino acids with characteristic taste are formed. Further degradation of the amino acids results in the formation of quality-degrading compounds, such as biogenic amines. An understanding of the formation of biogenic amines is provided by the scheme of amino acid degradation.

The scheme of amino acid degradation

After the hydrolysis of proteins by different kinds of peptidases and proteases, there are four levels of the degradation of amino acids. The first step in the degradation of amino acids is transamination, an important intermediate of which is aldimine formed from the amino acid and coenzyme pyridoxal phosphate.

Aldimines are the starting materials of biogenic amines by decarboxylation followed by hydrolysis. Aldimines from some amino acids (e.g. serine) transfer their side chain to a tetrahydrofolate (THF) coenzyme producing glycine from the amino

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acids. The major biogenic amines involved in the storage of food raw materials are described below.

Other reactions from aldimines derived from amino acids

Three of them (transamination, oxidative deamination of glutamate, urea cycle) are connected with the removing of the -amino groups and produce -keto carboxylic acids while the fourth level is the oxidative degradation of -keto carboxylic acids to pyruvate (Ala, Thr, Gly, Ser, Cys, Trp), acetyl-CoA (Trp, Leu, Ile), acetoacetyl-CoA (Phe, Tyr, Trp, Leu, Lys), -ketoglutarate (Glu, Gln, His, Arg, Pro), succinyl CoA (Met, Val, Ile), fumarate (Tyr, Phe, Asp) and oxaloacetate (Asp, Asn). The formulas of the protein-forming amino acids are presented at the end of the text.

3. Biochemistry of protein based foodsFood biochemistry deals with the biochemical processes that play a role in

food production and storage. At this stage, the influence of the enzyme pool of the living organism from which the food commodity is derived is often negligible. However, microorganisms play a very important role in the food industry, which is both useful (e.g. cheese maturation, alcoholic fermentation, etc.) and harmful (e.g. meat degradation, lactic acidification, fatty acidification, yellowing, etc.).

Meat products and dairy products are the most important foods that are predominantly protein-rich. However, we should not neglect that other foods and food raw materials also contain proteins, and we should not forget the own protein supply of the microorganisms that play a major role in the food industry.

We mainly deal muscle tissue. In relation to muscle tissue, we also outline the biochemical processes that take place in protein-rich foods during degradation. Other types of foods also contain proteins or amino acids. When these are degraded, the description of the degradation products resulting from the protein content will only refer to their formation.

Biochemistry of muscleMuscle is generally understood to mean muscle tissue, but meat as a food

contains tendon, connective tissue, adipose tissue and often bone tissue. In biochemistry, we distinguish between biochemistry of living muscle tissue (primarily biochemistry of muscle function, i.e., contraction and relaxation) and biochemistry of muscle tissue after cutting, which has two major stages, meat maturation and meat degradation.

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The structure of muscleMuscle tissue contains about 75% water and 19% protein. Protein components

are generally structural proteins, 10% of which are myofibrils (myosin, - and -actinin, M-protein, actin), plus 6% of sarcoplasm, the cytoplasm of muscle cells (myogens, globulins, myoglobin, hemoglobin) and 3% connective tissue (elastin, collagen). Muscle tissue contains still approx. 3% fatty tissue, and a great many other biomolecules e.g. carbohydrates, vitamins, monovalent and divalent cations, lactic acid and other molecules.

The striated muscle bundles, which make up the bulk of the muscle tissue, consist of muscle fibers that contain myofibrils, the space between which is filled by the sarcoplasm, the protoplasm of the muscle fiber. The repeating units that build up muscle fibers are called sarcomeres. Thick and thin filaments are arranged in a regular, comb-like manner inside the fibril. Bridges connect the two kinds of filaments. During muscle contraction, thin and thick filaments slide into each other. The thick filament is composed of myosin, protein C and M-line proteins, therefore it is often called as myosin filament or Z-filament. The thin filament is composed of - and -actinin, F-actin, tropomyosin and troponin complex, therefore it is often called as actin filament or I-filament.

The structure of the thick and thin filaments and the structure of myosine.

About 60% of the thick (in Hungarian vastag) filament is myosin. Myosin has a molecular weight of 460,000 D and is composed of two heavy (200-200,000 D) and three light chains (subunits) (15-30,000 D). The peptide chains of myosin contain several post-methylated amino acids (3-methyl histidine, N-N-methyl and -N-trimethyl-lysine). Myosin consists of head (sometimes heads) and tail. The tail is two super helical twisted heavy chains of -helix structure. The head consists of two globules heavy chains of equal size with a globular structure. Light chains are attached to the head.

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According to myosin function, ATPase, which is strongly activated by calcium ions and less activated by magnesium ions, is also activated by monovalent cations, unlike other ATPase enzymes. Myosin ATPase has an active center in the head that contains two -SH groups. Light chains are also likely to play a role in the conformation of the active site. There is also an actin binding site in the head which can bind actin to actomyosin complex. This complex is dissociated by ATP.

The major constituent of the thin (in Hungarian vékony) filament is about 60% actin. Actin is a 376 amino acid containing polypeptide chain that is a monomeric globular protein (G-actin) that forms a fibrillar polymeric form (F-actin) in a muscle cell. In the F-actin, the monomers are linked to form a double helix, which represents seven pairs of monomers per turn. The incorporation of each monomer requires an ATP-ADP conversion, i.e. the energy of a macroerg bond.

The structure of thin filament

The thin filament still contains two protein components, the total amount of which does not exceed 10%. Tropomyosin, a fibrous structure containing two folded -helices, is located in the super-helix groove of actin and always co-occurs with F-actin.

The troponin is a globular molecule located along the thin filament such that every seven pairs of actin monomers have two troponin molecules. Troponin consists of three subunits, each containing one active site. Troponin-C (TnC) binds calcium ions. TnC exhibits structural similarity to the protein called calmodulin, which is also used to bind calcium ions in other tissues. Calmodulin is used to bind calcium ions released by cells through the action of hormones. Troponin-I (TnI) contains the actin-binding site. At relaxed status, the troponin-I subunit is attached to the troponin-C subunit in the myofibril, thereby inhibiting ATPase activity. Troponin-T (TnT) contains the tropomyosin binding site.

The process of muscle functionMuscle contraction, which occurs in a very complex way due to nerve

impulse, is an energy-consuming process called ATPADP. It should be noted here that ADP is regenerated to ATP by phospho-creatine kinase. The phosphorus transporter is creatine phosphate, which is considered to be the storehouse of muscle cell energy. ATP is probably only able to bind to myosin in the form of a magnesium complex.

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The formula of creatine phosphate

In the relaxed muscle, the fluid surrounding the filaments has a low calcium ion content and no relationship between actin and myosin. In this state, myosin does not form actinomyosin complex with actin because actin is in an inactive conformation. In these cases, the tropomyosin is located between actin and myosin. ATP (presumably in the form of an Mg-ATP complex) is attached to the head of myosin containing ATPase but temporarily this complex does not hydrolyze to ADP and inorganic phosphate.

As a result of the nerve impulse, the sarcolemma releases calcium ions that bind to the troponin-C subunit (two calcium ions per subunit) entering the myofibril, thus increasing the concentration of calcium ions in the myofibril by one order of magnitude (from 10-7 M to 10-6 M). Binding of calcium causes a change in the conformation of troponin-C, which causes a series of conformational changes, as a result of which the relationship between troponin-C and troponin-I is broken. Conformational changes due to calcium ions thus release myosin ATPase activity from inhibition. ATPase activity results in hydrolysis of myosin-bound ATP. Its energy is used to activate the tail of myosin. These changes lead to the formation of a link between the myosin head and the actin monomer. This state can be compared to an elbow bow or a secured revolver.

