PRESERVATION FROM MICROORGANISMS

As noted in chapter 1, one set of techniques for the preservation of food is designed to kill microorganisms, to reduce or stop their growth, or to prevent them from reproducing. The methods used are generally either physical or chemical.

Physical methods of food preservation are designed to alter the environment in which microorganisms live, making it difficult or impossible for them to survive. Most microorganisms have certain common requirements for their survival: the presence of oxygen (for aerobic microorganisms), moisture, heat, and a certain optimal level of acidity. Physical methods of food preservation deprive mi­croorganisms of one or more of these conditions. For example, heat­ing food to some minimum temperature (pasteurization) can kill the microorganisms present in the food, preventing the food from spoiling or reducing the rate of spoilage. Freezing is less effective as a method of food preservation than pasteurization because it does not necessarily kill microorganisms, although it greatly reduces their rate of metabolism. Drying can be an effective method of food preservation because it deprives microorganisms of the moisture they need to live and reproduce. As with all methods of food preser­vation, each physical technique is more effective with some types of food and less effective with others. One of the most promising forms of food sterilization is radiation, discussed in chapter 5.

Chemical methods of food preservation act directly on microor­ganisms by altering their biochemical structure or the biochemical reactions used in their metabolism and reproduction. Chemical methods can be divided into three major categories:

1.  methods that change the permeability of a microorganism's cell membrane, preventing it from obtaining the nutrients it needs for its survival and thus causing its death;

2.  methods that interfere with a microorganism's biochemical reac­tions, usually involving the disruption of a specific enzyme activ­ity and thus causing the microorganism's death; and

3.  methods that block or interfere with the biochemical reactions involved in reproduction, preventing the growth of new micro­organisms.

A key factor in many of these methods is pH. pH is a measure of the acidity of a solution and is equal to the negative logarithm of the hydrogen ion concentration of the solution, or: pH = - log [H+].

The pH scale ranges from 0 to 14, with low pH numbers representing acidic solutions and high pH numbers representing alkaline solu­tions. The pH of pure water and any neutral solution is 7.0.

Scientists have determined that relatively few microorganisms can survive at a pH of less than 4.6, and for many of the most virulent microorganisms, the optimal pH is much higher. The table shows the optimal pH range for the survival of some common bacteria.

Organic and inorganic acids that retard or prevent spoilage by lowering the pH of food are some of the most widely used chemical preservatives. In many cases, these acids also interrupt one or more of the microorganism's biochemical reactions. Such compounds are sometimes known as microbial antagonists because their molecular structure is sufficiently similar to a second molecule to allow it to compete for positions on a microbe's chemical receptors. Some ex-

< OPTIMAL pH FOR VARIOUS TYPES OF MICROORGANISMS >
MICROORGANISM	pH
Bacteria	about 7.0
E. coli	6.0-8.0
Salmonella	6.8-7.5
Streptococci	6.0-7.5
Staphylococci	6.8-7.5
Clostridium	6.0-7.5
Fungi	about 5.6
Protozoa	6.7-7.7
Algae	4.0-8.5

amples of microbial antagonists currently in wide use are benzoic acid, sorbic acid, propionic acid, paraben, compounds of sulfur, and nitrates.

Benzoic acid (C6H5COOH) and its salts, the benzoates, are found naturally, most commonly in cranberries, prunes, and cinnamon. In addition to lowering the pH of food, benzoic acid and the benzoates interfere with the action of microbial enzymes that catalyze oxida-tive phosphorylation; that is, they prevent the microorganisms from storing the energy released when it metabolizes food. They also bind to and inhibit substances in the microorganism's cell membrane, reducing its ability to transport essential substances into the cell interior. Benzoic acid is most effective against molds, somewhat less effective against yeasts, and differentially effective against bacteria. It is used most commonly for the preservation of fruit juices, syrups, soft drinks, relishes, and margarine. The substance is usually used in the form of one of its salts, such as sodium or ammonium.

Sorbic acid (CH3CH = CHCH = CHCOOH) and the sorbates reduce the pH of food, react with chemicals in the cell membrane to reduce membrane transport, and interfere with a variety of enzymes in­volved in the cell's metabolism, especially the enolase and dehydro-genase enzymes. The foods to which sorbic acid is most frequently added are dairy products, primarily cheeses, meats, baked goods, prepared salads, pies and cakes, and pickled products. It is usually added in the form of calcium or potassium sorbate.

An interesting by-product of the use of benzoates and sorbates as food additives was announced in 2000. Scientists at the University of Rochester Medical Center discovered that these compounds may reduce the rate of tooth decay. Rats fed a diet of fluorides and benzo-ates or sorbates had fewer cavities than those whose diets contained fluorides only. The discovery was a happy surprise because these additives are so widely used today that everyone gets the benefits they provide—apparently including fewer cavities—without making any special effort.

