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Critical Reviews in Food Science & Nutrition
ISSN: 1040-8398 (Print) 1549-7852 (Online) Journal homepage: https://www.tandfonline.com/loi/bfsn20
Water activity, water glass dynamics, and thecontrol of microbiological growth in foods
Jorge Chirife , María del Pilar Buera & Dr. Theordore P. Labuza
To cite this article: Jorge Chirife , María del Pilar Buera & Dr. Theordore P. Labuza (1996) Wateractivity, water glass dynamics, and the control of microbiological growth in foods, Critical Reviewsin Food Science & Nutrition, 36:5, 465-513, DOI: 10.1080/10408399609527736
To link to this article: https://doi.org/10.1080/10408399609527736
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Critical Reviews in Food Science and Nutrition, 36(5):465-513 (1996)
Water Activity, Water Glass Dynamics, and theControl of Microbiological Growth in Foods
Jorge Chirife and María del Pilar Buera1
Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de BuenosAires, (1428) Buenos Aires, Argentina
Referee: Dr. Theordore P. Labuza, Dept. of Food Sci. and Nutrition, Ciudad Univ., Nunez, Buenos Aires, Argentina
1 Member of Consejo Nacional de Investigaciones Científicas y Técnicas, República Argentina.
ABSTRACT: Water is probably the single most important factor governing microbial spoilage in foods, and theconcept of water activity (aw) has been very valuable because measured values generally correlate well with thepotential for growth and metabolic activity. Despite some drawbacks (e.g., solute effect), the concept of aw hasassisted food scientists in their effort to predict the onset of food spoilage as well as to control food-borne diseasehazards in food products. In the last decade the concept of aw has been challenged. It has been suggested thatreduced-moisture food products (e.g., low and intermediate) may be nonequilibrium systems and that most ofthem are in the amorphous metastable state, which is very sensitive to changes in moisture content andtemperature. It has been proposed that the glass transition temperature Tg (temperature at which the glass-rubbertransition occurs), is a parameter that can determine many product properties, the safety of foods among them.The concept of water dynamics, originating in a food polymer science approach, has been suggested instead ofaw to better predict the microbial stability of intermediate-moisture foods. The usage of aw to predict microbialsafety of foods has been discouraged on the basis that (1) in intermediate-moisture foods the measured watervapor pressure is not an equilibrium one, and because aw is a thermodynamic concept, it refers only toequilibrium; and (2) the microbial response may differ at a particular aw when the latter is obtained with differentsolutes.
This review analyzes these suggestions on the basis of abundant experimental evidence found in theliterature. It is concluded that nonequilibrium effects (e.g., inability of water to diffuse in a semimoist food)appear to be in many cases slow within the time frame (food's shelf life) of the experiments and/or so small thatthey do not affect seriously the application of the aw concept as a predictor of microbial stability in foods.
The claims that a food science polymer approach to understanding the behavior of aqueous sugar glassesand concentrated solutions may be used to predict the microbial stability of food systems is not substantiated byexperimental evidence. This approach does not offer, at the present time, a better alternative to the concept ofaw as a predictor of microbial growth in foods.
It is also recognized that aw has several limitations and should be always used carefully, and this mustinclude precautions regarding the possible influences of nonequilibrium situations. This aspect may be summa-rized by simply saying that anyone who is going to employ the term water activity must be aware of theimplications of its definition.
KEY WORDS: water activity, water glass dynamics, microbiological growth.
I. INTRODUCTION
Water is probably the single most importantfactor governing microbial spoilage in foods andthe concept of water activity ( a j has been veryvaluable in physiological studies of microorgan-isms principally because measured values gener-ally correlate well with the potential for growth
1040-8398/96/S.50© 1996 by CRC Press, Inc.
and metabolic activity (Gould, 1985). The a , con-cept has assisted food scientists and microbiolo-gists in their efforts to predict the onset of foodspoilage as well as to identify and control food-borne disease hazards that might exist in variousfood products. The usefulness of a^ has beensomewhat diminished by the fact that measuredSLv, levels may not always be totally predictive of
465
microbial growth because the microbial responsemay differ at a particular a , when the latter isobtained with different solutes. Despite this occa-sional behavior, the aw concept has remained use-ful and the manipulation of a,, is an importantfactor for the control of microbial growth in food.Thanks to this concept, many processes could besuccessfully adapted and new products could bedesigned (van den Berg, 1991).
Despite its usefulness, the concept of aw as adeterminant of microbial growth in foods has beenchallenged (Slade and Levine, 1987; Franks,1991a,b; Slade and Levine 1991a,b). It has beensuggested over the last decade that certain foodproducts (mainly of the low- and intermediate-moisture type) are nonequilibrium systems andthat most of them are maintained or brought intoa state of thermodynamic metastability duringprocessing. However, pseudostability, because itmay last longer than the normal lifetime of the
product, would be preferable (van den Berg andBruin, 1981). This approach focused increasingattention on the dynamic aspects of food systems,rather than on equilibrium properties (Slade andLevine, 1991a). In many foods and biologicalmaterials the solids (either biopolymers or low-molecular-weight carbohydrates) are in an amor-phous metastable state that is very sensitive tochanges in moisture content and temperature(Slade and Levine, 1987; Levine and Slade, 1992;Roos and Karel, 1991a). This amorphous matrixmay exist either as a very viscous glass or as amore liquidlike, rubbery structure, as shown inFigure 1. The characteristic temperature, Tg, atwhich the glass-rubber transition occurs has beenproposed as a physicochemical parameter thatcan determine product properties, such as stabil-ity and safety of foods (Slade and Levine, 1987,1991a,b, Levine and Slade, 1992). Slade andLevine (1987) proposed new guidelines for more
Ä5-1
Tm dry
Melt
Rubbery state
Glassy state
Moisture content
FIGURE 1. A portion of a phase diagram showing the melting temperatures (Tm) and glass transition temperatures(Tg) of a crystallizing component as a function of moisture content.
466
credible criteria of stability and viability in inter-mediate-moisture systems to replace a , by usingtheir concept of water dynamics. Franks (1991a)stated that the assumption of a good correlationbetween measured a,,, and potential for microbialgrowth has led to "disastrous consequences in thereformulation of intermediate moisture foods tosome predicted 'safe' a , value, merely becauseglucose was replaced by fructose."
Slade and Levine (1987) and Franks (1991a)stated that in many product situations, equilib-rium thermodynamic descriptions are inappropri-ate, because the measured physical properties aretime dependent; thus, a,,, must not be used todescribe the attributes of these systems. The mea-sured a , relies on the existence of a liquid-vaporequilibrium; this situation may not occur whenthe substrate is a quasi-solid dispersion such as alow-moisture or even a semimoist food product.The high viscosity of the food system is related toa much slower rate of attainment of equilibrium;what is measured in such cases is a nonequilibriumrelative vapor pressure (RVP) and not a^. van denBerg and Bruin, 1981 suggested that in food sys-tems true equilibrium may not exist, but rathersome pseudoequilibrium exists, which is notclearly definable, but which may remain formonths in that condition. As a consequence, thethermodynamic a,, cannot be defined throughoutthe system, but rather an empirical pseudo-a^, isreflected in the system properties.
This review examines various aspects: (1) thesuggestions of Slade and Levine (1987, 1991a)that water dynamics may be used instead of a^ topredict the microbial stability of concentrated andintermediate-moisture food systems, (2) the sug-gested failure of a , as a predictor of microbialstability when glucose is replaced by fructose,and the behavior of fructose as an aw-loweringsolute in foods, and (3) the practical consequencesof nonequilibrium situations on a , values of foodsin the microbiological growth range.
II. EQUILIBRIUM IN WATER SORPTIONMEASUREMENTS
The main criticism of the utilization of aw inthe food area may be summarized as follows: Inlow-moisture and intermediate-moisture foods, the
concept of aw becomes meaningless because themeasured vapor pressure of water is no longer theequilibrium vapor pressure. At best, a stationarystate is reached under a given set of environmen-tal conditions and mistaken for equilibrium. Arecorded low value for water vapor pressure islikely to be due to the inability of water to diffuserapidly (within the observation period) through-out the substrate and to equilibrate with the vaporabove it (Slade and Levine, 1991a; Franks, 1991a).In moisture sorption studies the situation may befurther complicated if the amorphous materialundergoes a glass-rubber transition during thecourse of the measurement (Franks, 1991a). Thisis because the rates of diffusion-limited changes(e.g., moisture equilibration) are more rapid in therubbery liquid state than in the glassy solid state(Slade and Levine, 1991a). The analysis of vari-ous literature data may throw some light on theseaspects. However, in order to evaluate the litera-ture data, it is first necessary to have an idea of theusual precision in the experimental determinationof sorption isotherms. In a comprehensive col-laborative study within the framework of theEuropean Corporation in the Field of Scientificand Technical Research (COST), precision data(e.g., repeatability/reproducibility) in the deter-mination of sorption isotherms were determined.In a study with 24 laboratories the following pre-cision data were obtained for the adsorption iso-therms of macrocrystalline cellulose (MCC) andpotato starch. In an a„ range of interest to micro-bial growth (0.60 to 0.90) the average standarddeviation of all data was 2.6% on the equilibriummoisture content for MCC and 3.8% for potatostarch (Wolf et al., 1984). The repeatability (testsperformed at short intervals at one laboratory byone operator using the same equipment) was 2.0%for both MCC and potato starch. The reproduc-ibility (tests performed at different laboratories,which implies different operators and differentequipment) was 8.2 and 12.9% for MCC andpotato starch, respectively. Other authors deter-mined the repeatability of their isotherm data fordifferent foods and reported similar values(Andrieu et al., 1985; Macchia and Bettelheim,1964; Jayas and Mazza, 1991).
The definition of the equilibrium between foodsamples and the water vapor source is a critical
467
aspect in performing sorption isotherms. Practi-cally, the sorption process could be stopped whenthe weight difference after the final equilibriumbecomes less than the sensitivity of the balanceused. But even this stage may require a very longwaiting time; therefore, an apparent equilibriumstate is commonly defined in terms of a maximumtolerable weight change during an arbitrary time(Gal, 1981). This is the procedure usually fol-lowed in the literature. It is noteworthy that theequilibrium time will depend not only on waterdiffusion characteristics in the substrate, but alsoon mass transfer characteristics in the head spaceover the sample (Spiess and Wolf, 1985). Theheat and mass transfer within the equipment usedto determine the isotherms may or may not belower than in the product itself (King, 1968).
Lomauro et al. (1985) reported on a study totest an objective criterion for determining theequilibrium moisture content when sorption iso-therms are prepared. Foods of different physicalcharacteristics (flour, freeze-dried apple, freeze-dried turnip, freeze-dried ground beef, oatmealcookie, shredded wheat, and raisins) were ex-posed to an atmosphere of 75.2% RH at 25°C innonevacuated desiccators and the moisture con-tent recorded as a function of time. These authorsconsidered that a pseudoequilibrium was reachedwhen the moisture content (dry basis [d.b.]) didnot change more than 0.5% during three consecu-tive sample periods at not more than 7-d intervals.This criterion for equilibrium moisture contentwas compared with the values obtained after6-month storage in closed mason jars, which wereconsidered to be very close to the equilibriummoisture content. Lomauro et al. (1985) concludedthat the foods tested reached (or were very closeto) equilibrium within 1 month, based on theabove criterion. Also, the values compared well(differences between 0.5 and 3.4%) with the6-month-storage study. The differences betweenthe 1- and 6-month studies are in the range re-ported for the repeatability of isotherm determi-nations, as noted before.