The active tail of myosin pushes the complex with a powerful blow, the two filaments move in opposite directions, the angle of the head containing ADP and inorganic phosphate relative to the tail changes ("shrinks"), so the chemical energy from hydrolysis of ATP is converted into mechanical energy. established. The energy of the macroerg bond is also sufficient to cause the actinomyosin to cleave, causing the head to rise again. After contraction, ADP and inorganic phosphate leave the head, replaced by another Mg-ATP complex, which is hydrolyzed again, and the contraction process is repeated until the calcium ion concentration is high enough. When the calcium ion concentration decreases, the muscle relaxes.

Actin and myosin play a role in the process of relaxation-contraction, that is, in actual muscle work, tropomyosin and troponin are involved in regulating muscle function. In their relaxed state, they inhibit the formation of the myosin-actin bond, only the calcium ions, as signaling particles (calcium signal), undergo a conformational change that allows the binding of myosin to actin.

Skeletal and myocardial cells are characterized by the regular arrangement and sarcomere pattern of myofibrils. In smooth muscle cells, there is virtually no order and the structure of myofibrils is slightly different.

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The chemical mechanism of muscle contraction

The energy background of muscle functionMany ATP molecules are required for muscle function. Under aerobic

conditions, these nutrient degradation processes are provided by terminal oxidation such that the ATP produced here phosphorylates creatine into creatine phosphate, which regenerates ATP.

creatine phosphate + ADP creatine + ATPPart of ADP regenerates ATP with adenylate kinase:2 ADP ATP + AMPHowever, there is an early stage in the early phase of muscle function, when

nutrient degradation is not fast enough, the required ATP is produced anaerobically

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by lactic fermentation following glycolysis (two molecules ATP per glucose). For a long time, this accumulating lactic acid was earlier attributed to muscle fever. According to our latest knowledge, muscle fever is caused not by lactic acid, but by minor injuries in the unheated muscles. The resulting lactic acid does not decompose to carbon dioxide and water in the muscle. From the muscle, lactic acid passes through the bloodstream into the liver, where lactate is oxidized to pyruvate, which produces glucose through gluconeogenesis. This cycle is called the Cori cycle. Only the heart muscle can produce energy from the pyruvate through the citrate circuit.

Post-mortem biochemistry of muscle tissueAfter the death of a living organism with muscle tissue, in the muscle tissue

biochemical processes can cause changes. In the food industry, these changes are examined from the point of view of the influence on the quality of meat of slaughter animals. Post-slaughter metabolic changes may include:

Primary changes:-No more oxygen in the muscle tissue-No connection between muscle tissue and other tissues, especially the liver.

Primary changes lead to secondary changes.Secondary changes:We distinguish four phases of secondary changes, the first three of which are

significant in meat processing. The phases are: the pre-rigor phase, the rigid phase, the post-rigid phase, in which meat is not degraded yet, and the meat deterioration. In the pre-rigor phase the ATP and creatine phosphate content of the fresh cut

meat is reduced and the anaerobic processes become more prominent. If the blood remains in the tissues after slaughter, the oxygen supply to the muscle tissue is reduced more slowly and more rapidly during bleeding. The amount of ATP is also reduced by the fact that sarcoplasmic ATPase is active all the time. At this stage, ATP is mainly supplied by creatine phosphate and to a small extent by the action of adenyl kinase.

In rigor mortis, muscle stiffening occurs, pH is shifted to acidic direction and protein denaturation occurs. At this stage, the creatine phosphate pool is depleted and the amount of ATP will be so low that the established actin-myosin linkage cannot be broken down. This permanent connection causes rigor mortis (its other name is corpulent state). Lactic acid (lactate) formed during the anaerobic decomposition of carbohydrates, mainly glucose, remains in the muscle tissue due to the loss of the organ-to-organ linkage, which results in acidification of the pH in the tissue (pH 5.3-5.5). These changes can strongly affect the workability of the meat (consistency, water retention).

During the post-rigid state, changes in the flesh, taste and organoleptic qualities gradually develop. The release of rigor mortis is primarily due to the action of protein-degrading enzymes, which increase the concentration of peptides and free amino acids in muscle tissue. The activity of protein-degrading enzymes varies in different regions of the muscle tissue. Lysosomes of sarcolemma contain high levels of proteases, primarily cathepsin, whose temperature optimum is that of a living animal but that of its pH optimum is the acidic medium of rigor mortis (pH 5.5). As a result of the action of cathepsin, mainly sarcolemma proteins are degraded, while the connective tissue proteins and the myosin-actin complex are virtually unaffected.

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The three stages discussed so far are the normal process of normal meat maturation during meat processing when the meat reaches its optimal nutritional and nutritional qualities.

Over the last 30 to 40 years, it has been recognized that in some cases the quality of meat obtained as a result of maturing meat is significantly lower. This is because the amount of carbohydrates in the muscle tissue may be very different from the average, and consequently the rate of glycolysis, and thus the degree of acidification, may vary considerably. Normally, due to the degradation of glycogen in muscle tissue and glucose released from it, the pH drops to 6.0 over a few hours, and then acidification continues steadily, with a value of about 5.5 after 24 hours. If muscle tissue initially contained significantly more than average glucose, the rate of glycolysis was significantly faster and the pH was reduced to 5.0 within one hour. The rapid acidification is followed by a slight increase in pH, but after 24 hours the pH remains below normal (about 5.2). These meats are pale, soft in consistency, and watery, i.e. they have a low water retention capacity, and are therefore referred to as PSE meats (pale-soft-exsudative). Data on the incidence of PSE meat are rather uncertain, but according to some surveys, they may amount to 10-20% of the meat processed.

If muscle tissue initially contains very little glycogen, then little glucose is produced and acidity is low. Such meat is almost neutral after 24 hours. These meats are characterized by dark color, firm consistency and dry touch, hence the name DFD meats (dark-firm-dry).

Changes in the pH of different pork during maturation

The occurrence of PSE and PFP meats is probably related to the inadequate breeding of so-called meat animals. Understanding the biochemistry of meat maturation can help eliminate some of the poor quality meat. Efforts to accelerate the maturation process, in particular the artificial addition of proteases, also serve to improve the quality of the meat.The biochemistry of meat deterioration

The fourth phase of the post-mortem biochemistry of muscle tissue is meat deterioration. In this phase different microbial infections play a very important role. In this process. The spoilage of meat is primarily the degradation of the constituent proteins and the conversion of amino acids resulting from the hydrolysis of the proteins. The four phases of amino acid degradation resulting from protease function (transaminization, glutamate dehydrogenase function, urea cycle and amino acid side chain degradation) have been previously studied. The side chain of the amino acids

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can be involved in the degradation processes of various biomolecules, eventually converting them to carbon dioxide and water, while the energy generated is incorporated into the macroerg bond of ATP molecules. Alanine, threonine, glycine, serine and cysteine can produce pyruvate, while phenylalanine, tyrosine, tryptophan, leucine and lysine give rise to acetoacetyl coenzyme A. Glutamic acid, glutamine, histidine, arginine and proline provide α-keto-glutarate, methionine, valine and isoleucine succinyl coenzyme A, aspartic acid and asparagine oxalacetate.

