Antimicrobial agents have long been researched for their effectiveness to kill or inhibit growth of microorganisms in and on foods. This has been done in an effort to increase food safety for the consumer, as well as to increase the shelf life of food products. As part of process control activities in the food manufacturing plant, antimicrobial agents have been successfully applied, both in the product formulation stage as direct food additives designed to reduce or eliminate pathogens or spoilage organisms and as processing aids or secondary food additives during the food production process. Formulating “safety,” or microbial inhibitors, into the product can be considered step one as part of the company’s multi-hurdle approach to process control.

The ability of the antimicrobials to be used to decontaminate food is described in the U.S. Department of Agriculture’s Food Safety and Inspection Service (FSIS, USDA) Directives, and defined by the U.S. Food and Drug Administration (FDA). Antimicrobial agents can be classified into three categories: processing aids, secondary direct food additives or direct food additives. As defined by FDA, in order for an antimicrobial to be considered a processing aid, it should be added to the food during processing, and is either removed, converted into normal food constituents or functional additives that leave insignificant nonfunctional residuals [21 CFR 101.100 (a) (3) (ii)]. Secondary direct food additives are added during the manufacture or processing for functionality but are removed from the final food. Residuals of such additives cannot have any technical effect. This is consistent with the FDA definition of a processing aid, so no labeling is required [21 CFR Part 173]. Agents that are considered direct food additives do provide technical effects to the final food product and should be listed on the food label by their common (or usual) name [21 CFR Part 172].

The use of any antimicrobial depends on several factors, such as desired effect, legal limits of use and effect on the food. Certainly, the direct and secondary direct food additive antimicrobials discussed here are by no means intended to be viewed as a comprehensive list; nor are all of the uses listed for each antimicrobial to be viewed as the only applications possible. But those listed are certainly recognized as some of the most effective and/or promising agents used today as part of process control in food manufacturing operations.

Acid-Antimicrobial
Some of the more widely researched antimicrobials include organic acids, such as lactic, acetic and citric acids. As the name would imply, these acids also can enhance or contribute to the flavor of acidified or fermented foods, such as sausages, cheeses, pickles, or sauerkraut. The effectiveness of organic acids as antimicrobials differs widely based on concentration, pH, molarity and the concentration of the non-dissociated form. These acids, when used in combination with other compounds, have exhibited synergism. Potassium sorbate with lactic acid and citric acid have been reported to inhibit Salmonella, Pseudomonas fluorescens and Yersinia enterocolitica, as well as some lactic acid bacteria and osmophilic yeasts. Benzoic acid and sodium benzoate also are used to prevent yeast and mold growth in fruit juices and fruit products, and is currently allowed at 0.1% [21 CFR 184.1021]. The organic acids potassium sorbate and sorbic acid, used in bakery products, cheese and salad dressing, are antimicrobially effective at pH 4.0-6.0.

Currently, organic acids are allowed to be used at < 2.5 % of solution for pre-chilled carcass washing (FSIS Notice 49-94). Lactic acid is also allowed to be used at 5% of solution (55C) as a pre- and post-chill wash for beef carcasses. If used as stated with regard to beef or poultry carcass washing, use of 2.5 % of organic acids or 5% of hot lactic acid should have no residual effect on meat fabricated from the carcass. The maximum allowable usage is to be determined by the percent of the organic acid in aqueous solution prior to use and should not be determined by residual organic acid levels on the carcass. Exposing parsley leaves to an acetic acid wash (2%) for 15 minutes has been reported to reduce inoculated Yersinia enterocolitica from 10 CFU/g to <1 CFU/g. Much like the food regulations, organic acids may be used continuously on conveyor belts with no additional labeling as long as there are no residual technical effects to the meat and poultry. If residual technical effects are present, labeling is required. Approved antimicrobial agents other than organic acids may also be used on meat and poultry conveyor belts, but must be followed by a potable water rinse of the products.

