Growth of Micro-Organisms in Food | Microbiology

In this article we will discuss about the extrinsic as well as intrinsic factors affecting the growth of micro-organisms in food.

Extrinsic Factors:

1. Relative Humidity:

Relative humidity and water activity are interrelated, thus relative humidity is essentially a measure of the water activity of the gas phase. When food commodities having a low water activity are stored in an atmosphere of high relative humidity water will transfer from the gas phase to the food.

It may take a very long time for the bulk of the commodity to increase in water activity, but condensation may occur on surfaces giving rise to localized regions of high water activity. It is in such regions that propagules which have remained viable, but unable to grow, may now germinate and grow.

Once micro­organisms have started to grow and become physiologically active they usually produce water as an end product of respiration. Thus they increase the water activity of their own immediate environment so that eventually micro-organisms requiring a high a_w are able to grow and spoil a food which was initially considered to be microbiologically stable.

Such a situation can occur in grain silos or in tanks in which concentrates and syrups are stored. Another problem in large-scale storage units such as grain silos occurs because the relative humidity of air is very sensitive to temperature.

If one side of a silo heats up during the day due to exposure to the sun then the relative humidity on that side is reduced and there is a net migration of water molecules from the cooler side to re-equilibrate the relative humidity.

When that same side cools down again the relative humidity increases and, although water molecules migrate back again, the temporary increase in relative humidity may be sufficient to cause local condensation onto the grain with a localized increase in a_w sufficient to allow germination of fungal spores and subsequent spoilage of the grain.

This type of phenomenon can often account for localized caking of grain which had apparently been stored at a ‘safe’ water content.

The storage of fresh fruit and vegetables requires very careful control of relative humidity. If it is too low then many vegetables will lose water and become flaccid. If it is too high then condensation may occur and microbial spoilage may be initiated.

2. Temperature:

Microbial growth can occur over a temperature range from about — 8 °C up to 100^oC at atmospheric pressure. The most important requirement is that water should be present in the liquid state and thus available to support growth. No single organism is capable of growth over the whole of this range; bacteria are normally limited to a temperature span of around 35 °C and moulds rather less, about 30 °C.

A graph showing the variation of growth rate with temperature illustrates several important features of this relationship (Figure 3.13). Firstly, each organism exhibits a minimum, optimum and maximum temperature at which growth can occur.

These are known as cardinal temperatures and are, to a large extent, characteristic of an organism, although they are influenced by other environmental factors such as nutrient availability, pH and a_w. Micro-organisms can be classified into several physiological groups based on their cardinal temperatures.

This is a useful, if rather arbitrary, convention, since the distribution of micro-organisms through the growth temperature range is continuous. To take account of this and the effect of other factors, it is more appropriate to define cardinal temperatures as ranges rather than single values (Table 3.11).

In food microbiology mesophilic and psychrotrophic organisms are generally of greatest importance. Mesophiles, with temperature optima around 37 °C, are frequently of human or animal origin and include many of the more common foodborne pathogens such as Salmonella, Staphylococcus aureus and Clostridium perfringens.

As a rule mesophiles grow more quickly at their optima than psychrotrophs and so spoilage of perishable products stored in the mesophilic growth range is more rapid than spoilage under chill conditions. Because of the different groups of organisms involved, it can also be different in character.

Among the organisms capable of growth at low temperatures, two groups can be distinguished: the true or strict psychrophiles (‘cold-loving’) have optima of 12- 15°C and will not grow above about 20 °C. As a result of this sensitivity to quite moderate temperatures, psychrophiles are largely confined to polar regions and the marine environment.

Psychrotrophs or facultative psychrophiles will grow down to the same low temperatures as strict psychrophiles but have higher optimum and maximum growth temperatures. This tolerance of a wider range of temperature means that psychrotrophs are found in a more diverse range of habitats and consequently are of greater importance in the spoilage of chilled foods.

Thermophiles are generally of far less importance in food microbiology, although thermophilic spore formers such as certain Bacillus and Clostridium species do pose problems in a restricted number of situations.

Another feature evident from Figure 3.13 is that the curve is asymmetric – growth declines more rapidly above the optimum temperature than below it. As the temperature is decreased from the optimum the growth rate slows, partly as a result of the slowing of enzymic reactions within the cell.

If this were the complete explanation however, then the change in growth rate with temperature below the optimum might be expected to follow the Arrhenius Law which describes the relationship between the rate of a chemical reaction and the temperature.