Various authors reported their equilibriumtimes for isotherm determinations of different foodsystems using the gravimetric, static, method oversaturated salt solutions. Most authors utilized a
small amount of sample (1 to 3 g) to diminishwater transport resistances in the substrate andtheir equilibrium times ranged mostly between 1and 3 weeks, depending on temperature and rela-tive humidity (Lagoudaki et al., 1993; Valdez-Niebla et al., 1993; Hill and Rizvi, 1982; Engelset al., 1987; Hansen, 1971; Wedzicha and Quine,1983; Lima and Cal-Vidal, 1983; Kumar, 1974;Rasekh et al., 1971; Chhinnan and Beuchat, 1985;Hutchinson and Often, 1984; Mazza and Jayas,1991; Piñaga and Lafuente, 1965; Tsami et al.,1990; Bolin, 1980; Saravacos et al., 1986; Ayranciet al., 1990; Hayakawa et al., 1978). The datareported by Bizot et al. (1985) for starch are rel-evant to the present analysis. Bizot et al. utilizeda practical equilibration time of about 7 d (±0.02%water per 24 h) for a 1-g sample, but also storedtheir starch samples over saturated salt solutionsfor 2 years. They noted a slow drift of desorptionpseudoequilibria, but it was only 1% water con-tent (d.b.) after such a long time. On the otherhand, adsorbing samples remained stable, van denBerg (1991) reported that a 0.15-g sample of starchin a sorption balance under high vacuum exhib-ited equilibration times of 0.5 to 3 d, producingstates that were steady for up to several months.
From the above considerations it may be as-sumed that sorption determinations performed withthe usual precautions regarding a practical weightconstancy are likely to be close to equilibrium.The differences are probably within the uncer-tainties associated with the experimental determi-nation of isotherms (Wolf et al., 1984).
III. SORPTION ISOTHERMS OF FOODSCONTAINING CRYSTALLIZABLESUGARS
Dried fruits (e.g., raisins, figs, prunes, etc.)are good examples of nonequilibrium, intermedi-ate-moisture systems. Sugars (glucose, fructose,and sucrose) constitute about 50 to 83% of the drymatter (Tsami et al., 1990) and in the intermedi-ate-moisture range they are likely to be in anamorphous, metastable, rubbery state when storedat microbial incubation temperatures (Roos, 1987,1993; Chirife and Buera, 1994a). Sorption iso-
468
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İI5
•32 C ; Réf. (1)•
25 C ; Réf. (1)El
30 C ; Réf. (2)•
36 C ; Réf. (3)X
30 C ; Réf. (4)
0.4 0.5 0.6 0.7 0.8 0.9Water activity
FIGURE 2. Water adsorption isotherms in raisins: comparisons of several literature data. References: (1) Bolin,1980; (2) Saravacos et al., 1986; (3) Ayranci et al., 1990; (4) Tsami et al., 1990. (From Chirife, J. and Buera, M. P.,1995. J. FoodEng.)
therm data for raisins (glucose + fructose =83% d.b.) as reported by different workers arecompared in Figure 2. The agreement between alldata is good (considering the usual precision inthe reproducibility of isotherms) despite the factthat raisins constitute a nonequilibrium systemwith a high sugar concentration. Slade and Levine(1987) suggested, and Roos and Karel (1991a)confirmed, that sugar (sucrose) crystallization ispossible only above the Tg, and that the rate ofcrystallization depends on the difference betweenthe storage temperature and Tg (T-Tg). Crystalli-zation of amorphous sugars results in release ofmoisture and this increases product a , (Karel andRoos, 1993). However, the absence of dis-continuities in the isotherm of raisins is notice-able, suggesting that sugars remained amorphous,even at very large T-T , which would correspond
to moisture-temperature conditions for the micro-biological growth range (Chirife and Buera, 1994).This may be attributed to the presence of high-molecular-weight compounds in the raisins ma-trix, which delay (or inhibit) crystallization(Iglesias and Chirife, 1978).
Sorption isotherms of other fruits reported inthe literature do not show discontinuities (Tsamiet al., 1990), suggesting the absence (in the timeof observation) of sugar crystallization. Bolin(1980) reported a,, and equilibrium moisture val-ues for raisins sealed in glass jars and held at 21or 32°C for up to 12 months. His data for 21°C areplotted in Figure 3; it can be seen that storingraisins in sealed jars for several months had littleeffect on the moisture isotherm. This suggeststhat nonequilibrium effects (inability of water todiffuse rapidly) are very slow (in the time frame
469
60-
£ 50-•
TJ*«•$+J 40
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RAISINS, 21 C
Sugars : 83 % d.b.
0.2 0.3 0.4 0.5 0.6 0.7
Water activity0.8 0.9
Initial time
3 months
6 months
12 months
FIGURE 3. Effect of storage time at 21 °C on the adsorption isotherm of raisins. Based on data from Bolin, 1980.(From Chirife, J. and Buera, M. P., 1995. J. Food Eng.)
of these experiments) and/or small. Canelas et al.(1993) stored raisins (initial a^, = 0.61) at 4, 11,and 23°C for up to 11 months. Samples stored at11 and 23 °C showed an increase in aw from 0.61to 0.65 after 11 months. The sample stored at 4°Chad a lower variation, and after 8 months of stor-age it remained constant. It is noteworthy that anyobserved time dependency of measured RVP insemimoist systems may not necessarily indicatethe inability of water to diffuse rapidly within thefood. It may be also due to chemical changes thatmay occur during the time of observation. Canelaset al. (1993) stated that in stored raisins, the sug-ars (glucose and fructose) are involved in theMaillard reaction, and consequently a decrease insugar content was observed during storage. Thiswould increase the a ,; moreover, during thebrowning reaction, water is formed and this couldalso increase the aw. Because Maillard reactionshave a strong temperature dependence (Hendel et
al., 1955; Resnik and Chirife, 1979; Buera et al.,1987) it is expected that at 4°C reactions would beslower and a^ would remain almost constant, aswas experimentally observed.
IV. PREDICTION OF aw IN FOODS INTHE MICROBIOLOGICAL GROWTHRANGE
Franks (1991a) and Slade and Levine (1991a)stated that the high viscosity of certain semimoistsystems causes a very slow rate of attainment ofequilibrium, and what is measured in such casesis a nonequilibrium RVP and not aw. Franks(1991a) noted, however, that in simple solutions(liquid systems) the assumption of equilibrium(and hence the use of a,,) can be valid, because thediffusion rate of water molecules is high com-pared to the time scale of the measurement of
470
vapor pressure. Thus, it is interesting to compareexperimentally determined a^ in some semimoistfoods (nonequilibrium values) with predictionsmade from knowledge of the a , of simple solu-tions (equilibrium values) representative of theirmain soluble substances. In semimoist foods con-taining a relatively high proportion of solublesubstances (usually NaCl and/or low-molecular-weight sugars), a^ is mainly determined by themolality of solute(s) in the aqueous phase (Lupinet al., 1981). Predicted and measured a^ values inraisins in the intermediate-moisture range arecompared in Figure 4. The sugar content of rai-sins was (dry basis) 85.3% (Tsami et al., 1990),and the main sugars were glucose and fructose.Predicted values were calculated by applying theRoss equation (Ross, 1975; Chen, 1990) to thedata reported by Chirife et al. (1981a) for the a^
of glucose and fructose solutions. Good agree-ment between prediction and reality is observed.The goodness of this agreement should be judgedon the basis of experimental error in the determi-nation of sorption values. The comparisons wereextended to a wide variety of semimoist foodscomprising many physical states: solids (fish),pastes (ketchup and milk jam), jellies (fruit pre-serves and marmalades), emulsions (mayon-naises), and very concentrated liquid food solu-tions (sugar molasses and honey). The results areshown in Figure 5. A satisfactory agreement isobserved; slight discrepancies may be attributed,among other factors, to some inaccuracies in theuse of electronic hygrometers mostly used for theexperimental determination of a,, in semimoistfoods (see below). The observed agreement be-tween aw measurements in semimoist foods
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90-
80-
70-
60-
50-
40-
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Predicted (Glucose + Fructose)
00.3 0.4 0.5 0.6 0.7
Water activity0.8 0.9
•
Experimental - Adš.
Experimental - Des.
FIGURE 4. Comparison of predicted and measured water sorption in raisins at 30°C. (From Chirife, J. and Buera,M. P., 1994. J. FoodEng.)
471
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0.96-
0.92-
0.88-
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Ketchup/Mayonnaises•Milk jam
Moist salted fish13
Honey ("synthetic")
0.6 0.64 0.68 0.72 0.76 0.8 0.84 0.88 0.92 0.96 1Measured water activity
FIGURE 5. Predicted and measured aw values in various semimoist and concentrated foods. (From Chirife, J. andBuera, M. P., 1995. J. Food Eng.)
(nonequilibrium systems) and predictions madefrom equilibrium situations (liquid solutions) mayindicate that nonequilibrium effects (slow rate ofwater equilibration) are relatively small. At leastthe variations are comparable with the usual errorin the experimental determinations of a^
V. STABILITY OF METASTABLESYSTEMS
Some questions that may arise are (1) why, ifthe concept of a„ has been misleading, this pa-rameter has been such a helpful tool in predictingmicrobial stability in foods, and (2) why, ifsemimoist foods are nonequilibrium systems thatmay exist in different states of energy at differenttimes, their sorption isotherms also serve as valu-able information.
Equilibrium thermodynamics deals with theproperties of the macroscopic component in thestate of thermodynamical equilibrium. The ther-modynamical conditions corresponding to equi-librium lead to a minimum Gibbs free energy (G)and can be expressed by the equations
dG= 0;
92G>0
where T|G = order parameter (any parameter thatmeasures the order of the system).
In the case of the absolute minimum of G, wespeak of the stable equilibrium; in the case of alocal minimum, we talk about metastable equilib-rium. A schematic representation of true equilib-rium and metastable conditions is given in Figure6. The lifetime of metastable phases is related to
472
G (arbitrary units)
100
80
60
40
20
\ ™
\Melastable \phase \
I.."' /
VJStable
phase
00 10 20 30 40 50 60 70 80 90 100
^ o (arbitrary units)
FIGURE 6. Schematic representation of true equilibrium and metastable conditions. G, Gibbs free energy; T|G;order parameter (any parameter related to the order of the system); E , energy barrier.
the height of the energy barrier, E^, that the sys-tem must overcome to get from the local extremeto the true equilibrium. In many cases this life-time can be as long as thousands of years (e.g.,
glasses, frozen metastable phases of solid states;diamond [a metastable phase of graphite]), andmetastable phases that have specific physical prop-erties can be used in technical practice. These
473
states are stable with respect to small fluctuationsof external conditions. If the given system is in anonequilibrium state in consequence of externalconditions, those macroscopically observable pro-cesses take place within this object, and, thus, thestate variables are a function of time and spatialcoordinates. The process assuring that the systemreaches the state of thermodynamic equilibrium(within a certain interval of time) is called relax-ation. For various systems and processes the re-laxation time may considerably differ, for ex-ample from 1 x 10"6 s (realization of thethermodynamical equilibrium in ideal gases) toseveral years (impurity reached by diffusion inthe solid state). The mechanism of relaxation ofmetastable states is related to relatively large fluc-tuations and these states are stable with respect tosmall fluctuations. The metastable phase is in astate of partial thermodynamical equilibrium andit does not depend on the history of the system ifthe following relationship holds:
x i = L / V i « t e x p < x n
where L is the linear dimension of the system; V¡is the velocity of relaxation to the local equilib-rium relative to the i-th parameter, T¡ is the char-acteristic time of relaxation, texp is the character-istic observation time of the experiment, and tn isthe mean value of the creation of a new meta-stable nucleus (Chvoj and Kozisek, 1991).
