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PRESERVATIVES | Permitted Preservatives – Natamycin
Permitted Preservatives – Natamycin J Delves-Broughton, DuPont Health and Nutrition, Beaminster, UK Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Jacques Stark, volume 3, pp 1776–1781, Ó 1999, Elsevier Ltd.
Introduction
Structure and Properties
Natamycin is a polyene macrolide antimycotic produced by strains of the Actinomycetes, such as Streptomycetes natalensis, S. chattanoogenesis, and other closely related Streptomyces species. As an antimycotic, it exhibits strong antimicrobial activity against yeasts and molds. Prevention of fungal spoilage of foods is an important issue for the food industry as economic losses due to spoilage can be considerable. Apart from the visual and organoleptic spoilage of foods, many molds can produce mycotoxins that have carcinogenic properties. Natamycin shows no activity against bacteria. Such a selective antimicrobial activity has led to its use in bacteria fermented foods, whereby its selective action has no negative effect on the bacteria culture responsible for the desired fermentation but will have a positive antimycotic effect against contaminating yeasts and molds. Thus, it is used often on the surface of cheese, fermented meats or in yogurt, fermented creams, and similar products. It is marketed commercially as the NatamaxÔ family of products by Danisco and as the DelvocidÒ family of products by DSM. China also has manufacturers. To date, natamycin is the only microbially derived antifungal compound that is used in the food industry.
Natamycin is a polyene macrolide with a molecular weight of 665.7 Da and the empirical formula C33H47 NO13. Its structure has been determined and is shown in Figure 1. As dry powder it can be stored for several years with minimal loss of activity. Aqueous suspensions are less stable, particularly if exposed to light, certain oxidants, and heavy metals, but they remain sufficiently stable during practical use. Thus compounds such as peroxides or chlorides, often used as cleaning or disinfectant agents, should be used with care in the proximity of natamycin. Although solutions are more unstable in acid or alkaline conditions, the pH of most food products is not normally at levels that cause problems. Like similar polyene macrolides, natamycin is amphoteric containing one basic and one acidic group. Natamycin has low solubility in water (approximately 40 mg ml1) and is almost insoluble in nonpolar solvents, but it shows good solubility in strong polar organic solvents such as glycerol, methylpyrrolidone, and glacial acetic acid. The low solubility in water can be an advantage for the surface treatment of food as it will stay on the surface where it is needed instead of migrating into the food. Natamycin has numerous advantages over other antimycotic preservatives such as sorbates and these are summarized in Table 1.
History Natamycin was first produced in 1955 from a culture filtrate of a Streptomycetes isolated from a soil sample in South Africa. Its name is in fact derived from the South African province, Natal, from where it was originally isolated. Other names used in the past are pimaracin and tennectin. Commercial preparations are produced by fermentation of S. natalensis in a medium containing a carbon source (typically starch or molasses) and a fermentable nitrogen source (typically corn steep liquor, casein, soya). Fermentation is aerobic and mechanical agitation and antifoaming agents can aid the process. The temperature range is 26–30 C and the pH range is 6–8. Due to its low solubility, natamycin will accumulate mainly as crystals, and these can be extracted following separation of the biomass by solvent extraction. The natamycin content of most commercial preparations is 50% with the incipient being lactose, glucose, or salt. There are also natamycin-based products that contain food-grade polymers that aid the adherence of natamycin for the surface treatment of foods. Recently DuPont introduced NatamaxÒ plus B, which is natamycin complexed with cyclodextrin. This has increased solubility compared with standard natamycin preparations. Other natamycin-based products are used for topical veterinary and pharmaceutical applications to treat fungal infections, such as ring worm in horses and fungal eye infections (keratitis) in humans. Recently, the biosynthetic gene cluster for natamycin production has been characterized for S. chattanoogenesis.
Encyclopedia of Food Microbiology, Volume 3
Figure 1
Table 1
The structure of natamycin.
Advantages of natamycin over sorbate
Natamycin
Sorbate
Natural Fungicidal No effect on bacteria No migration into food No flavor Effective at 1–40 mg kg1 Effective at pH 3–9
Chemical Fungistatic Bactericidal Penetrates into food Bitter flavor Effective at 1000–2000 mg kg1 Effective only at acidic pH
http://dx.doi.org/10.1016/B978-0-12-384730-0.00269-X
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PRESERVATIVES j Permitted Preservatives – Natamycin
Mode of Action and Antimicrobial Activity Natamycin acts by combining with ergosterol and other sterols present in the cell membranes of yeasts and vegetative mycelium of molds. It was thought that this interaction resulted in pore formation, resulting in leakage of cellular material, but recently it has been shown that this is not the case. Rather, the interaction of natamycin with ergosterol results in the prevention of cell division and loss of enzyme function. Less is known about the action of natamycin against mold spores, but it is thought to inhibit their germination. Ergosterol is not found in the cell membranes of bacteria and hence their resistance. It is found in algal cell membranes and thus algae are also sensitive. There are no reports of development of resistance to natamycin in vivo. Studies undertaken in cheese and fermented sausage factories that have used natamycin for several years showed no increase in the levels of natamycin-resistant yeasts and molds compared with similar factories not using natamycin. Most molds (Table 2) are sensitive to natamycin at concentrations of 40 mg ml1 or less. Yeasts (Table 3) are even more sensitive with minimum inhibitory concentrations of 5 mg ml1 or less. A few species of molds such as Penicillium discolor that have no or low levels of ergosterol in their membranes can have reduced sensitivity. However, such reduced sensitivity rarely if ever causes problems in practical food preservation. Laboratory experiments to induce resistance have been unsuccessful.
