Intrinsic parameters

1.2.2.1 Low pH

Microorganisms may encounter stressful conditions in foods during manufacturing, during fermentation and in the gastrointestinal tract after the consumption of the contaminated foods. The natural pH of most foods ranges from neutral to highly acidic, as alkaline pH often renders the taste of the products rather unpleasant (soapy). In addition, food industries may regularly use acidic compounds (mostly organic acids) to improve the sensory characteristics of the final product. Given that the low pH conditions have been reported to have either bacteriostatic or bactericidal effect, depending on the pH value, the understanding of the response of microorganisms to acidic conditions may be of crucial importance for the food industries.

The mode of action of low pH against bacteria is generally focused on the lowering of the internal pH of the cell and/or the damage of the cell membrane. However, it may differ, depending on the type of the acidified agent (organic or inorganic acid). On the one hand, inorganic acids are not able to penetrate the cell membrane. Therefore, the acids increase the membranes permeability to protons (H+) by denaturing the enzymes present in its outer membrane (Beales, 2004). By these means, the internal pH of the cell decreases and the cell metabolic activity and function are gradually reduced until the complete cessation of active metabolism and subsequently death of the cell. Organic acids, on the other hand, may be used either as taste improvers (e.g. citric or acetic acid) or as preservatives in the form of salts (e.g. salts of sorbic or benzoic acid). Depending on the external pH, these acids usually exist both in undissociated and dissociated form, with the

concentration of undissociated acid being higher at lower levels of pH. In the dissociated form, organic acids may not penetrate the cell membrane, while in the undissociated one they may freely pass to the cell interior. Due to the high pH in the internal environment of the cells, the undissociated acid form dissociates. However, the charged ions may not leave the cells through the membrane, resulting in the accumulation of high amounts of anions in the inner of the cell and thus, decrease of the internal pH could be expected (Figure 1-5). Such conditions are inhibitory for the microbial metabolism and gradually exhaust the cell (Theron and Lues, 2007; Lambert and Stratford, 1999). Organic acids have been also reported to affect the viability of the cells by causing damage on the phospholipid membrane (Stratford and Anslow, 1998).

Figure 1-5 Representation of the mode of action of weak acids against bacteria. The low external pH favors the dissociation of the organic acid and the undissociated form is able to penetrate the cell membrane. When exposed to the high pH of the cytoplasm, the undissociated organic acid dissociates, while in parallel releases a proton. Since H+ cannot naturally exit the cell, the H+ATPase uses energy from the hydrolysis of ATP in order to pump the protons out of the cell (from Lambert and Stratford, 1999).

In an attempt to survive the acidic environmental conditions, microorganisms aim to maintain their internal pH (pH homeostasis; Beales, 2004). This may be achieved in many ways such as changes in membrane composition, induction of enzymes to repair macromolecules damages or discarding of protons from the cytoplasm (Yousef and Courtney, 2003; Foster, 2001). Regarding the latter, H+ATPase is an enzyme which utilizes energy from the hydrolysis of ATP in order to transfer the protons to the exterior of the cell and thus, maintain the internal pH (Figure 1-5; Gandhi and Chikindas, 2007). In addition, the response of bacteria to moderate acid stresses included also the synthesis of proteins, named acid shock proteins (ASPs) which promote survival at extremely low pH values (Abee and Wouters, 1999).

1.2.2.2 Water activity (aw)

Water activity is a parameter for the quantification of freely available water in a food or else, the ratio between the water pressure of the environment above the food and the vapour pressure of distilled water under the same temperature (Jay et al., 2005). This parameter is frequently used to ensure the safety and quality of foods, as without available water the microbial growth is inhibited. By decreasing the aw of a food the bacteria may perceive

an osmotic stress or shock. As osmotic stress is termed any change in the concentration of compatible solutes or salts around a cell. In foods, osmotic stress conditions may be caused due to the addition of salts, sugars or other water-binding solutions such as starch or gelatine (Yousef and Courtney, 2003). Under such conditions, the phenomenon of passive osmosis takes place. In particular, water is drawn from the cytoplasm out of the cell in order to equilibrate the concentration of solutes in both environments. However, this procedure limits the available water inside the cell and inhibits the transport of nutrients from outside the cell. Thus, the microbial activity is suppressed. Under extreme conditions osmotic stress may also lead to

structural damages and finally kill the cells (Wesche et al., 2009; Csonka, 1989).