The fact that this is not observed in practice is, on reflection, hardly surprising since microbial growth results from the activity of a network of interacting and interrelating reactions and represents a far higher order of complexity than simple individual reactions.

A most important contribution to the slowing and eventual cessation of microbial growth at low temperatures is now considered to be changes in membrane structure that affect the uptake and supply of nutrients to enzyme systems within the cell.

It has been shown that many micro-organisms respond to growth at lower temperatures by increasing the amount of unsaturated fatty acids in their membrane lipids and that psychrotrophs generally have higher levels of unsaturated fatty acids than mesophiles.

Increasing the degree of unsaturation in a fatty acid decreases its melting point so that membranes containing higher levels of unsaturated fatty acid will remain fluid and hence functional at lower temperatures.

As the temperature increases above the optimum, the growth rate declines much more sharply as a result of the irreversible denaturation of proteins and the thermal breakdown of the cell’s plasma membrane. At temperatures above the maximum for growth, these changes are sufficient to kill the organism – the rate at which this occurs increasing with increasing temperature.

3. Gaseous Atmosphere:

Oxygen comprises 21% of the earth’s atmosphere and is the most important gas in contact with food under normal circumstances. Its presence and its influence on redox potential are important determinants of the microbial associations that develop and their rate of growth. Since under redox potential, this will be confined to the microbiological effects of other gases commonly encountered in food processing.

The inhibitory effect of carbon dioxide (CO₂) on microbial growth is applied in modified-atmosphere packing of food and is an advantageous consequence of its use at elevated pressures (hyperbaric) in carbonated mineral waters and soft drinks.

Carbon dioxide is not uniform in its effect on micro-organisms. Moulds and oxidative Gram-negative bacteria are most sensitive and the Gram positive bacteria, particularly the lactobacilli, tend to be most resistant. Some yeasts such as Brettanomyces spp. also show considerable -tolerance of high CO₂ levels and dominate the spoilage microflora of carbonated beverages.

Growth inhibition is usually greater under aerobic conditions than anaerobic and the inhibitory effect increases with decrease of temperature, presumably due to the increased solubility of CO₂ at lower temperatures. Some micro-organisms are killed by prolonged exposure to CO₂ but usually its effect is bacteriostatic.

The mechanism of CO₂ inhibition is a combination of several processes whose precise individual contributions are yet to be determined. One factor often identified is the effect of CO₂ on pH. Carbon dioxide dissolves in water to produce carbonic acid which partially dissociates into bicarbonate anions and protons.

Carbonic acid is a weak dibasic acid (pK_a 6.37 and 10.25); in an un-buffered solution it can produce an appreciable drop in pH, distilled water in equilibrium with the CO₂ in the normal atmosphere will have a pH of about 5, but the effect will be less pronounced in buffered food media so that equilibration of milk with 1 atmosphere pCO₂ decreased the pH from 6.6 to 6.0.

Probably of more importance than its effect on the growth menstruum is the ability of CO₂ to act in the same way as weak organic acids, penetrating the plasma membrane and acidifying the cell’s interior.

Other contributory factors are thought to include changes in the physical properties of the plasma membrane adversely affecting solute transport; inhibition of key enzymes, particularly those involving carboxylation/decarboxylation reac­tions in which CO₂ is a reactant; and reaction with protein amino groups causing changes in their properties and activity.

Intrinsic Factors:

1. Nutrient Content:

Like us, micro-organisms can use foods as a source of nutrients and energy. From them, they derive the chemical elements that constitute microbial biomass, those molecules essential for growth that the organism cannot synthesize, and a substrate that can be used as an energy source.

The widespread use of food products such as meat digests (peptone and tryptone), meat infusions, tomato juice, malt extract, sugar and starch in microbiological media bears eloquent testimony to their suitability for this purpose.

The inability of an organism to utilize a major component of a food material will limit its growth and put it at a competitive disadvantage compared with those that can. Thus, the ability to synthesize amylolytic (starch degrading) enzymes will favour the growth of an organism on cereals and other farinaceous products.

The addition of fruits containing sucrose and other sugars to yoghurt increases the range of carbohydrates ‘available and allows the development of a more diverse spoilage microflora of yeasts.

The concentration of key nutrients can, to some extent, determine the rate of microbial growth. The relationship between the two, known as the Monod equation, is mathematically identical to the Michaelis-Menten equation of enzyme kinetics, reflecting the dependence of microbial growth on rate-limiting enzyme reactions:

where µ is the specific growth rate; µ_m the maximum specific growth rate; S the concentration of limiting nutrient; and K_s the saturation constant.