The exact and full equilibrium phase diagramsof a given material can exist only at a theoreticallevel. In fact, the real experimental conditions —maintained within the measured sample with lim-ited accuracy — may differ, more or less, fromunambiguous equilibrium conditions required forconstruction of equilibrium phase diagrams. Whenthe rates of elementary (molecular) processes —which determine the state of the system at themacroscopic level — the principle of local ther-modynamic equilibrium is applicable (Chvoj andKozisek, 1991).
VI. THE aw BEHAVIOR OF FRUCTOSE:WATER DYNAMICS AND MOLDGERMINATION
Slade and Levine (1987) stated that there is a"controversial, contradictory, and confusing state
of the intermediate moisture food (IMF) literatureon use of fructose vs. glucose to lower aw inmoisture management applications." They added,"it has not even been possible for agreement to bereached in the IMF literature on the correct rela-tive vapor pressures (RVP) values for 50 and 60%fructose solutions." Chirife and Buera (1994a)recently showed that this is not true. The e^ offructose and glucose solutions up to about 50 to60% (w/w) may be considered identical (withinthe uncertainty [about ±0.005aw] associated withusual experimental determinations of a ,), as con-clusively shown in Figure 7.
On the basis of the supposed contradictorystate of the literature on the a„, of fructose vs.glucose solutions, Slade and Levine (1987) ap-plied the water dynamics theory to explain ex-perimental results on germination of Aspergillusparasiticus in various intermediate moisture solu-tions. A comparison was made between the in-hibitory effects on conidia germination for a se-ries of concentrated solutions of selected glassformers, such as glucose, fructose, mannose,maltose, sucrose, glycerol, and others. The vari-ous glass formers were assayed in pairs, deliber-ately matched with respect to the individual pa-rameters of approximately equal RVP. It waspointed out that the solute system with a lowerratio of melting temperature to Tg (Tm/Tg) in thedry state (0% moisture content; see Figure 1)showed faster germination regardless of RVPvalues. In addition, due to the inherent lack ofmobility of a sugar in its glassy reference stateand the lower local viscosity (and so greater trans-lational mobility) in its rubbery state, water avail-ability was greater for fructose (dry TJTg = 1.06)than for mannose (dry Tm/Tg = 1.36), which wasgreater than for glucose (dry Tm/Tg = 1.42), whichin turn was greater than for glycerol (dry Tm/Tg =1.62). Water availability was also greater for mal-tose (dry Tm/Tg = 1.27) than sucrose (dry Tm/Tg =1.43). Slade and Levine (1987) thus indicatedantimicrobial stabilization decreased in the orderglycerol > glucose > mannose > fructose, andsucrose > maltose, and concluded that their re-sults demonstrated conclusively that the observedrates of germination (as a mechanical relaxationprocess) could be interpreted by a conceptualapproach based on mobility in the concentratedrubbery sugar-water systems. A very unusual
474
CO
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1-r
0.98-
0.96-
0.94-
0.92-
0.9-
0.88-
0.86-
0.84-
0.82-
0.8
Chen (1987)(fructose)
I25 C
Chlrifeetal. (1981)
(glucose & fructose)
Teng&Lenzi(1974)(glucose)
Ruegg& Blanc (1981)(glucose & fructose)
Tokuoka & lshitani (1991)(glucose & fructose)
¿&
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70Solute concentration, % w/w
FIGURE 7. The aw of glucose and fructose solutions at 25°C: comparison of several literature data. (From Chirife,J. and Buera, M. P., 1994. J. Food Sd.)
behavior of A. parasiticus in concentrated fruc-tose solutions was reported; the same fast germi-nation time (2 d) in solutions of 40 to 70% (w/w)was observed and this was attributed to the ex-traordinary mobility and water availability of fruc-tose rubbers, as suggested by its low dry TJTg
value. Overall, it was concluded that the results ofthese biological experiments demonstrated con-clusively that the observed rates of germinationcould be interpreted by an approach based onmobility transformations and that water dynamicsmay be used instead of a^ to predict the micŕobialstability of concentrated and intermediate mois-ture systems. Such an all-embracing conclusionabout microbial stability (of utmost importancefor food preservation) cannot be done on the basisof limited experimental data (e.g., testing of onlyone mold strain, A. parasiticus [Slade and Levine,1987]). Chirife and Buera (1994a) challenged then-
results and conclusions using available literaturedata for microbial stability in IM systems and didnot confirm the conclusions of Slade and Levine.For example, germination times at 25°C ofPenicillum implication spores in media with a„controlled by either glycerol or a 1:1 glucose/fructose mixture are compared in Figure 8. Theglucose/fructose mixture evidences greater stabil-ity (e.g., slower germination) than glycerol, whichis contradictory to the above predictions of Sladeand Levine. Figure 9 shows colony diameter ofMonilinia fructicola after 14 d (25 °C) at variousa , regulated by the addition of glucose, fructose,or mannose in a defined medium. At the same a^and in terms of mobility, inhibition of colonygrowth diameter should follow the order glucose> mannose > fructose; however, experimentalevidence shows the opposite, that is, glucose gavethe greatest diameter. Figure 10 shows the effect
475
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45
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15-
10-
5
0-0.78
Pénicillium implication
25 C
Water activity0.92 0.94
FIGURE 8. Solute effect (glycerol or 1:1 glucose/fructose) on germination time at 25°C of Penicillum implication.(From Chirife, J. and Buera, M. P., 1994. J. Food Sci.)
EE
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100
90-
80-
70-
60-
50-
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20-
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Monilinia fructicola
Glucose
Fructose
040.9 0.91
Mannose
25 C
0.92 0.93 0.94 0.95 0.96 0.97 0.98 0.99Water activity
FIGURE 9. Solute effect (glucose, fructose, or mannose) on colony diameter of Monilinia fructicola after 14 d ofincubation at 25°C. (From Chirife, J. and Buera, M. P., 1994. J. Food Sci.)
476
700
600-
500-
400-
300-<Ö
100-
0
Rhizopus sp.
Glycerol
25 C
0.9 0.91 0.92 0.93 0.94 0.95Water activity
0.96 0.97 0.98
FIGURE 10. Solute effect (glucose or glycerol) on radial growth at 25°C of Rhizopus sp. (From Chirife, J. and Buera,M. P., 1994. J.FoodSci.)
of different solutes on the radial growth of Rhizo-pus sp. colonies at 25°C in defined media. Thegrowth behaviors of P. funiculosum at 25°C onagar malt with the a^ controlled by the addition ofeither glucose or fructose are compared in Figure11. None of the results observed agree with thepredictions of Slade and Levine; instead, glucoseis more inhibitory (in terms of radial growth) thanglycerol (Figure 11) and fructose produced anequal or slightly more stable system (slowergrowth) than glucose (Figure 10). In order toverify the experimental results reported by Sladeand Levine (1987) for mold spore germination inIM solutions, Chirife et al. (1994) determined thegermination time at 28°C of various mold spores(A. flovus, A. niger, and Eurotium herbariorum)in concentrated solutions of glucose, fructose,mannose, glycerol, sucrose, maltose, and propy-lene glycol. The results did not agree with thefindings of Slade and Levine; the expected orderof antimicrobial stabilization, glycerol > glucose
> mannose > fructose, and sucrose > maltose, wasnot observed. For example, Figure 12 shows theeffect of solute type on germination time (at 28°C)of A. niger, germination did not occur more quicklyin solutions of fructose than in solutions of glu-cose, mannose, or glycerol, nor did it occur morequickly in solutions of maltose than in solutionsof sucrose, as expected from mobility (e.g., dryTm/Tg values) considerations. Chirife et al. (1994)also determined the effect of fructose concentra-tion on germination time of A. parasiticus ascompared with data previously reported by Sladeand Levine (1987). Their results are shown inFigure 13; a strong influence of fructose concen-tration on germination time is observed, and at70% (w/w) concentration, germination was notobserved after 70 d of incubation. These resultscontrast sharply with those of Slade and Levine(1987), who reported the same fast (2 d) germina-tion time for A. parasiticus in solutions of 40 to70% (w/w) fructose and attributed this behavior
477
u
5
4.5-
4-
3.5-
1-
0.5-
0
Pénicillium funiculosum
Glucose-
met
ed
iai
Ión
y
oo
3-
2.5-
2-
1.5-
25 C
Fructose
6 8 10 12 14Incubation time, day
16 18 20
FIGURE 11. Effect of glucose or fructose ön radial growth of Penicillum funiculosum in malt extract agar at 25°C.(From Chirife, J. and Buera, M. P., 1994. J. Food Sci.)
to the extraordinary mobility and water availabil-ity of fructose solutions.
VII. REPLACEMENT OF GLUCOSE BYFRUCTOSE AND GLYCEROL BYPROPYLENE GLYCOL AND THEMICROBIAL STABILITY OFINTERMEDIATE-MOISTURE FOODS
Franks (1991a) stated that the assumption ofa good correlation between measured aw and po-tential for microbial growth has led "to disastrousconsequences in the reformulation of intermedi-ate moisture foods to same predicted 'safe' a ,value, merely because glucose was replaced byfructose." However, no reference to substantiatethese statements is given. In the same line ofthought, van den Berg (1985) stated:
It is not surprising therefore that in recent years mis-conceptions have led to some difficulties in the pres-ervation of intermediate moisture products. New prod-ucts were formulated based on acquired experience,setting safety margins on the basis of a„. The newproduct, however, although having the same aw as itssafe predecessor but with a slightly different humec-tant composition, spoiled on the shelf by microbialaction.
This statement was not supported by refer-ences. Slade and Levine (1991a) also criticizedthe utilization of a,, to predict the microbiologicalsafety of intermediate moisture foods, saying that"misconceptions have led to some difficulties inthe preservation of intermediate moisture foods,"and adding:
For example consider an intermediate moisture petfood product that was originally formulated with amixture of solutes predominated by glucose and glyc-
478
co
IQ)
(D
Sucrose Maltose Mannose Glycerol Fructose GlucoseSolute
| I Aw = 0.85 H u Aw = 0.90
FIGURE 12. Solute effect (sucrose, maltose, mannose, glycerol, fructose, or glucose) on germination time ofAspergillus niger at 28°C. (From Chirife, J. et al., 1994. Food Res. Int.)
erol. This commercial product was empirically deter-mined to be microbiologically safe and stable at an a„of 0.92, which was then incorporated as a productspecification. Then for the purpose of cost reductionthe glucose-glycerol combination was replaced byfructose and propylene glycol, but the a„ specificationwas not lowered in a corresponding and appropriatefashion but rather naively kept at 0.92. The financiallydisastrous result required a recall of millions of dol-lars worth of spoiled product.