Excessive amino acid carbon chains are involved in animal organisms or are involved in gluconeogenesis (glucogen residues: alanine, arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine, glycine, histidine, methionine, proline, serine, threonine, tryptophan), ketogen amino acid is leucine. For some amino acids, incorporation can occur in both directions (glycogen and ketogen amino acids: isoleucine, lysine, phenylalanine, tyrosine).

Amino acid transformation - mostly in the presence of microorganismsIn the presence of some microorganisms, amino acids can undergo a very

versatile transformation. Some transformations may occur in the absence of microorganisms. The biochemical processes of the amino acid or protein content of different foods can be analogous, although not with the same intensity depending on the type of food and the circumstances. The results of the metabolism of the fermentation-producing microorganisms are also discussed. Among them cosmic oils from alcoholic fermentation are of great importance. The following describes the formation of some degradation products that has a negative effect on quality in food raw materials.

Biogenic aminesGenerally biogenic amines are flavour agents, often unpleasant flavour agents.

Earlier the biosynthesis of biogenic amines was presented by the decarboxylation of aldimines from amino acids followed by hydrolysis. The poor odor of decomposing meat is primarily responsible for some of the amino acids, including the previously presented biogenic amines formed during the decarboxylation of basic amino acids. Putrescine [H2N-(CH2)4NH2] from ornithine and cadaverine [H2N-(CH2)5NH2] from lysine are among the most unpleasant odorous diamines produced by the decay of proteins, previously thought to be toxic, are collectively referred to as ptomains. Later on it was found that ptomains are non-toxic materials

Decarboxylation of aspartic acid and glutamic acid can occur in two ways: when the -carboxyl group is removed, -aminocarboxylic acids are formed (-alanine from aspartic acid and -aminobutyrate from glutamic acid). Upon removal of the -carboxyl group of aspartic acid, alanine is formed.

Possibilities of decarboxylation of aspartic acidDuring cheese maturation, in the presence of Streptococcus faecalis, a

significant increase in thyramine content due to tyrosine decarboxylation occurs. Thyramine is the flavour agent of cheeses.

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Biosynthesis of thyramine

The -keto-carboxylic acid (4-hydroxyphenylpyruvic acid) formed by transamination of tyrosine from hops is first decarboxylated by . Reduction of the resulting aldehyde produces the bitter-tasting tyrosol, which plays a major role in the beer flavor.

Tyrosol biosynthesis

Eschericia coli and Clostridium strains contain not only tyrosine decarboxylase but also other amino acid decarboxylases, which are responsible for the small amount of cadaverine, putrescine. Agmatine produced from arginine by arginine decarboxylase.

Agmantine biosynthesis

Anaerobic bacteria can degrade glutamic acid in several ways. We now show the formation of the biogenic amine GABA, which is also formed in the human body. The frequent strong headaches of many glutamic acid users (known as unami in other name sodium glutamate is a spice in Chinese cuisine) can be indirectly in connection

17

with a derivative of glutamate. Because the biogenic amine of glutamic acid is -amino butyric acid (or 4-amino butyric acid) that is abbreviated as GABA. GABA is an important mediator (mediator) in the brain's function: it essentially inhibits the transmission of stimuli in certain parts of the brain, thereby regulating the transmission of stimuli – over-proliferation can therefore cause headache. GABA plays such a role not only in mammalian brains, but also in vertebrates in general. The presence of GABA can be detected in invertebrates, but their effect is unknown.

GABA biosynthesis

Due to special dehydratases containing coenzyme pyridoxal phosphate a water elimination occurs. In this way from serine pyruvate forms that produce acetyl coenzyme A and from threonine propionyl coenzyme A. From cysteine, cysteine sulfhydrase produces pyruvate and hydrogen sulfide. The smell of the latter indicates the deterioration of the egg (raccoon egg).

In the presence of anaerobic Clostridia the apolar amino acid side chains are decomposed oxidatively. The side chain becomes a fatty acid whose carboxyl group is originally -carbon, the original carboxyl group is converted to carbon dioxide and the amino group to ammonia. Finally, alanine is converted to acetic acid, valine to isobutyric acid (2-methylpropionic acid), and leucine and isoleucine to isomeric isovaleric acids (3-methylbutyric acid and 2-methylbutyric acid). Isovaleric acids, because of their unpleasant smell (in Hungarian büdös), have a deteriorating effect on quality.

Leucine can also produce 3-metil-butanol. In spirits industry it is called isoamyl alcohol. The decarboxylation of the -keto-carboxylic acid produced by the transamination of leucine produces the aldehyde, which is reduced to an alcohol. This produces the isoamyl alcohol in the alcoholic fermentation, which is one of the cosmic oils that is a quality deterioration factor.

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Isoamyl alcohol biosynthesis

Biochemical processes of myoglobin post mortemMyoglobin plays a key role in shaping the color of meat. Myoglobin contains

a heme ring system like hemoglobin, but myoglobin has only one protein unit (globin). Four of the six coordination bonds of the central ferro, i.e. iron (II) ion of the heme, are bound in a plane by the porphyrin skeleton, the fifth of the perpendicular to this plane binds to histidine, and the sixth to a water molecule by a hydrogen bridge; under appropriate conditions, oxygen may be bound here.

The structure of the heme

All heme derivatives containing Fe(II) in other words ferro ions are red. Under oxidative conditions an Fe(III) in other words ferri derivative is formed. The heme will become hematin, the hemoglobin will become brown methemoglobin, the myoglobin will become grayish-red metmioglobin, which has a sixth coordination bond attached to a hydroxide anion, none of which can bind oxygen. The formation of metmyoglobin is facilitated by uv radiation or acidification of the medium. Upon

19

exposure to heat, the protein component of myoglobin is denatured, and the resulting material is called myochromogen.

When the meat is degraded, the degradation of myoglobin, more precisely the heme inside, causes the meat to become discolored, since the individual degradation products are greenish and brownish in color. There are other the red coloured and oxygen-containing hemoglobin and myoglobin complexes but they are undesirable even dangerous. Among them the complex of myoglobin or hemoglobin with carbon monoxide, namely carboxy-hemoglobin and carboxy-myoglobin, they are produced in greater quantities upon carbon monoxide poisoning, but also binds carbon monoxide formed during oxidative cleavage of heme (see later). Nitroso-hemoglobin or nitroso-myoglobin may be formed during the pickling of meat or in the presence of

nitrated water. The N-nitroso group (N NO) can release the nitroso radical (•NO), which can cause many diseases, including cancer. Potassium nitrate (KNO3) is used to preserve the red color of the meat, but as you can see, this choice can be dangerous for consumers.

During the oxidative degradation of heme, the ring system is converted to a linear tetrapyrrol (biliverdin) between two rings containing a methyl and vinyl group (ring a and b) to form a linear tetrapyrrol (biliverdin) and carbon monoxide. The cleavage of the protein part may be before or after this oxidatíve degradation. For example, hemoglobin produces cleavage of green verdoglobin, which still contains iron, is bound to protein and contains hydroxyl in ring a and oxo in ring b. An analogous conversion can also occur in myoglobin. The blue-green biliverdin no longer contains protein and iron and the ring b also contains a hydroxyl group. Color degradation products are called bile dyes.

Biliverdin is reduced to orange-red bilirubin (the -CH = moiety between the c and d rings of biliverdin is reduced to -CH2-). Bilirubin is a very important bile dye, which, by partial saturation or oxidation, turns into dark brown mesobiline and stericobiline, which color the stools (shit). Bilirubin may be excreted by the kidney in the form of glycosides of two glucuronic acids. Such and similar degradation products are dyes in microorganisms, plants and animal (human) organisms. These compounds are often protein bound.

