The ability of microorganisms (except viruses) to grow or multiply in a food is determined by the food environment as well as the environment in which the food is stored, designated as the intrinsic and extrinsic environment of food, respectively. It is not possible to study the inﬂuence of any one factor on growth independently as the factors are interrelated. Instead, the inﬂuence of any one factor at different levels on growth is compared keeping other factors unchanged. The inﬂuence of these factors is discussed here.
INTRINSIC FACTORS OR FOOD ENVIRONMENT
Intrinsic factors of a food include nutrients, growth factors, and inhibitors (or anti- microbials), water activity, pH, and oxidation–reduction potential. The inﬂuence of each factor on growth is discussed separately. But, as indicated previously, in a food system the factors are present together and exert effects on microbial growth in combination, either favorably or adversely.
A. Nutrients and Growth
Microbial growth is accomplished through the synthesis of cellular components and energy. The necessary nutrients for this process are derived from the immediate environment of a microbial cell and, if the cell is growing in a food, it supplies the nutrients. These nutrients include carbohydrates, proteins, lipids, minerals, and vitamins. Water is not considered a nutrient, but it is essential as a medium for the biochemical reactions necessary for the synthesis of cell mass and energy. All foods contain these ﬁve major nutrient groups, either naturally or added, and the amount of each nutrient varies greatly with the type of food. Microorganisms normally present in food vary greatly in nutrient requirements, with bacteria requiring the most, followed by yeasts and molds. Microorganisms also differ greatly in their ability to utilize large and complex carbohydrates (e.g., starch and cellulose), large proteins (e.g., casein in milk), and lipids. Microorganisms capable of using these molecules do so by producing speciﬁc extracellular enzymes (or exoenzymes) and hydrolyzing the complex molecules to simpler forms outside before transporting them inside the cell. Molds are the most capable of doing this. However, this provides an opportunity for a species to grow in a mixed population even when it is incapable of metabolizing the complex molecules. Microbial cells, following death and lysis, release intracellular enzymes that can also catalyze break- down of complex food nutrients to simpler forms, which can then be utilized by other microorganisms.
B. Growth Factors and Inhibitors in Food
Foods can also have some factors that either stimulate growth or adversely affect growth of microorganisms. The exact nature of growth factors is not known, but they are naturally present in some foods. An example is the growth factors in tomatoes that stimulate growth of some Lactobacillus species. These factors can be added to raw materials during food bio processing or to media to isolate some fastidious bacteria from foods. Foods also contain many chemicals, either naturally or added, that adversely affect microbial growth. Some of the natural inhibitors are lysozyme in egg, agglutinin in milk, and eugenol in cloves. The inhibitors, depending on their mode of action, can prevent or reduce growth of and kill microorganisms.
C. Water Activity and Growth
The free water in a food is necessary for microbial growth. It is necessary to transport nutrients and remove waste materials, carry out enzymatic reactions, synthesize cellular materials, and take part in other biochemical reactions, such as hydrolysis of a polymer to monomers (proteins to amino acids). Each microbial species (or group) has an optimum, maximum, and minimum Aw level for growth. The Aw of food ranges from ca. 0.1 to 0.99. The Aw values of some food groups are as follows: cereals, crackers, sugar, salt, dry milk, 0.10 to 0.20; noodles, honey, chocolate, dried egg, <0.60; jam, jelly, dried fruits, parmesan cheese, nuts, 0.60 to 0.85; fermented sausage, dry cured meat, sweetened condensed milk, maple syrup, 0.85 to 0.93; evaporated milk, tomato paste, bread, fruit juices, salted ﬁsh, sausage, processed cheese, 0.93 to 0.98; and fresh meat, ﬁsh, fruits, vegetables, milk, eggs, 0.98 to 0.99. The Aw of foods can be reduced by removing water (desorption) and increased by the adsorption of water.
