Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 51 bacteria such as some members of the genus Bacillus Some aerobic bacteria actually grow better under slightly reduced conditions, and these organisms are referred to as microaerophiles Examples of microaerophilic bacteria are lactobacilli and campylobacters Some bacteria have the capacity to grow under either aerobic or anaerobic conditions Such types are referred to as facultative anaerobes Most molds and yeasts encountered in and on foods are aerobic, although a few tend to be facultative anaerobes With regard to the Eh of foods, plant foods, especially plant juices, tend to have Eh values of from +300 to 400 mV It is not surprising to find that aerobic bacteria and molds are the common cause of spoilage of products of this type Solid meats have Eh values of around −200 mV; in minced meats, the Eh is generally around 200 mV Cheeses of various types have been reported to have Eh values on the negative side, from −20 to around −200 mV With respect to the Eh of pre-rigor as opposed to post-rigor muscles, Barnes and Ingram2,3 undertook a study of the measurement of Eh in muscle over periods of up to 30 hours postmortem and its effect on the growth of anaerobic bacteria These authors found that the Eh of the sternocephalicus muscle of the horse immediately after death was +250 mV, at which time clostridia failed to multiply At 30 hours postmortem, the Eh had fallen to about 30 mV in the absence of bacterial growth When bacterial growth was allowed to occur, the Eh fell to about 250 mV Growth of clostridia was observed at Eh values of 36 mV and below These authors confirmed for horse meat the finding for whale meat: that anaerobic bacteria not multiply until the onset of rigor mortis because of the high Eh in pre-rigor meat The same is undoubtedly true for beef, pork, and other meats of this type Eh Effects Microorganisms affect the Eh of their environments during growth just as they pH This is true especially of aerobes, which can lower the Eh of their environment while anaerobes cannot As aerobes grow, O2 in the medium is depleted, resulting in the lowering of Eh Growth is not slowed, however, as much as might be expected due to the ability of cells to make use of O2 -donating or hydrogen-accepting substances in the medium The result is that the medium becomes poorer in oxidizing and richer in reducing substances.32 The Eh of a medium can be reduced by microorganisms by their production of certain metabolic byproducts such as H2 S, which has the capacity to lower Eh to −300 mV Because H2 S reacts readily with O2 , it will accumulate only in anaerobic environments Eh is dependent on the pH of the substrate, and the direct relationship between these two factors is the rH value defined in the following way: Eh = 2.303 RT (rH − 2pH) F where R = 8.315 joules, F = 96,500 coulombs, and T is the absolute temperature.34 Therefore, the pH of a substrate should be stated when Eh is given Normally Eh is taken at pH 7.0 (expressed Eh ) When taken at pH 7.0, 25◦ C, and with all concentrations at 1.0 M, Eh = Eho (simplified Nernst equation) In nature, Eh tends to be more negative under progressively alkaline conditions Among naturally occurring nutrients, ascorbic acid and reducing sugars in plants and fruits and –SH groups in meats are of primary importance The presence or absence of appropriate quantities of oxidizing—reducing agents in a medium is of obvious value to the growth and activity of all microorganisms 52 Modern Food Microbiology While the growth of anaerobes is normally believed to occur at reduced values of Eh, the exclusion of O2 may be necessary for some anaerobes When Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus were cultured in the presence of O2 , inhibition of growth occurred even when the medium was at a negative Eh of −50 mV.52 These investigators found that growth occurred in media with an Eh as high as 325 mV when no O2 was present With regard to the effect of Eh on lipid production by Saccharomyces cerevisiae, it has been shown that anaerobically grown cells produce a lower total level, a highly variable glyceride fraction, and decreased phospholipid and sterol components as compared to aerobically grown cells.41 The lipid produced by anaerobically grown cells was characterized by a high content (up to 50% of total acid) of 8:0 to 14:0 acids and a low level of unsaturated fatty acid in the phospholipid fraction In aerobically grown cells, 80–90% of the fatty acid component was associated with glyceride, and the phospholipid