Salts of lactic acid, or lactates such as sodium lactate and potassium lactate, have also been examined for their antimicrobial effectiveness. Lactates are able to inhibit gram-positive organisms more effectively than gram-negatives and have shown antifungal activity against some Aspergillus species. The use of lactates as antimicrobial agents is primarily due to their ability to reduce pH and water activity. Studies have differed with regard to the effectiveness of sodium lactate to lower water activity compared with sodium chloride. However, when both were used at 4%, sodium lactate was found to be more inhibitory than sodium chloride on the growth of common meat contaminants such as Brochotrix thermosphacta, Listeria monocytogenes, Staphylococcus aureus and Yersinia enterocolitica. Parameters of the food such as water activity will affect such comparisons due to the fact that higher concentrations of sodium lactate are needed in a more aqueous environment. Also, when considering the two, sodium lactate is reported to have five times less “salted flavor” than sodium chloride. The combination of sodium lactate and sodium diacetate has shown synergistic effect against Listeria monocytogenes and its application in ready-to-eat meat (RTE) products such as hot dogs has been widely accepted. Sodium diacetate, a molecular compound of acetic acid, is used to achieve an antimicrobial effect in baked goods, fats and oils, gravies and sauces, snack foods, meat products and soups and soup mixes, as well as to flavor these foods. Currently, sodium lactate and potassium lactate are allowed at 4.8% for the decontamination of meat, poultry and seafood products [21 CFR 184.1768, Sodium Lactate; 21 CFR 184.1639, Potassium Lactate].

Buffered sodium citrate (BSC) is currently being researched for its effectiveness as an antimicrobial. BSC is sodium citrate that has been buffered to a pH of 5.6 with citric acid. Research results on the ability of BSC as an antimicrobial have been conflicting. However, when examined as an inhibitory agent, BSC at 1% has been shown to inhibit the outgrowth of Clostridium perfringens spores in chilled marinated ground turkey breast, chilled roast beef and injected pork. Furthermore, BSC has also been reported to prevent the outgrowth of Listeria monocytogenes in vacuum-packaged frankfurters. These and similar studies indicate that BSC may not be effective as a bactericidal agent, but rather it is bacteriostatic and therefore an inhibitory agent. BSC is currently allowed in meat and poultry products at < 1.3% of product formulation [21 CFR 184.1751].

Acidified sodium chlorite (ASC) is being used for decontamination by the meat and poultry industry. ASC is a combination of an acid and sodium chlorite in aqueous solution. The antimicrobial species is chlorous acid that is formed in the mixture from the dissociation of sodium chlorite in the presence of acid. It primarily attacks by oxidizing sulfide and disulfide linkages and amino acid components of bacterial cell membranes, and therefore it has a broad antimicrobial spectrum against bacteria, mold and yeast. After treatment, ASC will be converted into much safer compounds, mainly sodium chloride and a small amount of sodium chlorate. When sodium chlorite was used with citric acid significant reductions in Escherichia coli O157:H7 and Salmonella Typhimurium have been observed on beef carcasses. ASC can be applied at ambient temperatures by spraying or dipping methods. It usually does not cause adverse organoleptic changes. ASC is allowed to be used on poultry carcasses and parts, and meat carcasses, parts and comminuted, formed or processed meat products [21 CFR 173.325]. The allowed level is 500-1200 ppm in combination with any Generally Recognized As Safe (GRAS) acid to a pH range of 2.3 to 2.9. The pH used is food dependent, since ASC also can be used for effective microbial reduction on fruits and vegetables.

Chemical-Antimicrobial
Trisodium phosphate (TSP) is used by the poultry industry as an antimicrobial for raw, unchilled carcasses and giblets. The allowable use is 8-12% solution used as a dip or spray, with 30 seconds allowed for giblets and 15 seconds for carcasses (65-85F) [21 CFR 182.1778]. The use of trisodium phosphate at 10% has been reported to reduce Salmonella Typhimurium by 2 log CFU/carcass. The use of trisodium phosphate to disinfect fruits and vegetables may be limited due to the high pH (11-12) of the solutions.