However, they did not support these state-ments with any scientific/technical evidence todemonstrate that solute replacement and no otherfactors were responsible for the claimed failure.As far as known, a soft-moist dog food formu-lated to a^ 0.92 using glucose and glycerol cannotbe "microbiologically safe and stable" as claimedby Slade and Levine (1991a); it will be spoiled by
molds and also by several bacteria, the patho-genic Staphylococcus aureus, among them (Haasand Heran, 1978; Boylan et al., 1976). In the foodindustry, practical control of microbial growthseldomly is achieved by a , alone. In order toachieve microbial stability, parameters other thana„ need to be utilized and controlled. The param-eters include pH, antimicrobials, mild heat treat-ment, oxygen partial pressure, etc. and may bebest visualized, as suggested by Leistner et al.( 1981), as hurdles that microorganisms must over-come or, in some way, circumvent in order tocause deterioration of a food. If this were the case,one cannot say that solute replacement was thecause for the failure unless all other hurdles werealso checked for any unexpected modification,and this might be reported and substantiated bysound experimental evidence before using this
479
90
80-
& 7 ( H
• " . 60HO
ï= 50Hcoï 4(H1 30-
° 20H
10-
Aspergillus parasiticusNo germination after 70 days
Slade & Levine (1986)x
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80Fructose concentration, % w
FIGURE 13. Effect of fructose concentration on germination time of Aspergillus parasiticus at 28°C. (From Chirife,J. et al., 1994. Food Res. Int.)
pet food episode as a demonstration that the con-cept of a , was invalid. Nelson (1993) pointed outsome potential reasons that may contribute to adifference in microbial stability when differentsolutes are used to achieve the same a^ and whichdo not mean that the concept of a,,, is invalid, assuggested by Slade and Levine (1991a). One ofthe arguments of Slade and Levine (1991a) wasthat propylene glycol has a much lower dry TJTg
ratio (1.27) than does glycerol (1.62), and, there-fore, according to their water dynamics approach,a lower RVP (or a,,) at an equivalent solute con-centration does not correlate with greater micro-biological stability for propylene glycol vs. glyc-erol. Chirife and Buera (1994) formulated an IMdog food (meat and bone meal, soybean flour,precooked corn, rice and wheat flour, oats, animalfat, minerals, and vitamins) using glucose/glyc-erol to depress aw in one case and fructose/propy-lene glycol in another. The samples were pack-
aged in moisture-proof pouches and stored at 34°C.After 3 d of storage, incipient mold growth wasvisually observed on the surface of samples for-mulated with glucose/glycerol and on the fifthday all the samples formulated with glucose/glyc-erol were totally spoiled by molds. On the con-trary, the samples formulated with fructose/pro-pylene glycol remained free of visible molds after20 d of incubation (Figure 14). These results areexactly the opposite of the predictions of Sladeand Levine (1991) made on the basis of the waterdynamics theory. Chirife et al. (1994) reportedstudies on the germination time of various moldspores in solutions of propylene glycol and glyc-erol at an aw of 0.90. Water dynamics predicts thata propylene glycol solution would have a poorermicrobiological stability than glycerol at anequivalent solute concentration. However, theresults of Chirife et al. (1994) showed exactly theopposite; all molds (A. niger, A. flovus, and
480
SEMI-MOIST DOG FOODStored 20 days at 34 C
Water Activity (Aw) - 0.89
Glucose/Glycerol Fructose/Prop.glycol
FIGURE 14. Mold spoilage in an IM dog food (aw = 0.89) formulated with glucose/glycerol or fructose/propylene glycol after 20 d of incubation at 34°C.
oo
Eurotium herbariorum) germinated readily inglycerol (1 to 2 d) but none of the molds were ableto germinate in propylene glycol after 70 d ofincubation at 28°C. This is simply because thebehavior of propylene glycol cannot be explainedsolely on the basis of mobility or aw effects alone,because this molecule possesses specific antimi-crobial effects largely known in the literature (Haasand Herman, 1978; Ballesteros et al., 1993).Gervais et al. (1992) also showed that Penicillumroquefortii and Trichoderma viride did not ger-minate in propylene glycol solutions of similara,,. These results are a dramatic demonstrationthat mobility effects per se are not able to explainthe biological response of a microorganism tochanges in its aqueous environment.
It may be concluded that the claims of Sladeand Levine that water dynamics may be used topredict microbial stability of concentrated andintermediate moisture food systems are not sub-stantiated by sound experimental evidence.
VIII. aw AS A DETERMINANT OFMICROBIOLOGICAL GROWTH
The definition of a moisture condition at whichpathogenic or spoilage microorganism cannotgrow is of paramount importance in food preser-vation and it has been widely recognized that theconcept of aw has been a valuable tool in thisaspect. Thanks to this concept, many processeshave been adapted successfully and new productsdesigned (van den Berg, 1991). It is accepted thatmeasured values of aw generally correlate well
with the potential for growth and metabolic activ-ity of microorganisms (Gould, 1985; 1988; Gouldand Christian, 1988). Its measurement has beenvery valuable for predicting the microbial stabil-ity (and safety) of foods (Leistner and Rodel,1975; Troller and Christian, 1978; Leistner et al.,1981; Silverman et al., 1983; Dodds, 1989; Glassand Doyle, 1991). It has been shown repeatedly inthe literature that each microorganism has a criti-cal a,, below which growth cannot occur (Brown,1974; Troller and Christian, 1978; Gould andMeasures, 1977; Troller, 1971; Leistner et al.,1981; Beuchat, 1983). For example, pathogenicbacteria cannot grow below an a^ of 0.85 to 0.86;yeasts and molds are more tolerant to reduced a ,,but usually no growth exists below a» of about0.62 (Scott, 1953; Hocking and Pitt, 1979;Beauchat, 1993; Silverman et al., 1983; Leistner,1987; Jermini and Schmidt-Lorenz, 1987;Ballesteros et al., 1993). Table 1 shows somecritical a„ values regarding the safety of foods(from various literature sources). Table 2 showsthe minimal a , for growth of food-borne bacterialpathogens at optimum pH, temperature, and sourceof nutrients. Many workers concluded that a„ is amain determinant of growth because it determinesthe osmotic stress and because the ability to growis determined by the degree of that stress and theosmoregulatory capacity of a particular microbialcell. A fundamental requirement for growth ofmicroorganisms on substrates of high osmolalityis the intracellular accumulation of solutes, eitherby transport or synthesis, to concentrations thatcounterbalance the osmolality of the externalmedium (Prior et al., 1987; Hocking, 1988; Chirife
TABLE 1Some Critical aw Values Regarding the Safety of Foods
Minimal aw
0.94
0.91
0.85-0.86
0.80
Growth and toxin production by alltypes (A, B, C, E, and G) of Clostridiumbotulinum
Anaerobic growth of Staphylococcusaureus
Aerobic growth of Staphylococcusaureus
Production of micotoxins
482
TABLE 2Minimal aw
a for Growth of Food-BornePathogens in Laboratory Media (at Optimum pHand Temperature)
Pathogen Minimal aw
Campylobacter jejuni 0.990Aeromonas hydrophilia 0.970Clostridium botulinum E 0.965Clostridium botulinum G 0.965Shigella spp. 0.960Yerslnia enterocolitica 0.960Clostridium perfringens 0.945Clostridium botulinum A and B 0.940Salmonella spp. . 0.940Escherichia coli 0.935Vibrio parahaemolyticus 0.936Bacillus cereus 0.930Listeria monocytogenes 0.920Staphylococcus aureus (anaerobic) 0.910
Staphylococcus aureus (aerobic) 0.860
a Usually adjusted by the addition of sodium chloride.
From Chirife, J., 1993. Food Control, 4:210-215.
et al., 1981b); this process is often referred to asosmoregulation. Chirife et al. (1981b) calculatedintracellular aw from the solute composition ofvarious bacterial cells (halophilic and nonhalo-philic) grown in media of a, between 0.85 and0.993 and found that the intracellular a^ was gen-erally equal or slightly lower than that of thegrowth medium. Their results are shown in Fig-ure 15. The biochemical basis of osmoregulationhas been the subject of numerous studies in thelast decade (Anderson and Witter, 1982; Csonka,1989; Landfald and Strom, 1986; Larsen et al.,1987; Gervais et al., 1992).
Franks (1991a) and Slade and Levine (1991a)have questioned the usefulness of specificationsof values of a,,, required for microbial safety andstability of semimoist foods on the basis of the so-called specific solute effect. Since the early re-ports by Scott (1953), it has been known that themicrobial response may differ at a particular aw
when the latter is obtained with different solutes;and it has been established that the aw of themedium is not the only determining factor regu-lating the biological response, but that the nature
of the aw-controlling solute also plays a role (Chris-tian, 1981; Gould, 1988; Ballesteros et al., 1993).Gould (1985) acknowledged that in some instancessolute effects may depend on the ability of thesolute to permeate the cell membrane. Glycerol,for example, readily permeates the membrane ofmany bacteria and so does not initiate the sameosmoregulatory response as nonpermeant solutessuch as sodium chloride and sucrose, and there-fore has a different (usually lower) inhibitory a^.Table 3 shows this effect for many pathogenicbacteria; with the exception of Staphylococcusaureus, for which the reverse is true, bacteria aresomewhat more tolerant to glycerol (lower mini-mal aw) than to sodium chloride. The situation ofS. aureus is discussed later in more detail. How-ever, when the solutes most often present in re-duced aw-preserved foods (e.g., sodium chloride,sucrose, glucose, and potassium chloride) are usedto control aw, specific solute effects are less evi-dent, as shown in Table 4. Christian and Scott(1953) studied the growth of Salmonellaoranienburg in B.H. medium, using three differ-ent methods for controlling a^: (1) adjusting the
483
0 8 0 0-85 090Medium a w
0 9 5
FIGURE 15. Comparison of intracellular and growth medium a„ for various halophilic and nonhalophilic bacteria.(From Chirife, J., et al., 1981. J. Appl. Bacterio!., 50:475-479.)
water contents to the desired a ,; (2) adding theappropriate amounts of an NaCl:KCL:Na2SO4
(5:3:2) mixture to a basal B.H. medium; and (3)adding sucrose to the basal medium. Growth wasinhibited in all three media at s^ values between0.95 and 0.94. These data are relevant to foodpreservation because the solutes for which thespecific effects are more evident, such as glyc-erol, propylene glycol, butylène glycols, polyeth-ylene glycols, and the like, have at present littlechance to be used for a,, control in human foods,either because of regulatory or of consumer de-mands for the so-called green label foods. Franks(1991a) and Slade and Levine (1991a), suggested
that because microbial growth is subject to spe-cific molecular and/or ionic interactions whetherthere is some other parameter or set of param-eters, it could form the basis of a criteria of cellactivity as influenced by water and the aqueousenvironment. In other words, specific effects mustbe taken into account in practical situations whereit is desired to relate microbial growth to thenature of the aqueous environment. Includedamong these factors are a parameter to measurethe permeability of the membrane to solutespresent and a factor that takes the specific toxiceffects of the molecules into account (Gould andChristian, 1988). Unfortunately, at present there
484
TABLE 3Solute Effect (Sodium Chloride vs. Glycerol) on the Minimalaw Supporting Growth of Pathogenic Bacteria in LaboratoryMedia8
Bacteria
Clostridium botulinum EClostridium botulinum GEscherichia coliClostridium perfringensSalmonella spp.Clostridium botulinum A and BVibrio parahaemolyticusBacillus cereusListeria monocytogenesStaphylococcus aureus
a , adjusted
NaCI
0.9660.9660.9490.9450.9410.9400.9320.9300.9200.860
with
Glycerol
0.943
0.9400.930
0.9300.9110.9200.9000.890
The a„ values quoted by different authors were checked using theoreti-cal models for the prediction of aw in aqueous solutions.
TABLE 4Minimal aw for Growth of Pathogenic Bacteria in Laboratory Media of aw
Adjusted with Salts (NaCI, KCI) or Sugars (Sucrose, Glucose)
Bacteria NaCI KCI
Listeria monocytogenes 0.92 —Vibrio parahaemolyticus 0.936 0.936Clostridium botulinum G 0.965 —Clostridium botulinum E 0.972 0.972Clostridium perfringens 0.945 —Staphylococcus aureus 0.864 0.864
From Chirife, J., 1993. Food Control, 4:210-215.
Sucrose
0.867
Glucose
0.920.9400.9650.972
———
0.9750.945
is no such parameter or set of them to replace (orto improve) the usage of aw.
