Error Catastrophe Theory of Senescence

The catastrophe theory

Mistakes at the translation and transcription level

• mRNA mistakes
• Missing of repair systems
• Cytokinins in the t-RNA adjacent to the anti-codon loop
• Nonsense protein formation (faulty translation)

Post-transcriptional protein folding

Bad DNA multiplication

DNA repair potential decreases

DNA in dry tissue (seeds) more susceptible to damage

mRNA life time changes

Changes at the DNA and RNA levels

oIncreases of RNAses

oDecrease of DNA-RNA polymerase

The error catastrophe theory of aging was proposed by Leslie Orgel in 1963 and states that -
in an individual, the aging (senescence) occurs as a result of accumulation of errors committed by the molecular genetic apparatus during protein synthesis. It claims that when the amount of error product exceeds a certain threshold value; malfunction, senescence and death results. Although these mistakes can occur in any protein made by the cell, when these mistakes occur in the enzymes and other proteins responsible for synthesizing DNA and RNA, or in the protein synthesizing machinery itself, this could lead to an increasing cascade of errors, referred to as an "Error catastrophe". 
The error could be at one or more sites along the protein production line, either at a transcriptional or translational level, resulting in the  "nonsense proteins" acccumulate. These are non-functional enzymes containing mistakes in the amino acid sequence. This escalating process could turn what is initially a very low error rate in young individuals into a significant rate of accumulation of errors in older individuals, and one would predict that the rate of error accumulation might continue to increase exponentially throughout the life of the individual. 

The genetic blueprint for each biological species occurs in the Deoxyribo nucleic acid (DNA) in the nucleus of each cell. When the cell divides, an enzyme known as DNA polymerase makes a new copy of the DNA by combining the appropriate building blocks known as deoxyribonucleotides in the correct sequence in a process known as DNA replication. The genetic sequences in the DNA are then transcribed by another kind of copying enzyme, known as RNA polymerase, into a ribonucleic acid (RNA) molecule called messenger RNA. Specific messenger RNAs contain the instructions for synthesizing individual proteins of the correct amino acid sequence, corresponding to the original blueprint in the DNA. This final protein synthesizing process is called translation.

During DNA replication, many DNA polymerases possess the ability to recognize mismatched bases, then back up and correct their own mistakes. In addition, very robust DNA repair systems are present to correct mistakes made during synthesis, or afterward by chemicals able to damage DNA. Thus, the error frequency in DNA replication is usually extremely low, perhaps less than one in a million bases. Studies to demonstrate age-related changes in the copying fidelity of polymerases or DNA repair capacity have not provided convincing support for the error catastrophe theory.

In general, RNA polymerases also combine ribonucleotide building blocks to make RNA with high sequence fidelity, but lower than that exhibited by DNA polymerases. However, the overall instability and turnover of messenger RNA tends to attenuate the impact of any mistakes made during messenger RNA synthesis. Protein synthesis is also generally carried out with high fidelity, and there is little evidence to suggest that this changes with age.

Gershon 1985, however challenged this theory and claimed that-

the process most prone to ageing is post-transcriptional protein folding and the most probable candidate being proteases. Erroneously folded non-functional proteases therefore lead to erratic cell metabolism and senescence.

Proteins do become randomly altered after they are synthesized; a variety of such processes is collectively referred to as post-translational modification because it occurs after synthesis of the protein and use of the messenger RNA as a template has been completed. Although distinct from the damage hypothesized in the original error catastrophe theory, post-translational modification of proteins could functionally resemble an error catastrophe. These modifications include oxidation of amino acid sidechains, racemization of certain amino acids, and condensation of the lysine side chain amino group with aldehyde groups such as those found in glucose. This latter process is known as non-enzymatic glycation. Biochemical mechanisms exist to repair the damage caused during some of these processes, suggesting they could have biological significance with implications for aging. However, there is a dearth of evidence unequivocally indicating that damage-inducing processes, damage accumulation, or repair processes are casually related to aging in mammalian species.

In summary, although altered proteins do accumulate with increasing age in mammals, the error catastrophe theory itself is no longer regarded as a viable theory. Nevertheless, there remains a healthy research interest in determining what roles damaged proteins, and the processes that either destroy the damaged protein or repair the damage, might play as casual factors in aging.

Types of Senescence

Leopold in 1975 has proposed four kinds of senescence patterns:

Whole plant (Overall) senescence:

 The complete plant dies soon after ripening of the seeds.This is found in monocarpic plants that generate fruit and flower only once in their life cycle. The plants might be annual (example: rice, gram, mustard, wheat, and so on), biennials (example: cabbage, henbane) or perennials (example: certain bamboos and so on).

