Presentación de resultado y prueba de hipótesis

As indicated in Fig. 5.25 and noted by numerous reports in the literature, the activity of microorganisms shows a definite limiting water activity below which the probability of microbial growth is considered to be zero (Troller and Christian, 1978). Different microorganisms shows different lower limits of water activity allowing growth, but most bacteria do not grow below water activity of 0.85, and no bacterial pathogens are known to grow below than water activity, even when the other environmental parameters (e.g., pH, temperature, nutrient content) are optimal. The growth of xerophilic fungi and of osmophilic yeast does occur at somewhat lower activity, but even their activity becomes insignificant in the range of water activities in the vicinity of 0.7.

The following critical values of water activity are generally accepted as providing adequate margins of safety with respect to microbiologically generated health hazards (Chirife and Buera, 1996).

Growth and toxin production by all types of Clostridium botulinum: 0.94 Anaerobic growth of Staphylococcus aureus 0.91

Aerobic growth of Staphylococcus auerus 0.85 Production of toxins by molds 0.80

The extensive studies on the subject of microbial safety have been used by the Food and Drug Administration in the formulation of its regulations requiring thermal processing of foods. Under these regulations the thermal processing for T^ABLE5.6 Crystallization of Sucrose in Freeze-Dried Systems at

54% RH at 358C

Composition Induction time kCR(h^{2 1})^c

Sucrose Hours 0.11

Sucrose þ 4.8% CMC^a Hours 0.06

Sucrose þ 9.1% CMC Hours 0.04

Sucrose þ 23% CMC Days 0.005

Surcrose þ 43% Avicel^bþ 13% CMC Weeks 0.0007

aCMC is carboxymethyl cellulose.

bAvicel is microcrystalline cellulose.

cKCRis crystallization rate constant.

Source: From Iglesias and Chirife (1978).

shelf stable foods does not apply to food with an equilibrium water activity of 0.85 or less.

There has been a great deal of literature dealing with the fundamental basis for the existence of fairly sharp limits of microbial activity (Booth, 1998). While some controversy persists with respect to the subject, it is the author’s view that the mechanism of prevention of microbial growth in low-moisture foods which is outlined below represents the current consensus view among both food microbiologists and food physicists (Gould and Christian, 1988). Figure 5.38 shows diagrammatically the essence of the mechanisms limiting microbial growth. The membrane surrounding cells is permeable to water, but largely impermeable to most solutes. For this reason equilibration between cell interior and the surrounding medium with respect to the chemical potential of water occurs readily. If a cell in its physiologically normal water activity is placed in a medium with a lower water activity, there exists a water activity difference across the membrane. This difference generates an osmotic pressure P that is related to the water activity difference as shown in Fig. 5.38. The osmotic pressure provides a driving force for transfer of water from the inside of the cell to the surrounding medium. If this process continues without any counteracting influences cell shrinkage is the result, and the shrinkage may result in irreversible damage to the cell membrane, and to death of the organism. Organisms have metabolic defenses against such an outcome. The most widely occurring type of defensive mechanism is the accumulation of solutes within the cell. There is a wide range of solutes, which may be so accumulated, including in particular salts, amino acids, and polyols, such as glycerol.

The osmotic pressure is related to concentration. The ideal relation for this concentration dependence in the Vant Hoff equation:

P ¼ cRT ð7Þ

F^IGURE5.38 Mechanism of inhibition of microbial growth at low water activities.

where P is osmotic pressure (KPa), c is molar concentration (mole/m³), R is gas constant [(KPa)(m³)/(K)(mol)], and T is absolute temperature (K).

It should be noted that in the Vant Hoff equation the quantity c, in common with Raoult’s law, freezing point depression, and boiling point elevation, refers to kinetic units in solution. Dissociated ionic compounds will therefore have c equal to concentration of ions in solutions.

The increasing intracellular concentration of accumulating solutes decreases the outgoing water flux; and when the intracellular water activity is depressed to the activity level in the surrounding material, water loss ceases, and cell shrinkage is minimized. However, the high internal concentration of solutes has an inhibiting effect on metabolic activities necessary for growth, and the growth ceases when that concentration reaches a critical level corresponding to a critical water activity for growth of the particular organism.

The question remaining unanswered at this point is, what are the mechanisms by which a given solute concentration within the cells results in complete inhibition of growth. A detailed discussion is beyond the scope of this book, and the reader is referred to recent reviews (Chirife and Buera, 1996). The mechanisms seem to include both specific inhibitory effects of low water activity effects on enzymes essential to cell function, and the excessive metabolic cost of the continuing effort to maintain a high solute concentration. It must be noted that while the membrane is very much more permeable water that to solutes, some loss of the accumulating solutes does occur, and the solutes have to be replaced continuously. A very recent paper in Nature deals with the mechanisms by which water transport through cell membranes is maintained at a much higher level than that of other small molecules (Murata et al., 2000). It should be noted here also that there is a dissenting view, proposing that the primary growth-inhibiting effect is due to reduced mobility, which in turn is due to the increase of T_gas the water activity decreases. This mechanism, in our opinion, may be of some secondary importance in special circumstances, such as growth of large mold colonies on surfaces, where diffusion of nutrients may become limiting, but the overwhelming weight of evidence suggests that osmotic effects are the dominant effect in the water limitation of microbial growth. Since the osmotic effects are directly related to water activity, any changes in water activity affect the stability of foods with respect to microbial activity. These changes do not necessarily involve overall moisture content changes. Thus, water activity may change due to hysteresis effects.

Another case resulting in water activity changes of a component sensitive to microbial attack may result in a mixture, which is not equilibrated when the storage is initiated, and in which there is water transfer between components, but no net change in the total water content of the mixture. Both cases have important practical consequences, since they may change shelf life.

The following two examples may illustrate these situations. Figure 5.39 shows the consequence of crystallization in food product containing a crystallizable sugar. Initially sugar in the product is amorphous, and the product is microbiologically stable and also has a moisture content which is sufficiently high to assure product softness. If crystallization occurs: two extreme possibilities exist for its consequences. If an unpackaged product is stored in a constant humidity environment, line B is followed and the product loses moisture and becomes hard but remains stable with respect to microbial deterioration. If the product is in a hermetically sealed package there is no moisture change, but because the crystals have a low hygrocapacity (low moisture content at a given water activity), the water activity increases into the range which permits mold growth. In the present example it is assumed that the softness of the texture of product is unaffected, because the water released by the sugar as it crystallizes plasticizes other texture-determining components, but of course this is not always the case, and crystallization may lead to texture defects as well. A well-known example of such defects is crystallization of lactose in ice cream, which leads to grittiness or sandiness. A situation in which components are initially not at equilibrium is illustrated in Fig. 5.40 (Larumbe et al., 1991). A composite candy product consists of caramel jam and chocolate. The initial water activity is 0.85, and the chocolate is 0.46. At an activity of 0.85 the jam develops mold growth within 10 days of storage. However during storage there is a tendency for slow equilibration, and the jam loses moisture to the chocolate. Chocolate has F^IGURE5.39 Effect of crystallization of an amorphous sugar on mold growth and physical state of an amorphous, sugar-containing system. A, crystallization at constant m; B, crystallization at constant a.

a very low hygrocapacity, but enough water is transferred from the jam to extend its mold-free shelf life from 10 to 18 days by lowering its water activity below 0.8.