Ingredients To Raise the Microbial Bar

  Many factors affect microbial growth in foods - temperature, oxygen, presence of salts, microbial load and competition, antimicrobial agents, water activity and pH are among the most important. Some of these relate directly to food ingredients. Pickled cucumbers use many of these elements: salt, acid, sugar, fermentation and spices. While pickling dates back 4500 years, many of today's ingredients provide the same effects in a much wider range of foods.

  These ingredients work via a number of different and often intertwined mechanisms and, in some cases, the exact effect is difficult to pinpoint. They may react with cell membranes by changing permeability or interfering with movement of chemicals; inactivate enzymes; disrupt genetic functions; or disrupt nutrient utilization or synthesis. Many can work in concert for increased safety. This information can aid a product designer in selecting an ingredient or group of ingredients that will best prevent a microbial time bomb.

Phixing the pH

  Depending on the microorganism, a low pH can act as a bacteriostat (inhibit microbial growth) or a bacteriocide (kill microorganisms). In most cases the reduced pH acts in concert with other factors - such as increasing the effectiveness of heat treatments or optimizing preservatives - and provides a hurdle to limit growth rather than providing complete safety. That reduced pH doesn't provide complete safety has become painfully evident in the past several years, as acidic beverages such as unpasteurized apple and orange juices have been found to harbor E. coli 0157:H7 and Salmonella.

  These acid environments might fall outside the microbial-growth norm, but low-acid foods are ideal for supporting microbial growth. The FDA defines low-acid foods as any that have a finished equilibrium pH greater than 4.6 and a water activity (A_w) greater than 0.85. Without complete sterilization to prevent microbial growth, particularly that of Clostridium botulinum in hermetically sealed packages, foods must have an acid pH. Some, such as tomatoes and oranges, come that way naturally. For those that don't, numerous food-grade acidulants can drop the pH.

  The most commonly used acids in the food industry are citric, lactic, malic, acetic (vinegar), fumaric, tartaric, and phosphoric acids. With the exception of phosphoric, these are all organic acids and occur naturally in many foods. Other acidic ingredients, such ascorbic acid, citric acid-containing lemon or benzoic acid-containing cranberry juices, might be added. Additionally, acid-producing fermentations by friendly bacteria might also help achieve a target pH.

  Fatty acids have also demonstrated the ability to prevent microbial growth. Lactic and acetic are considered short-chain fatty acids, and they inhibit gram-negative and gram-positive bacteria. Medium-chain saturated fatty acids (C8 to C14), especially lauric acid, restrict the growth of gram-positive microbes. Increased chain lengths, branching and other structural variations typically decrease their potency. Esterification often positively influences the antimicrobial effect; in many cases the monoglyceride is more effective than the fatty acid alone.

  The antimicrobial activity of some acids does not come strictly from pH reduction. They also work through the antimicrobial effects of the undissociated ions in solution. Short-chain undissociated acids can pass through cell walls and disrupt cellular metabolism.

  Quick chemistry tutorial: Weak acids, a category that includes the organic aids previously mentioned, do not dissociate completely in water. The weaker the acid, the smaller its Ka (acid dissociation constant, a term that describes the extent of the dissociation) and the less it will dissociate. Ka is often expressed as pKa, which equals -log Ka.   When the pH of a system approaches the pKa of an organic acid, it allows more unassociated short-chain acids through the walls, so short-chain acids can be more effective at a lower pH.

  All acids are not effective against all organisms; certain bacteria might have a built-in defense. For example, Enterobacteriaceae and certain other species can metabolize benzoate. Formulation might also influence how well they work. The pKa of most organic acids limits their optimum pH range, and fats can inhibit the action of some, such as sorbic.

  Using acids as antimicrobials is also circumscribed by their effect on the finished product. A pH of 2.3 might stop all growth, but it would also stop most consumers from putting the product in their mouths due to extreme acid taste. The pH can also promote unacceptable reactions, including color changes or precipitation.

  Most food acids also complement certain flavors, generally in combinations that occur naturally, such as propionic and cheese, citric and citrus, malic and apples and lactic and meat. "Acetic acid has a very pungent, vinegar-like flavor," says Bert DeVegt, national sales manager, Purac America, Inc., Lincolnshire, IL. "You can only use a certain level before it gets overpowering. Lactic acid is much milder."