The effect of the solute used to adjust the a ,on the minimal a^, for growth of Staphylococcusaureus has been reviewed recently by Chirife(1994). For some solutes such as sodium chlorideand sucrose the minimal SL^ supporting growth isin the vicinity of 0.86; however, the minimal a„allowing growth was well above 0.86 when sol-utes such as alcohols, diols, or polyethylene gly-cols were used to control a^ This effect is shownin Table 5. Values range from as high as 0.975 forethanol to as low as 0.86 for sucrose and sodiumchloride. For various substrates (sodium chloride,
salts mixture, sucrose and salts, dried soup, anddried milk) in which the minimal a , for growth isrelatively close to 0.86, aw is certainly a muchbetter indicator than moisture content, becausethe latter shows a pronounced variation in thedifferent systems, as also shown in Table 5.
Slade andLevine (1991a) strongly questionedthe usefulness of specifications of values of a,,required for microbial safety of IMF, for example,a^ = 0.85 for the growth of pathogenic bacteria(S. aureus) on the basis of specific solute effects.Chirife (1994) also examined the growth behav-ior of 5. aureus in solutions whose a , values werecontrolled using 16 different solutes comprising
485
TABLE 5Minimal aw (awm) for Growth of S. aureus at 30 to 37°C
Moisture content for awm = 0.86(g H2O/100 g solids)
Ethanol1,3-Butylene glycolPropyleneglycolPEG-200PEG-400GlycerolSodium acetateDried meatGlucose + saltsSucroseSucrose + saltsSalts mixtureDried milkDried soupNaCI
0.9750.970.960.930.930.890.880.880.880.8670.860.860.860.860.86
74.2312
15.162.2
445.3
From Chirife, J., 1994 J. Food Eng., 22:409-419.
inorganic and organic salts, sugars, polyols, etc.,including solutes that are unlikely to be ever usedin foods. He reported that although the minimala^, for growth depended in various cases on thesolute used to adjust it (as noted above; Table 5),S. aureus could not grow below the current widelyaccepted minimal a,, = 0.86; this is a result of theutmost importance regarding food safety and ac-tual food regulations. Ballesteros et al. (1993)studied specific solute effects on S. aureus cellssubjected to reduced aw activity controlled by theaddition of solutes such as sodium chloride, su-crose, propylene glycol, butylène glycol, and vari-ous polyethylene glycols. As a first approxima-tion they considered other modifications broughtabout by dissolution of solutes (in the growthmedium) in addition to lowering of a , and spe-cific interactions between cell and solute. Theyreported that there is not a clear relationship be-tween the S. aureus response to solute dissolutionand the modification of certain physical proper-ties of the medium (viscosity, dielectric constant,oxygen solubility, and oxygen diffusivity). A trans-mission electron microscopic study revealed thatthe inhibitory effects of sucrose and sodium chlo-ride (important food solutes) on S. aureus wereprimarily ascribed to their aw lowering abilities,
showing no significant specific solute effects.However, the other solutes examined showedspecific antibacterial activity against S. aureus,which may be compatible with cell wall attack.
IX. USE OF aw AS A PREDICTOR OFMICROBIAL STABILITY IN ACTUALFOODS: CHALLENGE OF MINIMAL aw
LIMITS FOR GROWTH
Minimal aw values for growth of differentmicroorganisms have been mostly determined inliquid laboratory media, using sodium chloride todepress the a„ (Baird-Parker and Freame, 1967;Briozzo et al., 1986; Emodi and Lechowich, 1969).As discussed in other sections of this review,there is no doubt that measured RVP in liquidlaboratory media are equilibrium ones and thus^ = p/Po> because the viscosity of NaCI solutionsis very small compared with the values corre-sponding to the onset of rubbery, nonequilibriumbehavior (Soesanto and Williams, 1981). How-ever, if we accept the idea that in a solid semimoistfood, equilibrium may not be established duringthe time of observation (Slade and Levine, 1991a),the recorded vapor pressure will be mistaken forequilibrium, and this will create some concerns
486
about the safety of measured a„, values in foodswith regard to microbial growth inhibition. Thissituation may be explored using literature data.
It has been well established that the minimuma^ for the growth of Clostridium botulinum typesA and B in liquid broth media adjusted with NaClis 0.94 to 0.95 (Baird-Parker and Freame, 1967;Ohye and Christian, 1966). Glass and Doyle (1991)confirmed this minimum value of 2^ for solidfoods in a study of the relationship between aw offresh pasta (meat- or cheese-filled tortellini andflat noodle linguine or fettucini) and toxin pro-duction by Clostridium botulinum. Four types offresh pasta were prepared with different a, inocu-lated with C. botulinum, packaged under modi-fied atmosphere, and stored at 30°C for 8 to 10weeks. The pH of all samples was favorable toC. botulinum growth. No toxin was detected intortellini with an a„ of 0.94 when held at 30°C for10 weeks. Toxin was produced at 2 weeks inlinguine at an a,, of 0.96 held at 30°C, whereaslinguine or fettucini at an a„ of 0.93 or 0.95 andheld at 30°C did not become toxic during 8 to 10weeks incubation. Glass and Doyle (1991) con-cluded that the a„ of fresh pasta is a principalfactor in preventing botulinal toxin production intemperature-abused products. These results are ingood agreement with predictions made from thebehavior of C. botulinum in liquid broth media ofknown aw and suggest that, at least within the timeframe of the experiments, nonequilibrium effectsin the determination of a , of pasta are not signifi-cant. Dodds (1989) reported a study of the effectsof a , on toxin production by C. botulinum incooked, vacuum-packaged potatoes whose a, , wascontrolled by the addition of NaCI and which wasincubated up to 60 d at 25°C. Toxin was producedat an a , of 0.96, but no toxin was detected whenthe aw was 0.955, in good agreement with predic-tions made from the behavior of the bacterium inliquid broth media. These results also suggest thatin the time frame of the experiments (which insome cases may be coincident with the shelf lifeof the product) nonequilibrium effects do not sig-nificantly influence the determination of aw inpotatoes, and, consequently, the bacterial responsemay be predicted adequately. Valik and Gorner(1993) studied the growth of S. aureus in pastadough in relation to its a , and found that thebacterium appeared to multiply until the a^ was
below 0.86, at which stage it decreased; this is ingood agreement with the minimal water activity(a«, = 0.86) for growth of S. aureus determined inliquid broth media adjusted with NaCl (Vaamondeet al., 1982; Chirife, 1994). Giannuzzi and Parada(1991) studied the behavior of S. aureus (threestrains) in dehydrated milk, beef, and pork equili-brated at aw values of 0.84 and 0.90 and incubatedat 30°C for up to 30 d. No growth was observedin any of the foods at aw = 0.84; however, growthoccurred in all systems at a , = 0.90; this agreeswith the known behavior of S. aureus in liquidbroth media. Silverman et al. (1983) reported thata„ was the main parameter in controlling 5. aureusgrowth in precooked bacon. They reported thatthe limiting a, for growth of S. aureus A-100 inbacon sealed in cans and stored up to 28 d at 37°Cwas 0.87 in good agreement with known behaviorin liquid broth media, as noted above. King et al.(1984) studied the effect of a,, on mold growth instored almond nutmeat. The nutmeats were equili-brated at different a,,, values by placing in closedjars over saturated salt solutions; equilibrationtime was 1 month. No mold growth was observedafter 18 months on almonds stored at a, , = 0.70,which is in good agreement with predictions madefrom the behavior of molds in laboratory media ofcontrolled a^ (Beuchat, 1983).
X. THE SAFETY OF aw ADJUSTMENT/MEASUREMENT FOR THE CONTROLOF BACTERIAL GROWTH IN FOODS
For foods in which the a„ is a main factorcontrolling development of microbial hazard orspoilage, one must be certain that samples do notexceed a specified aw. In this regard it is importantto examine some aspects concerning the safety ofaw adjustment/measurement for the control ofmicrobial growth in foods. These include the ef-fect of processing and storage conditions on sub-sequent a^, the utilization of proper values for thelimiting e^ (solute effect; already discussed), andthe error in the measurement of a,, (Chirife, 1993).For years now, mechanical, electrolytic, capaci-tance, or dew-point hygrometers have been usedmostly to measure the a„ of foods. Recently, anew instrument for determining a^ in meat prod-ucts has been introduced (Rodel et al., 1990). It isbased on the thermometric determination of the
487
initial freezing point and is marketed in Germany(NAGY aw-Kryometer). Another instrument alsointroduced recently to the market is the WaterActivity Meter manufactured by Ottawa Instru-mentation Ltd. (Canada). It consists of a thermo-metric device that unequivocally shows whethersamples complied with a standard (Sharp et al.,1991). It is claimed that neither devices (NAGYaw-Kryometer and Ottawa a^Meter) are affectedby nonaqueous volatiles from foods (Rodel et al.,1990; Sharpe et al., 1991). For most electricalhygrometers, the confidence interval (with threeto four replicate measurements) is in the range of±0.005 (Labuza et al., 1976; Troller, 1977; Favettoet al., 1983; Kitic et al., 1986). However, collabo-rative studies of aw measurements in differentfood systems showed a larger discrepancy, forexample, in the range ±0.01 to ±0.02 (Labuza etal., 1976; Aguilera et al., 1990). It was generallyagreed that a,, results must be reported to only twodecimal places (Troller and Scott, 1992). Severalfactors are responsible for this error in the mea-surements, including (1) sensor contamination withnonaqueous food volatiles such as propylene gly-col, ethanol, acetic acid, etc. (Pollio et al., 1986);(2) lack of agreement between different laborato-ries on values to be assigned to certain referencestandards of saturated salt solutions; and (3) sen-sor calibration problems. Resnik et al. (1984)performed a world survey of s^ at 25 °C of se-lected saturated salt solutions used as standards inthe range of microbial growth; their results showedthat although good agreement was observed onthe value assigned to certain salts, a significant
discrepancy in the values assigned to others ex-isted. Chirife et al. (1983) used a theoretical ap-proach based on the thermodynamic properties ofstrong electrolyte aqueous solutions to predict thea, of saturated solutions. Table 6 shows calcu-lated values for some selected salt solutions. In anattempt to reach a worldwide agreement on the a ,of standards used for calibration, Resnik andChirife (1988) compiled and proposed a set oftheoretical aw values for saturated salts, sulfuricacid, and sodium chloride solutions to be used asstandards in the range of microbial growth. Someof their results are shown in Table 7.
Sensor contamination with nonaqueous foodvolatiles is also a problem in a , measurements.Although some manufacturers provide filters toprotect the sensor, the use of these filters usuallyincreases the equilibrium time for a , readings dueto additional resistance to mass transfer in the gasspace between the sample and the sensor.
In order to take into account the error in-volved in the measurement of a„, in foods, it isconvenient to set a safety margin in the selectionof the working aw value, for example, ±0.01 to±0.02.
XI. GLASS TRANSITION IN SOLIDAMORPHOUS FOODS ANDBIOPOLYMERS: DOES GLASSY STATEPREVENT THE GROWTH OFMICROORGANISMS?
The characteristic temperature, Tg, at whichthe glass-rubber transition occurs has been pro-
TABLE 6The aw (at 25°C) of Selected Saturated Salt Solutions Used as Standards in the Range ofMicrobial Growth
Salt
NaBrNaCI(NH4)2SO4
KCIBaCI2KNO3K2SO4
Robinson and Stokes, 1965
0.5770.7530.8010.8430.9020.925
a From Resnik et al., 1984.b From Chirife et al., 1983.