The response of microorganisms to hyperosmotic conditions is primarily based on either the intracellular production or import from the environment of compatible solutes such as carnitine, glycine betaine, proline, ectoine and trehalose. The accumulation of these compounds increases their concentration in the cytoplasm and thus, equilibrates with the concentration of those outside the cell, without however, affecting normal cellular functions (Jay et al., 2005; Yousef and Courtney, 2003). If the solutes are not imported from the cellular environment, their production is regulated by the activation and expression of specific genes or by modifying enzyme activity. The coding of such genes is generated by specific sensors and signal transduction networks providing information to the cell about the osmolarity of its surroundings (Kültz and Burg, 1998).

1.2.2.3 Nutrient availability and food structure

The availability of nutrients in foods plays a crucial role in the growth and survival of spoilage or pathogenic bacteria. Microorganisms require adequate amounts of essential nutrients to utilize for reproduction or at the very least maintenance of cellular composition. However, energy sources may either be absent or limited and thus, microbial proliferation is itself absent or limited. The continuous exposure of cells to low nutrient conditions produces the condition of starvation stress (Jones, 2012; Spector, 1998). Starvation stress may mostly occur on abiotic but contaminated surfaces such as kitchen counters, cutting boards, kitchen sponges, walls and floors, but also in water, on the surface of animal carcasses or fresh produce and other foods. Under these conditions, the energy sources are rapidly exhausted or are unavailable and cells limit their growth activity. The mechanism by which cells respond to starvation stresses is called stringent response. First cells enter the stationary phase where they are more capable to withstand further harsh conditions. At this phase, the size of the cells is reduced in order to

constrain their need to nutrients, but after exposure to optimum conditions the size and the functionality of the bacteria is restored. In parallel, the expression of specific genes is induced in order to code starvation stress proteins, which may further enhance the resistance of the cells and/or promote the utilizing of alternative sources of nutrients (Ravishankar and Juneja, 2003). Stressed cells responding to carbon exhaustion may also cross protect to others stresses, but this is not evident in nitrogen or phosphorus starved cells (Moat et al., 2002).

Food structure does not possess any antimicrobial activity by itself, but it may indirectly affect the growth potential of microorganisms by controlling the availability of nutrients and the diffusion of toxic metabolites (Ongeng et al., 2007). Depending on their rheological properties, foods may be distinguished into two categories; (i) liquid and (ii) structural (Theys et al., 2008). In liquid foods, nutrients are equally diffused in the whole substrate and they may be easily accepted by the microorganisms. Likewise, the products of the bacterial metabolic activity diffuse away, without affecting the surrounding micro-environment, where growth occurs. On the contrary in structural foods, cells are immobilized and may not easily reach the available nutrients and thus, starvation stress conditions are more common. In addition, the accumulation of toxic metabolic products may create adverse conditions for the growth or even the survival of the microorganisms (Noriega et al., 2010a; Wilson et al., 2002). However, cells which grow in liquid foods are more susceptible to antimicrobial compounds compared to those which grow in structured foods (Skandamis et al., 2000). In addition, in solid products bacteria grow in colonies and therefore, interactions between closely located cells may affect the growth kinetics of the population (e.g. crowding effect; Noriega et al., 2010a; Thomas et al., 1997). It has to be stressed though, that in a real food system both liquid and solid forms may be present, which makes the understanding of microbial behaviour in such products more complex.