  Certain acids may also exhibit a synergistic effect when used in combination. For example, combining acetic and lactic acids can provide a potent combination, according to DeVegt. When used alone, acetic acid levels of 2.0% or lactic acid of 2.5% are required to inhibit the growth of Lactobacillus plantarum, a spoilage indicator in sauces and dressings. But when using a combination of these two acids, a total level of 1.5% acid is all that is required to achieve the same effect. "If you have a salad dressing with acetic acid and replace 50% of the acetic acid by lactic acid, you have a milder flavor and a more microbial-effective formula," DeVegt notes. "You can even use a higher level of lactic acid so the entire acid content of the system can be higher and still have a milder flavor profile." Because lactic acid is a stronger acid than acetic, adding the same weight actually produces a lower pH.

  Many acids and their salts can be formulated alone or with other antimicrobials, to make a sanitizing wash. These are typically used for meat products, but may also have some value for other foods such as fresh-cut vegetables. In one study, conducted by researcher Susan Summer, department of food science and technology at Virginia Polytechnic Institute and State University, Blacksburg, fruits and vegetables were contaminated with Salmonella, Shigella and E. coli and then washed with a spray of hydrogen peroxide followed by acetic acid. While the H2O2 alone was fairly effective, the second spray of acetic acid improved the results tenfold.

  Lactic and other acids can make effective washes for meat and poultry to keep the microbial count down. "Lactic acid is able to kill bacteria, giving a reduction between log one and log three," says DeVegt. "Manufacturers use about 2% lactic acid in a water solution as a final wash, before the carcass is chilled. It will kill up to about 99.9% of the bacteria. And if you leave it on the carcass surface, it will also have a bacteriostatic effect and inhibit the growth of bacteria." This same sort of treatment could also be used for cooked sausages, such as frankfurters, applied as a spray before peeling, he says.

Shake 'em with salts

  When some salts dissolve in water, the ions can react with the water, allowing them to act as weak acids. They therefore follow the same behavior as the organic acids. Commonly used salts include the lactates (calcium, sodium and potassium), the benzoates (sodium and calcium), the sorbates (potassium, calcium and sodium) and the propionates (sodium and calcium). In solution, these transform to lactic acid, benzoic acid, sorbic acid and propionic acid, respectively. Formulators can add the acid form but, particularly in the case of sorbates and benzoates, the salt form provides increased solubility over the dry acid.

  Salts are useful in meat products, where acids can adversely affect the product. Meats are a pH-neutral product, DeVegt says. "Acids promote protein denaturation. By lowering the pH, you're also decreasing the yield of the product by lowering the water-holding capacity of the meat protein." Still, the right pH will increase the effectiveness of the salts, so there may be a balancing act. With sodium lactate (an ingredient widely used to extend shelf life and increase safety of processed meat products), for example, "If you can lower the pH of a meat product by 0.1 to 0.3 units, you end up with a more effective hurdle," he continues.

  Salt (NaCl), on the other hand, works by inducing plasmolysis, or cell dehydration. Because nature subscribes to the theory that all things seek equality, the high salt level on the outside of the cell causes osmosis. Water passes from inside the cell through the membrane into the solution in a vain attempt to achieve equilibrium. Most bacteria, with the exception of extreme halophiles that can live in as much as 30% salt, are more susceptible to elevated salt levels than are yeast and molds. Bacteria that grow best in the absence of NaCl, but can grow at moderate salt concentrations, such as S. aureus, are called halotolerant.

A_w - go on

  Any solute can affect the product's A_w, including salt. But unless one is a human halophile, the levels of salt required to reduce it to effective levels would make most foods unpalatable. So, the most common food ingredients to use for A_w reduction are sugars and related compounds - think jellies, fruit fillings, sugary soft bars and other high-sugar, high-solids items. A_w effect is related closely to osmotic pressure, which sugar solutions also exhibit. The ultimate effect of a given weight of solute depends on its molecular weight - the lower the molecular weight, the more moles per gram, and greater the reduction in A_w.

Better living through chemistry

  Although many claim that these compounds only give the meat a pleasant reddish color, in fact they suppress bacteria, particularly C. botulinum. The exact mechanism is unclear, but may result from reaction compounds that cannot be metabolized under anaerobic conditions.

  The problem is that nitrates/nitrites also result in small amounts of compounds that might be toxic to larger organisms, namely humans. When nitrite combines with secondary amines, a reaction promoted by very high cooking temperatures, it forms nitrosamines, which are carcinogenic. For this reason, manufacturers often look to other antimicrobial agents to reduce the level of nitrites required.