Most accepted8
0;5770.7530.8010.8430.9020.9250.972
Predicted"
0.7510.8030.8430.903
0.975
488
TABLE 7Predicted aw
Cone.(% w/w)
0.51.01.52.02.53.03.54.04.55.05.56.06.5
Values of Saturated and Nonsaturated Solutions
Predicted aw Values
0.9970.9940.9910.9890.9860.9830.9800.9770.9730.9700.9670.9640.960
Cone.(% w/w)
7.07.58.08.59.09.5
10.010.511.011.512.012.513.0
of NaCI
a«
0.9570.9540.9500.9460.9430.9390.9350.9310.9270.9230.9190.9150.911
Solutions between
Cone.(% w/w)
13.514.014.515.015.516.016.517.017.518.018.519.019.5
15 and
aw
0.9060.9020.8970.8920.8880.8830.8780.8730.8670.8620.8570.8510.845
50°C
Cone.(% w/w)
20.020.521.021.522.022.523.023.524.024.525.025.526.0
aw
0.8390.8330.8270.8210.8150.8080.8020.7950.7880.7810.7740.7660.759
Predicted aw Values of Selected Saturated Salt Solutions between 10 and 37°C
Temp.NaCI (NH4)2SO4 KCI BaCI, K2SO4
10151719212325272931333537
0.7540.7530.7530.7520.7520.7510.7510.7500.7500.7500.7490.7490.748
0.8090.8080.8060.8050.8040.8030.8030.8020.8010.8000.7990.7980.797
0.8670.8590.8560.8520.8490.8460.8420.8400.8360.8330.8300.8270.823
0.9130.9100.9090.9070.9060.9050.9030.9020.9000.8990.8980.8950.894
0.9800.9790.9780.9770.9770.9760.9750.9750.9740.9730.9730.9720.971
From Resnik, S. L. and Chirife, J., 1988. J. Food Protect, 51:419-423.
posed as a physicochemical parameter that candetermine product properties, stability, and safetyof foods (Slade andLevine, 1987; 1991a; Levineand Slade, 1992). In a glass (at T < Tg), the ratesof all diffusion-limited processes are much lowerthan in the rubbery state (at T > Tg). van den Berg(1991) noted that according to Slade and Levine'sarguments, a food taken to below Tg should beperfectly stable in terms of any ordinary foodchain from producer to the consumer, and the useof the Tg-water content relationship for the con-sidered food product could eliminate the neces-sity of using aw to predict food stability. Gould
and Christian (1988) stated, however, that althoughhigh-viscosity states would greatly interfere withthe growth of microorganisms in foods (for in-stance, through the restriction of diffusion of nu-trients), this is an area where much remains to bedone in order to identify the potential for deliber-ate use of high-viscosity states to inhibit micro-bial growth. In recent years a considerable amountof data have been reported on the effect of mois-ture content on the Tg of food systems. These dataoffer the opportunity to challenge the role of theglassy state on microbial growth inhibition, be-cause above Tg, mobility is greatly increased. The
489
difference between the storage temperature andTg (T-Tg) has been used to correlate changes inmechanical properties above Tg (Soesanto andWilliams, 1981). Moisture content profoundlyaffects Tg in foods (Slade and Levine, 1987), andthe rates of diffusion-limited processes would bemuch more rapid in the rubbery liquid state (gov-erned by WLF kinetics [Karel et al., 1993]) thanin the glassy solid state (governed by Arrheniuskinetics [Slade and Levine, 1991a]). The behav-ior of Tg vs. a^ for some plant materials (carrots,strawberries, potatoes, onions, and apples) andbiopolymers (wheat starch, wheat gluten, andgelatin) is shown in Figure 16. Figure 17 showsthe effect of moisture content on Tg of apple andonion. It is noticed that values for biopolymersare much higher than for plants (Figure 16). Al-though some biopolymers such as starch havehigh Tg values even at high values of aw, it islikely that in many real foods the presence ofrelatively small amounts of low-molecular-weight
solutes (e.g., sugars) would depress the Tg. Figure18 shows the effect of low-molecular-weight sol-utes on the Tg values of some biopolymers. TheTg of amylopectin and maltodextrin DE 10 arereduced by the addition of 9% fructose or 7.5%(total solid basis) of D-xylose and lysine. Forexample, the addition of 7.5% of lysine and D-xylose reduced the Tg of maltodextrin by about 15to 23°C. The glass curves of potato and starch arecompared in Figure 19; despite the fact that starchis the main component of its dry matter, the Tg ofpotato is much lower than that of starch. This maybe attributed to low-molecular-weight solublecomponents (glucose, fructose, sucrose, and min-erals) present in potato tissue. It is interesting tonote that the behavior of sorption isotherms is notinfluenced in the same manner; as shown in Fig-ure 20, the adsorption isotherms of potato starchand potato tissue are not very different.
Chirife and Buera (1994) combined literaturedata on sorption isotherms with the particular
Wheat Starch
Potatoes
Carrots
0.3 0.4 0.5 0.6 0.7 0.8 0.9Water activity
FIGURE 16. Relationship between aw and Tg of various plant materials and biopolymers. (From Chirife, J. andBuera, M. P., 1995. J. Food Eng.)
490
üÓ)
30-
20-
10-
0
-10-
-20-
-30-
-40-
-50-
-600
Apple
Aw = 0.56
Onion
Aw = 0.60
10 15 20Moisture content, d.b, %
25 30
FIGURE 17. Effect of moisture content on Tg of apple and onion. (From data reported by Nelson, 1993 and IglesiasandChirife, 1982).
Tg-moisture content curve of different food sys-tems and the corresponding T-Tg was calculatedfor a typical incubation temperature (30°C) andfor selected ranges of a,,,. Their results are shownin Table 8; additional data for celery (Karel et al.,1993), bread (Le Meste et al., 1992), casein(Kalichevsky et al., 1993), onion, and apple(Nelson, 1993) are included. First of all, it shouldbe indicated that it is not easy to separate theindividual effects of aw and physical state (glassyor rubbery) on microbial growth because in manyfoods the matrix is glassy at a m.c. at which the a,,is below the limiting value for growth. For mois-ture contents (or a ,) typical of the intermediate-moisture range, all foods examined (cabbage,potato, horseradish, strawberries, carrot, celery,onion, and apple), with the exception of bread, arein the rubbery state, as indicated by the corre-sponding values of (T-Tg). In fact, the values ofT-Tg may well be approaching 90 to 100°C for a,,
of about 0.8, a value at which many microorgan-isms cannot develop due to the osmotic stress.The exception is bread, which is in the glassystate even at a,, as high as 0.85. For the isolatedbiopolymers the behavior is diverse. Collagenand maltodextrin are in the rubbery state in prac-tically the entire intermediate-moisture range, butfor casein and starch, the glassy state covers mostof the IM range. Other biopolymers are in theglassy state in the vicinity of a^ = 0.65, but theyare rubbery above an a„ of about 0.8.
Figure 21 shows a comparison of the sorptionisotherms of strawberries (Roos, 1987) and prunes(Tsami et al., 1990); some selected values of T-T„ for strawberries at 25 °C (calculated from T.values measured by Roos [1987]) are indicatedon the sorption isotherm for strawberries. Bothisotherms are similar and this is expected becausestrawberries and prunes have about the same sugarcontent (50 and 54% d.b., respectively). Dried
491
Ü
23
120-110-
. 100-90
era
D.E<u1-cg» •
nsii
(0ŕW(0(S
80
70
60
50
40
3020
10
0-10
Amylopectin
Maltodextrin +
xylose (3.5%) +
lysine (3.5%) Amylopectin + 9% fructose
0 10 15 20 25 30Moisture content, % d.b.
35 40
FIGURE 18. Effect of the addition of low-molecular-weight compounds on the glass curves of amylopectin-waterand maltodextrin DE 10-water. (From Chirife, J. and Buera, M. P., 1994. J. Food Eng.)
fruits are nonequilibrium systems because thesugars are in an amorphous metastable state thatis very sensitive to changes in moisture contentand temperature; however, they are usually main-tained in a state of pseudostability because it maylast longer than the product's lifetime. Crystalli-zation of the amorphous sugars are probably pre-vented by the nonsugar biopolymer matrix(Iglesias and Chirife, 1978). It may be assumedthat the Tg-moisture content relationship for prunesand strawberries are similar; thus one may expectthat the value of T-Tg for prunes at, for example,20% d.b., which corresponds to a,, = 0.62 (seedotted line in Figure 21), should be higher than59°C. At this value the food matrix is highlyplasticized by water and mobility effects (e.g.,diffusion) are greatly enhanced. Nevertheless, atthis moisture content prunes are resistant to mi-crobial growth (Pitt and Christian, 1968) so it is to
be concluded either that (1) if molecular mobilityis determined by T-Tg, this effect plays little roleon microbial growth inhibition (a , seems to bethe controlling parameter) or (2) molecular mo-bility is still reduced at high values of T-Tg, sogrowth does not occur, in which case T-Tg doesnot determine mobility. This situation is illus-trated in Figure 22 in which "transformed" (Sladeand Le vine, 1991a) selected water sorption datafor prunes and the glass curve for strawberries(which is likely to be similar for prunes [Karmaset al., 1992]) are plotted; it can be seen that acombination of moisture-temperature at whichmicrobial growth in prunes was inhibited is lo-cated well above the glass curve, that is, in therubbery state.
The glass transition behavior of dried milk asa function of moisture content (and a ^ has beenreported recently by Jouppila and Roos (1994)
492
o3
2Q.
Bco
"OTC(0
135-125:
115:
105:
95:
85:
75:
65:
55:
45;
35;
25:
15:
5-5
-15-25-35
Amylopectin
Composition (áprox.)(on dry basis)
Starch 73 % ; sugars 4 % ;minerals 4 %
0
Starch
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30Moisture content, % d.b.
FIGURE 19. Comparison of Tg temperatures of potato (from data reported by Karmas et al., 1992) to that of starch(from data reported by Zeleznak and Hoseney, 1987) and amylopectin. (From data reported by Kalichevsky andBlanshard, 1993.)
and is shown in Figure 23. Giannuzzi and Parada(1991) studied the behavior of Staphylococcusaureus (three strains) in dehydrated milk at a,,values of 0.84 and 0.90 and incubated at 30°C forup to 30 d. Their results are shown in Figure 24.No growth is observed for any of the three strainsat a^ = 0.84. Nevertheless, at this a,,, dried milkshould have passed through the rubbery state (es-timated value of T-Tg above 82°C), and lactoseprobably should have recrystallized due to theincreased mobility (Jouppila and Roos, 1994). Inany case, mobility effects cannot be consideredthe sole determinants of bacterial growth inhibi-tion, and it is likely to be controlled by a ,. It isnoteworthy that in the S. aureus growth experi-ments performed by Giannuzzi and Parada (1991),a , should have remained constant despite somewater loss due to lactose crystallization, becausedry milk was kept in a closed desiccator over asaturated salt solution (KC1), a^ = 0.84).
In his pioneering work on water activity, Scott(1953) also found that S. aureus cannot grow indried milk at aw < 0.86 (e.g., moisture content16% d.b.), and this indicates inhibition of growthin a highly plasticized dried milk matrix (Jouppilaand Roos, 1994).