D. pH and Growth
pH indicates the hydrogen ion concentrations in a system and is expressed as –log [H+], the negative logarithm of the hydrogen ion or proton concentration. It ranges from 0 to 14, with 7.0 being neutral pH. [H+] concentrations can differ in a system, depending on what acid is present. Some strong acids used in foods, such as HCl and phosphoric acid, dissociate completely. Weak acids, such as acetic or lactic acids, remain in equilibrium with the dissociated and undissociated forms:
[HCl]à[H+] + [Cl–], pH of 0.1 N HCl is 1.1
CH3 COOHà [H+] + [CH3COO–], pH of 0.1 N CH3COOH is 2.9
Acidity is inversely related to pH: a system with high acidity has a low pH, and vice versa.
Depending on the type, the pH of a food can vary greatly. On the basis of pH, foods can be grouped as high-acid foods (pH below 4.6) and low-acid foods (pH 4.6 and above). Most fruits, fruit juices, fermented foods (from fruits, vegetables, meat, and milk), and salad dressings are high-acid (low-pH) foods, whereas most vegetables, meat, ﬁsh, milk, and soups are low-acid (high-pH) foods. Tomato, however, is a high-acid vegetable (pH 4.1 to 4.4). The higher pH limit of most low-acid foods remains below 7.0; only in a few foods, such as clams (pH 7.1) and egg albumen (pH 8.5), does the pH exceed 7.0. Similarly, the low pH limit of most high-acid foods remains above 3.0, except in some citrus fruits (lemon, lime, grapefruit) and cranberry juice, in which the pH can be as low as 2.2. The acid in the foods can either be present naturally (as in fruits), produced during fermentation (as in fermented foods), or added during processing (as in salad dressings). Foods can also have compounds that have a buffering capacity. A food such as milk or meat, because of good buffering capacity, does not show pH reduction when compared with a vegetable product in the presence of the same amount of acid.
The pH of a food has a profound effect on the growth and viability of microbial cells. Each species has an optimum and a range of pH for growth. In general, molds and yeasts are able to grow at lower pH than do bacteria, and Gram-negative bacteria are more sensitive to low pH than are Gram-positive bacteria. The pH range of growth for molds is 1.5 to 9.0; for yeasts, 2.0 to 8.5; for Gram-positive bacteria, 4.0 to 8.5; and for Gram-negative bacteria, 4.5 to 9.0. Individual species differ greatly in lower pH limit for growth; for example, Pediococcus acidilactici can grow at pH 3.8 but normally Salmonella cannot. The lower pH limit of growth of a species can be a little higher if the pH is adjusted with strong acid instead of a weak acid (due to its undissociated molecules). Acid-resistant or tolerant strains can acquire resistance to lower pH compared with the other strains of a species (e.g., acid-resistant Salmonella). When the pH in a food is reduced below the lower limit for growth of a microbial species, the cells not only stop growing but also lose viability, the rate of which depends on the extent of pH reduction.
E. Redox Potential, Oxygen, and Growth
The process involves the loss of electrons from a reduced substance (thus it is oxidized) and the gain of electrons by an oxidized substance (thus it is reduced). The electron donor, because it reduces an oxidized substance, is also called a reducing agent. Similarly, the electron recipient is called an oxidizing agent. The redox potential, designated as Eh, is measured in electrical units of mill volts (mV). In the oxidized range, it is expressed in +mV, and in reduced range in –mV. In biological systems, the oxidation and reduction of substances are the primary means of generating energy. If free oxygen is present in the system, then it can act as an electron acceptor. In the absence of free oxygen, oxygen bound to some other compound, such as NO3 and SO4, can accept the electron. In a system where no oxygen is present, other compounds can accept the electrons. Thus, presence of oxygen is not a requirement of O–R reactions.
The redox potential of a food is inﬂuenced by its chemical composition, speciﬁc processing treatment given, and its storage condition (in relation to air). Fresh foods of plant and animal origin are in a reduced state, because of the presence of reducing substances such as ascorbic acid, reducing sugars, and –SH group of proteins.