Biochemistry of milk

Milk proteinsApart from animal meat, milk, especially cow's milk, is the most important raw material in foods rich in protein. Milk proteins include many groups of proteins. These are usually not homogeneous compounds, but mixtures of similar types of proteins. The most important of these is casein, but there are also other milk proteins: -lactoglobulins, -lactalbumins and serum albumins, lactoferrin and serum transferrin in milk. There are milk proteins with special biological effects, as well: the enzyme proteins, the biologically active glycoproteins and the membrane proteins of fat globules.

CaseinCasein is a heterogeneous group of phosphoproteins precipitated from

skimmed milk (low-fat milk) at pH 4.6 at 20 oC. It actually consists of 3 components (-, - and -casein). These components, which themselves are heterogeneous, can be separated by electrophoresis. For example, -casein can be separated into three components: s-casein sensitive to calcium ions (easily precipitated) and -kappa

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casein non-sensitive to calcium ions. The latter protein contains phosphoric acid and carbohydrate component, as well, so it is actually a glycophosphoprotein and plays an important role in stabilizing the micelles of milk casein. The great diversity of caseins is due to the relatively small number of casein components which have many genetic variants that are very close in amino acid composition and structure.

Biochemistry of milk coagulationEnzymes that break down proteins cause the coagulation of milk, that is, mass

precipitation of milk proteins. Young mammalian stomachs produce the grafting enzyme (rennin or chymosin), which plays a major role in cheese production. Rennin (EC 3.4.4.3) can be isolated from the stomach of the calfs in an inactive form (prerennin). It has three components (A, B and C rennin). All three types can cause milk coagulation. Rennin has an optimum pH of 3.8, but it can slightly work at pH 6.7. The action of rennin, that is, the coagulation of milk, is made possible by acidification property of lactic fermentation by microorganisms in the milk. Pasteurized milk does not coagulate because no lactic acid formation is inside. During coagulation, rennin first cleaves a glyco-macropeptide from -casein to form para--casein. The cleavage is always the hydrolysis of a phenylalanine-methionine bond. The glyco-macropeptide will initially be the C-terminal moiety of -casein. The carbohydrate moiety of the macropeptide is attached to a threonine. The directly linked carbohydrate moiety is a galactosamine and the carbohydrate terminus is N-acetyl neuramic acid. After a longer period of time, rennin also cleaves additional peptide bonds, increasing the non-protein nitrogen content of the milk. More recently, other microbial preparations for milk coagulation (often microbial rennin, MR) are also used. Cheese is also made in Hungary with an MR product made with the help of Endothia parasitica.

During the preservation (boiling, pasteurization) of the milk the natural bacterial flora of the milk is killed. Therefore, it is not possible to make coagulated milk (e.g. curd milk) from boiled milk or from pasteurized milk. These milks can also become contaminated during storage. In this case the introduced bacteria are not lactic acid producers, but various wild strains that synthesize compounds with an unpleasant smell and taste, in which case the milk becomes smelly.

Summary of microbial biochemical processes in food containing protein

Protein degradation by microorganisms can be divided into two broad groups:1. To induce hydrolysis of proteins, formation of peptides and amino acids.2. Formation of low molecular weight compounds resulting from amino acid degradation.The two types of process described above are important in two respects:1. Preparation of protein-based food products (eg cheese maturation) 2. Deterioration of protein-based foods (eg meat products, eggs, milk).We distinguish three phases of degradation processes:1. The early stages of decay, when harmful microorganisms multiply in food, 2. Metabolism of low molecular weight nutrients (amino acids, dipeptides, lactic acid, sugars) to some intermediate (e.g. formation of amines, acids, carbon dioxide, hydrogen sulfide, etc.), 3. Infectious microorganisms adapt to the conditions by initiating the hydrolysis of proteins with their proteases and continuing the conversion of peptides and amino acids. As the process progresses, the concentration of odorous and health-damaging intermediates increases, and the food rots.

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In the food industry, the aim is to prevent food spoilage following the action of microorganisms by the use of various preservatives and / or technologies.

Changes in protein content of vegetable raw materials

In point of view of the food industry, the protein content of plant raw materials can be divided into two broad categories. Vegetables and fruits, sugar beet and some other raw materials are low in protein, high in water and contain relatively large amounts of nitrogen-based low molecular weight biomolecules (such as amino acids and their degradation products). These raw materials contain virtually no spare proteins. Cereals and oilseeds have a relatively high protein content, some of which have a low protein content and low moisture content. The amount of free amino acids in these raw materials is negligible. Because these two types of raw materials can undergo different chemical and biochemical processes, they also have different storage requirements.

Changes in protein and amino acid content of vegetables and fruitsVegetables and fruits have a relatively high content of free amino acids. Only some of these are protein-forming amino acids, and many contain rare amino acids and their derivatives. The biological role of these rare amino acids is not always clear. Some of these derivatives can reserve as nutrients, while others are considered to have a regulatory role in certain plant families. Another assumption is that certain amino acid derivatives can provides protection against parasites. There are amino acid derivatives that occur only typically in one or some plant family. Most often, special derivatives of glutamic acid can be found, but serine derivatives are also not rare.

The following protein-forming and rare amino acids are found in higher amounts in ripe fruits. In apples, asparagine, aspartic acid, glutamic acid, serine, alanine and -alanine, and 4-hydroxymethyl proline. Asparagine and aspartic acid in pears are particularly important in the high nitrogen species and 4-hydroxymethyl proline. In strawberries, raspberries and currants, glutamine, glutamic acid, asparagine, aspartic acid, serine and alanine are present in greater amounts. In the cherry, 16 amino acids have been detected, including asparagine, glutamine, aspartic acid, arginine and 4-aminobutyric acid. Asparagine is the most abundant in apricots, the most abundant in peaches, but less than apricot. Similar to the others, the free amino acid composition of lemon and orange is similar: aspartic, aspartic, glutamic, arginine, but proline is also high. Other studies have shown that citrus fruits also have high levels of alanine, tyrosine and leucines.

Many scientists have also studied the free amino acid composition of grapes and wine products. Their amino acid composition was found to be very similar to that of lemon and orange. The relationship between the free amino acid content and aroma of the wines has also been intensively studied. It is well known that the free amino acid content of Hungarian wines is higher than the average, but it is still below the internationally permitted maximum effect, so it does not hinder the sale. The reason for this is not the grape varieties, but the excessive nitrogen fertilizer use and technology.

As a consequence of the high amino acid content, the cosmic oil content of the steamed spirits in Hungary (mainly isoamyl alcohol) is unfavorably high. The high content of cosmic oils in Hungarian spirits is also caused by the general practice of making brandy from cared waste fruit. Such fruits have a high microbial contamination, which also favors the formation of cosmic oils. The quality

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degradation by-products of alcoholic fermentation do not come from the decomposition of carbohydrates, but from other compounds derived from the fermented plant or additives, in particular proteins or amino acids derived from their degradation. For example, various grapes produce unwanted by-products in winemaking, barley brewing, or molasses refining, known as cosmic oils. Cosmic oils are formed by the biochemical processes described for microbial degradation of proteins.

Cosmic oils contain predominantly isoamyl alcohol (see earlier), but also contain other higher alcohols as well as butyric acid and other low-carbon fatty acids, succinic acid, multi-ester compounds and bitter substances (e.g., tyrosol). The bitter taste of beer is partly caused by the tyrosol produced from tyrosine.