 Figure: Types of senescence

Shoot (TOP) senescence:

The above-ground plant organs die off seasonally, to be renewed by growth from sub-terranian organs. This kind of senescence is found in some perennial plants that possess underground perennating structures such as rhizomes, corm, bulbs, and so on. The ground portion of the shoot dies each year after flowering and fruiting, however the underground portion (stem and root) survives and puts out new shoots again next year. Example: banana, ginger, gladiolus, and so on.

Sequential (Progressive) Senescence:

This is found mostly in perennial plants in which the tips of main shoot and branches remaining a meristematic state and carry on to generate new buds and leaves. The older leaves and lateral organs such as branches exhibit senescence and die and new ones gradually substitutes for them. Sequential senescence is obvious in evergreen plants. Example: Eucalyptus, Pinus and so on.

Simultaneous or Synchronous (Deciduous) senescence:

This is found in temperate deciduous trees like elm and maple. Such plants show seasonal (summer or winter) foliage senescence depending on the local stress. Pome and drupe deciduous fruit tress usually manifest winter shedding, while xerophytic species such as Zygophyllum shed foliage during the hot dry summer. Such a senescence of leaves or plant organs is termed as synchronous.

Senescence Curves

Several essentially different death curves can be observed for different plant population.
1. In the case of seasonal, annual, and monocarpic species, senescence can be dramatic. Until a certain stage, plants develop, elongate and produce grain/fruit and menifest a very low incidence of mortality. However, after the sensing of certain death signal, all individual cells die off with military parade precision. Curve E is typical of such behavior and is designated as a square wave.

Curve C is typical of human and domestic animal mortility. The more progressive the society the smaller the peak indicating infant mortality. After a period of relative stability, maximal mortality is observed in the latter period of life span.

Under certain circumstances of regulated diet and hygiene, the sygmoidal curve C, may be deflected to the square wave trend i.e. curve D, which has a definite advantage.

Curve A,B are believed to be typical to the perennial and polycarpic trees and shrubs. Also called as "an ecological mortility curve" typical of wild animals where very few individuals live to die of natural causes and not prey to younger or stronger animals, disease or natural disasters.

Protein Gelation

Gels consist of 3D networks (with junctions that can be polimer or polisacharides) of a biopolymer in which water is trapped and retained (Gelatine, pudin, boiled egg). These biopolimers are polysaccharides or proteins, or combinations of both. The junctions are neded in order to be able to build the network.

The properties of the gel are largely dominated by two types of interaction forces: protein protein interactions and water-protein interactions. In fact these two proteins are to some extend opposing each other a bit, so they should be in good balance in order to ensure a good gel network. A good gel network consists of a gel wich over the time retains its water. So the water does not leak out of the gel network, this process is known as syneresis. Typically this process is not desirable.

As mentioned, protein-protein and water-protein interactions should be balanced. It p-p interactions dominate, then proteins will form clusters, which grow bigger and finally will become insoluble in the aqueous solutions. The result is an agglomerations and coagulation of proteins, resulting in bigger protein particles which precipitate from the solution. Sometimes these coagulated proteins may still contain some water, but in general considerably lower amounts compared to those present in the original protein-water mixture. A typical example would be a casein curd which is obtained during cheese making. The curd obtained in this case though can still be considered as a gel, since sufficient protein-water water interactions are still present too. If protein water interactions are dominations then the proteins will not show a tendency to interact with each other. As a result the 3D network needed to form the 3D structure of gel will not be formed, or only to a limited extend. Possible viscosity of the protein solution can be increased because some water can be retained  and temporary a 3D network between the protein can be formed, which is however not stable and will be disturbed by the Brownian motion of all molecules present in the aqueous protein. 

These aspects are illustrated in the figures:

1: Represents  individual protein particles which are sometimes interacting with each other, but mainly interact with water, thus a 3D network is not created.

2And 3: The protein interactions increase and as it can be observed protein strings are being created which are interactions at some points thus creating juctions, it is a 3D structure so the water is entrapped into the structure.

4 the proteins interact so strongly that in fact the water is being expelled out of the protein-water system, giving rise to protein clumps which become unsoluble in the aqueous system and thus precipitate. 

Protein-protein Interation

The protein-protein interaction and water protein interaction play a major role in determining gel strength and quality. It is obvious that by changing particular parameters (both intrinsic and extrinsic) the gel properties can be changed). Therefore it could be discussed first how protein interactions originate. A part from peptide bonds linking up aminoacids, only one type of covalent bond may impact the interaction between proteins: these are the disulphide bonds between two residues (SH group). As aaminoacids and their side groups can be charged (due to protonation + charge, due to proton loss: -charge), obviously the carge intermediation are also potentially important; moreover these can be attractive or repulsive.  H-bridge formation between  a H donating (eg. OH group) and a H- acceptor (eg. C=O group) support protein protein interaction too. Temporary dipole interactions between phenyl group from an aromatic amino acid such as tryptophan, tyrosine or phenilaanine are also relevant, but already considerably weaker. So are the lipophilic interactions between the side changes of a-polar amino acids such as leucine, alanine, iso leucine, etc.