When S>>K_s, a micro-organism will grow at a rate approaching its maximum, but as S falls to values approaching K_s, so too will the growth rate. Values for K_s have been measured experimentally for a range of organisms and nutrients; generally they are extremely low, of the order of 10^-5M for carbon and energy sources, suggesting that in most cases, nutrient scarcity is unlikely to be rate-limiting.

Exceptions occur in some foods, particularly highly structured ones where local microenvironments may be deficient in essential nutrients, or where nutrient limitation is used as a defence against microbial infection, for example the white of the hen’s egg.

2. pH and Buffering Capacity:

As measured with the glass electrode, pH is equal to the negative logarithm of the hydrogen ion activity. Activity is proportional to concentration and the proportion­ality constant, the activity coefficient, approaches unity as the solution becomes more dilute. Thus:

where (a_H) is the hydrogen ion activity and [H⁺] the hydrogen ion concentration.

For aqueous solutions, pH 7 corresponds to neutrality (since [H⁺][OH^–] = 10^-14 for water), pH values below 7 are acidic and those above 7 indicate an alkaline environment. It is worth remembering that since pH is a logarithmic scale differences in pH of 1, 2 and 3 units correspond to 10-, 100- and 1000-fold differences in the hydrogen ion concentration.

The acidity or alkalinity of an environment has a profound effect on the activity and stability of macromolecules such as enzymes, so it is not surprising that the growth and metabolism of micro-organisms are influenced by pH. Plotting microbial growth rate against pH produces an approximately symmetrical bell- shaped curve spanning 2-5 pH units, with a maximum rate exhibited over a range of 1-2 units.

In general, bacteria grow fastest in the pH range 6.0-8.0, yeasts 4.5-6.0 and filamentous fungi 3.5-4.0. As with all generalizations there are exceptions, particularly among those bacteria that produce quantities of acids as a result of their energy-yielding metabolism. Examples important in food microbiology are the lactobacilli and acetic acid bacteria with optima usually between pH 5.0 and 6.0.

Most foods are at least slightly acidic, since materials with an alkaline pH generally have a rather unpleasant taste (Table 3.2). Egg white, where the pH increases to around 9.2 as CO₂ is lost from the egg after laying, is a commonplace exception to this.

A somewhat more esoteric example, which many would take as convincing evidence of the inedibility of alkaline foods, is fermented shark, produced in Greenland, which has a pH of 10-12.

The acidity of a product can have important implications for its microbial ecology, and the rate and character of its spoilage. For example, plant products classed as vegetables generally have a moderately acid pH and soft-rot producing bacteria such as Erwinia carotovora and pseudomonads play a significant role in their spoilage.

In fruits, however, a lower pH prevents bacterial growth and spoilage is dominated by yeasts and moulds. As a rule, fish spoil more rapidly than meat under chill conditions. The pH of post-rigor mammalian muscle, around 5.6, is lower than that of fish (6.2-6.5) and this contributes to the longer storage life of meat.

The pH-sensitive genus Shewanella (formerly Alteromonas) plays a significant role in fish spoilage but has not been reported in normal meat (pH<6.0). Those fish that have a naturally low pH such as halibut (pH ≈ 5.6) have better keeping qualities than other fish.

The ability of low pH to restrict microbial growth has been deliberately employed since the earliest times in the preservation of foods with acetic and lactic acids. With the exception of those soft drinks that contain phosphoric acid, most foods owe their acidity to the presence of weak organic acids.

These do not dissociate completely into protons and conjugate base in solution but establish an equilibrium:

Equation (3.13) is known as the Henderson-Hasselbach equation and describes the relationship between the pH of a solution, the strength of the acid present and its degree of dissociation. When the pH is equal to an acid’s pK_a then half of the acid present will be un-dissociated.

If the pH is increased then dissociation of the acid will increase as well, so that when pH = pK_a+1 there will be 10 times as much dissociated acid as un-dissociated. Similarly as the pH is decreased below the pK_a the proportion of un-dissociated acid increases.

This partial dissociation of weak acids, such as acetic acid, plays an important part in their ability to inhibit microbial growth. It is well established that, although addition of strong acids has a more profound effect on pH pro rata, they are less inhibitory than weak lipophilic acids at the same pH.

This is because microbial inhibition by weak acids is not solely due to the creation of a high extracellular proton concentration, but is also directly related to the concentration of un-dissociated acid.

Many essential cell functions such as ATP synthesis in bacteria, active transport of nutrients and cytoplasmic regulation occur at the cell membrane and are dependent on potential energy stored in the membrane in the form of a proton motive force. This force is an electrochemical potential produced by the active translocation of protons from the cell interior to the external environment.

