Commonly employed methods of preservation

This section describes preservation strategies that can be regarded as 'inpack' methods of preservation. These include the use of chemical additives (organic and inorganic), adjustment of pH, control of the reduction-oxidation potential, control of water activity, use of natural preservatives, microbial antagonism, low temperature and modification of the gaseous atmosphere.

These methods of preservation may be used singly or in combination - in which case they are termed a 'preservation system'. This approach gave rise to the 'hurdle' concept (Leistner and Rödel, 1976). Here, the prevention of growth of micro-organisms is accomplished by using a combination of preservative factors that would not be inhibitory if used singly.

In practice, the formulation or method of manufacture or packaging of many co-products or end-products - and the conditions used in their storage, distribution and sale - lead to the application of just such a preservation system. For example, acidulants (such as acetic, lactic or citric acid) may be combined with other preservatives (such as benzoic acid or sorbic acid). Additionally, sodium chloride and sucrose may be present. These ingredients each have the potential to act as a preservative and can act singly or in combination, and their effectiveness will vary according to the storage temperature of the product.

In order to use such methods of preservation in the most beneficial way, however, it is important to understand their characteristics, and how they interact with each other and with other components.

This is a measure of the concentration of protons, [H+], in a system, expressed as a negative logarithm: pH = -log[H+]. When water dissociates, the acidic portion (H+) is present in an equal proportion to the alkaline portion (HO-):

and hence pure water is neither acidic nor alkaline, but neutral. Its dissociation constant indicates that it has a concentration of H+ of 10-7 moles/litre. Expression of this as a negative logarithm gives the pH as 7.0, and this is, therefore, the pH of a neutral system. The further the pH falls below 7 the more acidic it is, and as the pH rises above 7 it becomes alkaline.

In many complex matrices, such as those found in foods, it is difficult to appreciate what a measured pH really means. Heterogeneity of the matrix can result in localised differences in pH, and certain food components can exert a considerable buffering capacity which prevents changes in pH. Although pH is frequently cited as a major characteristic in relation to microbiological stability, the pH should not be considered in isolation, but together with the type and concentration of acidulant used to adjust the pH, and the interaction with other preservatives if applicable.

Organic acids

Preservatives used in food matrices are predominantly weak acids, such as acetic, lactic, citric or sorbic acids (Fig. 6.2). Their preservative action is a combination of their effect on pH and the antimicrobial properties of the undissociated form of the molecule. Their antimicrobial effect is modulated by the thermodynamic characteristics of dissociation and partition, which are discussed below.

For any given concentration of these acids a proportion exists as the acidic undissociated form, and a proportion as the dissociated anionic form. It is the undissociated form that has the predominant antimicrobial effect (Baird-Parker, 1980; Sofos and Busta, 1981; Eklund, 1983).


This is the characteristic of a chemical compound to separate into certain component parts. In the case of weak organic acids it is an ionisation reaction. This is important in preservation because the preservatives that undergo these reactions are usually more active in one form than the other. A general equation for compounds that dissociate by ionisation in this way is:

OH Acetic acid


Lactic acid


CO OH Citric acid

Sorbic acid O

Fig. 6.2 Structural chemical formulae for acetic, citric, lactic and sorbic acids.

130 Handbook of waste management and co-product recovery HA ^ A- and H+

where A- is the anion and H+ the proton. For example, acetic acid dissociates according to the equation:

Acetic acid Acetate Hydrogen

(undissociated) (dissociated) ion


The antimicrobial form of weak organic acids is the undissociated form, and it is the concentration of this form that is important in the processes that rely on these acids as preservatives.

The dissociation is an equilibrium, and the concentration of each side of the equilibrium equation is dictated by the dissociation constant (Ka) that is a characteristic of the compound. The Ka is typically a small number, and published values are available (Windholz, 1983). For convenience we tend to express the dissociation constant in the form of its negative logarithm and give it the term pK where pk = -log Ka

Hence, the Ka of acetic acid at 25 °C of 1.76 x 10-5 moles/litre gives a pK of 4.76 (Table 6.3). The pK is characteristic of each compound. It varies with temperature, but only slightly (Robinson and Stokes, 1959), and the equation to determine this variation is:

where T is the temperature in degrees Kelvin (K); the constants A, B and C for some common food acids are shown in Table 6.4. The effect of tem-

Table 6.3 pK values of some organic acids used as preservatives (from Windholz, 1983)


pK at 25 °C
















perature change on pK is shown in Table 6.5. When the pH is equivalent to the pK, the proportions of the acid and its salt are equal. If the pH is decreased, then the concentration of the undissociated form is increased. If the pH increases, however, the concentration of the undissociated form declines. Some compounds possess more than one functional group that is able to dissociate. For example, citric acid has three carboxylic acid groups. Each of these dissociates, and each has a different pK.