Having these interactions in mind it is clear that for instance, by changins the PH of a protein solution, protein-protein interactions may change considerably and thus conduce to gel formation. A typical example can be found in the fresh cheese (cottage cheese) production of yogurt. In fact a combination of other charge interactions and an increased lipophilic interaction between the casein proteins are at the origin of this gelation.

By denaturing proteins, new disulphide bonds can be produced between proteins, ot disulphide bonds I proteins can be broken, thus facilitating the interaction with other proteins (because the protein chain has increased its flexibility, it can move freely in the 3D space). Tus gels can be induces. A typical example is boiled egg, resulting from gelation of ovoalbumin.

As the concentration of ions (salts) impacts protein protein interactions (the higher the salt  concentration the higher the protein-protein interaction) the concentration of salt can have an impact on gel formation.

Upon protein degradation (during cheese fermentation) in which part of the proteins ae hydrolised, shorter peptide chains are produced, thus lowering protein protein interactions. These results in gel losing its strength: the interior of the cheese becomes liquid.

Another example of Gel is gelatin: it is a special case because it is produced by parcial hydrolisis of collagen, increasing its solubility in water and therefore decreasing protein-protein interaction in binding tissue.

Protein-protein interactions can also be influenced by adding crosslinking agents, thus influencing gel formatin potential. 

Protein-Water Interaction

Solubility of proteins depends on the Ph. Caseins precipitates at ph of 4, caseins then are unsoluble. Why? Because At 4.5 the caseins reach the isoelectric point (ph at which the proteins have a neutral charge zero). The results is that phosphate groups are neutralized, and calcium bridges fall out of  each other, and the caseins  micelle fall out of each other, and the prteins will start to act differently until they precipitate.

It is possible to enhance the protein solubility by decreasing the ph. Why? Because we give them strong charge. When we protonate, by giving acid, we get positive charge. For casein it is not possible because when the micelles are disintegrated, then it is irreversible.

Water binding capacity: amount of water that can be bound in an amount of protein. Very important for meat products (weight is money). If you cook ham (pink or purple in color), during boiling we lose water binding capacity of the product because of protein denaturation. The apolar group is exposed so the water goes away. If I boil a pig leg the water will be evaporated, so we need to reduce this water loses. For this we need to increase the water binding capacities. It depends on the ph, at high ph we will have more water binding capacity (more water, but less quality)

Interfacial Properties of Protein: Emulsion and Foam

Emulsion: Mix of water and oil. Water and oil normally cannot be mixed. 2 kinds

a. Mixtures Were water is the continuous phase: oil in water emulsion (eg. simple sauce, mayonnaise )

b. Mixtures were oil is the continuous phase: water in oil emulsion (eg. margarine)

Why is it not stable? In milk for example there is cream at the top, the fat separates from the milk. What happens here? Small droplets on surface. In milk there are small casein micelles. In raw milk what happens is that the fat droplets are bigger and because there is a density difference between fat and water the droplets go upwards. The bigger the droplets the higher the density difference and the faster the speed at which the droplets go up.  The higher the density difference, the faster the droplet goes up. When we have creaming or a layer of oil on the top it means that it is unstable.

The important is what happens in the interphase between the water and the oil. The interphase must be stable. To make the interphase between the water and oil stable, we need to the polar and apolar part of the molecule together (anphiphilic part). The apolar par will be in the fat phase and polar par in the water phase. The oil droplet will be covered by water, the polar head will be attracted water, and it means that the water won’t feel the oil. Same with the oil, it will be only attracted by oil phase. In this way the interphase will be stabilized. To let this stable interphase happen we need an emulsifier. So for this we can use either a random coil protein (casein, don’t have tertiary structure) or globular.

When we have casein (random coil), some parts will be in the polar phase, others in the apolar phase, and other right on the interphase. This happens because the R group (leucine, phenil alanine are apolar), (cerine, aspartic acid, glutamic acid, this group is polar group). When we see at the primary structure we will se that there are parts were there is polar concentration, other apolar concentration , and in other a mix of polar and apolar. This is because the protein doesnt have a tertiary structure, it is a random coil, it is extremely flexible. In the primary structure there are this local zones of apolar, polar amino acids, then the caseins are good emulsifiers.

Globular protein is normally present in an aqueous environment and is not normally good emulsifier. So the surphase of the protein is in majority oil, because it is soluble in water.  If we bring this protein in an interphase between water and oil,  we will have a problem because they won’t like to be in the oil (if whoe surface is polar). The protein be defolded, it can undergo denaturation. When It is denatured, then it can enter in the interphase an d act as emulcifier. So normally globular proteins (with 3 rd structure) must to undergo denaturation (or partial denaturation). Before they become good emulsifiers. It can be done when we have the interphase and we mix water and oil, by adding mechanical energy, the protein can go out of each other , the dimer will be lost. The apolar and poalr zone will be separated and the emulsifier will be stabilized. Also in the globular protein there can be some more apolar zones and some more polar zones. It can act as an emulsifier depending on the distribution of the apolar zones, so sometimes we need denaturation and sometimes not.