  Meat has been smoked through the ages to increase shelf life. Several studies conducted at Kansas State University, Manhattan, show that liquid smoke inhibits pathogens such as E. coli 0157:H7, Salmonella, Listeria and Streptococcus. Phenolic compounds in smoke act as bacteriocides.

  Sulfur dioxide and sulfates are another example of compounds that, although used for other preservative effects, also might provide protection against bacteria, especially the gram-negative variety. When sulfur compounds go into solution in an acid environment, they can form ions that reportedly prevent microbial growth. These ingredients also have acceptance problems in some quarters - some people suffer severe reactions when exposed to them and their presence must be noted on the product label.

  Hydrogen peroxide (H202) finds use in the dairy and starch industries as a direct additive and as a package sterilizer, particularly in aseptic applications. The FDA restricts its use to a maximum of 0.05% in milk intended for cheese-making, and 0.04% in whey during the preparation of modified whey. In starch, the limit is 0.15%. H202 can also occur as a fermentation byproduct of lactic acid starter cultures. It acts against bacteria - especially anaerobes and facultative anaerobes, yeasts, molds and viruses.

  Although not food additives, the sterilizing epoxides ethylene oxide (EO) and propylene oxide destroy all microorganisms, including viruses. These are typically applied as a gas on products such as spices and some nuts. The government has established tolerances for ethylene oxide residues in 40 CFR 180.151 of 50 ppm "when used as a post-harvest fumigant in or on the following raw agricultural commodities: black walnut meats, copra, whole spices." However, EO is regulated by OSHA as a cancer and reproductive hazard.

  Another chemical used as an antimicrobial treatment, rather than an ingredient, is ozone (O3). Ozone is an unstable gas that decomposes into oxygen fairly quickly, especially in water. It acts against many microbial contaminants - including E. coli, Salmonella, Giardia, and Cryptosporidium. Ozone was declared as GRAS for treatment of bottled water in 1982 and in food processing in 1998. Potential food applications include increasing yields of certain crops, protecting raw products during storage and transit, sanitizing packaging materials, or adding to wash water for foods. Also, Electric Power Research Institute, Palo Alto, CA is investigating ozone control of insect infestation during food storage as an alternative to fumigants currently being phased out, such as methyl bromide, which is scheduled to be banned by the U.S. government in 2001.

Microbe vs. microbe

  Antibiotics are microbial or synthetic substances that inhibit other microbial species by targeting the bacterial cell wall, membrane and ribosomes. The only antibiotic approved as a direct food additive in this country is natamycin (pimarcin), for use on the surface of cheese products. However, this substance only works against molds and yeasts, not bacteria. It does appear to suppress mycotoxin production, though, so it does contribute to a safer food supply.

  Last year, the FDA approved a combination of powdered cellulose and natamycin for use in cheeses in response to a petition by St. Louis-based Fiber Sales and Development Corporation that will allow its use in dry applications.

  Nisin, a natural antimicrobial, is a polypeptide produced by Lactobacillus lactis. It works over a pH range of 3.5 to 8.0 and has been found to work well against gram-positive bacteria such as Clostridium and Bacillus. "Gram-negative bacteria can be made more sensitive to Nisaplin® (the brand name for Cultor Food Science's nisin-based preservative) through disruption of their cell walls by exposure to chelating agents, sub-lethal heat treatments, and freeze/thaw cycles," says Joss Delves-Broughton, R&D manager, Cultor Food Science, Trobridge, UK. "All established uses are against gram-positive bacteria. It is particularly effective against gram-positive, heat-resistant spores and has uses in foods that are pasteurized, but not fully sterilized. Nisaplin is considered to be a natural preservative.

  "Nisin is allowed in the U.S. in standard-of-identity processed cheese spreads, non-standard-of-identity pasteurized liquid egg products, non-standard-of-identity salad dressings and sauces, and pasteurized soups," Delves-Broughton continues. "Typical levels are 2.5 to 12.5 ppm nisin - equivalent to 100 to 500 ppm Nisaplin. "The effect of nisin on a number of different foods has been tested, and some of these uses are approved in different countries throughout the world. For example, nisin has been used to decrease the heat treatment required for canned foods, and appears to decrease botulism toxin production in fish stored under modified-atmosphere packaging. It may also have benefits in some baked goods, meats and alcoholic beverages.

  "Nisaplin is often used as a factor in hurdle technology," Delves-Broughton says. "For example, in processed-cheese preservation, it can be used in combination with lower moisture, low pH, increased salt content, as well as low storage temperatures to produce long shelf life and safe products."