Calculation of dissociation

The Henderson-Hasselbalch equation can be used to calculate the proportions of the undissociated and dissociated forms of weak acids. The equation states that the pH is equal to the sum of the pK of the acid and the logarithm of the ratio of the proportion of dissociated acid to undissociated acid (Wilson et al, 2000), thus:

[acid ]undissociated

Table 6.4 Values of A, B and C for use in equation [6.1] for the calculation of the effect of temperature on pK

Acid ABC

Acetic 1170.48 Citric

K1 1255.6

K2 1585.2

K3 1814.9

Lactic 1286.49

Benzoic 1590.2

3.1649 0.013 399

4.5635 0.011 673

5.4460 0.016 399

6.3664 0.022 389

4.8607 0.014 776

6.394 0.017 65

Table 6.5 Calculated effect of temperature on pK using equation [6.1]


Acetic 4.76 4.77 Citric

Lactic 3.86 3.87

Benzoic 4.20 4.23

This equation can be rearranged to give the concentration of weak acid in its undissociated form:

where [HA]aq is the concentration of undissociated organic acid in the aqueous phase and [HA]T is the total concentration of organic acid. As a 'ready reckoner' Table 6.6 gives the precentage undissociation for acetic acid over the range of pH 4.0-5.6, assuming a pK of 4.76 (20 °C).

A combination of low pH and organic acids can be used to inhibit the growth of, or kill, micro-organisms. The cell membrane of micro-organisms has a gradient of H+ across it. The concentration is low inside the cell and high outside it. If the pH outside the cell falls (and hence the concentration of H+ increases) the gradient of H+ across the membrane increases, and the H+ leaks into the cell. The lower the pH, the steeper the gradient becomes, and the more H+ leaks into the cell. The micro-organism must expend energy in the transport of H+ out of the cell. As the gradient of H+ increases, a large amount of energy is expended, and the cell's metabolism will slow down, and eventually stop. Undissociated, lipophilic organic acids - such as acetic acid - function as antimicrobial agents because of their solubility in the cell membrane where they may aid in the passage of H+ into the cell.

Sorbic acid and benzoic acids have the additional ability to inhibit a variety of metabolic processes directly (Leuck, 1980). Citric acid is inhibitory to the growth of micro-organisms because of its ability to chelate divalent cations such as calcium (Rammell, 1962).

Buffer systems

Acidulants and their salts can be combined to construct buffer systems so that any tendency of the pH to shift is resisted. Typically, the buffer system

Table 6.6 Effect of pH on the calculated proportion of dissociated and undissociated acetic acid in solution at 20 °C

Calculated % undissociated acetic acid

85.19 78.41 69.61 59.11 47.71 36.52 26.64 18.64 12.63

is composed of a weak acid and its salt (e.g. acetic acid and sodium acetate; citric acid and trisodium citrate). These buffer systems function according to the equation:

Acid Anion/salt

(undissociated) (dissociated)

If the acidity increases (i.e. an increase in the concentration of H+ occurs), then the H+ combines with the anion to produce undissociated acid. If the system becomes less acidic, then the acid dissociates to release the anion and H+, and hence the pH remains constant. In order to achieve the desired pH, a known quantity of acid and salt are combined so that a reservoir of both the undissociated and dissociated molecules is established. The pH is then a function of the pK.

However, changes to the chemistry are reiterative. For example, in aqueous solution organic acids will dissociate. This is dependent upon local pH, but dissociation will then perturb this pH. Dissociation is dependent on the local buffering capacity of the system, which is extremely difficult to predict. Wilson et al. (2000) developed calculations describing the reiterative dissociation of organic acids, and these calculations can be used to predict the true chemical composition. The method requires a characterisation of the buffering behaviour of the system by titration with a strong (i.e. completely dissociating) acid, and then using knowledge of the dissociation constants of weak acid preservatives to predict their concentrations and the pH.