Chlorine dioxide is being used by the poultry industry to treat poultry processing water. It also can be used in wash water for fruits and vegetables. The allowable usage is that the chlorine dioxide residue must not exceed 3 ppm in the processing or wash water [21 CFR 173.300]. Fruits and vegetables must be washed with a potable water wash following chlorine dioxide treatment.

Peracetic acid (peroxyacetic acid) has been found to be an effective antimicrobial against vegetative cells, endospores, yeast and mold spores because of its high oxidizing potential and a low pH of 2.8. The acid is prepared using acetic acid with hydrogen peroxide. Peracetic acid is useful as a sanitizer for contact surface decontamination [21 CFR 178.1010] and as a fruit, vegetable, meat and egg wash [21 CFR 173.315]. Peroxyacetic acid, when used in wash water, cannot exceed 80 ppm, and treated products have to be followed by a potable water rinse [21 CFR 173.370].

Sodium nitrite and nitrate have long been used in meat as “red” color stabilizers. They also have been used to effectively control and inhibit growth of Clostridium species. Unfortunately, heating nitrites at cooking temperatures can cause the formation of nitrosamines, which are carcinogenic. Still, it was determined that the benefit outweighed the risk in this case; but the use of nitrites is strictly specified depending on the food in question. As a color fixative in smoked, cured tunafish, sodium nitrite cannot exceed 10 ppm. When used as a preservative and color fixative in smoked and cured sablefish, salmon, and shad, sodium nitrite cannot exceed 200 ppm; with sodium nitrate not greater than 500 ppm. (Consult the CFR for regulations on specific cured meat products.) When as a preservative and color fixative with sodium nitrate, sodium nitrite cannot exceed 200 ppm and sodium nitrate no more than 500 ppm in the final cured meat, poultry, or wild game product. The use of these additives and directions for proper food preparation must be added on the retail label of such products.

Ovo-Antimicrobial
Lysozyme is a naturally occurring enzyme in both the animal and plant kindoms that plays an important role in the natural defense mechanism. It has the ability to lyse the cell walls of gram-positive bacteria by hydrolyzing the peptidoglycan polymers. Lysozyme is a natural food preservative because it is endogenous to many foods, and specific to bacteria cells and harmless to human cells. Commercial lysozyme is made from hen egg albumin. Gram-negative bacteria also may be killed by lysozyme if their outer membrane is disrupted prior to exposure. Organic acids are one example of compounds that can disrupt the Gram-negative outer membrane. Lysozyme is used in casings and cooked RTE meat and poultry products. The allowable usage is 2.5 mg/lb. of casings and 2.0 mg/lb. of RTE meat or poultry products [GRAS Notice No. 000064].

Lacto-Antimicrobial
Lactoferrin is a glycoprotein found in milk that has iron-chelating properties. It is this ability that causes a lack of available iron to bacteria, thereby inhibits bacterial growth. The antimicrobial activity of lactoferrin is highly dependant on the three-dimensional or tertiary structure of the protein, and it can be significantly increased by immobilization of the structure, resulting in a new product called “activated lactoferrin.” The activated lactoferrin is able to detach microorganisms from biosurfaces and also inhibits growth of food pathogens by absorbing free irons. It is effective against both Gram-positive and Gram-negative bacteria. However, it does not kill bacteria like other cidal intervention technologies. It functions mainly as bacteriostatic and has a microbial blocking effect. This may prove useful in addressing the problems associated with tightly bound or adherent pathogens in raw meat that are difficult to eliminate using traditional cleaning or intervention processes.