Unlike protons and other charged molecules, un-dissociated lipophilic acid molecules can pass freely through the membrane; in doing so they pass from an external environment of low pH where the equilibrium favours the un-dissociated molecule to the high pH of the cytoplasm (around 7.5 in neutrophils).

At this higher pH, the equilibrium shifts in favour of the dissociated molecule, so the acid ionizes producing protons which will tend to acidify the cytoplasm and break down the pH component of the proton motive force.

The cell will try to maintain its internal pH by expulsion of the protons leaking in but this will slow growth as it diverts energy from growth-related functions. If the external pH is sufficiently low and the extracellular concentration of acid high, the burden on the cell becomes too great, the cytoplasmic pH drops to a level where growth is no longer possible and the cell eventually dies (Figure 3.2).

3. Redox Potential, E_h:

An oxidation-reduction (redox) reaction occurs as the result of a transfer of electrons between atoms or molecules. In the equation below, this is represented in its most general form to include the many redox reactions which also involve protons and have the overall effect of transferring hydrogen atoms.

where n is the number of electrons, e, transferred.

In living cells an ordered sequence of both electron and hydrogen transfer reactions is an essential feature of the electron transport chain and energy generation by oxidative phosphorylation.

The tendency of a medium to accept or donate electrons, to oxidize or reduce, is termed its redox potential (E_h) and is measured against an external reference by an inert metal electrode, usually platinum, immersed in the medium. If the balance of the various redox couples present favours the oxidized state then there will be a tendency to accept electrons from the electrode creating a positive potential which signifies an oxidizing environment.

If the balance is reversed, the sample will tend to donate electrons to the electrode which wall then register a negative potential – a reducing environment. The redox potential we measure in a food is the result of several factors summarized in Table 3.3.

The tendency of an atom or molecule to accept or donate electrons is expressed as its standard redox potential, E₀‘. A large positive E₀‘ indicates that the oxidized species of the couple is a strong oxidizing agent and the reduced form only weakly reducing.

A large negative E₀‘ indicates the reverse. Some redox couples typically encountered in food materials and their E₀’ values are shown in Table 3.4. The measured E_h will also be influenced by the relative proportions of oxidized and reduced species present.

This relationship for a single couple is expressed by the Nernst equation:

where E_h and E₀’ are both measured at pH 7; R is the gas constant; T, the absolute temperature; n, the number of electrons transferred in the process and F is the Faraday constant.

Thus, if there is a preponderance of the oxidant over its corresponding reductant, then this will tend to increase the redox potential and the oxidizing nature of the medium.

With the notable exception of oxygen, most of the couples present in foods, e.g. glutathione and cysteine in meats, and to a lesser extent, ascorbic acid and reducing sugars in plant products, would on their own tend to establish reducing conditions.

From the Nernst equation, it is clear that the hydrogen ion concentration will affect the E_h, and for every unit decrease in the pH the E_h increases by 58 mV. The high positive E_h values registered by fruit juices (see Table 3.5) are largely a reflection of their low pH.

As redox conditions change there will be some resistance to change in a food’s redox potential, known as poising. This is analogous to buffering of a medium against pH changes and is, like buffering, a ‘capacity’ effect dependent on, and increasing with, the concentration of the couple. Also, like buffering, poising is greatest when the two components of a redox couple are present in equal amounts.

Oxygen, which is present in the air at a level of around 21%, is usually the most influential redox couple in food systems. It has a high E₀ and is a powerful oxidizing agent; if sufficient air is present in a food, a high positive potential will result and most other redox couples present will, if allowed to equilibrate, be largely in the oxidized state.

Hence the intrinsic factor of redox potential is inextricably linked with the extrinsic factor of storage atmosphere. Increasing the access of air to a food material by chopping, grinding, or mincing will increase its E_h.

This can be seen by comparing the values recorded for raw meat and minced meat, and for whole grain and ground grain in Table 3.5. Similarly, exclusion of air as in modified vacuum packing or canning will reduce the E_h. Microbial growth in a food reduces its E_h. This is usually ascribed to a combination of oxygen depletion and the production of reducing compounds such as hydrogen by the micro-organisms.

Oxygen depletion appears to be the principal mechanism; as the oxygen content of the medium decreases, so the redox potential declines from a value of around 400 mV at air saturation by about 60 mV for each tenfold reduction in the partial pressure of oxygen.