The quantity of acid and salt on each side of the above equation can be quite high in order to increase the size of the reservoir. This can allow the formulation of a product in such a way that it contains a high concentration of weak acid in a form that is microbicidal at a given pH (Debevere, 1987), providing that the organoleptic properties of the co-product or end-product do not suffer.


Organic acids in their undissociated form are lipophilic, although to differing extents. The oil : water partition coefficient is the ratio of the solubility of a compound in oil to its solubility in water. It is important to consider this coefficient in the application of co-products and end-products in systems that contain both a lipid and an aqueous phase, such as emulsions (e.g. mayonnaise). Here the micro-organisms are restricted to the aqueous phase (Tuynenburg Muys, 1971). Preservatives that have a high partition coefficient, however, are present in their lipophilic undissociated antimicrobial form in the aqueous phase at lower concentrations than in the lipid phase. Their effective concentration is, therefore, decreased. This is particularly true of sorbic and benzoic acids, which have partition coefficients of 3.1 and between 6 and 13, respectively (Von Schelhorn, 1964). Acetic acid, however, has a partition coefficient of between 0.03 and 0.07 (Gordon and Reid, 1922; Bodansky, 1928; Leo et al., 1971), and therefore exists in its antimicrobial undissociated form predominantly in the aqueous phase.

If the pH of the system to be preserved using organic acids is in a region where weak organic acids are present in both the undissociated and the dissociated form, then calculation of the residual concentration of the undissociated form following partition is difficult. This is because its concentration is subject to partition, and to the dissociation equilibrium dictated by the new pH of the system and its new residual concentration of undissociated acid.

The Henderson-Hasselbalch equation can be modified to take these effects into account. It gives the proportion of the total weak acid in an aqueous phase/lipid-phase system that is present in the undissociated form in the aqueous phase, given the pH, the volume fraction of oil and the partition coefficient for the undissociated weak acid. The equation is:

where Kp is the partition coefficient and 0 is the fraction volume of the oil phase (Wilson et al, 2000).

Partition can be affected by a variety of factors including: soluble components - such as glucose, sucrose and sodium chloride - which influence the solubility of the organic acid in the aqueous phase; and acidulants which influence pH and hence affect the concentration of the undissociated molecule.

Inorganic preservatives

The most well known inorganic preservatives are probably sulphur dioxide and nitrite, which are used widely. The inhibitory effect of nitrates is derived from their conversion to nitrite in foods. Both sulphur dioxide and nitrite are inhibitors of the growth of micro-organisms and of certain sensory changes, particularly those due to oxidation or enzymatic deterioration. Sulphur dioxide exists in various forms according to the pH of the matrix, and may be present as dissolved sulphur dioxide gas, or it may ionise to form undissociated sulphurous acid (the reaction product of sulphur dioxide in water).

Sulphurous acid Bisulphite




This is the most active antimicrobial form but it exists in equilibrium with bisulphite and sulphite ions. The inhibition of the growth of microorganisms is largely as a result of the inhibition of respiratory enzymes (Lueck, 1980), particularly those enzymes that contain sulphydryl groups, with which sulphur dioxide and its associated products are particularly reactive. The reactivity with aldehydes produced as integral parts of metabolic pathways is an additional site of inhibition (Gomez and Herrero, 1983).

Nitrite is thought to be inhibitory to the growth of micro-organisms due to the formation of nitrous acid and associated oxides. Although the biochemical effects have not been fully determined, they include inhibition of metabolism by reaction with dehydrogenases, with other respiratory enzymes, with cytochromes and with sulphydryl groups (Lueck, 1980; McMindes and Siedler, 1988). Nitrous acid has a pK of 3.4 (Windholz, 1983), and its effectiveness increases as the pH declines, and where it exists predominantly as the undissociated form.

As is the case for organic acids, the antimicrobial properties of each form of any compound that dissociates in these ways will differ. The pH, therefore, has a considerable influence on the state and the effectiveness of the chemical preservatives used. A disadvantage of the use of such inorganic compounds as preservatives in co-product streams is that both sulphur dioxide and nitrite can react with food ingredients. Accordingly, the residual concentration is often considerably lower than the initial concentration. However, when nitrite is heated with a culture medium that contains meat, the products of the reaction with the meat have antimicrobial properties greater than those due to nitrite alone (Perigo et al., 1967).