Lactoferrin is allowed to be used at < 2% as a water-based spray for beef carcasses and parts, but must be listed in the ingredients statement of the product [GRAS Notice No. 000067]. If used as a beef carcass spray (1 g/carcass), washed with tempered water, and then rinsed with lactic acid, no listing on the label is required [GRAS Notice No. 000130].

Bacto-Antimicrobial
Nisin is a small antimicrobial peptide produced by certain strains of Lactococcus lactis subsp. lactis, a common lactic acid-producing bacterium. It is composed of 34 amino acids and has a molecular weight of approximately 3,510 Dalton. Nisin is also referred to as a bacteriocin, but possesses a wider antimicrobial spectrum than most bacteriocins. It is active against a wide range of Gram-positive bacteria, and is particularly effective against heat-resistant bacterial spores. Nisin inhibits the growth of Gram-positive bacteria by disrupting cell membrane and causes leakage of cytoplamic materials. It is not active against Gram-negative bacteria, molds and yeasts because it is unable to penetrate these cells to gain access to the cytoplasmic membrane.

Purified nisin has been commercially available, and due to its effectiveness in pathogen and spoilage control, it has gained great popularity in food applications. In fact, nisin is the only bacteriocin so far approved by FDA for food applications. FDA has affirmed that nisin derived from certain strains of Lactococcus lactis subsp. lactis is GRAS for use as an antimicrobial agent in various cheese products such as cheese spreads and process cheese when used at a level that delivers a maximum of 250 ppm of nisin in the finished product (21 CFR 184.1538). Nisin also can be used in meat and poultry at 600 ppm when the meat is fully cooked and used as a component in sauces. Under these conditions, the meat and poultry cannot be more than 50% of the product. Nisin can also be used as a component of meat and poultry soups at a much lower concentration of < 5 ppm of the product formulation. More recently, nisin is allowed to be used in casings at 3.15 mg/lb., and in cooked meat and poultry products at 2.5 mg/lb. [GRAS Notice No. 000065]. In all cases listed here, nisin must be declared on the ingredient statement.

There are many other bacteriocins that have been demonstrated as antimicrobials. Examples include pediocin, sakacin, reuterin, lacticin, macedocin and colicin. Most of these are gram-positive inhibitors, and in general, they have much narrower antimicrobial spectrums than nisin. Their application in purified form in foods has yet to be approved.

Phyto-Antimicrobials
Much research has been performed in an effort to discover additional natural agents with antimicrobial properties. One of the promising areas of this research is the work with spices. Usually, spices are added to food or food formulations based on flavor or aroma profiles. However, many spices also possess a certain degree of antimicrobial ability based on their inherent active compounds. The use of spices and herbs in combination are reported to have synergistic effects, as do spice blends that combine several spices together. Some of the antimicrobial active compounds derived from plant essential oils include eugenol, carvacrol, thymol, and vanillin. Some of the spices most effective for bacterial inhibition include allspice, bay leaves, capsicums, cinnamon, cloves, cumin, garlic, lemon grass, onion, oregano, rosemary, tarragon, and thyme. Each of these spices has more than 10 biologically active compounds. Unfortunately, in order to achieve a high amount of the active compounds, the formulator would likely need to add an unreasonable amount of the spice to the food. Therefore, the active compounds need to be isolated and concentrated, with research performed on the concentrates. While showing an increase in antimicrobial ability, some concentrated compounds may lend adverse flavor or odor depending on the food product in question.

Some common spices have been shown to have these desired antimicrobial effects. The addition of cinnamon to apple juice enhances the lethal effect of potassium sorbates or sodium benzoate against Escherichia coli 0157:H7, as shown in a Kansas State University study. For apple juice producers that don’t have thermal treatment, the addition of cinnamon to the juice along with sorbate, plus temperature control, may eliminate E. coli 0157:H7 as required. The amount of cinnamon (0.3%) plus 0.1% potassium sorbate did the job.