Foams: Mixture of water and air. Composed of air bubles and they are surrounded by water. There is an interphase between air and water, the air is the apolar matrix and water is the polar matrix. It is chemicallya problem in protein solutions because they are foeaming when they have surface active properties (casein solutions, ovalbumine). 

Protein Oxidation

# Tyrosine oxidation: When there are 2 tyrosine molecules together, the 2 radicals can also form a crosslink D-tyrosine, it is a natural process happening during the aging of meat. When the animal is getting older there is more production of D-tyrosine, and as result of it the muscles become tougher. It is an example of a protein oxidation reaction.

# Methionine can be oxidized into a sulfoxide and get homocysteine. Sulfoxide in the body can be back reduced into methionine (cost a lot of energy). At the moment you get the sulfone then it is not nutritionally relevant. Metionine is essencial.

# Cysteine is the most vulnerable amino acid towards oxidation, because of the free sufridril group. The free sulfridril group can be further oxidized by another sulfridril group, so can get a dimer (disulfride brigde, cysteine). It can be stepwise oxidized into the mono or disulfoxide and sulfone. Until disulfoxide it can g back, but when it gets to sulfone, there is no way back.

The formation of cysteine (disulfide bridge) is important during bread making. The Polymerization reactions of wheat protein (glutelins and gliamines, which are prolamines) produce a protein network, which is largely base on the production of the disulfite bridges. This chemistry (dimerization of cysteine) is important to improve the dough structure, and build up the gluten network during bread making.   So oxidants are used as bread improvers such a bromates (now prohibited due toxicity, were used in UK)

# Tryptophan is the 2^nd most important amino acid with regards to oxidation. It is rather unstable. After a some of steps, it can produce Formilkiruninie and qinurinine which is toxic.

Protein Cross-linking: Enzyme Resistant Bond in Proteins

It happened due to action of high temperatures and drying. And there is production of non-peptide bonds, highly resistant to proteolysis (unnatural covalent bonds), resulting in lower digestibility and bioavailability products.

DEHIDROALANINE is a very important product, because it is a very reactive molecule. It is a strong electrophile (lacks electron). So when you introduce into the reaction a molecule with excess of electron with the molecule that lacks an electron, you will produce a covalent bond.  A compound with excess of electron (nucleophile) would be Nitrogen, meaning that a protein can be a good electron donating (lysine). So lysine (N-Standing amine group, with excess of electrons) can react with dehydroalanine, creating an adduct and producing LISINOALANINE, it is not a peptide (not a dipeptide because there isn’t a peptide bond), it is a dimer or di amino acid, but nod a peptide, meaning that it cannot be digested (enzymes cannot break it).

Ornitine can react also with dehidroalanine and get ornithoalanine.

Cysteine can react with the sulfridril group and produce LANTIONINE (Adduct between Cysteine and dehydroalanine). So by doing this you are producing protein crosslinks, producing dimers, trimmers, polymers.

Consequences: produce non peptide bonds, so it becomes undigestable. Loosing essencial aminoacids (loose alanine). So nutritional value decreses (1. Reduce digestability, and 2. reducingessencial aminoacids). Lisinoalanine is shown to be toxic for red blood cells in animal studies.

There is another kind of protein crosslinking reaction. For example glutamin can react with lysine (amonia is lost) producing an adduct lisinoglutamate, this is an amine bond but not a peptide bond. This is produced by heat and drying.  It also can happen with asparagine. The consequences are the same.

The reaction can also happen enzymatically, transglutaminase (meat glue). It is possible to glue together pieces of meat by crosslinking proteins using transglutaminese.

By making bigger these proteins, it is possible to increase its allergenicity.

In maillard reaction we will also be confronted with protein crosslinking

Protein Racemization

Racemization is a conversion of an L amino acid into a D amino acid. Amino acids are asymmetric, so L amino acids can be deprotonated by creating a very unstable charged carbon ion (flat structure, so the proton can either come from front or back). Out of the carbon ion, alanine and cysteine (they can be kicked out) and can produce hydrogen gas and H2S, and produce an intermediated (dehydroalanine). The enzymes cannot work on the R amino acids so the proteins stay there undigested. R amino acids in very high concentrations can be toxic.

It occurs when we are heating foods, It can occur during sterilization experiments (such as boiling).  Since we are losing essential amino acids, nutritional value is reduced. Since it is on protein structure, not amino acid, the body might not recognize the protein, so will not recognize it and will have a problem digesting it, thus has an impact on bioavailability. R amino acids can also be toxic.