  Lactic acid bacteria - the same microorganisms used to make fermented dairy and meat products - produce other bacteriocin peptides during fermentation. While they are not currently available or approved as food additives, these metabolites might add to a fermented food's safety. In addition to nisin, these include compounds such as lactococcins, pediocins, lactacins and leucocin A. Many of these act against Listeria.

Food fights

  Lysozyme, an enzyme found in the white of hen eggs, exhibits bacteriocidal properties by hydrolyzing certain polysaccharides in bacterial cell walls. Approximately 3.4% of the albumen protein in egg consists of lysozyme. The FDA has approved it for use in cheese to prevent "late-blowing," an undesirable gas formation by Clostridium during cheese ripening. Researchers have also investigated its use as an antimicrobial agent in several other applications, including vegetables, tofu, sausage and seafood. It is believed to act against gram-negative species such as Bacillus and Micrococcus.

  "Lactoferrin presents a new opportunity in formulating foods by providing a multifunctional ingredient with antimicrobial and probiotic characteristics," says Loren Ward, Ph.D., research scientist, Avonmore Waterford Ingredients, Monroe, WI. Lactoferrin, an iron-binding glycoprotein, occurs in milk and bodily fluids such as tears, blood and saliva. It can be commercially isolated from cow's milk for use in food products. The ingredient is currently used in infant formulas or functional foods for its positive effect on intestinal flora. Research shows that lactoferrin inhibits E. coli, Salmonella typhimurium, Shigella dysenteria and other bacteria, and may inhibit some viruses also.

  Lactoferrin inhibits bacterial growth by at least two different mechanisms: by chelating iron, which makes this element unavailable for microbial growth, or by directly binding to the cell membrane, which alters membrane permeability. "Both antimicrobial mechanisms would be active with an iron-free lactoferrin product such as Bioferrin(tm)1000," notes Ward.

  Plants can also be sources of microbial barrier compounds. Spices have been used for centuries as food preservatives. Some are currently being touted for antioxidant capacities, but certain spice compounds also control microbial growth. Most of these are phenolic compounds that are typically not present in concentrations high enough to have an effect when the spices are used normally. Cinnamon contains cinnamic aldehyde, which acts against bacteria and inhibits mold and mycotoxin production. Other spice-based antimycotics include vanillin, citral, citronellol, geraniol and menthol. Eugenol, found in clove oil and allspice, suppresses mold and bacteria such as Salmonella, Vibrio and Staphylococcus. Thymol, contained in thyme, oregano savory and sage, inhibits a wide range of pathogenic bacteria and molds that produce mycotoxins.

  Vegetables also might be utilized in the fight against bacteria. They contain a wide range of compounds, many of which are phenolic or flavonoid molecules that have evolved as defensive antimicrobials in the plants themselves. For example, tannins can denature microbial protein. Other plant-based pathogen preventatives include alkaloids, lactones, glucosides, anthocyanins, phytoalexins, caffeine and protein-like compounds, such as zeamatin.

  Garlic, onion and similar plants provide allicin, a sulfur compound with considerable antimicrobial activity against both gram-positive and gram-negative organisms. The compound inhibits bacterial enzyme activity. Allicin isn't formed until the plant tissue is disrupted through cutting or crushing, when the plant's allin hydrolyzes. Numerous studies have shown this compound's effectiveness against many food-poisoning organisms, yeasts and molds, although it does not appear to inhibit toxin production of C. botulinum. Since botulism has been reported after the consumption of both fried onions and garlic in oil, this should come as no surprise, however. One 1973 study by Tynecka and Gos (published in Acta Microbiologica Polonica Series B, a Polish journal) showed a 1:128 dilution of garlic juice inhibits E. coli and a 1:64 dilution inactivates the organism.

  Another potential natural preservative occurs in high levels in horseradish and mustard oil. Researchers with USDA's Agricultural Research Service and Oklahoma State University (OSU), Stillwater, have looked at allyl isothiocyanate (AITC), a chemical that inhibits the growth of food pathogens such as L. monocytogenes, E. coli and S. aureus. ARS food technologist Henry Fleming, at the Raleigh, NC, Food Science Research Unit, and OSU food chemist Brian Shofran found that mustard oil contains 93% AITC, while horseradish has 60%. Other isothiocyanates turn up in cole vegetables, cabbage, spinach, radishes and related plants.

  Continued investigation of these types of compounds and others will become increasingly important in the future. The types of foods we eat, their method of preparation and the seriousness of the effects of microbial contamination all require increased vigilance and improved methods to defeat the microbial marauders that plague the food supply.