The decrease in E_h as a result of microbial activity is the basis of some long- established rapid tests applied to foods, particularly dairy products. Redox dyes such as methylene blue or resazurin are sometimes used to indicate changes in E_h which are correlated with microbial levels.

Methylene blue is also used to determine the proportion of viable cells in the yeast used in brewing. A cell suspension stained with methylene blue is examined under the microscope and viable cells with a reducing cytoplasm appear colourless. Non-viable cells fail to reduce the dye and appear blue.

Redox potential exerts an important elective effect on the microflora of a food. Although microbial growth can occur over a wide spectrum of redox potential, individual micro-organisms are conveniently classified into one of several physiolo­gical groups on the basis of the redox range over which they can grow and their response to oxygen.

Obligate or strict aerobes are those organisms that are respiratory, generating most of their energy from oxidative phosphorylation using oxygen as the terminal electron acceptor in the process. Consequently they have a requirement for oxygen and a high E_h and will predominate at food surfaces exposed to air or where air is readily available.

For example, pseudomonads, such as Pseudomonas fluorescens, which grows at an E_h of +100 to +500 mV, and other oxidative Gram-negative rods produce slime and off-odours at meat surfaces.

Bacillus subtilis (E_h — 100 to + 135 mV) produces rope in the open texture of bread and Acetobacter species growing on the surface of alcoholic beverages, oxidize ethanol to acetic acid to produce either spoilage or vinegar Obligate anaerobes tend only to grow at low or negative redox potentials and often require oxygen to be absent.

Anaerobic metabolism gives the organism a lower yield of utilizable energy than aerobic respiration, so a reducing environment that minimizes the loss of valuable reducing power from the microbial cell is favoured.

The presence or absence of oxygen can naturally affect this, but for many anaerobes, oxygen exerts a specific toxic effect of its own. For example, it has been observed that Clostridium acetobutylicum can grow at an E_h as high as +370 mV maintained by ferricyanide, but would not grow at +110mV in an aerated culture.

This effect is linked to the inability of obligate or aero-intolerant anaerobes to scavenge and destroy toxic products of molecular oxygen such as hydrogen peroxide and, more importantly, the superoxide anion radical (0₂^–) produced by a one- electron reduction of molecular oxygen. They lack the enzymes catalase and superoxide dismutase, which catalyse the breakdown of these species as outlined below.

Obligate anaerobes, such as Clostridia, are of great importance in food microbiology. They have the potential to grow wherever conditions are anaerobic such as deep in meat tissues and stews, in vacuum packs and canned foods causing spoilage and, in the case of C. botulinum, the major public health concern: botulism.

Aero-tolerant anaerobes are incapable of aerobic respiration, but can nevertheless grow in the presence of air.

Many lactic acid bacteria fall into this category; they can only generate energy by fermentation and lack both catalase and superoxide dismutase, but are able to grow in the presence of oxygen because they have a mechanism for destroying superoxide based on the accumulation of milli-molar concentrations of manganese.

4. Antimicrobial Barriers and Constituents:

All foods were at “some stage part of living organisms and, as such, have been equipped through the course of evolution with ways in which potentially damaging microbial infections might be prevented or at least limited. The first of these is the integument; a physical barrier to infection such as the skin, shell, husk or rind of a product.

It is usually composed of macromolecules relatively resistant to degradation and provides an inhospitable environment for micro-organisms by having a low water activity, a shortage of readily available nutrients and, often, antimicrobial compounds such as short chain fatty acids (on animal skin) or essential oils (on plant surfaces).

The value of these physical barriers can be clearly seen when they are breached in some way. Physical damage to the integument allows microbial invasion of the underlying nutrient-rich tissues and it is a commonplace empirical observation that damaged fruits and vegetables deteriorate more rapidly than entire products, and that this process is initiated at the site of injury.

Consequently it is important to the farmer and food processor that harvesting and transport maintain these barriers intact as far as possible.

As a second line of defence, the product tissues may contain antimicrobial components, the local concentration of which often increases as a result of physical damage. In plants, injury can rupture storage cells containing essential oils or may bring together an enzyme and substrate which were separated in the intact tissue.

The latter occurs in plants such as mustard, horseradish, watercress, cabbage and other brassicas to produce antimicrobial isothiocyanates (mustard oils) and in Allium species (garlic, onions and leeks) to produce thiosulfinates such as allicin (Figure 3.3).

A class of antimicrobials known collectively as phytoalexins are produced by many plants in response to microbial invasion, for example the antifungal compound phaseollin produced in green beans.

