Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
IMPROVING THE STABILITY OF NATAMYCIN ON SHREDDED CHEESE TO PREVENT MOLD GROWTH
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0201471
Grant No.
(N/A)
Project No.
VA-135727
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2004
Project End Date
Sep 30, 2009
Grant Year
(N/A)
Project Director
Marcy, J. E.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
FOOD SCIENCE AND TECHNOLOGY
Non Technical Summary
Dollar sales of shredded cheese increased 7.8% to $1.8 billion in 2002, which composes a 24.4% share of the retail cheese business The most difficult problem that shredded cheese manufacturers face is product return as a result of mold growth. Mold spoilage is commonly attributed to the increased surface area of the cheese shreds and the extra handling and exposure that the shreds experience in the cut and packing facility. The purpose of this work is to evaluate packaging and processing techniques which could lower the cost of shredded cheese packaging and prevent mold spoilage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
5013430200080%
7123430110220%
Goals / Objectives
Previous research has shown that the spoilage of shredded cheese is greatly influenced by UV light destruction of the prevalent anti-mold agent (natamycin) used on cheese. The object of this research is to determine the type and amount of UV absorber in polymer packaging required to maintain the stability of natamycin on cheese under fluorescent lighting. Currently, shredded cheese is modified atmosphere packaged (MAP. We wish to determine if the shelf life of cheese can be extended using UV protective packaging while removing the modified atmosphere from the packaging. We also wish to quantify the effect of the delivery system of natamycin as a dry powder, an aqueous suspension, and as an aqueous solution when complexed with cyclodextrin. Our studies to date have shown that ineffective delivery of natamycin is one of the principle causes of mold spoilage on shredded cheese. In addition we wish to quantify the effect of the initial mold population in shredded cheese on the duration until visible mold growth occurs for different concentrations of natamycin.
Project Methods
A spectral scan of a natamycin sample in water between the wavelengths of 250 and 400 nm will be performed on a Shimadzu UV-2101PC UV-VIS Scanning Spectrophotometer. Samples of commercially available poly(ethylene) or poly(ethylene terephthalate) packaging containing various amounts or types of UV absorber will be prepared. Based on the results of spectral scans of UV absorbing polymers the six most promising materials will be selected for additional testing. Packages will be formed with each material and tested at light intensities of 500-2,000 lux until 90% of an aqueous suspension of natamycin is destroyed. Light sources will include fluorescent, day-light, and halogen vapor lights. Cheddar or mozzarella cheese will be shredded and applied with 2% (w/w) cellulose anticaking agent. Shredded cheese will be treated with 20 mg/kg natamycin sprayed as an aqueous suspension. These cheese samples will then be transferred to polymer packaging bags containing different UV absorber amounts and types and stored at 4C under ambient air atmosphere. These samples will be exposed to continuous fluorescent lighting by cool white 40-watt T12 fluorescent bulbs mounted 16 cm above the samples to receive an illumination intensity of 1000 lux. Cheese samples will be quantified for natamycin content by extraction and HPLC analysis at weekly storage intervals until visible mold growth is observed. The natamycin present in treated shredded cheese samples will be extracted with methanol. Natamycin content in the filtrate will be determined by HPLC. Solid inclusion complexes of natamycin with alpha-cyclodextrin, hydroxypropyl gamma-cyclodextrin, and beta-cyclodextrin will be obtained by complexation in aqueous solution. Solutions of 16 mM alpha-cyclodextrin, 70 mM hydroxypropyl beta-cyclodextrin, and 70 mM gamma-cyclodextrin will be prepared in HPLC water. These concentrations were chosen for their relatively high complexation efficiency based on previous phase solubility studies. Sample filtrate will be quantified by UV differential spectrophotometry. Cheddar or mozzarella cheese will be shredded and applied with 2% (w/w) cellulose as the anticaking agent. Shredded cheese will be treated with 20 mg/kg natamycin with several delivery systems: dusted as a dry powder with the anticaking agent, sprayed as an aqueous suspension, and sprayed as an aqueous solution when complexed with cyclodextrins (alpha-cyclodextrin, hydroxypropyl gamma-cyclodextrin, and beta-cyclodextrin). These treated cheese samples will then be stored in the polymer packaging with UV absorber that was selected as the most efficient at preserving natamycin stability based on the earlier study. Cheese samples will be quantified for natamycin content by extraction and HPLC analysis at weekly storage intervals until visible mold growth is observed. Due to the very limited solubility of alpha-cyclodextrin in methanol-water mixtures and the practical insolubility of natamycin in water, the system will be designed to undergo a series of purges.