Methods of Assay The natamycin contents of food products can be determined by microbiological, immunological, mass spectrophotometric (MS), liquid chromatographic (LC), and high-performance liquid chromatographic (HPLC) methodologies. Minimum detection limits for these methods are approximately 0.5 mg g1. Recently an LC-MS/MS method was developed that has a minimum detection limit of 0.0003 mg ml1.
Toxicology and Legislation Toxicology studies have been undertaken using mice, rats, and guinea pigs. Natamycin was least toxic if administered orally (LD50 ¼ 1500 mg kg1 body weight in rats and mice) or subcutaneously (LD50 ¼ 5000 mg kg1 body weight) and most toxic if administered intravenously (LD50 ¼ 5–10 mg kg1). No natamycin was absorbed from the intestinal tract after 7 days’ feeding of up to a maximum 500 mg natamycin per day. Feeding studies have been conducted in rats, rabbits, and dogs. The acceptable daily intake (ADI) was set at 0.3 mg kg1 of body weight per day in 1976 by the Food and Agricultural Organization/World Health Organization. It should be noted that no ADI has been set by the European Union. Specification of natamycin in the United States (21 CFR 172.55) requires purity of the anhydrous compound to be 97 2% containing less than 1 ppm arsenic and no more than 20 ppm heavy metals. Natamycin is approved as a food preservative in 32 countries worldwide. In the European Union, it has the E number, E235, and is permitted for surface treatment of hard, semihard,
Table 2
Sensitivity of molds to natamycin MIC (mg ml 1) a
Byssochlamys fulva 040021 Penicillium candidum S66 P. chrysogenum S138 P. commune ABC118 P. cyclopium S124 P. nalgiovense S125 Aspergillus chevalieri 4298 A. clavatus A. nidulans A. ochraceus 4069 Cladosporium cladosporioides Gloeosporium album Penicillium chrysogenum P. islandicum P. verrucolosum var. cyclopium Sclerotinia fructicola Botrytis cinerea Aspergillus niger CBS733.88 A. versicolor 108959 B. nivea 163642 Fusarium solani S200 P. roqueforti S44 Absidia sp. Acremomium sclerotigenum Alternaria sp. Aspergillus flavus CBS 3005 A. flavus BB 67 A. flavus Madagascar A. flavus Port Lamy A. niger A. versicolor Mucor mucedo Penicillium digitatum P. expansum P. notatum 4640 P. nigricans P. viridicatum Westling Scopulariopsis asperula Aspergillus oryzae Fusarium sp. Geotrichum candidum Penicillium roqueforti var. punctatum 6018 Rhizopus oryzae 4758 P. discolor 547.95 P. discolor 549.95 P. discolor 551.95
0.1–1.25
0.1–2.5
1–25 2.5
4.0–8.0
10
>40
Minimum inhibitory concentration (defined as no growth after 5 days at 25 C. Inoculum of ~104 spores in center of agar plate).
a
and semisoft cheese and dry sausages at a maximum surface concentration of 1 mg dm2, and penetration is restricted to 5 mm below the surface. A more general use is approved in South Africa where it is approved in wine (principally to prevent secondary fermentation by yeast), fruit juices and pulp, various cheeses, yogurt, canned foods, processed meat, and various fish and shellfish products. In the United States, natamycin is permitted on a weight basis of 20 mg kg1 in certain cheeses and shredded cheese, on the surface of baked goods, and in cottage cheese, cream cheese, and sour cream. It can also be used in yogurt in the United States provided the yogurt is labeled as ‘nonstandard of identity.’
PRESERVATIVES j Permitted Preservatives – Natamycin Table 3
Sensitivity of yeasts to natamycin MIC (mg ml
Brettanomyces bruxellensis Candida albicans C. krusei H66 C. pseudotropicalis H3 C. valida H74 C. vini Debaryomyces hansenii H42 Dekkera bruxellensis CBS2796 D. bruxellensis CBS4459 D. bruxellensis CBS6055 Hanseniasporum uvarum CBS5074 Hansenula polymorpha Pichia membranaefaciens H67 Rhodotorula mucilaginosa CBS8161 Saccharomyces (Zygosaccharomyces) bailii S. bayanus S. bayanus IO18-2007 S. carlsbergensis CRA6413 S. cerevisiae ATCC9763 S. cerevisiae CRA124 S. cerevisiae H78 S. cerevisiae 8021 S. cerevisiae var. ellipsoideus S. exiguus S. ludwigii 0339 Torulopsis candida Z. bailii CRA229 Z. rouxii CBS1640 Candida guilliermondii C. kefyr H2 C. paralopsilosis NCYC458 C. utilis H41 Kloeckera apiculata Kluyveromyces lactis H17 Rhodotorula gracilis Saccharomyces cerevisiae S. cerevisiae var. paradoxus H103 S. exiguus Rees CBS1514 S. florentinus H79 S. unisporus H104 S. (Zygosaccharomyces) rouxii 0562 S. sake 0305 Torulopsis lactis-condensi Torulaspora rosei Zygosaccharomyces barkerii
1 a
)
1.0–2.5
3.0–10.0
a
Minimum inhibitory concentration (defined as inhibition of growth for 14 days at 25 C. Inoculum level at ~103 cfu ml1).