In cysteine rich food it can be smell (H2s), for example in milk pasteurization (dehydroalanine). This reaction is produced under high temperatures, however alkaline environments enhance this reaction (enhance formation of dehydroalanine). Soy proteins are extracted in alkaline environment, so during production of soy milk will produce some of these degradation products. So in proteins extracted in alkaline environments, there will be higher concentrations of R amino acids and Dehydroalanine.

Protein Pyrolysis

Pyrolysis is an incomplete burning reaction. It is produced due to an extensive heating. This compound are produced due to ciclization of tryptophan and glutamine resulting in production of very toxic CARBOLINES.

 Produce imidazole quinolones, which are heterocyclic amines, which are mutagenic substances, meaning that they can change the genetic material of a cell, so they can change a cell into a cancer cell. They all have mutagenic properties. They are also called the hamburger mutagens, produced during grilling of meat due to very high temperatures (more than 100°). To produce it, very high temperatures are needed, usually on surface of grilled meat. 

Protein Putrification

It is the microbial or enzymatic deterioration / degradation process of proteins on the amino acid level. It occurs as a result of microbial spoilage (eg. tuna), or fermentations (eg. camembert cheese), or in fruits and vegetables (enzymatic, pinaple)

Decarboxylation (remove co2 group) and Deamination (produce ammonia and Co2) are happening. For this we need to have a free amino acid. When we have a free amino acid we can obtain a decarboxylation or a deamination.

Deamination: is not so important because produce carboxylic acid, which are intermediate products of the amino acid metabolism, so they will be converted into energy in the body, burned in creb cycle. Not toxic, not produce taste.  

Decarboxylation: Production of CO2 and Biogenic amines (amines produced a biological way, being the enzymatic decarboxylation of a free amino acid). This process is typically linked to microbial spoilage. Biogenic amines are considered to be the most relevant in this reaction, because some of them might be toxic (histamine), while others have an impact on the sensorial properties of the food (typical ammonia like odor). Biogenic amines originate from an amino acid so they can produce ethyl amine (from ethyl amine) and short chain amines. So it can produce volatile amines which have ammonia like odors (eg. typical aroma of fermented or ripe cheese such as camembert. During the fermentation the proteins are hydrolyzed and free amino acids are produced, giving rise to the typical aroma).
So there will be sensorial effects (degradation of taste and smell) which can be desirable or undesirable, eg. Rotten fish smell, camembert cheese.  (sensorial effect, with regards to a particular smell and taste due to action of the biogenic amines).
Another important effect is the Safety; a particular amino acid (histidine) which gives rise to  a biogenic amine known as histidine, which is toxic. It is a small hormone in our body and regulates muscles responsible for respiration can be controlled by histamine. When take histamine (exogenouse histamine) from the food, due to the increase presence of this molecule in the body you will develop an intoxication (allergy like effect) by exogenously taking it. Gives asthma, skin rush, irritation vomiting, etc. typical foods are fish products, like fermented fish, fish sauce.

Other important biogenic amines that can be produced are putricine (derived from ornithine) and cadaverine (derived from lysine) which give a very unpleasant odor, like dead bodies, so they can also give very strong taste deterioration.

 Some biogenic amines are naturally occurring (eg. in grape fruit, pinaple), they are adrenaline analogs like serotonin, so they have a similar effect in our body. Antidepressants inhibit the degradation of biogenic amino acids, so people who are under medication of antidepressants are more vulnerable. Biogenic amines are also responsible for bad smell of some cheese (putrecine and cadaverine)

Proteolysis

Cut the peptide bond. Hydrolysis of the peptide bond and need water for it. Can do it:

-          Enzymatic protein hydrolysis: basis of protein digestion (peptidases or proteases like trypsin, chemotropism). The enzyme will cut the protein at a specific place. Important with respect to digestion and fermentation. Eg. soy sauce and fish sauce usually fermented, obtained from protein hydrolysis. It is more than denaturation; you disintegrate de protein to some extend. Eg. in cheese making important, camembert cheese, the interior part of cheese becomes liquid because the protein is being disintegrated after the fermentation. This is because there are microbiological enzymes present fermenting the cheese.

ü  Endogenously enzyme present: eg. Raw ham. The enzymes of the meat are slowly digesting the meat. There is no outgrowth of bacteria, it is an auto degradation.

ü  In industry food are also enzimaticaly hidrolised in reactors.

-          Chemical hydrolysis: add sodium hydroxide, not used. Hydrochloric acid hydrolysis is largely use (collagen is chemically partially hydrolyzed in production of Maggi cubes), also in production of gelatin. Proteins are chemically hydrolyzed with hydrochloric acid. This is done to enhance the flavors. Acid and alkaline also use to determine the amino acid composition of a protein. Advantage of this method is that some proteins are degraded and split off (asparage into xxx).