Many natural constituents of plant tissues such as pigments, alkaloids and resins have antimicrobial properties, but limited practical use is made of these. Benzoic and sorbic acids found in cranberries and mountain ash berries respectively are notable exceptions that are used in their pure forms as food preservatives.

Considerable attention has been directed to the antimicrobial properties of those plants used as herbs and spices to flavour food (Table 3.6).

Analysis of their volatile flavour and odour fractions, known as essential oils, has frequently identified compounds such as allicin in garlic, eugenol from allspice (pimento), cloves and cinnamon, thymol from thyme and oregano, and cinnamic aldehyde from cinnamon and cassia which have significant antimicrobial activity (Figure 3.4).

As a consequence, herbs and spices may contribute to the microbiological stability of foods in which they are used.

It has, for example, been claimed that inclusion of cinnamon in raisin bread retards mould spoilage. Usually, however, their role in preservation is likely to be minor and, in some cases, they can be a source of microbial contamination leading to spoilage or public health problems.

Outbreaks of botulism associated with crushed garlic in oil and home canned peppers demonstrate that even in relatively high concentrations plant antimicrobials are not a complete guarantee of safety.

Antimicrobial components differ in their spectrum of activity and potency, they are present at varying concentrations in the natural product, and are frequently at levels too low to have any effect. Hops and their extracts are ubiquitous ingredients in beer.

Humulones contained in the hop resin and isomers produced during processing, impart the characteristic bitterness of the product but have also been shown to possess activity against the common beer spoilage organisms, lactic acid bacteria.

When first introduced into brewing, hops probably contributed to microbiological stability, but this is less likely nowadays with the relatively low hopping rates used. In fact the ability of lactic acid bacteria to acquire resistance to hop resins means that the brewery environment probably acts as a very efficient natural enrichment culture for humulone-tolerant bacteria, thus negating any beneficial effects.

A rather different example of the importance of plant antimicrobials is provided by oleuropein, the bitter principle of green olives. In the production of Spanish-style green olives, it is removed by an alkali extraction process, primarily for reasons of flavour.

However oleuropein and its aglycone are also inhibitory to lactic acid bacteria; if not removed at this early stage, they would prevent the necessary fermentation occurring subsequently.

Animal products too, have a range of non-specific antimicrobial constituents. Probably the supreme example of this is the white or albumen of the hen’s egg which possesses a whole battery of inhibitory components. Many of the same or similar factors can also be found in milk where they are present in lower concentrations and are thus less effective.

Both products contain the enzyme lysozyme which catalyses the hydrolysis of glycosidic linkages in peptidoglycan, the structural polymer responsible for the strength and rigidity of the bacterial cell wall. Destruction or weakening of this layer causes the cell to rupture (lyse) under osmotic pressure.

Lysozyme is most active against Gram-positive bacteria, where the peptidoglycan is more readily accessible, but it can also kill Gram-negatives if their protective outer membrane is damaged in some way.

Other components limit microbial growth by restricting the availability of key nutrients. Ovotransferrin in egg white and lactoferrin in milk are proteins that scavenge iron from the medium. Iron is an essential nutrient for all bacteria and many have evolved means of overcoming iron limitation by producing their own iron-binding compounds known as siderophores.

In addition, egg white has powerful cofactor—binding proteins such as avidin and ovoflavoprotein which sequester biotin and riboflavin restricting the growth of those bacteria for which they are essential nutrients, see Table 3.7.

Milk also has the capacity to generate antimicrobials in the presence of hydrogen peroxide. The milk enzyme lacto-peroxidase will catalyse the oxidation of thiocyanate by hydrogen peroxide to produce inter alia hypo-thiocyanate. This can kill Gram-negative bacteria and inhibit Gram-positives, possibly by damaging the bacterial cytoplasmic membrane (Figure 3.5).

5. Water Activity:

Water is a remarkable compound. Considered as a hydride of oxygen (H₂O) it has quite exceptional properties when compared with the hydrides of neighbouring elements in the periodic table such as ammonia (NH₃), methane (CH₄), hydrogen sulfide (H₂S), and hydrofluoric acid (HF), see Table 3.8. Life as we know it is totally dependent on the presence of water in its liquid state.

The reactions which take place in the cytoplasm do so in an aqueous environment and the cytoplasm is surrounded by a membrane which is generally permeable to water molecules which may pass freely from the cytoplasm to the environment and from the environment to the cytoplasm.