Natamycin as a Cheese Preservative Use on the surfaces of cheeses is the largest application for natamycin. Experience has shown that it is most effective if it is evenly applied at sufficient concentration (0.6–1 mg g1). The three main methods of surface treatment are spraying the surface of the cheese with a natamycin suspension, dipping or showering the cheese in a suspension, and applying natamycin in a polyvinyl acetate suspension coating to the cheese surface. Suitable spraying equipment is critical to successful application of natamycin onto the cheese surface. A number of companies specialize in such equipment. Suspensions of
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natamycin can support the growth of bacteria over time, and to prevent this, 8–10% salt can be added to the suspension. For shredded cheese, pneumatically driven spray guns are recommended to spray the shredded cheese as it is being tumbled, thus ensuring homogenous application to the surface. It is essential that the spraying system is well maintained, with properly designed spray nozzles positioned correctly in the spraying drum. The recommended concentrations of the natamycin suspension are 1250–2500 mg l1. The suspension should be sprayed at approximately 6 l ton1 to achieve a target level of 7–15 mg kg natamycin on the cheese shreds. Due to its low solubility, it is important to keep natamycin preparations in suspension by stirring or agitation; otherwise, it will gradually settle out. Successful application of natamycin onto shredded cheese can delay or protect against yeast and mold spoilage in modified atmosphere packs that acquire leaks (a common problem) and also extend the shelf life of the product once the pack has been opened. Natamycin can be used in the production of blue cheese to prevent excessive development of the mold, Penicillium roqueforti, on the cheese surface. The desirable level of natamycin on the cheese surface is 12 mg cm2 or more. This can be achieved by using shower-type saturation spraying with natamycin preparation (as a 1250–2500 mg l1 natamycin suspension) using a recirculation system to optimize economical use and keep the natamycin preparation in suspension. Blue cheeses can be treated with natamycin either before or after punching (piercing). Treated cheeses have been shown to have superior interior blue mold development compared with untreated cheese where undesirable surface growth can block the opening of the punch hole, limiting oxygen availability. The best way to treat cheese blocks is by using spray equipment that employs spinning disc technology or pneumatically driven nozzles. A very fine even spray should be applied to all six surfaces of the block of cheese in conjunction with a moving conveyor belt system. Excess spray suspension can be recirculated for further use. As mentioned, the addition of 8–10% salt is recommended for such use to prevent bacterial growth during prolonged run times. Block cheeses also can be treated by simple dipping into natamycin suspensions for a few seconds. Use of natamycin combined with polymers can increase the adherence of the natamycin to the cheese surface. Cheeses treated with natamycin must be allowed to dry before packing or wax coating. Many cheeses are susceptible to unsightly surface growth of molds during ripening. Ripening typically takes place at 10 C or above, with the cheeses stored on large shelves in large ripening rooms. Cheeses, such as parmesan, require a long ripening period and during this period can be subject to mold contamination and subsequent spoilage. Polyvinyl acetate (PVA) and water-based emulsion coatings are plastic-type coatings in liquid form that dry on the surface of the cheese to form a protective film. The film can be removed at the end of the ripening period. PVA coatings that contain natamycin preparations are commercially available from coating manufacturers. The natamycin content of the coating ranges from 250 to 1000 mg kg1. The coating can be applied to the cheese surface by dipping, spraying, or painting either manually or mechanically. Often several coats are applied at regular intervals during ripening, the cheese being turned at regular intervals to achieve thorough and complete protection.
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PRESERVATIVES j Permitted Preservatives – Natamycin
Various feta-type cheeses are often soaked or stored in brine (8–20% salt). Salt-tolerant (halophilic) yeasts and molds are a potential spoilage problem. Natamycin added to the brine at 10–20 mg ml1 will prevent or delay their growth. Natamycin at concentrations of 10–30 mg g1 can be mixed into soft cream cheeses and cottage cheese dressings to provide protection against yeast and mold spoilage.
Natamycin for Fermented Sausages Fermented sausages are prepared by stuffing casings with ground meat and fat inoculated with an acidifying bacterial starter culture or allowing natural contaminant fermenting organisms to grow. A wide variety of cured or fermented sausages are popular with consumers in many countries. Fermented sausages are popular in mainland Europe. Examples include Bresaola, Morttadella, Salami, Pastirma, Pepperoni, Saucisson Sec, and Summer Sausage. The fermentation process can last for variable periods of time ranging from 1 day to 1 month at 15–25 C depending on the size and type of sausage. Fermented sausages are prone to spoilage by the growth of yeasts and molds, resulting in unsightly surface mycelium or colonies. During ripening, the pH falls and this reduces the water-holding capacity of the meat, resulting in an increase in surface moisture. This provides ideal conditions for surface mold growth. Later, during wholesale distribution or retail storage, there is further potential for unwanted fungal growth. A wide variety of molds can be implicated in storage, including Aspergillus and Penicillium spp. The recommended dosages for dipping or spraying sausages are 2500–4000 mg ml1 in water. Thorough agitation is required to keep the natamycin in suspension, and spraying of the sausages must be even and complete. A further method of treating the sausages is to pretreat the casings before stuffing. This can be best achieved by soaking the casing in a 500–1000 mg ml1 natamycin suspension. It is more effective, however, to treat the sausages after stuffing.
Natamycin as a Yogurt Preservative Yogurts because of their low pH can be prone to spoilage by yeasts and molds. A preservative is required that has no negative effects on the viability and fermentation performance of the bacterial starter cultures used in yogurt production, but it shows amtimicrobial activity against yeasts and molds. The selective antimicrobial action of natamycin meets this criteria and natamycin is an effective preservative in both set and drinking yogurts. The natamycin can be added to the milk before or after pasteurization at the same time as the inoculation of the starter cultures. The effect of natamycin at concentrations ranging from 0 to 20 mg ml1 against an inoculated yeast is shown in Figure 2.