Importantly, it should be noted that both sulphur dioxide and nitrite, and some of their reaction products, raise serious toxicological questions.

Reduction-oxidation potential

Otherwise known as redox potential, or Eh, the reduction-oxidation potential is a measure (cited in millivolts) of the balance of chemically reduced and oxidised compounds within a system, rather than of the partial pressure of oxygen within or around the system. The Eh is influenced by pH and temperature, and may change as micro-organisms multiply or metabolise. In combination with pH and the composition of the gaseous atmosphere it may influence the ability of micro-organisms to grow.

Aerobic micro-organisms grow best at a positive Eh (an oxidized environment), and anaerobes at a negative Eh (a reduced environment), although the range of Eh in which a given organism (including anaerobic bacteria) can multiply is usually wide.

In oxidized environments (i.e. positive Eh) an accumulation of free radicals produced by interaction with oxygen (e.g. peroxides, superoxides) can prevent the growth of those bacteria that do not possess enzymes (catalase, superoxide dismutase) capable of destroying these radicals. Exclusion of oxygen, and a decline in the Eh, decreases the threat to these bacteria, and strict anaerobes may begin to grow.

Total exclusion of oxygen, however, may not result in a negative Eh (for example, in plant tissues as noted below). Then, the anaerobic bacteria must use their own chemical reducing power to decrease the Eh of the environment in order to grow. This stress may not be sustainable for some bacteria and they will fail to multiply. Some clostridia, however, can multiply at a positive Eh, providing that oxygen is absent (Jones, 1989).

The Eh of matrices varies widely. The Eh of minced fresh meats, packed loosely and hence oxygenated, is about +200 mV. That of cheeses ranges from -20 to -200 mV. The liquid in plant tissues has Eh values of around +300 to +400 mV at a pH of 4-5. Some chemical additives have a marked influence on Eh; for example, ascorbic acid decreases Eh and nitrite increases it (Brown and Emberger, 1980).

Water activity

There has been a recent tendency to move away from the use of the term 'water activity' and to instead use the term 'water potential'. This term describes the difference between the chemical potential of water at any point in a system and that of pure water under standard conditions. It involves a calculation using the temperature and the ideal gas constant together with a knowledge of the vapour pressure of water in the system and the vapour pressure of pure water at the same temperature. Water potential can be expressed in terms of energy or pressure units and is a more precise description of the state of water in a system than is water activity.

Nevertheless, the term 'water activity' is still used widely and is generally accepted as being a useful tool in the understanding of the principles behind the interaction of water and solutes in preservation. As so many descriptions of foods and the growth of micro-organisms are constructed in terms of water activity this terminology is retained here. Water activity is a measure of the concentration of the available water in a system and can be defined as the tendency of water to escape from a solution relative to its ability to escape from pure water at a specific temperature. It is equal to the equilibrium relative humidity divided by 100. Pure water, therefore, has a water activity (aw) of 1.00, and an environment where water is absent has an aw of 0.00 (Troller, 1983).

It is possible to calculate aw from first principles using a variety of equations, such as Raoult's law (Christian, 1980; Labuza and Bell, 1984), which was derived by Christian (1980) as:


where m is the molal concentration of the solute, v is the number of ions generated by each molecule of the solute and 0 is the molal osmotic coefficient.

All biochemical reactions inside the microbial cell take place in an aqueous system. Any decrease in the amount of available water in the cell will decrease the efficiency of these reactions; a severe loss of water will cause metabolism to cease and may cause structural damage, such as the collapse of the cell membrane due to plasmolysis (Troller, 1983). Synthesis of compatible solutes inside the cell (solutes produced and retained by the cell) is required in order to attempt to balance the osmotic effect of the external environment and equalise the osmotic gradient across the membrane. This is an energy-requiring process that places further stress on the micro-organism (Christian, 1981; Troller, 1983).