was found to be 16:1 and 18:1 acids Unlike aerobically grown cells, anaerobically grown S cerevisiae cells were found to have a lipid and sterol requirement Nutrient Content In order to grow and function normally, the microorganisms of importance in foods require the following: water source of energy source of nitrogen vitamins and related growth factors minerals The importance of water to the growth and welfare of microorganisms is presented earlier in this chapter With respect to the other four groups of substances, molds have the lowest requirement, followed by Gram-negative bacteria, yeasts, and Gram-positive bacteria As sources of energy, foodborne microorganisms may utilize sugars, alcohols, and amino acids Some microorganisms are able to utilize complex carbohydrates such as starches and cellulose as sources of energy by first degrading these compounds to simple sugars Fats are also used by microorganisms as sources of energy, but these compounds are attacked by a relatively small number of microbes in foods The primary nitrogen sources utilized by heterotrophic microorganisms are amino acids A large number of other nitrogenous compounds may serve this function for various types of organisms Some microbes, for example, are able to utilize nucleotides and free amino acids, whereas others are able to utilize peptides and proteins In general, simple compounds such as amino acids will be utilized by almost all organisms before any attack is made on the more complex compounds such as high-molecular-weight proteins The same is true of polysaccharides and fats Microorganisms may require B vitamins in low quantities, and almost all natural foods have an abundant quantity for those organisms that are unable to synthesize their essential requirements In general, Gram-positive bacteria are the least synthetic and must therefore be supplied with one or more of these compounds before they will grow The Gram-negative bacteria and molds are able to synthesize most or all of their requirements Consequently, these two groups of organisms may be found growing on foods low in B vitamins Fruits tend to be lower in B vitamins than meats, and this Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 53 fact, along with the usual low pH and positive Eh of fruits, helps to explain the usual spoilage of these products by molds rather than bacteria Antimicrobial Constituents The stability of some foods against attack by microorganisms is due to the presence of certain naturally occurring substances that possess and express antimicrobial activity Some plant species are known to contain essential oils that possess antimicrobial activity Among these are eugenol in cloves, allicin in garlic, cinnamic aldehyde and eugenol in cinnamon, allyl isothiocyanate in mustard, eugenol and thymol in sage, and carvacrol (isothymol) and thymol in oregano.47 Cow’s milk contains several antimicrobial substances, including lactoferrin (see below), conglutinin, and the lactoperoxidase system (see below) Raw milk has been reported to contain a rotavirus inhibitor that can inhibit up to 106 pfu (plaqueforming units)/ml It is destroyed by pasteurization Milk casein as well as some free fatty acids have been shown to be antimicrobial under certain conditions Eggs contain lysozyme, as does milk, and this enzyme, along with conalbumin, provides fresh eggs with a fairly efficient antimicrobial system The hydroxycinnamic acid derivatives ( p-coumaric, ferulic, caffeic, and chlorogenic acids) found in fruits, vegetables, tea, molasses, and other plant sources all show antibacterial and some antifungal activity Lactoferrin is an iron-binding glycoprotein that is inhibitory to a number of foodborne bacteria and its use as a microbial blocking agent on beef carcasses is discussed in Chapter 13 Ovotransferrin appears to be the inhibitory substance in raw egg white that inhibits Salmonella enteritidis.4 Cell vacuoles of cruciferous plants (cabbage, Brussels sprouts, broccoli, turnips, etc.) contain glucosinolates, which upon injury or mechanical disruption, yield isothiocyanates Some of the latter possess antifungal as well as antibacterial activity More on antimicrobials in foods can be found in Chapter 13 Lactoperoxidase System This is an inhibitory system that occurs naturally in bovine milk, and it consists of three components: lactoperoxidase, thiocyanate, and H2 O2 All three components are required for antimicrobial effects, and Gram-negative psychrotrophs such as the pseudomonads are quite sensitive The quantity of lactoperoxidase needed