Natamycin as a Preservative on the Surface of Baked Goods Surface mold growth on baked goods, which includes bread, tortillas, muffins, and cakes, restricts the shelf life of these products and can have a significant economic impact. Application of natamycin to the surface of baked goods using fine sprays using either spray gun or spinning disc technology as described has proved to be an effective method in increasing shelf life. As with the surface spraying of cheeses and sausages, it is important that the natamycin be applied evenly to all surfaces. Surface levels of natamycin that have been proved to be effective are 0.5 mg cm2 and above. Natamycin is approved in the United States at levels in bread up to 14 mg g1, tortillas and English muffins up to 20 mg g1, and cakes and U.S.-style muffins at 7 mg g1. In China, it can be used on the surface of moon cakes and baked goods when applied by spraying or dipping in a suspension of concentration of 200–300 mg kg1, providing that the residues in the treated product are less than 10 mg kg1.
Natamycin Control of Yeast Spoilage in Wine Although wine is produced by the fermentative action of yeasts, this same metabolic activity can result in spoilage. Unwanted
9 8
Log 10 CFU g−1
7 6 5 4 3 2 1
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
Day 0 ppm
Figure 2
5 ppm
7.5 ppm
10 ppm
The effect of natamycin on the growth of Saccharomyces cerevisiae H78 in live yogurt at 8 C.
20 ppm
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PRESERVATIVES j Permitted Preservatives – Natamycin yeast growth can cause several wine defects: ester taints, volatile acidity, phenolic off-flavors, deacidification, turbidity, and unwanted secondary fermentation of semisweet wines. This spoilage can be caused not only by contaminant yeasts but also by those used for wine fermentation if their growth is unchecked or is restarted by the addition of further nutrients. Yeast spoilage can result in serious economic loss and is a worldwide problem. It is only in South Africa, however, where such use in wine is authorized. In that country, natamycin is allowed in wine, alcoholic fruit beverages, and grapebased liquors at a maximum level of 30 mg ml1. Natamycin usually is added after fermentation is completed, the wine has been racked, and free sulfur-dioxide levels have been adjusted to 37 mg ml1. The wine is then filtered and natamycin added at 5–10 mg ml1 before bottling. It is used particularly in semisweet wine to reduce secondary fermentation and can be employed when chemical preservatives, such as sorbate and sulfur dioxide, fail to control the growth of spoilage yeasts. It has been determined that the half-life of natamycin in wine under typical storage conditions is around 20 days.
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See also: Alternaria; Aspergillus; Aspergillus: Aspergillus oryzae; Aspergillus: Aspergillus flavus; Bread: Bread from Wheat Flour; Brettanomyces; Byssochlamys; Candida; Cheese: Microbiology of Cheesemaking and Maturation; Food Packaging with Antimicrobial Properties; Confectionery Products – Cakes and Pastries; Debaryomyces; Fermented Meat Products and the Role of Starter Cultures; Fermented Milks and Yogurt; Fusarium; Geotrichum; Characteristics of Hansenula: Biology and Applications; Intermediate Moisture Foods; Kluyveromyces; Mucor; Penicillium andTalaromyces: Introduction; Preservatives: Classification and Properties; Preservatives: Permitted Preservatives – Sorbic Acid; Permitted Preservatives – Propionic Acid; Rhodotorula; Saccharomyces – Introduction; Spoilage of Plant Products: Problems caused by Fungi; Spoilage Problems: Problems Caused by Fungi; Streptomyces; Wines: Microbiology of Winemaking; Zygosaccharomyces; Resistance to Antimicrobials; Fruit and Vegetable Juices.
Further Reading Natamycin to Control Spoilage of Fruit Juices Natamycin has been shown to be an effective preservative in both pasteurized and unpasteurized fruit juices, preventing the growth of yeasts and molds. Additional levels typically used are 6–12 mg ml1 and efficacy in delaying or preventing yeast and mold spoilage is usually superior to sorbate at 1000 mg ml1 or higher. Furthermore, yeasts and molds are becoming increasingly resistance to sorbate, and the use of high levels of sorbate can have a bad taste effect. Retention of natamycin in orange juice pasteurized at 80 C for 10 min is around 70%.
Potential Applications Potential applications include uses on the surface of such fruits as strawberries, use in tomato pureé, and use in black olive production to prevent the growth of molds on the surface of the brine during the fermentation process, without interfering with the desired lactic acid bacteria fermentation. Although not a food use, natamycin has been proposed as a selective antifungal agent in microbiological agar media.
Delves-Broughton, J., Steenson, L., Dorko, C., Erdmann, J., Mallory, S., Norbury, F., Thompson, B., 2010. Use of natamycin as a preservative on the surface of baked goods: a case study. In: Doona, C.J., Kustin, K., Feeherry, F.E. (Eds.), Case Studies in Novel Food Processing Technologies. Woodhead Publishing, Oxford, pp. 303–330. Delves-Broughton, J., Thomas, L.V., Doan, C.H., Davidson, P.M., 2005. Natamycin. In: Davidson, P.M., Sofos, J.N., Branen, A.L. (Eds.), Antimicrobials in Food, third ed. CRC Press, pp. 275–288. Stark, J., Tan, H.S., 2003. Natamycin. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives. Kluwer Academic, London, pp. 179–195. Thomas, L.V., Delves- Broughton, J., 2001. Applications of the natural food preservative natamycin. Research Advances in Food Science 2, 1–10.
