The polyene macrolide antibiotic natamycin (Antibiotic A-5283) is commonly used to retard the growth of surface molds on various cheese varieties. Natamycin is commonly applied to the surface of cheese by dipping or spraying, using an aqueous dispersion containing 200 to 300 ppm of the additive. The large molecular weight of natamycin, 666 g/mol, and conjugated double bond structure causes it to be extremely insoluble in water and most food grade solvents. The inability to apply natamycin in true solution creates void non-treated areas on the food surface. These non-treated areas promote the growth of fungal organisms. A water soluble N-alkyl semisynthetic derivative of natamycin was synthesized by the Michael addition reaction of the parent with a N-substituted malemide. A comparative study investigating the effectiveness of the semisynthetic derivative of natamycin and the parent antibiotic in suppressing mold growth on one month aged shredded Cheddar cheese modified atmosphere packaged (MAP) was performed. A 20 ppm natamycin treatment effectively suppressed visible mold growth (<104 CFU/g) in MAP samples for up to 30 days after opening. The 20 ppm semisynthetic derivative performed similarly to the 10 ppm natamycin treatment in retarding mold growth. Visible mold growth did not occur for these treatments in MAP samples until 20 days after opening. Analysis of storage conditions revealed that an outgrowth of mold in shredded cheese occurred in MAP packages stored longer than 15 days. This bloom in mold growth was attributed to the degradation of natamycin and the semisynthetic derivative throughout storage. The stability and degradation of natamycin and the derivative were monitored throughout the study. Antibiotic concentration on the cheese surface was quantified by molecular absorption spectrometry. Results from this study showed, heavily contaminated samples caused the rate and loss of natamycin and the derivative to increase. Antibiotic concentration decreased at a similar rate in MAP and open package conditions. Natamycin and derivative were found to have similar degradation properties. / Master of Science

Natamycin is an antimycotic with very low water solubility and extremely high photosensitivity, which is used to extend the shelf life of shredded cheese products. The objectives of this research are: (a) to find a new delivery system for natamycin, which increases its aqueous solubility and (b) to increase the chemical stability of natamycin so that it has a prolonged antifungal effect on the surface of the shredded cheese. Molecular inclusion complexes of natamycin were formed with β-, hydroxypropyl β-, and γ- cyclodextrins (CDs) which allowed large increases in aqueous solubility without the use of organic co-solvents or surfactants. The water solubility of natamycin was increased 16-fold, 73- fold, and 152-fold with β-CD, γ-CD, and hydroxypropyl β-CD, respectively. The natamycin:CD inclusion complexes resulted in nearly equivalent in vitro antifungal activity as natamycin in its free state. Nuclear magnetic resonance (NMR) was utilized to prove the formation of true inclusion complexes. 1H NMR shift titrations of N-(3 -N-dimethylaminosuccimido) natamycin with β- and γ-CDs enabled determination of the stoichiometry of both complexes as 1:1. Aqueous solutions of natamycin (20 mg/L) were found by quantitative HPLC to be completely degraded after 24 hours of exposure to 1000 lux fluorescent lighting at 4 °C. After 14 days of storage in darkness at 4 °C, 92.2% of natamycin remained in active form. Aqueous solutions of natamycin:β-CD complex and natamycin:γ-CD complex were significantly more stable (p < 0.05) than natamycin in its free state when stored in darkness at 4 °C. Clear poly(ethylene terephthalate) packaging with an ultraviolet light absorber allowed 85.0% natamycin to remain after 14 days of storage under 1000 lux fluorescent lighting at 4 °C. Such dramatic increases in water solubility and light stability will enable natamycin to function as a more effective antimycotic in the food industry. / Master of Science

Natamycin is an antimycotic compound that is widely used in the cheese industry to increase the shelf life of cheeses, especially shredded cheeses, by inhibiting the growth of molds. Natamycin is applied to the surface of cheese as an aqueous suspension or as a powder. However, natamycin is not readily water soluble making it harder to distribute evenly over shredded cheese Natamycin is degraded by ultraviolet (UV) light at wavelengths of 350 nm and below. Typical packaging applications do not provide adequate UV protection causing natamycin to degrade. This work was undertaken to determine the efficacy of UV absorber film to prevent UV light degradation of natamycin on the surface of shredded cheese. Current accepted methods to determine concentration of natamycin were evaluated for appropriateness in natamycin degradation studIes. The use of cyclodextrins to increase water solubility was tested to see if a uniform distribution of natamycin over the shredded cheese could be done effectively. Furthermore, a known application of mold was performed to see how well natamycin and each of its applications could prevent visible mold growth from occurring. The International Dairy Federation recognizes two methods to quantify natamycin on shredded cheese: high performance liquid chromatography (HPLC) and spectrophotometry. Concentrations of natamycin in aqueous suspensions were determined using both methods. Results show that spectrophotometry is flawed when quantifying the amount of active natamycin because the method gives erroneously high results. The amount of active natamycin is not accurately quantified using spectrophotometric techniques because it cannot separate the active form from the inactive form of natamycin. Polymer packages containing a UV absorber (11.4% light transmission at 350 nm) allow significantly less UV-associated degradation of natamycin than those packages that lacked a UV protectant (90.0% light transmission at 350 nm) (p<0.05). Incorporating a UV absorber into a package helps protect natamycin and its various complexes from UV light degradation, which can increase the shelf life of shredded cheese. However, even with a UV absorber, natamycin is still able to degrade. Natamycin was complexed with different cyclodextrins to help better solubilize natamycin â β-cyclodextrin, hydroxy-propyl β-cyclodextrin and γ-cyclodextrin. Using cyclodextrins to apply natamycin more uniformly onto shredded cheese did not significantly increase the consistency of distribution (p<0.05). Variability was uniform throughout all treatments with the exception of HPBCD complex. After 27 days, all of the UV packages treated with each of the cyclodextrin treatments containing shredded cheese began to show visible mold growth. Those packages stored in total darkness remained mold free through the duration of the experiment ending on day 62. When untreated with natamycin and an initial concentration of 101-102 spores/gram of Penicillium roqueforti, shredded cheese remained free from visible mold growth for 24 days in total darkness at 4°C. Samples treated with one of the natamycin treatments were able to remain mold free for at least 9 more days, showing visible signs of mold growth at day 33. There was no statistical difference between the treatments of dry natamycin, aqueous suspension natamycin, β-cyclodextrin-natamycin complex, and γ-cyclodextrin-natamycin complex (p<0.05). However, there was a difference with the use of hydroxy-propyl β-cyclodextrin-natamycin complex. Hydroxy-propyl β-cyclodextrin-natamycin complex allowed the shredded cheese to last for 41 days, 17 days longer than the control sample. / Master of Science