Propionic acid (CH3CH2COOH) occurs naturally in certain fruits, such as apples and strawberries, and in tea and violets. When added to foods, it binds to molecules on the surface of microorganism cell membranes, reducing and stopping the flow of materials into and out of the cell. Inside the cell, it reduces pH, interfering with the organism's ability to metabolize normally. It acts most effectively against molds but can be used against certain types of spore-form­ing bacteria called rope-formers. Propionic acid and the propionates are used in baked goods, cheeses, and dairy products. It is usually added to foods in the form of its calcium or sodium salts, calcium or sodium propionate.

The term paraben refers to any of the alkyl esters of para-hydroxybenzoic acid. These compounds are especially effective in preventing the growth of molds and yeasts. Some of the esters that have been used as food additives are methylparaben, ethylparaben, propylparaben, butylparaben, and heptylparaben. Heptylparaben finds some limited use as a preservative in malt beverages and soft drinks where it appears to inhibit the growth of spores in bacteria such as Bacillus and Clostridium. The most common preservative formulation consists of a mixture of methyl and propyl esters. Such formulations are used in baked goods, jams and jellies, soft drinks, certain dairy products, and some kinds of fish and meat. Chemical structures for two paraben preservatives are shown below.

Compounds of sulfur called sulfites act both as microbial an­tagonists and as antioxidants, substances that prevent or retard the natural decay of foods. In the first role, they block the action of

ONa                                        ONa

COOCH3                           COOC2H5

Sodium                                    Sodium

methylparaben                      ethylparaben

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Two common parabens

two enzymes critical to the formation of ATP (adenosine triphos­phate), the "fuel" living cells use to produce energy. When these two enzymes, glyceraldehyde-3-phosphate dehydrogenase and alcohol dehydrogenase, are inhibited, the microorganism cell is unable to generate ATP, and it dies. Sulfites inhibit these two en­zymes by disrupting both the sulfur bonds in cysteine, one of the amino acids present in their molecular structure, and the disulfide bonds that hold the enzymes in their three-dimensional structure. This structure is what allows them to bond to food molecules, so when it has been destroyed, the enzymes are unable to continue functioning.

In common usage in the food industry, the term sulfite refers to a group of related chemical species that includes sulfur dioxide (SO2), sulfurous acid (H2SO3), the sulfite ion (SO32~), and the bisulfite ion (HSO3~). The form in which sulfur occurs depends on various fac­tors, the most important of which is pH. At low pH, the acid form (H2SO3) predominates and is most active.

Sulfur and its compounds are among the oldest chemical preser­vatives known. There is some evidence that the ancient Egyptians used such compounds to sterilize their wine barrels, and the burn­ing of sulfur among the Romans for the purpose of sterilization is well documented. According to the Food Additives and Ingredients Association, sulfites are the most widely used of all food preserva­tives today. Large amounts are used as preservatives in the produc­tion of wine and vinegar. Probably their most important use is in the treatment of fruits and vegetables that have just been harvested, to protect the products against attack by molds and yeasts.

Nitrates (NO3~) and nitrites (NO2~) are used primarily to cure meats. One function is to retain the red color that most people regard as a sign of meat that is fresh and healthful. The red color is produced by a series of reactions that occur when a nitrate or nitrite (such as potassium nitrate [KNO3] or sodium nitrite [NaNO2]) has been added to meat. When the additive is a nitrate, the first step in that process is the reduction of the nitrate to the nitrite:

NaNO3 k NaNO-

Microorganisms that occur naturally in meats, such as Micrococcus, catalyze this reaction. In the next step, the nitrite is converted to nitrous acid, which is then further reduced to nitric oxide (NO):

NaNO2 k HNO2 k NO

In the final step of this process, the nitric oxide reacts with myoglo-bin in meat, converting it to nitrosomyoglobin, a compound with a bright red color characteristic of fresh meat.

Nitrates and nitrites play a second critical role as additives to meat and meat products: They inhibit the production and germina­tion of Clostridium botulinum spores. This bacterium is the organism responsible for the deadly disease known as botulism, one of the most virulent diseases known to humans.

The agent thought to inhibit C. botulinum is nitrous acid, which oxidizes amino (NH2—) groups readily. Nitrous acid reacts with and deaminates cytosine, converting it to uracil (see the figure below). Since cytosine is a component of all DNA molecules, this reaction radically alters those molecules, converting them into a form that does not permit normal replication and transcription. This action of nitrous acid is thus thought to be responsible for the deactivation and inhibition of essential enzymes used by the C. botulinum bacte­rium, especially the dehydrogenases and oxidases.