-          When proteins are hydrolyze we get peptides and it is important because it give flavor. But it can be further degraded and putrefaction takes action (spoilage)

Protein Denaturation and Consequence

DENATURATION - Loss of native structure

Protein denaturation denotes the loss of the native protein structure due to a change of the physiological conditions to other conditions. Protein structure can be seen on four different levels: primary (amino acid sequence), secondary (presence of alfa helices or beta sheets substructures), tertiary (three dimensionally folded protein) and quaternary structure (protein clusters, dimers etc). Apart from the primary structure these structures originate from the following interactions between amino acids: SS bounds, electrostatic interactions, H-bounding, hydrophobic interactions and Vanderwaals forces (figures could be added to explain this better). If these interactions change due to a change in the environment of the protein (e.g. pH change will induce charge change in protein and thus may influence electrostatic interactions), this may have an effect on these structural elements. As long as the primary structure of the protein does not change (= changes on amino acid level), these changes are considered as protein denaturation. Denaturation can be induced by heat, Insostatic pressure (physically disrupting the structure), pH change, addition of salts (playing with salt concentration), removal of water (e.g. sublimation during freezing), physical shear, etc. (proteins have a isoelectric character, neutral charge). The protein structure is affected by the hydrogen bonds, the more water, the morw hydrogen bonds.

 Heat denaturation though is considered to occur mostly in the food industry. Each protein is characterised by a particular denaturation time and temperature, but this may be affected by the water activity of your product. Protein could also undergo denaturation during cooled storage due to a change in hydrophobic interactions at low temperatures. Since all the above mentioned interactions are not broken at once, denaturation is a step wise process and as such can be reversible or unreversible (figures could be added explaining this).

Denaturation is important because

-           It changes drastically the functionality of proteins (good or bad).

-          Solubility may be affected (coagulation of egg white) (other examples could be given).

-          It may also affect the biological activity of proteins, such as enzymes.

-          It may have a positive impact on the digestibility of proteins, since they become more accessible for digestive proteases.

-          As no changes on amino acids are involved, no negative impact on nutritional value is expected, on the contrary bio-availability increases.

Summary of consequences: 1. Inactivate antimicrobial factors; 2. Digestibility of proteins is higher (proteases can reach the protein in a better way when it is denatured); 3. Nutritional value increases; 4. Changes on functionality which could be good or bad (activation of enzymes, gelatinization, change of physical characteristics - cooked egg). Denaturation is important because it changes drastically the functionality of proteins (good or bad). Solubitlity e.g. may be affected (coagulation of egg white) (other examples could be given). It may also affect the biological activity of proteins, such as enzymes. It may have a positive impact on the digestibility of proteins, sine they become more accessible for digestive proteases. As no changes on amino acids are involved, no negative impact on nutritional value is expected, on the contrary even since bio-availability increases. 

Protein Interaction

Di sulphide bond: Covalent side chain cross link. Interaction between 2 cysteine recidues. They create a lot of rigidity. When we have 2 cysteines we create a disulphide bridge. The more cysteine the more disulphite bonds, the more bridges and the more rigid. This interactions are very strong. 

Ionic interactions: Lisine + aspartic acid (positive and negative attract each other). Attraction between positively and negatively charged. It is the second strongest.

Hydrogen bonds: responsible for peptide bond. It has a strong interaction. Interaction between hidrogent that is covalently bond to an electro-negative atom.

Hydrophobic interaction: not very strong, but exist. Fat lovers

Van der walse: at atomic level, they are weak but they can be at high numbers. They are the weakest.

Structure of Protein

Primary structure: Each protein has a unique sequence. Primary structure is like a finger print, each one has a unique structure. Peptide bond (hydrogen bonds)

Secondary structure: Random coil structure (beta casein, due to high amount of beta proline); Alpha helix structure (the more frecuent, the residues are oriented towards the exterior part of the helix); beta sheet structure (More stable).

Tertiary structure: protein folds again and forms a 3d structure, due to the action of the interactions

Quaternary structure: Oligomeric structure, polypeptide chain, combined proteins. Due to all  different interactions we can create the complex structure of the protein. 

Stability of Food: Water Activity and Glass Transition Theory

Stability is inversely related to reactivity. The more reactive the molecule, the more unstable it is. It is determined by the change of 2 molecules encountering each other (1. chance of encounter – diffusion controlled). When the mobility of the molecule increases also the chance of encountering will increase. When the molecules are close to each other, they need to get together in a particular place (2. Chance of collision – collision frequency), finally it needs to cross an energy barrier to reach another state (3. activation energy). When the barrier is low, the reactivity of a system is controlled by the diffusion coefficient.  When the reaction depends only on the molecular mobility, the glass transition theory will be good to explain if the reactions occur or not, because molecular mobility is part of the glass transition theory. Eg.proton exchange, radical combination reaction.

Diffusion coefficient depends on viscosity, mobility and the temperature dependency of the viscosity can be described via Argeniuos theory or the WLF. Viscosity included in equations is the viscosity that molecule feels locally, and sometimes it will deviate from the viscosity that we are able to measure. Local relaxation time is totally different than what we measure in practice.