Bothast et al. (1981) performed a study of theeffects of moisture content and temperature onmicrobiological stability of wheat flour duringstorage. They reported that the mold count (As-pergillus glaucus and A. candidus were predomi-nant) increased in flour with 17.6% moisture (d.b.)during storage at 25 or 34°C; the CO2 in theheadspace paralleled the increase in mold counts.Figure 25 shows the adsorption isotherms of wheatflour and wheat starch (Bushuk and Winkler,1957), which are very similar because starch is byfar the main component of the dry matter in flour.At a moisture content of 17.6% d.b. the a^ of flouris about 0.76, and this value will permit the growth
493
oo 0.1 0.4 0.5 0.6
Water activity0.9
FIGURE 20. Comparison of water adsorption isotherms of potato starch (from data reported by van den Berg, 1981 )and potato. (From data reported by Crapiste and Rotstein, 1982.)
of various xerophilic molds (Beuchat, 1983) asexperimentally observed by Bothast et al. (1981).The Tg-moisture content curve for wheat flour isnot available to us, but those of starch (maincomponent) and gluten are reported in the litera-ture. Figure 26 shows the effect of moisture con-tent on Tg for wheat starch (Zeleznak and Hoseney,1987; Roos and Karel, 1991b), gluten (Hoseneyet al., 1986), and also lignin (Slade and Levine,1991a), which is a minor component of dietaryfiber in flour. At 17.6% moisture (d.b.) the Tg ofstarch and gluten are 63 and 21 °C, respectively,but because starch is by far the main constituentof flour (starch, 80 to 82%; gluten, 12 to 14%d.b.), it may be assumed that wheat flour at thismoisture content and stored at 25°C is likely to beglassy. However, mold growth (and CO2 genera-tion) was observed (Bothast et al., 1981). King etal. (1984) demonstrated that Xeromyces sp. and
Chrysosporium sp. were able to grow in wheatflours during storage (25°C) at \ , values rangingfrom 0.66 to 0.68. Applying the same argumentsas above, one may also conclude that wheat flourat the corresponding moisture contents (Bushukand Winkler, 1957) is likely to be glassy. Thesesituations are better illustrated in Figure 27, whichshows "transformed" water sorption data in wheatflour and the relative location of the selected val-ues at which growth occurred (King et al., 1984)and the glass curve for wheat flour based on datafor starch. Cahagnier et al. (1993) studied theeffects of different ^ values on growth of storagemolds (A. candidus and P. implicatum) of maize.They reported mold growth, ergosterol accumula-tion, and development of fat acidity in maize ofa„= 0.82 stored at 30°C. Moisture content ofmaize at this a,, is about 18 to 19% d.b. (Iglesiasand Chirife, 1982). At this moisture content the
494
TABLE 8Approximate Values of T-Tg at aw Values in the Range ofMicrobial Growth for Various Food Systems at 30°C
FoodsAppleBread, white
Cabbage
Carrot
CeleryHorseradishOnionPotatoStrawberry
BiopolymersCasein
CollagenElastin
Gelatin
Gluten
Maltodextrin DE 36
DE 5
Starch, wheat Native
Gelatinized
0.550.750.850.640.750.640.750.630.750.600.640.650.75
0.750.850.620.650.930.650.800.650.810.750.850.750.840.650.890.637
68-20
- 884997591847488628095
-52-60
42-223
19-16
5-10
146582
-147
-329
-14
Sources of data for the effect of moisture content on Tg: Karmas et al.,1992; Roos, 1987; Paakonen and Roos, 1990; Hoseney et al., 1986;Kakivaya and Hoeve, 1985; Batzar and Krelbich, 1981; Slade et al.,1989; Zeleznak and Hoseney, 1987; Roos and Karel, 1991 b; Karel et al.,1993; Le Meste et al, 1992; Nelson, 1993; Kalichevsky et al., 1993.Sources of data for sorption isotherms: Karmas et al., 1992; Roos, 1987;Paakonen and Roos, 1990; Bushuk and Winkler, 1957; Bull, 1944; vanden Berg, 1981; Roos and Karel, 1991b; Iglesias and Chirríe, 1982.
Tg of starch (main component of maize) is about if transport of nutrients from the food matrix is50 to 55°C (Zeleznack and Hoseney, 1987) so required. However, based on previous observa-one might assume that maize stored at 30°C may tions, we may conclude either that (1) the glassybe in the glassy state or in its proximity. Mobility state (based on Tg data for the main component ofof food components may be a key factor govern- flour) does not preclude mold growth, or (2) al-ing microbial growth in reduced moisture foods, though the "average" food matrix could be glassy
495
60
50-
* 40•o
a>ou
30-
20-
10-
0
Sugar content
Strawberry : 50 % dbPrunes : 54.5 % db fr-Tg) = 90 c
(T-Tg) = 35 C
(T-Tg) = 59 C
Ó Olí 0İ2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Strawberries•
Prunes
Water activity
FIGURE 21. Sorption isotherms of strawberry (25°C; Roos, 1987) and prunes (30°C; Tsami et al., 1990) andselected Tg values for strawberries (Roos, 1987). Line represents moisture content-a,, at which microbial stability wasobserved.
(as determined by DSC), molds may grow insome non-glassy micro-regions of the substrate.The Tg for the gluten fraction is lower than thatfor starch, and, in wheat flour-based foods, forinstance, a two phase system could be formed inwhich one fracture (gluten) could be rubbery andthe other (starch) could be glassy. If small amountsof sugars are present, they could plasticize one ofthe phases selectively, or provide a separate phasein which molds may grow. If phase separationsoccur, each one could have its own T value. Evenassuming that they could be detected (which isnot easy if the component(s) is/are) in very lowconcentrations when compared with the mainmatrix), the Tg value that would serve as a refer-ence to predict microbial growth would remainundetermined.
In any case, Tg data currently available forcomplex foods does not allow forcasting micro-
bial stability, on the basis of mobility consider-ations, with confidence. Examples are given thatshow microbial growth in foods at glassy (ornearly glassy) conditions, and microbial inhibi-tion of foods in the rubbery state.
XII. COMMENTS ON SOMEINAPPROPRIATE GENERALIZATIONSOF FOOD APPLICATIONS OF GLASSTRANSITION-RELATED PHENOMENA
In trying to present the food polymer scienceapproach as an all-embracing one, Slade andLevine (1987, 1991a,b, 1992) made some inap-propriate generalizations of glass transition theoryin areas that, although not strictly related to mi-crobiology, also deserve some comments.
Slade and Levine (1992) stated:
496
Microbial growth inhibitionin the rubbery state
82 84 86 88 90 92 94Prune solids, weight %
96 98 100
FIGURE 22. 'Transformed" selected sorption data in prunes and the glass curve for strawberries (Roos, 1987),which is expected to be similar to that for prunes, to illustrate on microbial growth inhibition in the rubbery state.
Since starch and gluten are the major storage poly-mers of wheat endosperm, and they are synthesized,stored in the mature seed, and hydrolyzed by enzymesof germination in the same temperature and moistureenvironment, it appears biochemically and physiologi-cally logical that their aqueous glass curves should besimilar, if not identical.
They added that the results and conclusionsof many literature studies seem to support thissuggestion. As shown in Figure 28, the glass curvesof wheat starch (Zeleznak and Hoseney, 1987)and wheat gluten (Hoseney et al., 1986) are farfrom being identical; for example, at 25% mois-ture (d.b.) there is a difference of about 35°Cbetween their Tg values!!
Slade and Levine (1991a) proposed that con-ventional water sorption isotherms may be trans-formed into a water/glass dynamics map. Theytook combinations of moisture content and tem-
perature that give the same value of observedRVP and replotted these data (as a series of iso-RVP contours) in the two dimensions of tempera-ture and weight percent of solids compared to thecorresponding temperature-moisture content lo-cation of a hypothetical glass curve for the solid.They performed this procedure for milled hardwheat sorption isotherms at various temperaturesand compared the replotted sorption data to thecorresponding temperature-moisture content of ahypothetical glass curve for wheat flour. Becausethey did not have the glass curve for wheat, theysaid that the curve shown was based on data forstarch and gluten. They added that the resultsrevealed that combinations of temperature andmoisture content that fall below the glass curvegive observed RVPs approaching zero. The prob-lem is that the glass curve shown in their datadoes not correspond either to starch, gluten, or
497
o-4H
ü -1
co
sit
£ŕCO
CO
es
-20--24--28 :
-32 :
-36:
-40--44--48 :
-52 :
-56 :
-60
RH 66 %
10 12 14 16 18 20 22 24Moisture content, d.b. (non-fat)
Low-fat (10.7% fat)- S -Low-fat (18.6% fat)- * -Whole milk
26
FIGURE 23. Effect of moisture content on Tg of dehydrated milk powder. (From data reported by Jouppila and Roos,1994.)
amylopectin. The actual curve of native wheatstarch (and also of amylopectin) is completelydifferent from the glass curve they used, invali-dating their conclusions. Their approach was fur-ther tested using published literature water sorp-tion (at various temperatures) and glass transitiondata for wheat starch and wheat gluten. The re-sults, shown in Figures 29 and 30, do not indicatethat combinations of moisture content and tem-perature that fall below the glass curve give RVPs(or aw values) approaching zero as claimed bySlade and Levine (1991a).
Slade and Levine (1991a) suggested thatusing the food polymer science approach tointerpret results from analysis of conventionaldrying processes may lead to increased under-standing and insight. Ease of drying can beviewed as a functional manifestation of mobil-ity in a food system. It is known that a critical
moisture content separates the period of con-stant rate drying (where the rate of removal asa function of time is constant) from the periodof falling rate drying (where the rate of waterremoval as a function of time decreases dra-matically and is no longer constant). Theyclaimed that this critical point for rice and otherstarch-based products is about 27 to 30% waterand is related to W'g (maximally freeze-con-centrated glass), but do not give any seriousreferences to corroborate this statement. More-over, all drying literature demonstrates that thecritical moisture content is not solely a functionof the physical properties of the solid, but alsodepends on the drying conditions at the surface(e.g., air velocity, temperature, and humidity;Jason, 1958; Fornell et al., 1980). Results fromLeung and Steinberg (1979) and Bakshi andSingh (1980), for example, clearly contradict
498
1.0E+10:
1.0E+03¡
1.0E+02¡
1.0E+01Í
1.0E+00
Staphylococcus aureus, 30 C
•---ısı Aw = 0.90FDA-C243
0
Aw = 0.84
(T-Tg) > 82 C
10 15 20 25 30Incubation time, day
35 40
FIGURE 24. Inhibition of growth of Staphylococcus aureus (at 30°C) in rubbery dry milk. (From data reported byGiannuzzi and Parada, 1991.)
the statements of Slade and Levine (1991a) thatfor starch-based products the critical moisturecontent (which separates the periods of constantrate and falling rate) is 27 to 30% water.
In their criticisms to the aw concept, Slade andLevine (1991a) discouraged the usage of a„ todefine safe limits for microbial growth inhibitionin foods. They stated:
The possibility that a community of food scientistscould believe that specifying a maximum a,, value of0.85 (limiting value for growth of S. aureus which isthe most a^-tolerant pathogen) for a cheese cake fill-ing can guarantee product safety, without any consid-eration of the nature of the mixture of water-compat-ible solids used to produce a particular a^ value, isboth disheartening and potentially deadly.
For Slade and Levine (1991a), the choice ofthis example (cake filling) to substantiate their
"apocalyptical" statements was unfortunate be-cause it has been well established that a,, doesplay a key role on the growth behavior of S. aureusin cream fillings. Cream pie fillings are a perish-able product that can support rapid microbiologi-cal growth of this bacterium because of its highnutritional value, and the possibility for cream tobe contaminated with staphylococci is high(Cathcart et al., 1947; Hirooka et al., 1987; Preonaset al., 1969). Hirooka et al., (1987) inoculated acream filling of known composition (milk, su-crose, corn flour, yellow dye, and egg yolk) with5. aureus and incubated at>37°C. The s^ of thiscream was calculated from its compositional dataand found to be 0.97; its pH was 6.0 to 6.7.Schmidt and Gould (1969) also inoculated a creamfilling of known composition, but in this casedextrose was the soluble solid responsible for aw
lowering; the a^ was calculated to be also 0.97.