Green peppers and cucumbers contain relatively few free amino acids. In addition to the usual Asp, Asn, Gln and Glu, potatoes are rich in lysine, methionine and leucine. Tomatoes are also rich in Gln, Asn and Asp. Changes in free amino acid content during ripening was examined especially in vegetables. In the case of timely harvesting, the protein content is maximal and the free amino acid content is not so high. Delayed harvest increases the free amino acid content. These levels do not change much when stored. If the potato is rich in reducing sugars, the Maillard reaction (see later) is excessive when baked. Therefore, there should be no delay in picking potatoes, especially early potatoes.

The lysine, arginine and tyrosine contents are the most important for the Maillard reaction. The Maillard reaction is discussed in the biochemistry of carbohydrate-based foods, non-enzymatic browning (FIG. 37).In tomato measurement, glutamic acid and asparagine are mainly increased. Excessive nitrogen content in the soil favors an increase in the free amino acid content, which may result in the tomato concentrate becoming brown due to the Maillard reaction at a suitable reducing sugar content.

Vegetables and fruits contain little and varied protein composition. Many of these are enzymes. We deal with these enzymes in enzymatic tanning and fruit ripening. Each potato contains the same type of protein, but the amount may vary from one species to another. Of the potato proteins, tuberin is the most valuable. The protein content of a potato depends on the activity of the proteases it contains. Potatoes contain a variety of protease inhibitors, which inhibit excessive increases in free amino acid content.

Fruits contain relatively little protein (usually less than 1%, apples only 0.2% protein), but their total nitrogen content can be up to 80 percent protein. Most of these are enzymes.

Biochemical processes in cereal proteinsIn healthy, resting cereal grains, low water content virtually prevents

metabolic processes from taking place. The gluten content (gluten is a component of gliadin and glutenin in cereal proteins) has changed slightly, but so far no causal relationship has been established between the number of sulfhydryl groups and the deterioration in quality. In any case, it has been found that the reduced glutathione content of the stored flour is reduced during storage.

4. Other aspects of biochemistry of plant food raw materials

Food ingredients of plant origin generally contain not primarily proteins but other nutrient biomolecules, primarily carbohydrates, in addition to complex lipids.

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Foods containing carbohydrates contain polysaccharides on the one hand and oligo- and monosaccharides on the other. Processes of plant food raw materials during harvest, storage and processing can often involve degradation products from multiple biomolecules. The fermentation industry uses carbohydrate-based preparations as a starting material.

Non-enzymatic browning processes in foodThe effects or processes whereby food or food ingredients become brown due

to decomposition are called browning processes. Carbohydrates are primarily involved in browning processes, but amino acids also play a major role. According to the mechanism of degradation processes, two large groups are distinguished. Physical and chemical effects cause the so-called non-enzymatic browning processes. In enzymatic browning processes, oxidase enzymes in foods or food raw materials exposed to oxygen in the air undergo oxidation and, consequently, ring closure processes. Carbohydrates undergo heat treatment (roasting, baking, boiling, etc.) and undergo thermal decomposition (such as caramelization), but these processes are chemical and not biochemical reactions. Now, only the Maillard reaction is addressed. The browning of the red color of meat through heat treatment and the thermal decomposition of hemoglobin are not considered as food browning processes.

Maillard reactionWhen proteins (or amino acids or compounds containing at least a free amino

group) and carbohydrates (or simple sugars containing a carbonyl group) coexist in food, the functional groups of the two types of biomolecules upon heating condensation reactions (contact with the formation of water) eventually lead to brown pigments, melanoids. Maillard discovered this reaction in 1912. This process leads to the formation of crunchy brown breadcrumbs, which occurs during biscuit making, coffee roasting, and in part, it also plays a role in sunburn.

The theoretical basis of the Maillard reactionThe Maillard reaction plays a role in the formation of pleasant flavour agents

during cooking and baking, but it is also responsible for a number of quality-damaging effects (burnt bread, yellow milk due to overheating, etc.). Both sugars and amino acids involved in the reaction lose their biological value, which is why, for example, biscuits have a lower biological value than the starting material bread.

In the Maillard reaction, the simple sugars formed by the hydrolysis of carbohydrates are carbonyl (oxo) derivatives, and amino groups of amino acids can react with each other to form Schiff bases by condensation. Further reactions of Schiff bases produce brown melanoid pigments. Multiple pigments may contain aromatic rings. Melanoid pigments are heteroaromatic compounds containing oxo groups, like ring melanin, in which the ring nitrogen is derived from the amino group of the amino acids. Some pigments, also colored, containing no nitrogen and resulting from the Maillard reaction, are called melanoidins. In a microwave oven, only the water content of the food is excited, and at this lower temperature (100 ° C) the sub-processes of the Maillard reaction do not take place or are complete. This is the reason why the bread made in the microwave does not turn coloured and has no crust.

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The theoretical basis of the Maillard reaction

However, in the case of longer baking at higher temperatures (120-150 oC), such as french fries, the likelihood of formation of an unsaturated acid amide, acrylamide, is increased, especially from asparagine. Acrylamide has a health hazard because it is the starting material of the highly hepatic and carcinogenic glycidamide. This oxidation takes place in the liver by biotransformation for the oxidative degradation of so-called foreign bodies, cytochrome P450.

acrylamide glycidamide

Enzymatic browning processes in foodEnzymatic browning is defined as all browning processes that occur primarily

on the cutting surfaces of plant raw materials in the presence of oxygen. These processes are catalyzed by various plant oxidases. Many vegetables and fruits have enzymatic browning (e.g. potatoes, apples, pears, and more fruits and vegetables).

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The cause of the browning reaction is a defense mechanism of plant cells. The biomolecule-rich cytoplasm of damaged cells on cutting surfaces is an excellent nutrient medium, therefore, it is a great opportunity for microorganisms to attack, besides, the oxygen intensively acting on the damaged surfaces can be a danger to the plant.

Aromatic compounds formed temporarily during enzymatic browning contain phenolic hydroxyl groups and phenols are known to have antimicrobial (antiseptic) activity. The formation of phenolic derivatives and their conversion to colorful compounds requires significant oxygen utilization, in these way valuable biomolecules are saved from oxidation. Thus, enzymatic browning also seeks to counteract the effects of two hazards. The occurrence of brown color in food raw materials is often a factor of quality deterioration, so it is always desirable to prevent enzymatic browning, at least to reduce its speed.During enzymatic browning, plant phenols are converted to diphenols, then quinones, and finally to brown dyes by phenol oxidases. Phenol oxidase is a commonly used but not an exact term for these enzymes. Their official name is oxygenases. Oxygenases are those enzymes that do not oxidize the substrate by removing hydrogen (like dehydrogenases, which play a vital role in basic metabolic processes), but incorporate molecular oxygen into the substrate. Dioxygenases incorporate both of the oxygen molecules of the oxygen molecule into the substrate. Monooxygenases incorporate only one oxygen into the substrate and the other oxygen of the oxygen molecule forms water with hydrogen from a reduced coenzyme, usually (NADPH+H+). Accordingly, phenolic oxidases may also be mono- and diphenol oxidases. Monooxygenases are primarily involved in enzymatic browning. Thus, one oxygen in the oxygen molecule oxidizes phenol to diphenol and the other oxidizes (NADPH+H+) coenzyme.