is 0.5–1.0 ppm, whereas bovine milk normally contains about 30 ppm.6 Although both thiocyanate and H2 O2 occur normally in milk, the quantities vary For H2 O2 , about 100 U/ml are required in the inhibitory system, whereas only 1–2 U/ml normally occur in milk An effective level of thiocyanate is around 0.25 mM, whereas in milk the quantity varies between 0.02 and 0.25 mM.6 When the lactoperoxidase system in raw milk was activated by adding thiocyanate to 0.25 mM along with an equimolar amount of H2 O2 , the shelf life was extended to days compared to 48 hours for controls.6 The system was more effective at 30◦ C than at 4◦ C The antibacterial effect increases with acidity, and the cytoplasmic membrane appears to be the cell target In addition to the direct addition of H2 O2 , an exogenous source can be provided by the addition of glucose and glucose oxidase To avoid the direct addition of glucose oxidase, this enzyme has been immobilized on glass beads so that glucose is generated only in the amounts needed by the use of immobilized β-galactosidase.7 This system was effective in goat’s milk against P fluorescens and E coli where the growth of the former was controlled for days and the latter for days at 8◦ C.55 The lactoperoxidase system can be used to preserve raw milk in countries where refrigeration is uncommon The addition of about 12 ppm of SCN− and ppm of H2 O2 should be harmless to the consumer.44 An interesting aspect of this system is the effect it has on thermal properties In one study, 54 Modern Food Microbiology it was shown to reduce thermal D values at 57.8◦ C by around 80% for L monocytogenes and by around 86% for S aureus at 55.2◦ C.27 Although the mechanism of this enhanced thermal destruction is unclear, some interesting implications can be envisioned Biological Structures The natural covering of some foods provides excellent protection against the entry and subsequent damage by spoilage organisms In this category are such structures as the testa of seeds, the outer covering of fruits, the shell of nuts, the hide of animals, and the shells of eggs In the case of nuts such as pecans and walnuts, the shell or covering is sufficient to prevent the entry of all organisms Once cracked, of course, nutmeats are subject to spoilage by molds The outer shell and membranes of eggs, if intact, prevent the entry of nearly all microorganisms when stored under the proper conditions of humidity and temperature Fruits and vegetables with damaged covering undergo spoilage much faster than those not damaged The skin covering of fish and meats such as beef and pork prevents the contamination and spoilage of these foods, partly because it tends to dry out faster than freshly cut surfaces Taken together, these six intrinsic parameters represent nature’s way of preserving plant and animal tissues from microorganisms By determining the extent to which each exists in a given food, one can predict the general types of microorganisms that are likely to grow and, consequently, the overall stability of this particular food Their determination may also aid one in determining age, and possibly the handling history of a given food EXTRINSIC PARAMETERS The extrinsic parameters of foods are not substrate dependent They are those properties of the storage environment that affect both the foods and their microorganisms Those of greatest importance to the welfare of foodborne organisms are as follows: temperature of storage relative humidity of environment presence and concentration of gases presence and activities of other microorganisms Temperature of Storage Microorganisms, individually and as a group, grow over a very wide range of temperatures Therefore, it is well to consider at this point the temperature growth ranges for organisms of importance in foods as an aid in selecting the proper temperature for the storage of different types of foods The lowest temperature at which a microorganism has been reported to grow is −34◦ C; the highest is somewhere in excess of 100◦ C It is customary to place microorganisms into three groups based on their temperature requirements for growth Those organisms that grow well at or below 7◦ C and have their optimum between 20◦ C and 30◦ C are referred to as psychrotrophs (see Chapter 16) Those that grow well between 20◦ C and 45◦ C with optima between 30◦ C and 40◦ C are referred to as mesophiles, whereas those that grow well at and above 45◦ C with optima between 55◦ C and 65◦ C are referred to as thermophiles (Physiological properties of these groups are treated in Chapters 