This dynamic two way flow of water molecules is normally in a steady state and a living organism will only be stressed if there is a net flow out of the cytoplasm, leading to plasmolysis, or a net flow into the cell leading to rupture of the membrane, and the latter is normally prevented by the presence of a cell wall in the bacteria and fungi.

In our everyday lives we think of water as existing in its liquid state between its freezing point (0°C) and boiling point (100 °C) and we might expect that this would limit the minimum and maximum temperatures at which growth could possibly occur.

But, of course, the freezing point of water can be depressed by the presence of solutes and there are a number of micro-organisms which can actively grow at subzero temperatures because their cytoplasm contains one or more com­pounds, such as a polyol, which act as an antifreeze.

Similarly the boiling point of water can be elevated by increased hydrostatic pressure and, in nature, very high pressures exist at the bottom of the deep oceans. Under these circumstances the temperature of liquid water may be well above 100 °C and the relatively recent exploration of submarine volcanic vents has uncovered some remarkable bacteria which can indeed grow at such high temperatures.

Although the cytoplasm must be in the liquid phase for active growth (and it is important not to confuse growth and survival, for many micro-organisms can survive but not grow when their cytoplasm has been completely dried) water in the environment of the living organism may be present, not only in the liquid phase as pure water or a solution, but also in the atmosphere in the gaseous phase, or associated with what would be described macroscopically as the solid phase (Figure 3.6).

A useful parameter which helps us to understand the movement of water from the environment to the cytoplasm or from the cytoplasm to the environment is water activity, a_w.

The water activity of a substrate is most conveniently defined as the ratio of the partial pressure of water in the atmosphere in equilibrium with the substrate, P, compared with the partial pressure of the atmosphere in equilibrium with pure water at the same temperature, P₀.

This is numerically equal to the equilibrium relative humidity (ERH) expressed as a fraction rather than as a percentage:

This has important implications for the storage of low a_w foods.

In 1886 Francois Marie Raoult described the behaviour of an ideal solution by an equation which has since then been known as Raoult’s law:

where P_A is the partial vapour pressure of A above a solution, in which X_A is the mole fraction of the solvent A, and P_A0 is the vapour pressure of pure liquid A at the same temperature.

If the solvent A is water then Equations (3.18) and (3.19) can be combined to give:

Thus for an aqueous solution the water activity is approximately given by the ratio of the number of moles of water to the total number of moles (i.e. water + solute), i.e.:

It should be noted that water activity is a colligative property, that is to say it depends on the number of molecules or ions present in solution, rather than their size. Thus a compound like sodium chloride, which dissociates into two ions in solution, is more effective at reducing the water activity than a compound like sucrose on a mole-to-mole basis.

Physical chemists would prefer to work with the chemical potential of water (µ_w), which is a complex parameter made up of a reference state, a water activity term, a pressure term and a gravitational term:

which can be rearranged to give a new parameter, Ψ, known as the water potential having the same dimensions as pressure:

For situations associated with everyday life on the surface of the Earth it is possible to ignore the pressure and gravity terms and a good approximation of the relationship between the water potential and water activity is given by Equation (3.24):

where R (the gas constant )= 0.08205 dm³atm K^-1 mol^-1; and V_m (the molar volume of water) = 0.018 dm³ mol^-1 ‘

Thus at 25 °C (298 °K) a water activity of 0.9 would correspond to a water potential of -143 atm or — 14.5 MPa.

Water potential may contain both an osmotic component, associated with the effect of solutes in solution, and a matric component, associated with the interaction of water molecules with surfaces, which can be clearly demonstrated by the rise of water in a capillary tube. The latter might be particularly important in discussions about the availability of water in a complex matrix such as cake.

A parameter related to water activity is osmotic pressure which can be thought of as the force per unit area required to stop the net flow of water molecules from a region of high to one of low water activity. Cytoplasm is an aqueous solution and so must have a lower water activity than pure water; thus a micro-organism in an environment of pure water will experience a net flow of water molecules into the cytoplasm.

If it cannot cope with this it will increase in size and burst. Bacteria, fungi and algae cope by having a rigid strong wall capable of withstanding the osmotic pressure of the cytoplasm which may be as high as 30 atm (ca. 3 MPa) in a Gram-positive bacterium or as little as 5 atm (ca. 0.5 MPa) in a Gram-negative species.

Freshwater protozoa, on the other hand, cope with the net flow of water into the cell by actively excreting it out again with a contractile vacuole.

As water activity is decreased, or osmotic pressure is increased, in the environment it is essential that the water activity of the cytoplasm is even lower, or its osmotic pressure even higher. This is achieved by the production of increasing concentrations of solutes which must not interfere with cytoplasmic function.