A-H + O2 + (NADPH+H+) A-OH + NADP+ + H2OMonooxygenase-catalyzed reactionA + O2 AO2 or 2A + O2 2 AO

Dioxygenase-catalyzed reaction

Diphenol oxidases are monooxygenases (may be enzymes that catalyze the oxidation of ortho- and para-diphenols) which contain copper ions and these copper ions participate in the redox process instead of coenzyme. In this case, the oxidation takes place in two steps. Diphenol is oxidized by the enzyme itself to quinone, while the copper (II) ion (cupri) (Cu2+) dimer in the enzyme is reduced to dimer of copper(I) (cuprous) ion (Cu2

2+). The oxygen in the air to produce water regenerates the reduced enzyme.

(Cu2+)2-enzyme + diphenol (Cu+)2-enzyme + diquinone + 2 H+

(Cu+)2-enzyme + 1/2 O2 + 2 H + (Cu2+)2-enzyme + H2OFunction of diphenol oxidases

The dioxygenases may contain copper or iron ions. Iron-containing dioxygenases temporarily form an enzyme-iron-oxygen complex from which the redox reaction with the substrate regenerates the original E-Fe2+ complex.

E-Fe2+ + O2 E-Fe2 +–O-O = E-Fe3+–O-O– (+ substrate) E-Fe2+

Diphenol oxidases essentially have two functions. Catalysis of monophenoldiphenol conversion (cresolase activity) and diphenolquinone

26

conversion (catecholase activity). The second step in enzymatic browning is the oxidation of polyphenols, usually the first step, to the quinones. In the continuation of the oxidation process, coupled oxidation is common, in which the resulting quinones oxidize further polyphenols, including polyphenols that are not substrates for polyphenol oxidases.

Melanin biosynthesis

Also involved in the initial phase of enzymatic browning is the oxidative conversion of an amino acid containing a phenolic hydroxyl group, tyrosine, which is catalyzed by a monooxygenase called tyrosine hydroxylase with copper cofactor. Tyrosine is converted by a tyrosine hydroxylase to a diphenol (DOPA) (3,4-dihydroxyphenylalanine, the acronym for old name of this compound dioxy-

27

phenylalanine) which is oxidized by the oxygen in the air to dopakinone. Dopakinone is cyclized to leukodopaquinone and further oxidized to dopachrome. DOPA is also the starting material for the biosynthesis of noradrenaline and adrenaline. Dopachrome develops in several steps the red (brown) and black dyes known as the melanins, collectively. Melanin and its derivatives are also the coloring materials of the hair and the skin.

Since enzymatic browning is clearly a disadvantageous process for the food industry, it should be prevented. Among the preventive treatments, heat treatment enzyme inactivation technologies are the most widespread. Unfortunately, peroxidases are less sensitive to heat and additional treatment is often required. The activity of phenolic oxidases in the acidic medium is dramatically reduced and citric acid is often used. The use of citric acid has the dual advantage of not only having an acidifying effect but also having an excellent complexing property; it binds the copper content of phenolic oxidases. In the storage of food, the exclusion of air is often used as a preventive method. However, this is difficult to implement on an industrial scale. Antioxidants and the substrate concentration dilution method are often used to prevent enzymatic browning.

Breathing of vegetable food raw materialsIn the biochemical processes of vegetable origin, but already in food raw

materials, only the respiratory energy, that is, the ATP produced in the terminal oxidation, can be used, since there is no photosynthesis in these raw materials. Breathing produces carbon dioxide and water from the carbohydrate content of such food raw materials, which can be significant. Particular concern is the formation of heat in parallel with breathing and its secondary consequences. The primary goal when storing food raw materials of plant origin is to minimize respiratory losses.

General issues in the regulation of respiratory metabolismOxygen tension around plant tissues regulates the rate of anaerobic

(fermentation) and aerobic degradation in carbohydrate degradation processes. The more oxygen, the greater the percentage of pyruvate that is completely oxidized. The lowest oxygen concentration at which all pyruvate is aerobically degraded is the extinction point. In fruits, this is usually 2-5% in living tissues, but no data on post-harvest status. Increasing oxygen concentration can also reduce the amount of carbohydrate degradation up to a certain limit, a Pasteur effect that has been proven for picked apples. The specific surface of the harvested plant parts also influences respiratory rate, and even the rate of oxygen transport in tissues can be a regulator.

The distribution of glucose degradation between glycolysis and the pentose phosphate cycle depends on the state of the body. As the body ages, the intensity of the pentose phosphate cycle increases and the proportion of the latter in plant tissues attacked by pathogens increases. This rate shift is also characteristic of the human body. The proportion of the pentose phosphate cycle can be about 10% in young age, and up to 30% in old age. The ratio of glycolysis to pentose phosphate cycle is determined by the body's reduced coenzyme, or more specifically, by (NADPH + H +) levels and requirements. Breathing speed is not affected by this ratio.

The mechanism of respiratory regulation is complex, often with feedback. In terminal oxidation, ADP and inorganic phosphate levels affect ATP level, and some synthetic reactions are regulated by ATP level (e.g. glycolysis, citrate cycle, etc.). ATP inhibits phospho-fructo-kinase (PFK), pyruvate kinase, pyruvate dehydrogenase multienzyme complex and citrate synthesis. Citrate, which is accumulated in the

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mitochondria, enters the cytosol, inhibits the initial steps of glycolysis and promotes fatty acid synthesis. Thus, adenylate regulation is significant. The inhibitory effect of ATP is counteracted by AMP.

When discussing the breathing of cereals, it is necessary to observe not only the processes inside the grain (corn, seed), but also the microflora on the surface of the grain. During storage grain decomposition begins in the grains. This is done by the amylases, the activity of the -amylase is at rest. Sucrose may also be synthesized from degradation monosaccharides, glucose and fructose. In oilseeds, glucose is formed through the glyoxylate cycle. The amount of mono- and oligosaccharides formed during degradation is approximately equilibrium due to their degradation by respiration. On higher levels, but also equilibrium in the atmosphere of nitrogen or carbon dioxide, occur in the seeds.

Breathing during vegetable storageVegetable breathing intensity increases steadily during storage. Lowering

storage temperature reduces respiratory losses. The degradation of polysaccharides is not the same as the change in respiratory intensity. A good example is the storage of potatoes. It is well known that at too low a temperature (between 0 and +4 oC) the potatoes turn to more sweet and their mono- and disaccharide content increases about four-fold. This creates the opportunity for non-enzymatic browning, thereby reducing the quality of the food it contains (e.g. French fries). In the potato, the sucrose content mainly increases.

When the potatoes are placed in a warmer place, the mono- and disaccharide content decreases more rapidly and the starch resynthesis is restored more rapidly than the carbon dioxide produced by respiration. Therefore efforts were supported for cold-tolerant potato breeding.

Breathing is a heat producing process. Some vegetables and fruits have a sharp increase in respiratory rate above +10 oC (e.g. cauliflower, tomato, lettuce). In the case of apricots and sour cherries this is already at +5 oC. For other vegetables and fruits, the respiration measured at +5 oC is doubled at +10 oC.

5. The role of climacteric respiration in fruit storage

Ethylene (H2C=CH2) is a plant hormone that can accelerate to ripen in certain fruits. The fruits can be divided into two groups according to their maturity. Fruits producing ethylene are called climacteric fruits, and ethylene non-producing fruits are called non-climacteric ones. Climacteric and non-climacteric fruits also react differently to externally supplied ethylene. Each plant organ contains ethylene. In climacteric fruits (e.g. apples, pears, apricots, peaches, plums, tomatoes, bananas), mitochondrial activity, ethylene synthesis, and protein and RNA synthesis change periodically. The pre-climacteric minimum is found in immature fruit. In this case, mitochondrial activity and ethylene formation are low, protein and RNA synthesis is intense, and the pectic substances are in the propectin phase. During maturation, the intensity of ethylene synthesis increases and at the same time the breathing intensifies. As a result, maturation results in the formation of a climacteric peak, formation of pectin, minimization of protein and RNA synthesis, loss of ethylene production and mitochondrial activity at its peak. This is how the fruit reaches a state of overripe, whereby the pectic substances are converted to pectic acids. The biochemistry of pectin is a topic of another part of this course.