Most micro-organisms require a high water activity in order to grow. For most bacteria, an aw in excess of about 0.9 is required. Some yeasts and moulds can multiply at an aw of about 0.80 (Leistner et al, 1981), although osmotolerant yeasts and xerophilic moulds are capable of growth when the aw is decreased to 0.6 (Lueck, 1980).

The aw can be decreased by the addition of solutes, such as sodium chloride, sucrose or glycerol. In some cases the solute (humectant) itself may have toxic effects, and the inhibition of growth when sodium chloride is used to adjust aw can be greater than when glycerol is used, due to the tox-icity of high concentrations of sodium chloride (Baird-Parker and Freame, 1967; Gomez and Herrero, 1983; Troller, 1983). In practice, an appreciable decrease in aw can only be achieved by the use of high concentrations of solute (Table 6.7). For example, the concentration of sodium chloride, and of sucrose, in water that is required to effect an aw of 0.90 is 16.54 g per 100 g H2O and 144 g per 100 g H2O, respectively (Robinson and Stokes, 1959; Seiler, 1969; Lueck, 1980). Consequently, in systems that contain large quantities of water, the tool of decreased aw is often used as one component of a preservation system (Tuynenburg Muys, 1975).

Table 6.7 Quantities of sodium chloride and sucrose required to poise an aqueous solution to a given aw (from Lueck, 1980)

NaCl (g/100 g H2O)

Sucrose (g/100 g H2O)


































However, a high concentration of solutes, and hence a low aw, in the aqueous phase of emulsions or other two-phase foods decreases the solubility of some preservatives (e.g. sorbic acid, benzoic acid), and increases their partition into the lipid phase. This can have the effect of decreasing the degree of preservation of the food. An important effect of low aw is to increase, sometimes quite markedly, the heat resistance of microorganisms, and it can also lessen the inhibitory effect of low pH (Beuchat, 1973; Smith et al., 1982; Atherton and White, 1985).

Natural preservatives

Natural preservatives can include inhibitory compounds found naturally in plant tissues, or produced by micro-organisms. Many herbs and spices have antimicrobial properties, as do the extracts of many other plant tissues (indeed sorbic acid is found in, and was originally manufactured from, rowanberry oil (Lueck, 1980)). The active components vary in type considerably and include thiocyanates, sulphoxides, cinnamates, and a range of acids and phenolic compounds (Atherton and White, 1985). The concentration of these compounds in the tissues may vary considerably, and many have limited usefulness as preservatives because of their sensory qualities. For example, attempts to inhibit the growth of yeasts in coleslaw by using onion tissue required unacceptably high concentrations of onion (Brocklehurst et al., 1982).

Enzymes can also be inhibitory and contribute to preservation. Egg white lysozyme breaks down the cell wall of certain bacteria, and has been used to replace sorbate in food preservation (Atherton and White, 1985). The lactoperoxidase system found in milk requires the presence of the enzyme glucose oxidase, of glucose and of thiocyanate in order to function, and liberates hydrogen peroxide which is toxic to some micro-organisms.

The by-products of microbial metabolism can be inhibitory, but often quite specifically. Examples of inhibitors are the bacteriocins and antibiotics. Probably the most commonly employed example is nisin, an antibiotic produced by Lactococcus lactis, which has a narrow spectrum of activity against Gram-positive bacteria.

Microbial antagonism

The growth of certain micro-organisms can be inhibitory to the growth of others. The most closely studied are the lactic acid bacteria - including Lactobacillus, Lactococcus, Pediococcus and Leuconostoc - which can be inhibitory to a range of food-poisoning and food-spoilage bacteria (Daly et al, 1972; Park and Marth, 1972; Babel, 1977). The mechanism of inhibition is thought to be due to a number of effects in combination, including depletion of substrate, decrease in the redox potential, production of antibiotics and the decrease in pH due to the production of organic acids (Branen et al, 1975; Hurst, 1983).

Low temperature

Storage at refrigeration temperatures can be fundamental to the stability of co-products and end-products. The effect of a decrease in temperature is a retardation of chemical deterioration and of growth rate of microorganisms, thereby prolonging the period of microbiological stability.

