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)

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.

Water Sorption Isotherm

The sorption isotherm describes the water activity as function of the water content expressed on a dry matter basis. The sorption isotherm is a curve that can be determined empirically and can be predicted by the Law of Rault (however it is not precise). It gives us an idea about interactions between water and other food constituents; it also gives us information about the availability of the water as function of the water content, therefore different zones in the isotherms can also be distinguished (zone1, zone 2, zone3). If the isotherms are constructed under different temperatures, additional information about the heat of evaporation can be found (important in the design of drying equipment)

BET (brunour emet teller )Isotherm: By fitting experimental data to the empirical equation, the monolayer (potential) water content can be derived. It is a very interesting parameter, because when the moisture content equals to the monolayer water, the food reaches the highest stability. It is an interesting parameter to know, because when drying food it is not recommended to dry further than monolayer water. BET is only useful in the lower part of sorption isotherm (below value of 0.4).

GAB isotherm: it is more complex, and cannot be linearized. It needs to be solved withnonlinear regression equation with statistical software. Can be used over the whole water activity range. It is a more powerful equation that BET.

There are 3 different kinds of sorption isotherms (linking water activity and water content):

ü  Type I: typical for anticaking agents (eg. Sodium silicate). These components have a large moisture adsorption capacity. When binding sites are occupied, no water uptake is possible due to which reaches a plateau in moisture content.

GRAFICO

ü  Type II: Has an S shape, the shape is a result of several interaction forces between water and  food (contains bound water- constitutional, vicinal & molayer). Typical of polymeric of polymeric food ingredients such as proteins and polysaccharides. Foods with amorphous low molecular weight will show such an hygroscopic behaviour. Such products are vulnerable for caking at too high relative humidity. It has 2 inclination points, the 1^st one responds to the addition of multilayer water causing as well to the filling of pores and capillaries,  and the 2^nd inclination responds to further swelling and dissolution of some of its components.

ü  Type III: typical of foods rich in cristaline low molecular weight components like sugar and salt. Calles J shaped sorption isotherm. Moisture uptake is very restricted until the low molecular weight components start to dissolve in the absorved water (deliquescent point).

GRAFICO

We can also  distinguish 3 zones (link with the kinds of water) in the sorption isotherm, this zones can be related to the kinds of water:  Zone III: significant change in moisture content, results in a restricted change in a restricted change of water activity; Zone II: small change of moisture content, results in a big change in water activity; Zone I: is determined by the bound water in the food: constitutional, vicinal and monolayer.

GRAPH OF ZONES

Temperature dependency

Increasing the temperature will increase the water activity and availability of water also increase. In a SI with different temperatures (eg. 30, 45, 50° SI f potatoes) you see that at fixed moisture content, there will be a shift of the sorption isotherm to the right. So it has a drastic impact on the water activity. Meaning that the increase in temperature (in aqueous food) will have a dual effect on the stability of the food because: 1.The solvent will become more available and 2. There is a kinetic factor, browman motion, so reactivity of the molecules increase.  SI at higher temperature will start to cross, it is because at higher temperature the solubility of the components increase. From the temperature dependency and from clausious claperon equation, at particular moisture content, we can calculate the heat of evaporation, energy required to remove water out of the food (at that particular moisture content). The lower the moisture content the steeper the line, because at the high water conteng (40g /100g of DM), the amunt of multilayer water in the food on total amount is very high and this is the weakest part of the chai. So the heat of evaporation will not be that high. So the slope will not be high. So the slope will be almost as free water. When we remove water from the matrix, the water will be more and more bound resulting in a bigger curve. The curves are important with regards to drying foods in industry. it is needed to calculate how much heat we need. The heat required is not equal to the normal heat of free water, because as the water evaporates the heat of evaporation also increases (due to strong interaction of bond water).

So in general the heat of sorption as function of the moisture content: al low water content, the heat of sorption will be similar to the normal water, and when you lower the water content you will see it increases. Thus we will have much more water.

Consequences: dual effect.

1.      The higher the temperature the faster the browman motion. The molecules walk fatser at higher temperatures and when they walk faster there can be more reactions.

2.      The water becomes more available, so the water activity increases. Thus solvent capability of the water increase. This will also favor the reactivity of the food.

GRAPH

In view of the temperature dependency of Aw, the sorption isotherms are temperature dependent:

-          Generally at higher temperatures and similar water contents, the water becomes more available, the water is more available, but it is also important to consider that at higher water activities, an increase in temperatures will cause an improvement in the soluvility of the substances, which results in a decrease I the water activity. Hence, at higher water activities, SI may cross each other.

-          Satability of moisture content: the stability of a food is highest when its mpisture content equals the monolayer water content.

Hysteresis

It is the relationship between water content and water activity depends upon the fact whether you dry the food or make the food humid, so there is a relationship between desorption and adsorption. If you dry your food, it will follow the solid line (by experimentally doing the sorption), when you put it in a wet environment again the same dried products) it will follow the dotted line. Meaning that the SI at the same moisture content will show higher water activity. It happens because when you dry the food and remove the water out of the capiaries, it is very difficult to put the water back into the capillaries.  Therefore at the same water content it will reach easier the monolayer water content, so the water will be more available. Another explanation is related to the impact of water content on protein structure, because water is important to retain the protein structure (by freezing you but bound water into a cristal grid). It means that if temperature is low enough we are able to take bound water, this means that we can change the hydratation state of a protein. Due to this proteins can denature (they change structure and unfold). So the water biding capacities after freezing change (water binding capacities become lower) because we are unfolding the protein (by denaturation) and the interior part of the molecule is more hydrophobic than the exterior part. Consequence: the water activity also depends on the history of the product.