On the basis of their growth in the presence and absence of free oxygen, microorganisms have been grouped as aerobes, anaerobes, facultative anaerobes, or microaerophiles. Aerobes need free oxygen for energy generation, as the free oxygen acts as the ﬁnal electron acceptor through aerobic respiration. Facultative anaerobes can generate energy if free oxygen is available, or they can use bound oxygen in compounds such as NO3 or SO4 as ﬁnal electron acceptors through anaerobic respiration. If oxygen is not available, then other compounds are used to accept the electron (or hydrogen) through (anaerobic) fermentation. An example of this is the acceptance of hydrogen from NADH2 by pyruvate to produce lactate. Anaerobic and facultative anaerobic microorganisms can only transfer electrons through fermentation. Many anaerobes (obligate or strict anaerobes) cannot grow in the presence of even small amounts of free oxygen as they lack the super- oxide dismutase necessary to scavenge the toxic oxygen free radicals. Addition of scavengers, such as thiols (e.g., thiolglycolate), helps overcome the sensitivity to these free radicals. Microaerophiles grow better in the presence of less oxygen. Growth of microorganisms and their ability to generate energy by the speciﬁc metabolic reactions depend on the redox potential of foods. The Eh range at which different groups of microorganisms can grow are as follows: aerobes, +500 to +300 mV; facultative anaerobes, +300 to +100 mV; and anaerobes, +100 to –250 mV or lower. However, this varies greatly with concentrations of reducing components in a food and the presence of oxygen. Molds, yeasts, and Bacillus, Pseudomonas, Moraxella, and Micrococcus genera are some examples that have aerobic species. Some examples of facultative anaerobes are the lactic acid bacteria and those in the family Enterobacteriaceae. The most important anaerobe in food is Clostridium. An example of a microaerophile is Campylobacter spp.
III. EXTRINSIC FACTORS
Extrinsic factors important in microbial growth in a food include the environmental conditions in which it is stored. These are temperature, relative humidity, and gaseous environment. The relative humidity and gaseous condition of storage, respectively, inﬂuence the Aw and Eh of the food. The inﬂuence of these two factors on microbial growth has been discussed previously. In this section, the inﬂuence of storage temperature of food on microbial growth is discussed.
A. Temperature and Growth
Microbial growth is accomplished through enzymatic reactions. It is well known that within a certain range, with every 10rC rise in temperature, the catalytic rate of an enzyme doubles. Similarly, the enzymatic reaction rate is reduced to half by decreasing the temperature by 10rC. This relationship changes beyond the growth range. Because temperature inﬂuences enzyme reactions, it has an important role in microbial growth in food.
Foods are exposed to different temperatures from the time of production until consumption. Depending on processing conditions, a food can be exposed to high heat, from 65rC (roasting of meat) to more than 100rC (in ultrahigh temperature processing). For long-term storage, a food can be kept at 5rC (refrigeration) to –20rC or below (freezing). Some relatively stable foods are also kept between 10 and 35rC (cold to ambient temperature). Some ready-to-eat foods are kept at warm temperature (50r to 60rC) for several hours (e.g., in the supermarket deli). Different temperatures are also used to stimulate desirable microbial growth in food fermentation.
Microorganisms important in foods are divided into three groups on the basis of their temperature of growth, each group having an optimum temperature and a temperature range of growth: (1) thermophiles (grow at relatively high temperature), with optimum ca. 55rC and range 45 to 70rC; (2) mesophiles (grow at ambient temperature), with optimum at 35rC and range 10 to 45rC; and (3) psychrophiles (grow at cold temperature), with optimum at 15rC and range –5 to 20rC. Two other terms used in food microbiology are very important with respect to microbial growth at refrigerated temperature and survival of microorganisms to low heat treatment or pasteurization, because both methods are widely used in the storage and processing of foods. Psychrotrophs are microorganisms that grow at refrigerated temperature (0 to 5rC), irrespective of their optimum range of growth temperature. They usually grow rapidly between 10 and 30rC. Molds; yeasts; many Gram- negative bacteria from genera Pseudomonas, Achromobacter; and Gram-positive bacteria from genera Leuconostoc, Lactobacillus, Bacillus, Clostridium, and Listeria are included in this group. Microorganisms that survive pasteurization temperature are designated as thermodurics. They include species from genera Micrococcus, Bacillus, Clostridium, Lactobacillus, Pediococcus, and Enterococcus. Bacterial spores are also included in this group. They have different growth temperatures and many can grow at refrigerated temperature as well as thermophilic temperature. When the foods are exposed to temperatures beyond the maximum and minimum temperatures of growth, microbial cells die rapidly at higher temperatures and relatively slowly at lower temperatures. Microbial growth and viability are important considerations in reducing food spoilage and enhancing safety against pathogens, as well as in food bioprocessing. Temperature of growth is also effectively used in the laboratory to enumerate and isolate microorganisms from foods.