NH2

HN02

I

H

Cytosine

I

H

Uracil

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Conversion of cytosine to uracil by nitrous acid

A fairly recent addition to the tools used for food preservation is the modified atmosphere packing (MAP) concept. Researchers have learned that they can extend the shelf life of many foods by selecting appropriate packaging materials and the appropriate atmosphere in which to store them. This concept is hardly new. It dates to at least 1795 when the French confectioner Nicolas (Frangois) Appert (ca. 1750­1841) discovered vacuum packing of food. Without actually under­standing the scientific principles involved, Appert found that storing foods in a can void of air retards the rate at which they spoil. Today it is understood that vacuum packing deprives microorganisms of the oxygen they need to grow and reproduce, reducing the rate at which toxins are released into the stored food.

Some forms of MAP employ Appert's original design. Food is placed into a gas-impermeable bag, from which air is almost totally removed (vacuum packaging) or partially removed (low-pressure or hypobaric packaging). In either case, microbial action is reduced because of the low levels of oxygen available for their growth and reproduction.

In another form of modified atmospheric packaging, food is stored in a gas-impermeable bag to which is added a specific mixture of oxy­gen, nitrogen, and/or carbon dioxide. Mixtures high in oxygen con­centration (high-oxygen MAP) are used almost exclusively for the stor­age of meats. Such mixtures usually contain about 70 percent oxygen, 20-30 percent carbon dioxide, and 0-10 percent nitrogen. The high concentration of oxygen inside the package ensures that sufficient amounts of the gas will be available to combine with myoglobin in the meat, producing its characteristic "fresh" red color. Packaging con­taining mixtures low in oxygen and high in nitrogen and/or carbon dioxide is similar in some ways to vacuum and hypobaric packaging. Reduced levels of oxygen retard the rate of microbial growth, extend­ing shelf life of the product. In addition, carbon dioxide gas itself may also act as a microbial antagonist in at least two different ways. First, carbon dioxide dissolves in water to form the weak acid carbonic acid (H2CO3). Carbonic acid ionizes to form hydrogen, bicarbonate (HCO3-), and carbonate ions (CO32-), lowering the pH of the food:

co2 + h2o k h2co3 k h+ + hco3 - k h+ + co32-

As pH is lowered, the rate of bacterial survival, growth, and repro­duction declines. Second, all forms of carbon dioxide (the gas itself, carbonic acid, and the ions it produces) are thought to interfere with essential biochemical reactions carried out by microbial cells. Evidence suggests that these species may affect the permeability of microbial cell membranes, interfere with the action of certain amino acids in cells, and inhibit the action of certain enzymes in­volved in cell metabolism.

All of the modified atmospheric packaging systems just described make use of a passive packaging material, usually a chemically inert plastic, and depend on the gases injected into the package for their food preservation action. Another recently developed approach in­volves the use of an "active" or "intelligent" packaging material. Here the material itself contains one or more substances that are gradu­ally released into the package and reduce the rate of spoilage of the food.

In an active packaging system, some antimicrobial substance is incorporated into the packaging material itself during production. In one series of experiments, for example, sorbic acid and potas­sium sorbate were added to the wax used to wrap cheese, where the compounds helped to destroy molds that cause cheese to spoil. The additive is usually formulated such that it will be released from the wrapping material slowly over time. In some cases, it is designed to adhere permanently to the inner surface of the packaging material. When a food is wrapped in the treated packaging, the antimicro­bial agent slowly migrates out of the wrapping material and diffuses throughout the food, where it performs its preservative function. In addition to antimicrobial actions, active packaging systems may perform as oxygen scavengers (to reduce the rate of natural food decay), moisture scavengers (to reduce the concentration of mois­ture inside the package), and ethylene scavengers (to remove a gas released during the ripening of fruit).

Active packaging technology is less than two decades old, and many questions have yet to be answered, for instance: What antimi­crobial agents can be used and what wrapping materials are most suitable for their deposition? What are the best methods for deposit­ing an agent into a given wrapping material? Under what conditions are the agents best able to act on the foods? Thus far, researchers have tried a number of antimicrobial agent-wrapping combinations in a variety of physical formats. For example, some traditional or­ganic acids (sorbic, benzoic, and propionic) have been implanted into polyethylene wrapping, carbon dioxide gas into cellulose wrap­ping, and propionic acid into ionomer-polymer wrapping. One of the most interesting lines of research focuses on attempts to implant microbial agents into edible types of wrapping. For example, in one experiment, sorbates were injected into an edible biopolymer for use in food packaging. The challenge with this line of research, of course, is to find antimicrobial-wrapping combinations that are not only effective in reducing spoilage but also safe for human consumption.

Thanks to developments in the food sciences over the past cen­tury, food processors now have a number of chemicals available to them as food additives for protection against spoilage as a result of the action of microorganisms. In addition, researchers continue to explore new methods of food preservation, such as modified atmo­spheric packaging, which holds promise for revolutionizing methods by which foods are preserved.