They are thus known as compatible solutes and include such compounds as the polyols glycerol, arabitol and mannitol in the fungi and amino acids or amino acid derivatives in the bacteria. With a reduction of water activity in their environment the number of groups of micro-organisms capable of active growth decreases (Table 3.9).

The exact range of water activities allowing growth is influenced by other physicochemical and nutritional conditions but Figure 3.7 illustrates the range for a number of individual species of micro-organisms and Figure 3.8 demonstrates the interaction between temperature and water activity for Aspergillusflavus and Penicillium expansum.

Figure 3.9 shows the range of a_w values associated with a number of different food commodities.

Because low water activities are associated with three distinct types of food three terms are used to describe the micro-organisms especially associated with these foods:

(i) Halo-Tolerant:

Able to grow in the presence of high concentrations of salt

(ii) Osmo-Tolerant:

Able to grow in the presence of high concentrations of unionized organic compounds such as sugars

(iii) Xero-Tolerant:

Able to grow on dry foods. These terms do not describe rigidly exclusive groups of micro-organisms but are useful in the context of studies of particular food commodities. Some micro­organisms actually grow better at reduced a_w and may be described as halophilic, osmophilic or xerophilic, indeed the halo-bacteria are obligately halophilic and cannot grow in the absence of high concentrations of salt.

This group of bacteria, which includes such genera as Halo-bacterium and Halo-coccus, belong to the Archaebacteria and accumulate potassium chloride as their compatible solute. They are obligately halophilic because the integrity of their outer wall depends on a high concentration of sodium chloride in their environment.

They are usually associated with salt lakes or salt pans where solar salt is being made and may cause the proteolytic spoilage of dried, salted fish.

The limiting value of water activity for the growth of any micro-organism is about 0.6 and below this value the spoilage of foods is not microbiological but may be due to insect damage or chemical reactions such as oxidation.

At a water activity of 0.6, corresponding to a water potential of — 68 MPa, the cytoplasm would need to contain very high concentrations of an appropriate compatible solute and it is probable that macromolecules such as DNA would no longer function properly and active growth must cease.

However, it is important to note that, even if active growth is impossible, survival may still occur and many micro-organisms can survive at very low water activities and are frequently stored in culture collections in this form.

It is a relatively simple matter to determine the water content of a food commodity by drying to constant weight under defined conditions. The water content, however, may not give a good indication of how available that water is, i.e. what the water activity is, unless the relationship between these two properties has been established.

Thus, oil-rich nuts with a water content of 4-9%, protein rich legumes with 9-13% water content and sucrose rich dried fruits with a water content of 18-25% could all have the same water activity of about 0.7 and would thus be acceptably stable to spoilage by most micro-organisms.

The relationship between water activity and water content is very sensitive to temperature and may seem to depend on whether water is being added or removed from a substrate. An example of a water sorption isotherm is shown in Figure 3.10.

In this example the material has been allowed to equilibrate effectively at a known water activity before measuring the water content but Figure 3.11 demonstrates the differences which may be observed depending on whether a given water content is achieved by adding water to a dry commodity or removing it from a wet commodity.

The same water content seems to be associated with a higher a_w, in the former case than in the latter. This hysteresis phenomenon is a reflection of the long time that it may take for water to equilibrate with the constituents of a complex food matrix.

The measurement of water activity can thus be achieved by measuring the water content if the shape of the isotherm has been established. Water activity can be measured by measuring the equilibrium relative humidity of the atmosphere in contact with the sample. This can be done by the dew point method or with a hair hygrometer.

There are a number of instruments which measure relative humidity through its effect on the electrical properties, such as conductivity or resistivity, of materials. Thus the resistance of lithium chloride, or the capacitance of anodized aluminium, changes with changes of relative humidity.

A method known as the Landrock-Proctor method depends on gravimetrically measuring changes in water content of samples of the material after equilibration with atmospheres of known relative humidity which can be obtained using saturated solutions of a number of inorganic salts.

If the sample has a lower a_w than the atmosphere then it will gain weight, if it has a higher a_w then it will lose weight. By carrying out measurements of weight change over a range of relative humidity it is possible to extrapolate to the relative humidity which would cause no weight loss and thus corresponds to the a_w of the sample.

Figure 3.12 shows the result of such an experiment with samples of madeira cake and Table 3.10 shows the water activities of a variety of saturated salt solutions at 25 °C. Some of these salt solutions have large temperature coefficients and so the temperature needs to be very carefully controlled.