Relative Humidity of Environment
The RH of the storage environment is important both from the standpoint of aw within foods and the growth of microorganisms at the surfaces. When the aw of a food is set at 0.60, it is important that this food be stored under conditions of RH that do not allow the food to pick up moisture from the air and thereby increase its own surface and subsurface aw to a point where microbial growth can occur. When foods with low &„ values are placed in environments of high RH, the foods pick up moisture until equilibrium has been established. Likewise, foods with a high aw lose moisture when placed in an environment of low RH. There is a relationship between RH and temperature that should be borne in mind in selecting proper storage environments for foods. In general, the higher the temperature, the lower the RH, and vice versa. Foods that undergo surface spoilage from molds, yeasts, and certain bacteria should be stored under conditions of low RH. Improperly wrapped meats such as whole chickens and beef cuts tend to suffer much surface spoilage in the refrigerator before deep spoilage occurs, due to the generally high RH of the refrigerator and the fact that the meat-spoilage biota is essentially aerobic in nature. Although it is possible to lessen the chances of surface spoilage in certain foods by storing under low conditions of RH, it should be remembered that the food itself will lose moisture to the atmosphere under such conditions and thereby become undesirable. In selecting the proper environmental conditions of RH, consideration must be given to both the possibility of surface growth and the desirable quality to be maintained in the foods in question. By altering the gaseous atmosphere, it is possible to retard surface spoilage without lowering the RH.
Presence and Concentration of Gases in the Environment
Carbon dioxide (CO2) is the single most important atmospheric gas that is used to control microorganisms in foods. Ozone (O3) is the other atmospheric gas that has antimicrobial properties, and it has been tried over a number of decades as an agent to extend the shelf life of certain foods. It has been shown to be effective against a variety of microorganisms, but because it is a strong oxidizing agent, it should not be used on high-lipid-content foods since it would cause an increase in rancidity.
The physical and chemical environments control microbial growth within the growth range mainly by inﬂuencing their metabolic process associated with synthesis of energy and cellular components. Beyond the growth range, these factors, either individually or in combination, can be used to control microbial growth and even to destroy them. Actual growth is accomplished through the metabolism of various nutrients present in a food. The processes by which the food nutrients are transported inside the microbial cells and then metabolized to produce energy, cellular molecules, and by-products. Ozone was tested against Escherichia coli 0157:H7 in culture media, and at 3 to 18 ppm the bacterium was destroyed in 20 to 50 min- utes.16 The gas was administered from an ozone generator and on tryptic soy agar, the D value for 18 ppm was 1.18 minutes, but in phosphate buffer, the D value was 3.18 minutes. To achieve a 99% inactivation of about 10,000 cysts of Giardia lamblia per milliliter, the average concentration time was found to be 0.17 and 0.53 mg- min/L at 25°C and 5°C, respectively.77 The protozoan was about three times more sensitive to O3 at 25°C than at 5°C. It is allowed in foods in Australia, France, and Japan; and in 1997 it was accorded GRAS (generally regarded as safe) status in the United States for food use. Overall, O3 levels of 0.15 to 5.00 ppm in air have been shown to inhibit the growth of some spoilage bacteria as well as yeasts.