Theoretically the formulas exist, but in practice the glass transition theory is far from ideal.  Means that what we predict on theory is far from the practice. Potential explanation is that the viscosity included in the ecuation is the viscosity that the molecule feels localy, so it will deviate from what we are able to measure macroscopically. Eg. pudin (3d network of starch molecules), locally the molecules are entangled between polymer chains so the mobility is more restricted to what it is predicted on basis on macroscopic  viscosity. So local viscosity and mobility and relaxation time is different to what we measure in practice.

-          Impact of water activity on the microbiological development (below water activity of 0,6 foods are typically microbiologically stable), however the glass transition theory cannot predict the microbiological stability of foods. Bacteria are less resistant than yeast and moulds.

-          Enzymatic reactions, at high water activity, more enzymatic reaction. When we reduce the mobility, there will be less enzymatic reactions. In monolayer water content there are almost zero enzymatic reaction (no solvent capabilities).

-          Hydrolysis needs water as reactant. At higher water content more hydrolysis.

-          Non enzymatic follows similar trend. But sometimes at very high water activity it is reduced. Because for this kind of reaction you will need the reducing sugars to get in contact with the amin group, and if there is too much water. They will be diluted in it. So the reaction won’t happen due to a highly diluted solution.

-          Strange with lipid oxidation. At monolayer water content we don’t expect much chemistry happening (theoriticaly), but in lipid oxidation it continuous. It is logic because you don’t need water for lipid oxidation (only a oxygen and lipid). Another explanation is that the pro-oxidants (metal ions, cupper and ferrum) become available. Also the matrix is less accessible when it is dry than when it is moisture (dry meat), so penetrability of oxygen is higher at higher moisture content. Sometimes it is lower, or sometimes stabilizes; it depends from matrix to matrix.

 We also reach a minimum where there is an increase of lipid oxidation. An explanation could be that the hydartation layer around the food provokes the lipid oxidation, however when it is removed it will like to join  the fat even faster. Another explanation is that solubility in water is lower than the solubility in oil. Solubility of oxygen in water is 8 mg/lt particle. Solubility of oxygen in oil is 40 mg/lt. (40 times more). When food has a hydration layer, the molecule will have a difficulty to penetrate it. So basically the layer of water prevents the oxygen to enter the particle.

This is a theoretical scheme! In practice most food behaves more or less like here. But many foods don’t behave like this, so it is just a starting point. There are many deviations in practice:

-          Oat meal at 25°. Doesn’t correspond to the general curve of oxidation reaction.

-          For penuts (lipid oxidation deviation)

-          For musley (lipid oxidation deviation)

-          For enzymatic reactions: Low molecular weight matrix we see that the enzymatic reaction will start faster tha the high molecular weight. The high molecular weight can retain more water.

-          Non enzymatic browning: here it is a disaster. In a glucose fructose, glycine mixture.

Summarizing:

-          Microbiological stability is not supported by glass transition.

-          Physical stability can be explained using the glass transition theory (explain better the water uptake of powder). Eg. When amorphous lactose is a glass it will not take up water, when it is in rubber state it will take up water. Lactose hydride crystals are less hygroscopic, no tendency to take up water.

-          Chemical stability, the theory rarely supports it.

Conclusion:

Water activity tells us about interaction of water with the food matrix. In contrast

The Glass transition theory tells us something about the non-aqueous fraction of the food, and how this state is behaving. There is a link with availability of water. Also a link with chemical stability (but not always, some cases yes, other cases no). This theory has a lot of merits with the physical stability

Both theories are complementary to describe the impact of water on foods, but we don’t know enough to understand everything. 

Glass Transition Theory

Crystalline matter can originate from melt in which the first phase transitions take place and are characterized by an exchange of latent heat.

Amorphous substances can also crystalize from melt or from a solution. During a fast cooling they will be converted to a metastable glass state. Glass state is in fact a liquid state of which the viscosity is so high that the material acts as a solid without being a crystal. This vitification results in a reduction of transitional mobility of the components, which is the core element to make the link with the stability of foods. Thus the processes that are controlled by diffusion will be inhibited as a result of the drastic loss of molecular mobility.  However even in the glass state, molecular mobility is still occurring (especially on atomic level: vibrations, rotations). This so called relaxations will result in additional changes in a particular state.

Depending of the way of cooling various types of glass can be formed. These phase transitions are associated with temperatue changes. The glass transition does not occur at one particular temperature , but occurs over a temperature range. Still binary water-solid mixtures are being characterized by a particular glass temperature.

The glass state is induced by the process of vitrification and it can only happen in a particular temperature. It depends upon the circumstances (cooling, agitation, etc), this will have an impact on the moment when the glass transition will take place. Glass transition temperature is the temperature at which the food becomes glass.