Introduction
Structure and Properties
Natamycin is a polyene macrolide antimycotic produced by strains of the Actinomycetes, such as Streptomycetes natalensis, S. chattanoogenesis, and other closely related Streptomyces species. As an antimycotic, it exhibits strong antimicrobial activity against yeasts and molds. Prevention of fungal spoilage of foods is an important issue for the food industry as economic losses due to spoilage can be considerable. Apart from the visual and organoleptic spoilage of foods, many molds can produce mycotoxins that have carcinogenic properties. Natamycin shows no activity against bacteria. Such a selective antimicrobial activity has led to its use in bacteria fermented foods, whereby its selective action has no negative effect on the bacteria culture responsible for the desired fermentation but will have a positive antimycotic effect against contaminating yeasts and molds. Thus, it is used often on the surface of cheese, fermented meats or in yogurt, fermented creams, and similar products. It is marketed commercially as the NatamaxÔ family of products by Danisco and as the DelvocidÒ family of products by DSM. China also has manufacturers. To date, natamycin is the only microbially derived antifungal compound that is used in the food industry.
Natamycin is a polyene macrolide with a molecular weight of 665.7 Da and the empirical formula C33H47 NO13. Its structure has been determined and is shown in Figure 1. As dry powder it can be stored for several years with minimal loss of activity. Aqueous suspensions are less stable, particularly if exposed to light, certain oxidants, and heavy metals, but they remain sufficiently stable during practical use. Thus compounds such as peroxides or chlorides, often used as cleaning or disinfectant agents, should be used with care in the proximity of natamycin. Although solutions are more unstable in acid or alkaline conditions, the pH of most food products is not normally at levels that cause problems. Like similar polyene macrolides, natamycin is amphoteric containing one basic and one acidic group. Natamycin has low solubility in water (approximately 40 mg ml1) and is almost insoluble in nonpolar solvents, but it shows good solubility in strong polar organic solvents such as glycerol, methylpyrrolidone, and glacial acetic acid. The low solubility in water can be an advantage for the surface treatment of food as it will stay on the surface where it is needed instead of migrating into the food. Natamycin has numerous advantages over other antimycotic preservatives such as sorbates and these are summarized in Table 1.
History Natamycin was first produced in 1955 from a culture filtrate of a Streptomycetes isolated from a soil sample in South Africa. Its name is in fact derived from the South African province, Natal, from where it was originally isolated. Other names used in the past are pimaracin and tennectin. Commercial preparations are produced by fermentation of S. natalensis in a medium containing a carbon source (typically starch or molasses) and a fermentable nitrogen source (typically corn steep liquor, casein, soya). Fermentation is aerobic and mechanical agitation and antifoaming agents can aid the process. The temperature range is 26–30 C and the pH range is 6–8. Due to its low solubility, natamycin will accumulate mainly as crystals, and these can be extracted following separation of the biomass by solvent extraction. The natamycin content of most commercial preparations is 50% with the incipient being lactose, glucose, or salt. There are also natamycin-based products that contain food-grade polymers that aid the adherence of natamycin for the surface treatment of foods. Recently DuPont introduced NatamaxÒ plus B, which is natamycin complexed with cyclodextrin. This has increased solubility compared with standard natamycin preparations. Other natamycin-based products are used for topical veterinary and pharmaceutical applications to treat fungal infections, such as ring worm in horses and fungal eye infections (keratitis) in humans. Recently, the biosynthetic gene cluster for natamycin production has been characterized for S. chattanoogenesis.
Encyclopedia of Food Microbiology, Volume 3
Figure 1
Table 1
The structure of natamycin.
Advantages of natamycin over sorbate
Natamycin
Sorbate
Natural Fungicidal No effect on bacteria No migration into food No flavor Effective at 1–40 mg kg1 Effective at pH 3–9
Chemical Fungistatic Bactericidal Penetrates into food Bitter flavor Effective at 1000–2000 mg kg1 Effective only at acidic pH
http://dx.doi.org/10.1016/B978-0-12-384730-0.00269-X
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PRESERVATIVES j Permitted Preservatives – Natamycin
Mode of Action and Antimicrobial Activity Natamycin acts by combining with ergosterol and other sterols present in the cell membranes of yeasts and vegetative mycelium of molds. It was thought that this interaction resulted in pore formation, resulting in leakage of cellular material, but recently it has been shown that this is not the case. Rather, the interaction of natamycin with ergosterol results in the prevention of cell division and loss of enzyme function. Less is known about the action of natamycin against mold spores, but it is thought to inhibit their germination. Ergosterol is not found in the cell membranes of bacteria and hence their resistance. It is found in algal cell membranes and thus algae are also sensitive. There are no reports of development of resistance to natamycin in vivo. Studies undertaken in cheese and fermented sausage factories that have used natamycin for several years showed no increase in the levels of natamycin-resistant yeasts and molds compared with similar factories not using natamycin. Most molds (Table 2) are sensitive to natamycin at concentrations of 40 mg ml1 or less. Yeasts (Table 3) are even more sensitive with minimum inhibitory concentrations of 5 mg ml1 or less. A few species of molds such as Penicillium discolor that have no or low levels of ergosterol in their membranes can have reduced sensitivity. However, such reduced sensitivity rarely if ever causes problems in practical food preservation. Laboratory experiments to induce resistance have been unsuccessful.