16 and 17.) Intrinsic and Extrinsic Parameters of Foods That Affect Microbial Growth 55 With regard to bacteria, psychrotrophic species and strains are found among the following genera of those presented in Chapter 2: Alcaligenes, Shewanella, Brochothrix, Corynebacterium, Flavobacterium, Lactobacillus, Micrococcus, Pectobacterium, Pseudomonas, Psychrobacter, Enterococcus, and others The psychrotrophs found most commonly on foods are those that belong to the genera Pseudomonas and Enterococcus (see Chapter 16) These organisms grow well at refrigerator temperatures and cause spoilage at 5–7◦ C of meats, fish, poultry, eggs, and other foods normally held at this temperature Standard plate counts of viable organisms on such foods are generally higher when the plates are incubated at about 7◦ C for at least days than when incubated at 30◦ C and above Mesophilic species and strains are known among all genera presented in Chapter and may be found on foods held at refrigerator temperatures They apparently not grow at this temperature but grow at temperatures within the mesophilic range if other conditions are suitable It should be pointed out that some organisms can grow over a range from 0◦ C to >40◦ C One such organism is Enterococcus faecalis Most thermophilic bacteria of importance in foods belong to the genera Bacillus, Paenibacillus, Clostridium, Geobacillus, Alicyclobacillus, and Thermoanaerobacter Although not all species of these genera are thermophilic, they are of great interest to the food microbiologist and food technologist in the canning industry Just as molds are able to grow over wider ranges of pH, osmotic pressure, and nutrient content, they are also able to grow over wide ranges of temperature as bacteria Many molds are able to grow at refrigerator temperatures, notably some strains of Aspergillus, Cladosporium, and Thamnidium, which may be found growing on eggs, sides of beef, and fruits Yeasts grow over the psychrotrophic and mesophilic temperature ranges but generally not within the thermophilic range The quality of the food product must also be taken into account in selecting a storage temperature Although it would seem desirable to store all foods at refrigerator temperatures or below, this is not always best for the maintenance of desirable quality in some foods For example, bananas keep better if stored at 13–17◦ C than at 5–7◦ C A large number of vegetables are favored by temperatures of about 10◦ C, including potatoes, celery, cabbage, and many others In every case, the success of storage temperature depends to a great extent upon the relative humidity (RH) of the storage environment and the presence or absence of gases such as CO2 and O3 Temperature of storage is the most important parameter that affects the spoilage of highly perishable foods, and this fact has been emphasized by the work of Olley and Ratkowsky and their co-workers According to these investigators, spoilage can be predicted by a spoilage rate curve.34 The general spoilage curve has been incorporated into the circuitry of a temperature function integrator that reads out the equivalent days of storage at 0◦ C and thus makes it possible to predict the remaining shelf life at 0◦ C It has been shown that the rate of spoilage of fresh poultry at 10◦ C is about twice that at 5◦ C, and that at 15◦ C is about three times that at 5◦ C.18,22 Instead of using the Arrhenius law equation, the following was developed to describe the relationship between temperature and growth rate of microorganisms between the minimum and optimum temperatures.40 √ r = B(T − T0 ) where r is the growth rate, B is the slope of the regression line, and T0 is a conceptual temperature of no metabolic significance The linear relationship has been shown to apply to spoilage bacteria and fungi when growing in foods or when utilizing amino acids.40 The incorporation of growth data into mathematical equations to predict the behavior of microorganisms in food systems is discussed further in Chapter 20 ...52 Modern Food Microbiology While the growth of anaerobes is normally believed to occur at reduced values of Eh, the exclusion of O2 may be necessary... magnus were cultured in the presence of O2 , inhibition of growth occurred even when the medium was at a negative Eh of −50 mV.52 These investigators found that growth occurred in media with an Eh... glyceride fraction, and decreased phospholipid and sterol components as compared to aerobically grown cells.41 The lipid produced by anaerobically grown cells was characterized by a high content (up