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The breathing of non-climacteric fruits (e.g. strawberries, cherries, pineapples, oranges, lemons, cucumbers, grapes) is also stimulated by ethylene, but the extent of respiration depends on ethylene concentration and its effect decreases over time. There is no such dramatic change in the ripening processes of these types of fruits as in the case of climacteric fruits. In the case of raspberries and blueberries, the climacteric nature is still questionable.

Ethylene activates the synthesis of mRNA required for the biosynthesis of enzymes that initiate the fruit ripening process. These mRNA types are stable at the start of maturation.

Biochemistry of ethyleneThere is an exact timetable for the production of ethylene during the ripening

of climacteric fruits. Semi-ripe fruits have a maximum ethylene production, for example, in tomatoes the highest is when the fruit is pink. In overripe fruit, the rate of production is one tenth of the maximum. The place where ethylene is produced in fruits is the origin of this meaty crop. Its rate of formation has been observed to be related to the action of other plant hormones, particularly plant auxin (heteroauxin, indole-3-acetic acid, IES). It is common experience that young, auxin-rich, highly-growing plants produce little ethylene. In other parts of the plant, even some molds, ethylene is formed. This is the reason why moldy fruits ripen faster - and deteriorate.

Synthesis of ethylene from methionine

Ethylene in plants is usually synthesized from methionine, but since the starting material for methionine is oxalacetate, it can eventually be formed from any biomolecule that is degraded through the citrate circuit. In climacteric fruits, SAM (S-adenosylmethionine) is formed from methionine with ATP. Cleavage of SAM produces 1-aminocyclopropane-1-carboxylic acid (ACC) and 5'-methylthioadenosine.

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ACC decomposes to ethylene and cyanoformic acid (NC-COOH). Cyanoformic acid is converted to hydrogen cyanide at a loss of carbon dioxide. HCN is the starting material for cyanogenic glycosides. The nucleus of climacteric fruits, and in particular the inner skin of the bones, always contains cyanogenic glycosides, especially amygdalin.

Hydrogen cyanide produced alongside ethylene is toxic to all organisms because it blocks the function of the last complex of terminal oxidation, cytochrome oxidase. Climacteric fruits detoxify hydrogen cyanide in the form of amygdalin that is stored in the seeds.

Amygdalin is a benzaldehyde cyanohydrin whose hydroxyl group is linked to a disaccharide (gentiobiose) linked by a 1,6-glycosidic bond from two glucose. Amygdalin is hydrolyzed in the stomach of the human body to benzaldehyde cyanohydrin. Then this compound decomposed to benzaldehyde and hydrogen cyanide. The latter is one of the strongest poisons, causing 20 bitter almonds to die! The seeds of all climacteric fruits contain significant amounts of amygdalin.

benzaldehid(mandulaillat)

benzaldehid-ciánhidrin

CH

CN

O genciobiózgenciobióz

A keserûmandula szaganyaga

CH3C

HO

acetaldehid

H2NCH

COOH

R+

-aminosav

a fenntiek szerint

ANCH3 CH N C

COOH

HR

HCH3 CH N

R

COOHC

HCH3 CH N C

COOH

R

CH3 CH2 NR

COOHC

ketimin

H2O

H2O

CH3CH2 NH2 +COOH

CR

O

etil-amin -keto-karbonsavAldimin – ketimin tautomer átalakulás

H C N C

O

H

+C

H O–H

CN

amigdalin

Formation and degradation of amygdalin

Based on the impact studies so far, the mechanism of action of ethylene cannot be clearly established. Two fundamentally different mechanisms have been described. One is that ethylene binds only loosely to the appropriate receptor and exerts its regulatory activity in a cascade-like sequence. This means that it is not metabolized during ethylene action. The weakness of the ethylene receptor binding seems to be demonstrated by the immediate loss of its biological effect when ethylene is removed.

Alternatively, there is a reaction between ethylene and the receptor and ethylene is metabolized. Ethylene is thought to interact with a variable valence receptor. This assumption seems to be supported by the fact that the atmosphere of carbon dioxide inhibits the effect of ethylene.

6 Changes in the lipid content of foods – rancidity of fats and oils

The lipid content of food raw materials may undergo many changes as a result of processing and storage effects. These effects may be physical (e.g .effects of temperature, etc.), chemical effects (e.g. pH of the medium, oxygen in the air, etc.) and biological effects (e.g. degradation of activated enzymes in the food raw material, effects of microorganisms present in the environment, etc.) and combinations of these effects.

Changes in lipid content can basically be of two types: hydrolytic and oxidative degradation. Both types of changes are known to be enzymatic and non-enzymatic (under the influence of organic chemical reagents). The former can be

31

caused not only by the enzyme supply of the food raw material itself, but also by the microorganisms and their enzymes.

The enzymatic and non-enzymatic hydrolytic effects of the lipid components of food raw materials cause relatively little damage. Simple lipids cannot be hydrolyzed. Among the complex lipids, the hydrolytic enzymes for the ester groups of the neutral fats are the lipases, and the phospholipases for phospholipids but their activity is minimal under the storage conditions of the food raw material. Non-enzymatic hydrolysis can cause significant damage at most in highly alkaline media e.g. during hydrolysis of triglycerides by sodium hydroxide soaps (in Hungarian szappan) can be formed by boiling. The name of this process is soap boiling (in Hungarian szappanfőzés)

RCOOCH2

RCOOCH

RCOOCH2

+ 3 NaOH +CH2

CH

CH2

O

O

O

H

H

H

R CO

O3

Naszappan

glicerolA szappanfôzés összfolyamata

Hydrolysis of triglycerides by sodium hydroxide

Neutral triglycerides (triacylglycerols)are triesters of glycerol with fatty acids (C16-C18) that are highly concentrated energy stores in fat cells (adipose cells), water-repellent materials (e.g. on the skin) and heat-insulators in human and animals. Fats are solid and their fatty acid parts are saturated fatty acids are palmitate (CH3(CH2)14–COOH) and stearate (CH3(CH2)16–COOH) and unsaturated oleate. The liquid oils contains mostly polyunsaturated fatty acids and their major fatty acid component is linoleate e.g. sunflower oil contains about 80% linoleate as fatty acid component.

Formulas of oleate, linoleate and linolenate

Oxidation effects due to oxidation, especially of the oxygen content of the air, in which triglycerides are mainly exposed to the polyunsaturated fatty acids (linoleic acid and linolenic acid) can lead to a much greater deterioration in the quality of food raw materials and finished foods. The oxygen attack occurs on the methylene group (> CH2) between two cis-formed unsaturated carbon-carbon bonds.

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The mechanism of this oxidation processes is radical and their course is not significantly different in enzymatic (lipoxygenase-catalyzed) and non-enzyme-catalyzed (autooxidation) reactions. In addition, the two sets of processes can come together in multiple places so that the damage can occur together. Oxygen attack occurs at the saturated carbon atom between the two double bonds. In the first steps of this process, called lipid peroxidation, lipid hydroperoxides are formed. The process is facilitated by iron and copper ions. It is believed that one of the tasks of vegetable oils in plants is to reduce the increased oxygen concentration in lipid peroxidation in the event of injury or stress.