Methods of Assay The natamycin contents of food products can be determined by microbiological, immunological, mass spectrophotometric (MS), liquid chromatographic (LC), and high-performance liquid chromatographic (HPLC) methodologies. Minimum detection limits for these methods are approximately 0.5 mg g1. Recently an LC-MS/MS method was developed that has a minimum detection limit of 0.0003 mg ml1.
Toxicology and Legislation Toxicology studies have been undertaken using mice, rats, and guinea pigs. Natamycin was least toxic if administered orally (LD50 ¼ 1500 mg kg1 body weight in rats and mice) or subcutaneously (LD50 ¼ 5000 mg kg1 body weight) and most toxic if administered intravenously (LD50 ¼ 5–10 mg kg1). No natamycin was absorbed from the intestinal tract after 7 days’ feeding of up to a maximum 500 mg natamycin per day. Feeding studies have been conducted in rats, rabbits, and dogs. The acceptable daily intake (ADI) was set at 0.3 mg kg1 of body weight per day in 1976 by the Food and Agricultural Organization/World Health Organization. It should be noted that no ADI has been set by the European Union. Specification of natamycin in the United States (21 CFR 172.55) requires purity of the anhydrous compound to be 97 2% containing less than 1 ppm arsenic and no more than 20 ppm heavy metals. Natamycin is approved as a food preservative in 32 countries worldwide. In the European Union, it has the E number, E235, and is permitted for surface treatment of hard, semihard,
Table 2
Sensitivity of molds to natamycin MIC (mg ml 1) a
Byssochlamys fulva 040021 Penicillium candidum S66 P. chrysogenum S138 P. commune ABC118 P. cyclopium S124 P. nalgiovense S125 Aspergillus chevalieri 4298 A. clavatus A. nidulans A. ochraceus 4069 Cladosporium cladosporioides Gloeosporium album Penicillium chrysogenum P. islandicum P. verrucolosum var. cyclopium Sclerotinia fructicola Botrytis cinerea Aspergillus niger CBS733.88 A. versicolor 108959 B. nivea 163642 Fusarium solani S200 P. roqueforti S44 Absidia sp. Acremomium sclerotigenum Alternaria sp. Aspergillus flavus CBS 3005 A. flavus BB 67 A. flavus Madagascar A. flavus Port Lamy A. niger A. versicolor Mucor mucedo Penicillium digitatum P. expansum P. notatum 4640 P. nigricans P. viridicatum Westling Scopulariopsis asperula Aspergillus oryzae Fusarium sp. Geotrichum candidum Penicillium roqueforti var. punctatum 6018 Rhizopus oryzae 4758 P. discolor 547.95 P. discolor 549.95 P. discolor 551.95
0.1–1.25
0.1–2.5
1–25 2.5
4.0–8.0
10
>40
Minimum inhibitory concentration (defined as no growth after 5 days at 25 C. Inoculum of ~104 spores in center of agar plate).
a
and semisoft cheese and dry sausages at a maximum surface concentration of 1 mg dm2, and penetration is restricted to 5 mm below the surface. A more general use is approved in South Africa where it is approved in wine (principally to prevent secondary fermentation by yeast), fruit juices and pulp, various cheeses, yogurt, canned foods, processed meat, and various fish and shellfish products. In the United States, natamycin is permitted on a weight basis of 20 mg kg1 in certain cheeses and shredded cheese, on the surface of baked goods, and in cottage cheese, cream cheese, and sour cream. It can also be used in yogurt in the United States provided the yogurt is labeled as ‘nonstandard of identity.’
PRESERVATIVES j Permitted Preservatives – Natamycin Table 3
Sensitivity of yeasts to natamycin MIC (mg ml
Brettanomyces bruxellensis Candida albicans C. krusei H66 C. pseudotropicalis H3 C. valida H74 C. vini Debaryomyces hansenii H42 Dekkera bruxellensis CBS2796 D. bruxellensis CBS4459 D. bruxellensis CBS6055 Hanseniasporum uvarum CBS5074 Hansenula polymorpha Pichia membranaefaciens H67 Rhodotorula mucilaginosa CBS8161 Saccharomyces (Zygosaccharomyces) bailii S. bayanus S. bayanus IO18-2007 S. carlsbergensis CRA6413 S. cerevisiae ATCC9763 S. cerevisiae CRA124 S. cerevisiae H78 S. cerevisiae 8021 S. cerevisiae var. ellipsoideus S. exiguus S. ludwigii 0339 Torulopsis candida Z. bailii CRA229 Z. rouxii CBS1640 Candida guilliermondii C. kefyr H2 C. paralopsilosis NCYC458 C. utilis H41 Kloeckera apiculata Kluyveromyces lactis H17 Rhodotorula gracilis Saccharomyces cerevisiae S. cerevisiae var. paradoxus H103 S. exiguus Rees CBS1514 S. florentinus H79 S. unisporus H104 S. (Zygosaccharomyces) rouxii 0562 S. sake 0305 Torulopsis lactis-condensi Torulaspora rosei Zygosaccharomyces barkerii
1 a
)
1.0–2.5
3.0–10.0
a
Minimum inhibitory concentration (defined as inhibition of growth for 14 days at 25 C. Inoculum level at ~103 cfu ml1).