499
m
TJ
30
25-
20-
0)
o
1 1 °5-
0
30.1 C
Wheat Starch
moisture = 17.6%
Wheat Flour
Aw = 0.76
0 0.1 0.2 0.3 0.4 0.5 0.6Water activity
0.7 0.8 0.9
FIGURE 25. Comparison of water adsorption isotherms of wheat starch and wheat flour at 30.1°C. (From datareported by Bushuk and Winkler, 1957.)
Because the limiting aw for growth of S. aureus isabout 0.86 (Chirife, 1994), one may anticipatethat, other factors being adequate (pH, nutrients,and temperature), the cells may grow easily inthese cream fillings, as experimentally observedby Hirooka et al. (1987) and Schmidt and Gould(1969) and shown in Figure 31. Preonas et al.(1969) inoculated pie fillings (flour, shortening,sucrose, nutmeg, vanilla, salt, whole egg, skimmilk, corn syrup, and water) of a^ 0.96 to 0.97with S. aureus, and growth was observed whenincubated at 30°C. However, addition of dextroseto further lower the a,, prevented growth. Sillikerand McHugh (1967) studied the factors influenc-ing microbial stability of butter-cream type fill-ings and found that a,, of the fillings (adjusted
either with sucrose, dextrose, or invert sugar)played a key role in the inhibition of growth ofinoculated S. aureus. They reported that fillings(sugar, shortening, margarine, nonfat dry milk,salt, vanilla, water, and gelatin) containing 2.1 to3.0 parts sucrose to 1 of water ( ^ = 0.85 andlower) were inherently bactericidal to staphylo-cocci, as shown in Figure 32. In fillings contain-ing equivalent concentrations (in osmotic terms)of dextrose and invert sugar, the results weresimilar.
XIII. CONCLUDING REMARKS
Slade and Le vine (1991a) harshly critiquedthe use of a,, in the food industry, saying:
500
ü
ture
£Q .
tem
¡ion
"5>c03im.
+•«
COCO(GO
100-
90-
80-
70-
60-
50-
40-
30-
20--
10-
0-0 2 6 8 10 12 14 16 18 20 22 24 26 28 30
Moisture content, % dry basis
FIGURE 26. Effect of moisture content on Tg transition of wheat starch, gluten, and lignin. (From Tg—moisturecontent data reported by Zeleznak and Hoseney, 1987; Hoseney et al., 1986; Kalichevsky and Blanshard, 1993; andSlade and Levine, 1991a.)
The real danger of this careless and oversimplifiedusage (of a j relates to government defined and im-posed specifications for values of a„ required by lawfor microbial safety of IMF products for human con-sumption. The potential for disaster inherent in naivecompliance with such a rigid quantitative approach isfrightening.
As the only example, they mentioned thatvery similar compounds such as glucose and fruc-tose may have very different degrees of growthinhibition at the same measured RH. As men-tioned before, van den Berg (1985) and Franks(1991a) have also joined Slade and Levine (1991a)in mentioning "difficulties produced by the use ofaw in food preservation." However, their claimswere not supported by referenced experimental
evidence. Moreover, the present review detailsthe shortcomings of the supposed experimentalevidences reported by Slade and Levine (1987;1991a) as proof of their claims.
Slade and Levine (1991a) also pointed outthat the growth behavior of microbial cells de-pends not just on the pH, but also on the chemicalnature of the buffer. Thus, pH and aw are onlypartially diagnostic of cell growth, and the "mis-use of aw" is comparable to the misuse of pH. Itis well documented in the literature that the mi-crobial response to lowered pH is determined byboth the pH of the system and the acidulant usedin acidification. For example, acetic acid is recog-nized as a better inhibitor (at similar pH) of bac-terial growth than other acids used in food preser-
501
oor
ratu
iT
emp
ei90-
80-
70-
60-
50-
40-
30-
20-
10-
0
(Starch)
Tg
Microbial growth
in Glassy state
(Wheat Flour)
Growth ofA glaucus & A candidas
Growth of
Xeromyces sp.
Chrysosporium sp.
75 77 79 81 83 85 87Wheat Flour, weight %
89 91
FIGURE 27. Transformed" selected sorption data in wheat flour and the glass curve for wheat flour based on datafor starch (Zeleznak and Hoseney, 1987) to illustrate microbial growth in the glassy state.
vation such as citric, lactic, or malic acid(Abdul-Raouf et al., 1993; Tsang et al., 1985;Minor and Marth, 1972). The minimum pH re-
, quired to inhibit growth and toxin productionby Clostridium botulinum depends on theacidulant used (Minor and Marth, 1972; Smeltet al., 1982). Nevertheless, it is generally ac-cepted that the limiting pH of 4.6 provides agood margin of safety against the hazards ofbotulism in acidified foods, and such productsare given only a mild treatment (Post et al.,1985). The well-documented existence of asolute effect for pH did not preclude its wide-spread usage in food preservation. And this issimply because any specified safe pH limit usu-
ally does not correspond to the most inhibitoryacid, but to less inhibitory ones. Sorrels et al.(1989) studied the effect of pH and type of acidon growth of histeria monocytogenes. They re-ported that the overall antimicrobial action(when based on equal pH) was acetic acid >lactic acid > citric acid > malic acid. A pH of5.0 was sufficient to inhibit growth ofL. monocytogenes when acetic acid was used; alower pH was needed for the others. Neverthe-less, usual literature tabulations indicate a pHlower than 4.6 to inhibit the growth of thispathogen in accordance with a safe practice(Brown and Booth, 1991). The same happenswith the solute effect for aw limits for microbial
502
130
ü
CO
oCL
IcoOTc(Q
WW
İ2(5
0
Amylopectin
Starch (wheat)
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38
Moisture content, % d.b.
FIGURE 28. Comparison of the glass curves of wheat gluten (Hoseney et al., 1986; Kalichevsky et al., 1992) andwheat starch (Zeleznak and Hoseney, 1987; Kalichevsky and Blanshard, 1993).
growth. For example, literature tabulations ofminimal aw specify that the lowest aw permit-ting growth of 5. aureus is 0.86, even thoughthis bacteria may be inhibited with much higheraw values, using less conventional solutes(Chirife, 1994).
Despite the repeated claims of Slade andLevine (1987; 1991a,b, 1992), experimental evi-dence of mobility factors controlling the safetyand microbial stability of IM foods is still to beprovided. The analysis of experimental evidencedoes not substantiate their claims in the sensethat the "food polymer science approach to un-derstanding of aqueous sugar glasses and con-centrated solutions may be used to predict themicrobial stability of food systems." And, cer-tainly, this approach does not presently offer abetter alternative to the concept of aw as a pre-
dictor of microbial growth in foods. The mea-surement of a„ which has been long used forpredicting the safety and spoilage of foods,continues to prove empirically highly usefuland should become exploited to its fullest po-tential with a greater appreciation of its theo-retical (and sometimes practical) limitations(Duckworth, 1988). Gould (1988) indicated thatthe uncritical use of aw as a determinant of cellactivity had the effect of steering attention awayfrom some more fundamental aspects of thewater relations of microbial cells.
The authors are not saying here that mobil-ity factors, in addition to aw may not be usefulfor a better definition and prediction of micro-bial behavior in foods. However, much moreresearch is needed in order to define the condi-tions under which those factors (viscosity/
503
100
ü
o
1a
1
90-
80-
70--
60--
50-
-40-
30-
20-
1075 85 90
Starch, weight %100
FIGURE 29. Transformed" isotherms for water adsorption in wheat starch showing the relative locations of iso-a„contours and the glass curve for starch. (Glass curve for wheat starch from data reported by Zeleznak and Hoseney,1987; water adsorption data for wheat starch at various temperatures from data reported by Bushuk and Winkler,1957.)
diffusivity) may effectively inhibit (or retard)microbial growth. Opportunities that have akinetic rather than an equilibrium basis, such asthose that may derive from better-directed con-trol of viscosity/diffusivity, may play a role anddeserve to be investigated, but this does not meanthat the use of a„ should be disregarded. At thispoint it is useful to recall the following state-ments of van den Berg (1991):
Water activity and glass transition are two differententities; the former being a solvent property and thelatter a property related to the structure of the solid;they each relate rather complementary, to importantaspects and knowledge of both is needed for under-standing the food-water relationships of the systemunder consideration.
The nonequilibrium effects discussed in thepresent review (e.g., inability of water to diffusein semimoist foods) appear to be, in many cases,slow within the time frame (food's shelf life) ofthe experiments and/or so small that they do notaffect seriously the application of the a , conceptas a predictor of microbial stability. This is re-gardless of the rigorous theoretical meaning ofany measured a ,, for example, a thermodynamicactivity, a nonequilibrium RPV, or an empiricalpseudo-a,,. This does not mean that, becausechanges in nonequilibrium food systems mightbe slow and/or small, considerations of nonequi-librium effects, glassy and rubbery states, etc.are unnecessary or that the a,, concept is all thatone needs to ensure the safety of semimoist foods.
504
70 .60 .50 .30 .20
75 80 85 90Gluten, weight %
100
FIGURE 30. Transformed" isotherms for water adsorption in wheat gluten showing the relative locations of ¡so-aw
contours and the glass curve for gluten. (Glass curve for gluten from data reported by Hoseney et al., 1986; wateradsorption data for gluten at various temperatures from data reported by Bushuk and Winkler, 1957.)
This is not so. The a„, concept has several limi-tations and should always be used carefully; thismust include considerations (and precautions)regarding the possible influence of nonequilibri-um situations, such as those pointed out by Sladeand Levine (1987,1991a). These warnings, how-ever, are not new. Almost 20 years ago theywere clearly stated by Reid (1976) at an interna-tional symposium entirely devoted to intermedi-ate-moisture foods, held in Reading, England.He pointed out that because a,, is a thermody-namic concept, it refers only to equilibrium; thus,if equilibrium has not been attained, the defini-tion does not apply. Reid (1976) also discussedthe implications of a solution capable of super-saturation (pseudoequilibrium). The vapor pres-sure of the supersaturated solution is lower than
that of the saturated solution; thus the aw is alsolower. Whether this observation is meaningfuldepends on the stability of the supersaturatedsolution. His concepts were summarized as fol-lows: "Since activity is a thermodynamic con-cept anyone who is going to employ the termmust be aware of the requirements of the defini-tion."
The field of food science and technology hasrecently enjoyed an exponential growth of inter-est in glasses and glass transition in foods and theplasticizing effect of water. Slade and Levine werepioneers in this aspect and their efforts should befully appreciated and commended. However, thisapproach — which is certainly original and stimu-lating — should not be presented as an all-em-bracing one without adequate experimental evi-
505
Staphylococcus aureus
1.0E+01f5
1.0E+00
with Sucrose
with Dextrose
Cream Filling
Aw = 0.97
37 C
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80Incubation time, hour
FIGURE 31. Growth of Staphylococcus aureus in cream fillings of aw = 0.97. (From data reported by Hirooka, et al.,1987 and Schmidt and Gould, 1969.)
1.0E+06a
1.0E+05;
•1.0E+0İİ
1.0E+000
Staphylococcus aureus
Cream filling
Aw = 0.84(w/sucrose)
37 C
4 6 8 10 12
Incubation time, day14 16
FIGURE 32. Inhibition of growth of S. aureus in a cream filling of a^ adjusted to about 0.84 by the addition ofsucrose. (From data reported by Silliker and McHugh, 1967.)
506
dence. This will help the development of an areaof research that offers many challenging ques-tions and promises many opportunities for tech-nological development.
ACKNOWLEDGMENTS
The authors acknowledge financial supportfrom Universidad de Buenos Aires and ConsejoNacional de Investigaciones Científicas y Técnicasde la Argentina.
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