Some other areas of natural antimicrobials being studied are teas and prune extracts. Teas contain tannins and polyphenolic compounds such as catechin, catechin gallate, caffeine, chlorogenic acid, epicatchin, epicatechin gallate, epigallocatechin gallate, gallic acid, gallocatechin, theaflavin theobromine, and theophylline. These compounds have been shown to have bactericidal and bacteriostatic properties against a range of bacteria. Prune extracts have also been researched for their antimicrobial effectiveness. They have been shown to inhibit growth of aerobic bacteria in ground beef. In addition, prune extracts act as a humectant, which is reported to make the product more palatable. As a further benefit, prune extracts are cheaper than ground beef, thereby providing monetary savings to the meat producer.

Hop acids are compounds derived from hops and some of them, especially hops beta acids, have shown antimicrobial activities. The primary hops beta acids are lupulone, colupulone and adlupulone. They are obtained by extraction and separation processes from the hops flower. The purified hops beta acids have antimicrobial activity against certain Gram-positive spoilage bacteria, and inhibit the growth of L. monocytogenes in foods. Such hops beta acids can be used as an antimicrobial agent in casings for frankfurters at a concentration of 5.5 mg/kg of frankfurter and in RTE meats at a concentration of 4.4 ppm in the finished product (GRAS Notice No. 000063). A mixture of hops beta acids, egg white lysozyme and cultured skim milk can be used in salad dressings used in refrigerated meat and poultry deli salads with antimicrobial effect.

A Formula for Success
Of course, the effectiveness, or log reduction achieved, of an antimicrobial is greatly dependent upon the concentration used and the food product in question. Thus, although there have been many innovations in the use of antimicrobials in and on food products, continued research in the field is paramount. Formulating food safety into product, where possible, enhances the manufacturer’s efforts to control and reduce microbial hazards early on in the process.

Beth Ann Crozier-Dodson, Ph.D., is a research assistance and coordinates activities for Dr. Daniel Y.C. Fung’s BioHazard Level 2 Food Microbiology Laboratory at Kansas State University, Manhattan, KS. She has performed research in lactic acid multi-hurdle interventions, cinnamon and carbon dioxide as antimicrobials for apple juice, prune extracts for microbial reduction in ground beef, BSC, chlorine dioxide and aeromicrobiology, among others. Crozier-Dodson is the general laboratory coordinator for the annual KSU International Rapid Methods and Automation in Microbiology Workshop, for which she received a USDA Commendation for Contribution to Public Health, and designs and teaches microbiology workshops around the world.

Mark Carter is a Section Manager for Microbiology and Food Safety for Kraft Foods North America. His groups are responsible for developing and implementing food safety and testing programs for the Grocery, Dairy and Meals business sectors. These sectors include products such as Oscar Mayer meats and lunchables, DiGiorno meals and pizza, as well as Kraft cheeses and salad dressings.

Zuoxing Zheng, Ph.D., is Associate Principal Scientist with Kraft Foods North America, where he is responsible for development of novel antimicrobial systems for food applications to improve safety and quality of foods.

For More Information
To obtain a full listing of antimicrobial food additives and their allowances, please refer to the FSIS USDA Directives and Chapter 21 of the Code of Federal Regulations. To see a list of GRAS notices listing antimicrobial agents under review or approved since 2000, visit www.accessdata.fda.gov/scripts/fcn/fcnNavigation.cfm?rpt=grasListing.

To read more about fruit and vegetable decontamination, a detailed guide called “Surface Decontamination of Fruits and Vegetables Eaten Raw: A Review,” by Dr. Larry R. Beuchat (University of Georgia) is available. The article was prepared in conjunction with NSF International and the WHO (WHO/FSF/FOS/98.2).

For an extensive guide to spices and their effectiveness, refer to the article “Antimicrobial Activity of Spices,” by Dr. Erdogan Ceylan and Dr. Daniel Y. C. Fung in the Journal of Rapid Methods and Automation in Microbiology 12(2004) 1-55.