Natamycin as a Cheese Preservative Use on the surfaces of cheeses is the largest application for natamycin. Experience has shown that it is most effective if it is evenly applied at sufficient concentration (0.6–1 mg g1). The three main methods of surface treatment are spraying the surface of the cheese with a natamycin suspension, dipping or showering the cheese in a suspension, and applying natamycin in a polyvinyl acetate suspension coating to the cheese surface. Suitable spraying equipment is critical to successful application of natamycin onto the cheese surface. A number of companies specialize in such equipment. Suspensions of
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natamycin can support the growth of bacteria over time, and to prevent this, 8–10% salt can be added to the suspension. For shredded cheese, pneumatically driven spray guns are recommended to spray the shredded cheese as it is being tumbled, thus ensuring homogenous application to the surface. It is essential that the spraying system is well maintained, with properly designed spray nozzles positioned correctly in the spraying drum. The recommended concentrations of the natamycin suspension are 1250–2500 mg l1. The suspension should be sprayed at approximately 6 l ton1 to achieve a target level of 7–15 mg kg natamycin on the cheese shreds. Due to its low solubility, it is important to keep natamycin preparations in suspension by stirring or agitation; otherwise, it will gradually settle out. Successful application of natamycin onto shredded cheese can delay or protect against yeast and mold spoilage in modified atmosphere packs that acquire leaks (a common problem) and also extend the shelf life of the product once the pack has been opened. Natamycin can be used in the production of blue cheese to prevent excessive development of the mold, Penicillium roqueforti, on the cheese surface. The desirable level of natamycin on the cheese surface is 12 mg cm2 or more. This can be achieved by using shower-type saturation spraying with natamycin preparation (as a 1250–2500 mg l1 natamycin suspension) using a recirculation system to optimize economical use and keep the natamycin preparation in suspension. Blue cheeses can be treated with natamycin either before or after punching (piercing). Treated cheeses have been shown to have superior interior blue mold development compared with untreated cheese where undesirable surface growth can block the opening of the punch hole, limiting oxygen availability. The best way to treat cheese blocks is by using spray equipment that employs spinning disc technology or pneumatically driven nozzles. A very fine even spray should be applied to all six surfaces of the block of cheese in conjunction with a moving conveyor belt system. Excess spray suspension can be recirculated for further use. As mentioned, the addition of 8–10% salt is recommended for such use to prevent bacterial growth during prolonged run times. Block cheeses also can be treated by simple dipping into natamycin suspensions for a few seconds. Use of natamycin combined with polymers can increase the adherence of the natamycin to the cheese surface. Cheeses treated with natamycin must be allowed to dry before packing or wax coating. Many cheeses are susceptible to unsightly surface growth of molds during ripening. Ripening typically takes place at 10 C or above, with the cheeses stored on large shelves in large ripening rooms. Cheeses, such as parmesan, require a long ripening period and during this period can be subject to mold contamination and subsequent spoilage. Polyvinyl acetate (PVA) and water-based emulsion coatings are plastic-type coatings in liquid form that dry on the surface of the cheese to form a protective film. The film can be removed at the end of the ripening period. PVA coatings that contain natamycin preparations are commercially available from coating manufacturers. The natamycin content of the coating ranges from 250 to 1000 mg kg1. The coating can be applied to the cheese surface by dipping, spraying, or painting either manually or mechanically. Often several coats are applied at regular intervals during ripening, the cheese being turned at regular intervals to achieve thorough and complete protection.
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PRESERVATIVES j Permitted Preservatives – Natamycin
Various feta-type cheeses are often soaked or stored in brine (8–20% salt). Salt-tolerant (halophilic) yeasts and molds are a potential spoilage problem. Natamycin added to the brine at 10–20 mg ml1 will prevent or delay their growth. Natamycin at concentrations of 10–30 mg g1 can be mixed into soft cream cheeses and cottage cheese dressings to provide protection against yeast and mold spoilage.
Natamycin for Fermented Sausages Fermented sausages are prepared by stuffing casings with ground meat and fat inoculated with an acidifying bacterial starter culture or allowing natural contaminant fermenting organisms to grow. A wide variety of cured or fermented sausages are popular with consumers in many countries. Fermented sausages are popular in mainland Europe. Examples include Bresaola, Morttadella, Salami, Pastirma, Pepperoni, Saucisson Sec, and Summer Sausage. The fermentation process can last for variable periods of time ranging from 1 day to 1 month at 15–25 C depending on the size and type of sausage. Fermented sausages are prone to spoilage by the growth of yeasts and molds, resulting in unsightly surface mycelium or colonies. During ripening, the pH falls and this reduces the water-holding capacity of the meat, resulting in an increase in surface moisture. This provides ideal conditions for surface mold growth. Later, during wholesale distribution or retail storage, there is further potential for unwanted fungal growth. A wide variety of molds can be implicated in storage, including Aspergillus and Penicillium spp. The recommended dosages for dipping or spraying sausages are 2500–4000 mg ml1 in water. Thorough agitation is required to keep the natamycin in suspension, and spraying of the sausages must be even and complete. A further method of treating the sausages is to pretreat the casings before stuffing. This can be best achieved by soaking the casing in a 500–1000 mg ml1 natamycin suspension. It is more effective, however, to treat the sausages after stuffing.
Natamycin as a Yogurt Preservative Yogurts because of their low pH can be prone to spoilage by yeasts and molds. A preservative is required that has no negative effects on the viability and fermentation performance of the bacterial starter cultures used in yogurt production, but it shows amtimicrobial activity against yeasts and molds. The selective antimicrobial action of natamycin meets this criteria and natamycin is an effective preservative in both set and drinking yogurts. The natamycin can be added to the milk before or after pasteurization at the same time as the inoculation of the starter cultures. The effect of natamycin at concentrations ranging from 0 to 20 mg ml1 against an inoculated yeast is shown in Figure 2.