Micro-organisms can multiply over a very wide range of temperatures. Those capable of growth at low temperatures are described as either psy-chrophilic or psychrotrophic. The former description requires an optimum temperature for growth of about 15 °C or lower, a maximum temperature for growth of about 20 °C or lower and a minimum temperature for growth of 0 °C or lower (Olson and Nottingham, 1980). Psychrotrophic microorganisms, however, are described as having a minimum temperature for growth of between -5 and +5 °C, but do not meet the optimal and maximal temperature requirements of psychrophiles.

There is some risk in the control of the growth of micro-organisms by using low temperature alone. Some food-poisoning micro-organisms (e.g. Clostridium botulinum type E, Listeria, Yersinia and Aeromonas) can multiply in conditions of refrigeration (Schmidt et al, 1961; Alcock, 1983; Walker and Stringer, 1987). This may result in increased risk when the storage of a product at low temperature has retarded the growth of a product's normal spoilage flora, and prevented the development of visible signs of spoilage. Growth of the food-poisoning bacteria may be further enhanced if the competition afforded by the normal spoilage flora has been decreased.

Small changes in temperature can have a large effect on the growth rate of micro-organism at chill temperatures. Whereas at temperatures close to the optimum for their growth a change in temperature of 10 °C is generally reflected by a two-fold change in growth rate (i.e. temperature quotient, Q10, of 2), as the temperature approaches the minimum for growth the temperature quotient becomes larger. Thus, a small increase in storage temperature during refrigeration can lead to large differences in the growth rate of micro-organisms or, indeed, may allow growth of some microorganisms that had previously been unable to grow.

Table 6.8 displays the time taken for a population of the spoilage bacterium Pseudomonas fluorescens to double (doubling time) at a range of temperatures. By using the equation:

(where dt is the doubling time, t - to is total time, N is 106 and No = 1), it is possible to determine the time taken to give a population of 106/g of co-product, end-product or food from an initial population of 1/g.

If we assume that at 106/g this organism can cause spoilage then we can use these data to predict the shelf-life at these temperatures (Table 6.9). These factors emphasise the importance of well-controlled chill

Table 6.8 Approximate doubling time of Pseudomonas fluorescens

Temperature (°C)

Approximate doubling time (hours)















Table 6.9 Calculated time for an increase in the number of Pseudomonas fluorescens from 1/g to a concentration of 106/g (the number of micro-organisms likely to result in spoilage), calculated from the doubling times in Table 6.8

Storage temperature (°C)

Time to 106/g (hours)















temperatures throughout the preparation, storage, distribution and sale of co-products and end-products.

Modification of the gaseous atmosphere

This involves the introduction of a gas or mixture of gases in order to supplement or replace the existing mixture of carbon dioxide (CO2), oxygen (O2) and nitrogen (N2). The modification may be deliberate, often achieved by evacuation followed by flushing with the appropriate gas or gases, or by virtue of the respiration of a packaged commodity causing an increase in the concentration of CO2 and a decrease in the concentration of O2.

In non-respiring products, the atmosphere is poised at the required composition, and will not be modified further, unless the packaging material is sufficiently permeable to allow exchange of gases with the outside environment. Packaging of systems that are living and hence that contribute to the modification of the atmosphere by their respiration, leads to a dynamic state of modification. The composition of the atmosphere in such products changes continuously. The packaging material is usually chosen to allow exchange with the outside environment in order that an equilib rium can be reached, although this is not achieved easily, if at all, in products that have a high rate of respiration.

Atmospheres that consist solely of CO2 and hence replace O2 in contact with the product, have an antioxidative effect, and are used in the extension of the shelf-life of fruit juices and carbonated beverages, and in the prevention of microbiological and sensory changes in some dairy products. Similarly, atmospheres that consist solely of N2 have a preservative effect based on the exclusion of O2. These atmospheres are also inhibitory to the growth of obligately aerobic microorganisms.

The pH of an unbuffered medium can be decreased in an atmosphere of CO2, due to the production of carbonic acid. Although this decrease can be as much as 1 pH unit in the presence of 20% CO2, this effect does not fully explain inhibition due to CO2 (Clark and Takacs, 1980). Some degree of inhibition due to modification of the gaseous atmosphere is probably due to partial inhibition of enzymes used in oxidative metabolism (Gill and Tan, 1980). It is important to note that an absence of O2 in foods may represent a hazard, in that it presents the risk of growth of anaerobic food-poisoning bacteria, such as Clostridium botulinum.

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