The term food preservation refers to any one of a number of techniques used to prevent food from spoiling. The following are the general methods of food preservation: application of heat, such as canning and preserving, pasteurization, evaporation, sun-drying, dehydration and smoking, application of cold, as ill cold storage, refrigeration and freezing, the use of chemical substances such as salt, sugar, vinegar, benzoic and lactic acids, fermentation, examples being acetic, lactic, alcoholic, etc., such mechanical means as vacuum, filtration and clarification processes, devices or agents for preventing chemical deterioration or bacteriological spoilage (the use of oil, paraffin and water glass are included here), combinations of two or more of the above.
Food preservation has become an increasingly important component of the food industry as fewer people eat foods produced on their own lands, and as consumers expect to be able to purchase and consume foods that are out of season.
Food spoilage can be attributed to one of two major causes: the attack by pathogens (disease-causing microorganisms) such as bacteria and molds, or oxidation that causes the destruction of essential biochemical compounds and/or the destruction of plant and animal cells. The various food preserving methods are all designed to reduce or eliminate one or the other (or both) of these causative agents.
For example, a simple and common method of preserving food is by heating it to some minimum temperature. This process prevents or retards spoilage because high temperatures kill or inactivate most kinds of pathogens. The addition of compounds known as BHA (butylated hydroxyanisole) and BHT (butylated hydroxytoluene) to foods also prevents spoilage in another different way. These compounds are known to act as antioxidants, preventing chemical reactions which cause the oxidation of food resulting in its spoilage. Among the most primitive forms of food preservation that are still in use today are such methods as smoking, drying, salting, freezing, and fermenting.
Because most disease-causing organisms require a moist environment in which to survive and multiply, drying is a natural technique for preventing spoilage. Leaving foods out in the sun and wind to dry out is probably one of the earliest forms of food preservation. Evidence of the drying of meats, fish, fruits, and vegetables also go back to the earliest recorded human.
Vacuum drying is a form of preservation in which a food is placed in a large container from which air is removed. Water vapor pressure within the food is greater than that outside of it, and water evaporates more quickly from the food than in a normal atmosphere. Vacuum drying is biologically desirable since some enzymes that cause oxidation of foods become active during normal air drying. These enzymes do not appear to be as active under vacuum drying conditions, however. Two of the special advantages of vacuum drying are that the process is more efficient at removing water from a food product, and it takes place more quickly than air drying.
Spray drying is the process during which concentrated solution of coffee in water is sprayed through a disk with many small holes in it. The surface area of the original coffee grounds is increased many times, making dehydration of the dry product much more efficient. Freeze-drying is a method of preservation that makes use of the physical principle known as sublimation, the process by which a solid passes directly to the gaseous phase without first melting. Freeze-drying is a desirable way of preserving food because at low temperatures (commonly around –10°C to –25°C) chemical reactions take place very slowly and pathogens have difficulty surviving. The food to be preserved by this method is first frozen and then placed into a vacuum chamber. Water in the food freezes and then sublimes, leaving the moisture content in the final product of as low as 0.5%.
The precise mechanism by which salting preserves food is not entirely understood. It is known that salt binds with water molecules and thus acts as a dehydrating agent in foods. A high level of salinity may also impair the conditions under which pathogens can survive. The value of adding salt to foods for preservation has been well known for centuries. Sugar appears to have effects similar to those of salt in preventing spoilage of food. The use of either compound (and of certain other natural materials) is known as curing.
Freezing is an effective form of food preservation because the pathogens that cause food spoilage are killed or do not grow very rapidly at reduced temperatures. The process is less effective in food preservation than are thermal techniques such as boiling because pathogens are more likely to be able to survive cold temperatures than hot temperatures. One of the problems surrounding the use of freezing as a method of food preservation is the danger that pathogens deactivated (but not killed) by the process will once again become active when the frozen food.