Natamycin as a Preservative on the Surface of Baked Goods Surface mold growth on baked goods, which includes bread, tortillas, muffins, and cakes, restricts the shelf life of these products and can have a significant economic impact. Application of natamycin to the surface of baked goods using fine sprays using either spray gun or spinning disc technology as described has proved to be an effective method in increasing shelf life. As with the surface spraying of cheeses and sausages, it is important that the natamycin be applied evenly to all surfaces. Surface levels of natamycin that have been proved to be effective are 0.5 mg cm2 and above. Natamycin is approved in the United States at levels in bread up to 14 mg g1, tortillas and English muffins up to 20 mg g1, and cakes and U.S.-style muffins at 7 mg g1. In China, it can be used on the surface of moon cakes and baked goods when applied by spraying or dipping in a suspension of concentration of 200–300 mg kg1, providing that the residues in the treated product are less than 10 mg kg1.
Natamycin Control of Yeast Spoilage in Wine Although wine is produced by the fermentative action of yeasts, this same metabolic activity can result in spoilage. Unwanted
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Log 10 CFU g−1
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Figure 2
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The effect of natamycin on the growth of Saccharomyces cerevisiae H78 in live yogurt at 8 C.
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PRESERVATIVES j Permitted Preservatives – Natamycin yeast growth can cause several wine defects: ester taints, volatile acidity, phenolic off-flavors, deacidification, turbidity, and unwanted secondary fermentation of semisweet wines. This spoilage can be caused not only by contaminant yeasts but also by those used for wine fermentation if their growth is unchecked or is restarted by the addition of further nutrients. Yeast spoilage can result in serious economic loss and is a worldwide problem. It is only in South Africa, however, where such use in wine is authorized. In that country, natamycin is allowed in wine, alcoholic fruit beverages, and grapebased liquors at a maximum level of 30 mg ml1. Natamycin usually is added after fermentation is completed, the wine has been racked, and free sulfur-dioxide levels have been adjusted to 37 mg ml1. The wine is then filtered and natamycin added at 5–10 mg ml1 before bottling. It is used particularly in semisweet wine to reduce secondary fermentation and can be employed when chemical preservatives, such as sorbate and sulfur dioxide, fail to control the growth of spoilage yeasts. It has been determined that the half-life of natamycin in wine under typical storage conditions is around 20 days.
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See also: Alternaria; Aspergillus; Aspergillus: Aspergillus oryzae; Aspergillus: Aspergillus flavus; Bread: Bread from Wheat Flour; Brettanomyces; Byssochlamys; Candida; Cheese: Microbiology of Cheesemaking and Maturation; Food Packaging with Antimicrobial Properties; Confectionery Products – Cakes and Pastries; Debaryomyces; Fermented Meat Products and the Role of Starter Cultures; Fermented Milks and Yogurt; Fusarium; Geotrichum; Characteristics of Hansenula: Biology and Applications; Intermediate Moisture Foods; Kluyveromyces; Mucor; Penicillium andTalaromyces: Introduction; Preservatives: Classification and Properties; Preservatives: Permitted Preservatives – Sorbic Acid; Permitted Preservatives – Propionic Acid; Rhodotorula; Saccharomyces – Introduction; Spoilage of Plant Products: Problems caused by Fungi; Spoilage Problems: Problems Caused by Fungi; Streptomyces; Wines: Microbiology of Winemaking; Zygosaccharomyces; Resistance to Antimicrobials; Fruit and Vegetable Juices.
Further Reading Natamycin to Control Spoilage of Fruit Juices Natamycin has been shown to be an effective preservative in both pasteurized and unpasteurized fruit juices, preventing the growth of yeasts and molds. Additional levels typically used are 6–12 mg ml1 and efficacy in delaying or preventing yeast and mold spoilage is usually superior to sorbate at 1000 mg ml1 or higher. Furthermore, yeasts and molds are becoming increasingly resistance to sorbate, and the use of high levels of sorbate can have a bad taste effect. Retention of natamycin in orange juice pasteurized at 80 C for 10 min is around 70%.
Potential Applications Potential applications include uses on the surface of such fruits as strawberries, use in tomato pureé, and use in black olive production to prevent the growth of molds on the surface of the brine during the fermentation process, without interfering with the desired lactic acid bacteria fermentation. Although not a food use, natamycin has been proposed as a selective antifungal agent in microbiological agar media.
Delves-Broughton, J., Steenson, L., Dorko, C., Erdmann, J., Mallory, S., Norbury, F., Thompson, B., 2010. Use of natamycin as a preservative on the surface of baked goods: a case study. In: Doona, C.J., Kustin, K., Feeherry, F.E. (Eds.), Case Studies in Novel Food Processing Technologies. Woodhead Publishing, Oxford, pp. 303–330. Delves-Broughton, J., Thomas, L.V., Doan, C.H., Davidson, P.M., 2005. Natamycin. In: Davidson, P.M., Sofos, J.N., Branen, A.L. (Eds.), Antimicrobials in Food, third ed. CRC Press, pp. 275–288. Stark, J., Tan, H.S., 2003. Natamycin. In: Russell, N.J., Gould, G.W. (Eds.), Food Preservatives. Kluwer Academic, London, pp. 179–195. Thomas, L.V., Delves- Broughton, J., 2001. Applications of the natural food preservative natamycin. Research Advances in Food Science 2, 1–10.