> CH2 + O2 > CH • + HO2 •> CH • + O2 > CH – OO •

> CHO2 • +> CH2 > CH – OO – H +> CH •lipid hydroperoxide

Formation of lipid hydroperoxides

Lipid hydroperoxides, the concentration of which can be determined by iodometry, are capable of further degradation in a variety of ways. During further oxidation they are converted to diperoxides and then to polymers. Oxidative cleavage produces aldehydes, hydroxy compounds and then carboxylic acids. These lower aldehydes and carboxylic acids cause the characteristic odor of the rancid fat (e.g. butyric acid, valeric acid), so rancidity is a serious quality-deteriorating process.

Formation of degradation products during lipid peroxidation

During lipid peroxidation, dialdehydes, in particular malonic dialdehyde (OHC-CH2-CHO), which is otherwise odorless, are also formed and can be quantified with appropriate reagents (e.g. thiobarbituric acid). The dialdehydes react with the –CH2– moiety of thiobarbituric acid, resulting in a colorable pink compound.

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Formula of malondialdehyde and thiobarbituric acid

Lipid soluble free radicals produced by mass during the process of rancidity can lead to various diseases in the human body (e.g. atherosclerosis, cancer, etc.), therefore, rancidity of food is not only a quality deterioration for consumers but also a health damaging process.

Lipid peroxidation is also behind the drying nature of linoleic acid-rich drying oils, such as linseed oil. In this case, the oxygen-containing intermediates formed by the decomposition of the lipid hydroperoxide formed during lipid peroxidation will react with another lipylene acid to form an ether linkage.

Drying of linseed oil

When an ether bond is formed, one cis double bond is transformed. A similar transformation occurs when oils are hydrogenated (double bond saturation) to form trans-fatty acids that are dangerous to health. In this case, only one of the double bonds of linolenic acid is saturated, the other only undergoes cis-trans isomerization. Trans fatty acids adversely affect the balance of cholesterol in food biochemistry).

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Literature1. Luckner, M.: Secondary metabolism in microorganisms, plants and animals

Springer-Verlag Berlin Heigelberg New York London Paris Tokyo Hong Kong 1990.

2. Stryer, L.: Biochemistry (3rd Edition) W.H. Freeman & Company New York 1988.

Topics in Biochemistry of the storage of food raw materials

Three of questions, maximum 3×5=15. Marks: <7 = 1 elégtelen (unsatisfactory), 7-8 = 2 elégséges (sufficient), 9-10 = 3 közepes (satisfactory), 11-12 = 4 jó (good), 13-15 = 5 jeles (excellent).

1 Describe briefly the basic rules of life at the molecular level. 2 What is the first step in the breakdown of nutrients into the body is to name

the enzymes that catalyze it by the individual biomolecules.3 What are the stages of glucose degradation? Briefly describe the anaerobic

continuation of glycolysis. Write down the material balance for the processes.

4 What are the stages of glucose degradation? Describe briefly the two phases of breathing. Write down the material balance for the processes.

5 Briefly describe the degradation of amino acids.6 Describe briefly the degradation of lipids.7 What processes involve breathing, where they occur, and what happens

during it.8 Describe the location, essence, and material balance of the citrate circle.9 Describe the site, nature and material balance of terminal oxidation.10 Describe with equations what derivatives may be formed from the

aldimines of amino acids with pyridoxal phosphate. During the oxidative degradation what happens to the -keto carboxylic acids formed from amino acids.

11 Muscle structure, role of individual elements, conditions of movement.12 Biochemistry of muscle function.13 Biochemistry of the first three post-mortem phases of muscle tissue

and its effect on meat consumption.14 Biochemistry of the fourth post-mortem phase of muscle tissue and its

effect on the edibility of meat.15 The effect of microorganisms on the degradation of amino acid

residues. Describe the equation for the formation of at least two derivatives.16 Biochemistry of milk and other protein foods.17 What is the biological role of meat coloring and the fate of

postmortem.18 Causes and possibilities of non-enzymatic browning of vegetable food

raw materials.19 Biochemistry of enzymatic browning processes of vegetable food raw

materials.20 The role of respiration of plant food raw materials in storage.21 Ethylene biosynthesis and role of metabolism in fruit storage.

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22 Chemical name, cause and biological consequence of food rancidity. Write down the formula of at least one starting material for the process and the first step.

Units of the polypeptide chain, the L--amino acidsThey are the building blocks of proteins connected by peptide bonds. Standard

(protein, proteinogenous) amino acids build up proteins, non-standard (non-protein, non-proteinogenous) amino acids can be important metabolic intermediates. The name of standard amino acids is used generally in their abbreviated form. The modified Fischer conventions of the formulas of twenty standard amino acids and their abbreviations are presented in schemes. Ten of amino acids (Val, Leu, Ile, Phe, Lys, Thr, Trp, Met, Arg, His) are called essential amino acids, because the human body cannot synthesize them from other compounds at the level needed for normal growth, therefore they must be obtained from food.

Secondary interactions between the side chains of amino acids: hydrophobic interactions – glycine (Gly), alanine (Ala), valine (Val), leucine

(Leu), isoleucine (Ile), phenylalanine (Phe), proline (Pro) ionic interactions – aspartic acid (Asp) glutamic acid (Glu), lysine (Lys), arginine

(Arg) hydrogen bonds – serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn),

glutamine (Glu), tryptophan (Trp), histidine (His) disulphide bond – cysteine (Cys) dipole-dipole interactions methionine (Met);

H2N CH2 COOH H2NCH

COOH

CH3

H2NCH

COOH

CH

CH3 CH3

H2NCH

COOH

CH3 CH3

CH

CH2

glicin (Gly)

alanin (Ala)

valin (Val)

leucin (Leu)H2N

CHCOOH

CH

CH3CH2

CH3

H2NCH

COOH

CH2N COOHH

izoleucin (Ile) fenilalanin (Phe) prolin (Pro)

A hidrofób kölcsönhatásra alkalmas fehérjealkotó aminosavak

Amino acids of hydrophobic character

36

Az ionos kölcsönhatásra alkalmas fehérjealkotó aminosavak

H2NCH

COOH

(CH2)4

NH2

lizin (Lys)aszparaginsav (Asp)

H2NCH

COOH

COOH

CH2

glutaminsav (Glu)

H2NCH

COOH

CH2

CH2

COOH

H2NCH

COOH

(CH2)3

NH

C=NH

NH2

arginin (Arg)

Amino acids with ionic character

H2NCH

COOH

CH2OH

H2NCH

COOH

OHCH3

CH

H2NCH

COOH

CH2

H2NCH

COOH

CH2

CONH2

H2NCH

COOH

CH2

CH2

CONH2

OHtirozin (Tyr)

treonin (Thr)szerin (Ser)

aszparagin (Asn)glutamin (Gln)

triptofán (Trp) hisztidin (His)

H2NCH

COOH

CH2

NH

H2NCH

COOH

CH2

NNH

A hidrogénkötésre alkalmas fehérjealkotó aminosavak

Amino acids with hydrogen bonds

37

H2NCH

COOH

CH2

CH2

S–CH3metionin (Met)

A dipólus-dipólus kölcsönhatásra alkalmas fehérjealkotó aminosav

H2NCH

COOH

CH2SHcisztein (Cys)

A diszulfidkötésre alkalmas fehérjealkotó aminosav

Cysteine with disulphide bond and methionine with dipole-dipole interaction

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