If the system is given sufficient time, allowing a maximal amount of ice being formed, the remaining aquous solution will be able to reach the highes possible concentration of dissolved substances.

Change from liquid to crystal has a first order phase transition, meaning that there is a change in the enthalpy, but it does not occur due to change on temperature.eg. From liquid to ice, from liquid to vapor.

Second order phase transition, is when you have a change in enthalpy together with a change in temperature, these are metastable conditions.

The transition of the complementary  liquid state to th glass state is a transition zone in which the food acts as a rubber, hence indicates the rubber state. The rubber state is not completly liquid, nor completely solid, but an intermediate between both thus shows particular mobility. Both the rubber state and the glass state are essential and characteristic elements of the glass transition theory.

Summarizing: a liquid solution of a particular substance can be cooled down (o concentrated) until the product starts to act leathery, rubbery material which upon further cooling (or concentration) will be converted into glass, in which the molecular mobility is restricted significantly. Both states are NON EQUILIBRIUM STATES and consequently also time dependent. Glass and rubber transitions are also dependent on the speed at which water is removed from the system, by evaporation (drying, extrusion) or crystallization (freezing.

The glass and the rubber state describe the state of the non-aqueous fraction of the food. The role of the water in a food according to the glass transition theory becomes clear if the parallel with polymer science is made. Water will act as plasticizer and will reduce the interaction between the non-aqueous food components which will result in lower glass transition temperature. This process is called plasticization. The impact of moisture content on the properties of the food as function of the glass transition theory is shown in the state diagram.

What happens sequentially?

According to polymer science, you can have following phases: solution, eg. Sucrose solution, when in concentrate the solution I can have crystalline sucrose (equilibrium condition), when I have crystalline solution I can heat it and melt it (equilibrium condition). However rubber and glass transition are not stable, non-equilibrium conditions, glass transition is time dependent phenomenon, it depends upon circumstances of the food (time dependent, and also depends upon circumstances). When we have a solution we can concentrate it by evaporating the water and a particular point we expect the sucrose start to crystalize, when we remove the water very gently (evaporation) we will not obtain crystals, we will obtain over saturated solution. When you put it on fridge you will obtain a crystal (solid as a rock). What we did is turned the solution into a vitrified substance, into a vitrified glass. So by concentrating and by cooling you can turn it into a glass (vitrified).

The rubber is the intermediate state between the liquid solution and the vitrified state. There are a lot of foods that are in rubber state (dried meat), semi liquid sate.

State diagram:

As the glass and rubber state are non-equilibrium condition diagrams, the state diagram describes the properties of food as function of the moisture content.  The state diagram is a kind of map on which the changes in phase behavior of a particular substance is given as function of temperature and moisture content at a constant pressure.

From the state diagram it can be concluded that the glass transition depends largely upon the solute concentration. Because of its plasticizing effect the water will reduce largely the glass transition temperature. The impact is more intense at low moisture content.  The glass transition temperature of moisture rich foods are very low and typically lower than the freezing temperatures used in industry.  the glass transition temperature of binary mixtures can be estimated based on the empirical Gordon Taylor equation ( but it has some problems so in the literature it can be found various temperatures for the same food).

The glass transition temperature is also dependent upon the type of molecule which is dissolved in the water. Aqueous mixtures of low molecular weight sugars are characterized by a much lower glass transition temperature of aqueous mixtures of poly-saccharides as similar moisture content. Foods containing typically complex bio-polymers are characterized by higher glass transition temperatures, which are reached during normal freezing temperatures used in industry (eg: bread). Foods richer in low molecular weight compounds can be in the glass state upon concentration or drying (milk powder). Moreover there is a clear relationship between the polymerization degree of sugars and the glass transition temperature. It is clear that such a behavior can be related to the number of interactions ad the intensity of the interactions between water and the solutes. Apart from water also glycerol can act as plasticizer.

Factors determining Tg

• Moisture content

• Temperature

• Type of solute

Impact of temperature and moisture content on molecular mobility

The relaxation time (level of molecular mobility) is directly proportional to the viscosity of the aquous phase of the food. When viscosity is low the relaxation time will also be low. They are also dependent on temperature (according to Arrhenius kinetics).  

Arrhenius kinetics is not valid in rubber state (relation between temperature and molecular mobility is not valid here). Impact of temperature is much higher here. There is an Inverse relationship between relaxation time and molecular mobility.

When food is in rubber state, small variations in temperature will have a big variation on the stability of food when they are in the rubber state. When increase temperature the relaxation time is smaller, thus molecular mobility becomes lower. The course you get depends on the measuring technique. The temperature dependency of the relaxation time is extremely big in rubber state, much bigger than in glass state.

Moisture content and molecular mobility

Water is a plasticizer so it will increase molecular mobility thus reduces relaxation time. Also small variations on water content will have drastic variations on relaxation time, thus on stability of food.  Small variations on water content will have big impact on water activity.