GRAPH HISTERESIS

Practical consequences:

·         We can determine the amount of water in foods

·         Wc/Wa interaction

·         Availability of water as function of water content and as function of temperature.

·         Heat of evaporation

·         Derive monolayer water and stability of food.  

Criticism to concept of aw:

·          Thermodynamically – non equilibrium conditions. Theoretically there are problems because it is derived from thermodynamics. In thermodynamics we suppose that the foods are in equilibrium but foods are never in equilibrium. From thermodynamics, desorption and sorption should be the same. So the concept of water activity is still not solved. 

·          Hysteresis phenomenon- cannot explain it thermodynamically.

·          ‘strange’ observations. Eg. Amorphous lactose: the moisture content drops as function of time. It is not explainable. Also at higher water activity it collapse and becomes very dense, there is crystallization after 0.45 water activity. 

Selected effects on microbiological stability

Water Activity

Foods can contain different kinds of water, depending on the water content. Water content is a poor predictor of food stability. The stability of foos will be determined by the extent to which water is available as a solvent, as reactant or to support microbial development. Despite some foods have a low moisture content, the availability of water can be high because of poor interaction with other food matrix components. Hence it can act as free water.

Water activity is a way to describe in a quantitative manner the interaction between food and water, giving an idea of how strongly the water is bound to the food, thus indicating the availability of water in food.

It is the chemichal potential of the water in foods over the chemical potential of free water at the same temperature. It is practically equal to the partial vapour pressure just above the food matrix in a very small layer of air which is in full equilibrium with the food. (formula 4 de water activity)

Its value can vary between 0 and 1 (1 represents free water and 0 constitutional water).

Due to the fact that temperature has an impact on the mobility of the molecules, water becomes more available at higher temperatures because.

From the temperature dependence of water activity at constant water content, the heat of sorption can be calculated.

In order to measure stability in foods we need to identify the relationship between water content and water activity ( Eg. If water activity and water content are low, it means the water is bound, if water is bound there is high stability) this relationship is temperature dependent; therefore it can only be described under isotherm conditions.

Factors affecting aw

·         moisture content

·         temperature

·         ice/water

·         kind of compounds

·         history of the food

·         sorption isotherm

Kinds of Water

Foods contain different kinds of water. It’s important to note that there is a difference between free water and bound water:

1. Free water behaves physic – chemically as pure water. It is bulk water (about 96%).

2. Bound water can be further differentiated depending on the strength of its interaction with the food matrix

a. Constitutional water: it is part of the molecular structure of non water molecules in the food. It is very strongly bound to the food matrix, so strongly bound (to charged particles) that it is not available water. Typically we see these constitutional water in macromolecules like biopolimers, polisacharides and proteins. No solvent capacities. Very large evaporation enthalpy.

b. Vicinal water: mostly bound through hydrogen bonds and polar non charged molecules. It can be present (entraped) in the capillaries of the food matrix. It is able to hydrate complex food molecules forming monomolecular layer, hence de name Monolayer water. Monolayer water: it is formed by one water molecule layer around the food matrix (very strongly bound, strong interaction with food matrix).   These types of water have very reduced availability. No solvent capacities. Large evaporation enthalpy.

c. Multilayer:  It is an expanding hydration layer formed of several water layers  constituted through the attraction  between water molecules via hydrogen bonds. It is a transition zone towards free water and it can start behaving as a solvent, despite its lower availability.   Moderate to little evaporation enthalpy

Water Molecule

Water is the chemical substance with chemical formula H
2O: one molecule of water has two hydrogen atoms covalently bonded to a single oxygen atom. Water appears in nature in all three common states of matter (solid, liquid, and gas) and may take many different forms on Earth: water vapor and clouds in the sky, seawater in the oceans,icebergs in the polar oceans, glaciers in the mountains, fresh and salt water lakes, rivers, and aquifers in the ground.

Water molecules strongly interact with each other due to the hydrogen bonds in H2O. It creates a permanent dipole (with positive and negative charges, localization of charges, O negative and H positive). 1 water molecule can produce 4 hydrogen bonds, so 1 water molecule will attract 4 other molecules (very strong bond). When we want to remove these H bonds, by breaking the bond, it requires 11-24 KJ. The other water molecules are situated in a 3 D network, so we have 4 molecules forming a hydrogen bond, and the 3D orientation is also unique. So the bonds are formed in a 3D grid (not plane), this explains such strange properties. Water dissociates into protons and hydroxyl ions. Hydrogens and protons can be bound to oxygen. There is a constant exchange of protons over the water molecule. It is an extremely dynamic system, with the constant exchange of protons over the water molecule. The flexibility and mobility of the system is very high. This is why despite the bond is very strong its viscosity is low. In frozen foods, bigger ice crystals will attract smaller ones and they will grow due to this dynamic, due to the flexibility and mobility of protons, that build into the crystal grid of ice. Dissociation constant at 25° is 20 to the power of -14 (important concept). In the case of water, it dissociates in equilibrium reaction. The equilibrium is characterized by equilibrium constant. Dissociation constant is temperature dependent, when increase the temperature the dissociation constant is higher. A neutral solution at 100° has a PH of 6 (not 7). At higher temperatures, neutrality of ph is 7, so you favor the dissociation reaction at higher temperatures.

Water

Water is a transparent fluid which forms the world's streams, lakes, oceans and rain, and is the major constituent of the fluids of living things.
Water is one of the most important components of foodstuff on a quantitative basis. It is important in foods because it is works as a bulk ingredient (eg. it is important economically but can give rise to problems. Foods sold per kilogram so when the water content decrease due to low water binding, also decreases the industry loses money. So some ties it is added chemicals to increase the water binding capacities. Also some times the products have too much water and this is fraud like in frozen shrimp, or milk diluted in water), as a solvent and because of the water intake.