Fermentation is a naturally occurring chemical reaction by which a natural food is converted into another form by pathogens. It is a process in which food spoils, but results in the formation of an edible product. Perhaps the best example of such a food is cheese. Fresh milk does not remain in edible condition for a very long period of time. Its pH is such that harmful pathogens begin to grow in it very rapidly. Early humans discovered, however, that the spoilage of milk can be controlled in such a way as to produce a new product, cheese. Bread is another food product made by the process of fermentation. Flour, water, sugar, milk, and other raw materials are mixed together with yeasts and then baked. The addition of yeasts brings about the fermentation of sugars present in the mixture, resulting in the formation of a product that will remain edible much longer than will the original raw materials used in the bread-making process.
Heating food is an effective way of preserving it because the great majority of harmful pathogens are killed at temperatures close to the boiling point of water. In this respect, heating foods is a form of food preservation comparable to that of freezing but much superior to it in its effectiveness. A preliminary step in many other forms of food preservation, especially forms that make use of packaging, is to heat the foods to temperatures sufficiently high to destroy pathogens.
In many cases, foods are actually cooked prior to their being packaged and stored. In other cases, cooking is neither appropriate nor necessary. The most familiar example of the latter situation is pasteurization. During the 1860s, the French bacteriologist Louis Pasteur discovered that pathogens in foods could be destroyed by heating those foods to a certain minimum temperature. The process was particularly appealing for the preservation of milk since preserving milk by boiling is not a practical approach. Conventional methods of pasteurization called for the heating of milk to a temperature between 63 and 65°C for a period of about 30 minutes, and then cooling it to room temperature. In a more recent revision of that process, milk can also be "flash-pasteurized" by raising its temperature to about 71°C for a minimum of 15 seconds, with equally successful results. A process known as ultra-high-pasteurization uses even higher temperatures, of the order of 90–130°C, for periods of a second or more.
One of the most common methods for preserving foods today is to enclose them in a sterile container. The term canning refers to this method although the specific container can be glass, plastic, or some other material as well as a metal can, from which the procedure originally obtained its name. The basic principle behind canning is that a food is sterilized, usually by heating, and then placed within an air-tight container. In the absence of air, no new pathogens can gain access to the sterilized food. In most canning operations, the food to be packaged is first prepared in some way—cleaned, peeled, sliced, chopped, or treated in some other way—and then placed directly into the container. The container is then placed in hot water or some other environment where its temperature is raised above the boiling point of water for some period of time. This heating process achieves two goals at once. First, it kills the vast majority of pathogens that may be present in the container. Second, it forces out most of the air above the food in the container.
The commercial packaging of foods frequently makes use of tin, aluminum, or other kinds of metallic cans. The technology for this kind of canning was first developed in the mid- 1800s, when individual workers hand-sealed cans after foods had been cooked within.
The majority of food preservation operations used today also employ some kind of chemical additive to reduce spoilage. Some familiar examples of the former class of food additives are sodium benzoate and benzoic acid; calcium, sodium propionate, and propionic acid; calcium, potassium, sodium sorbate, and sorbic acid; and sodium and potassium sulfite. Examples of the latter class of additives include calcium, sodium ascorbate, and ascorbic acid (vitamin C); butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT); lecithin; and sodium and potassium sulfite and sulfur dioxide.
A special class of additives that reduce oxidation is known as the sequestrants. Sequestrants are compounds that "capture" metallic ions, such as those of copper, iron, and nickel, and remove them from contact with foods. The removal of these ions helps preserve foods because in their free state they increase the rate at which oxidation of foods takes place. Some examples of sequestrants used as food preservatives are ethylenediamine-tetraacetic acid (EDTA), citric acid, sorbitol, and tartaric acid.
II. MICROBIOLOGY OF FERMENTED FOODS
Food fermentation involves a process in which raw materials are converted to fermented foods by the growth and metabolic activities of the desirable microorganisms. The microorganisms utilize some components present in the raw materials as substrates to generate energy and cellular components, to increase in population, and to produce many usable by-products (also called end products) that are excreted in the environment. The unused components of the raw materials and the microbial by-products (and sometimes microbial cells) together constitute fermented foods. The raw materials can be milk, meat, ﬁsh, vegetables, fruits, cereal grains, seeds, and beans, fermented individually or in combination.