Progress 10/01/04 to 09/30/09

Outputs
OUTPUTS: We have followed our experiment with the natural antimicrobial, Natamycin, with an experiment looking at other natural antimicrobial compounds. In November 2005, the Food and Drug Administration (FDA) received a small study conducted by private laboratory where the presence of low levels of benzene was reported in a small number of foods and beverages that contained benzoate salts and ascorbic acid. As a follow-up, the FDA's Center for Food Safety and Applied Nutrition (CFSAN) conducted a study on beverages found in the market. Although the FDA has no standards for allowed levels of benzene, they have adopted the standards that the US Environmental Protection Agency (EPA) holds for drinking water, which dictates that the maximum contaminant level (MCL) should be below 5 micro g/L. It was hypothesized that sodium benzoate, a common preservative, was reacting with ascorbic acid to produce free benzene (FDA, 2006). Sodium benzoate is the sodium salt of benzoic acid which is present as benzoic acid in aqueous environments. A wide variety of foods and beverages use sodium benzoate as an antimicrobial additive. It is most suitable in naturally acidic or acidified foods and beverages in the pH range from 4.0 to 4.5. Due to its broad availability and low cost, it is used in many different types of foods, including carbonated and still beverages. The usage level ranges from 0.05 to 0.10 per cent. The mechanism by which benzene may be produced is described by Gardner and Lawrence, 1993. The study suggests that transition metals can catalyze a one-electron reduction of oxygen by ascorbic acid to produce the superoxide anion radical, "which undergoes spontaneous disproportionation to produce hydrogen peroxide" (Gardner and Lawrence, 1993). The transition metals needed to catalyze the reaction would be present in the water used for the preparation of the beverages. The study goes on to show that subsequent metal-catalyzed reduction of hydrogen peroxide by ascorbic acid can generate a hydroxyl radical. The study concludes by showing that the hydroxyl radical generated by the metal-catalyzed reduction of oxygen and hydrogen peroxide by ascorbic acid "can attack benzoic acid to produce benzene under conditions prevalent in many foods and beverages" (Gardner and Lawrence, 1993). The reaction is highly dependent on the concentration of ascorbic acid, optimum level of transition metal catalyst, and pH. Benzene production increases with increasing ascorbic acid concentration until the ascorbic acid concentration becomes too high, and then it, competes with benzoic acid for the hydroxyl radical (Gardner and Lawrence, 1993). The maximum amount of benzene is produced at pH 2, and constantly decreases as pH reaches 7. Benzene is not detectable in mixtures with pH above 7 (Gardner and Lawrence, 1993). Benzene production also increases with exposure to elevated temperatures (Kyoung and others, 2008). Consequently, soft drinks and beverages with ascorbic acid and added sodium benzoate that are subjected to intense heat can be susceptible to the production of benzene. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: The target audience is the food processing industry who are actively seeking to replace antimicrobial compounds with naturally occurring replacements. The results of this experiment have been reported at a national meeting and are being prepared for publication. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In the task to find a replacement for sodium benzoate, 28 chemical compounds isolated from natural sources known to have antimicrobial activity were selected for evaluation. All 28 compounds are currently ineffective as antimicrobials in beverages due to their low aqueous solubility. The first research objective is to accurately quantify the aqueous solubility of these compounds. The Minimum Inhibitory Concentration (MIC) of the compounds against Zygosaccharomyces bisporus, Saccharomyces cerevisiae, and Zygosaccharomyces bailli will be determined. Based on the solubility and MIC data, compounds with the best antimicrobial activity, but limited aqueous solubility, will be chosen to form molecular inclusion complexes with alpha-cyclodextrin (alpha-CD) and beta-cyclodextrin (beta-CD). Phase solubility analyses was performed to determine if the compounds' complexation with alpha-CD and beta-CD improves their aqueous solubility. Subsequently, solid inclusion complexes were prepared for further characterization. The stability of the inclusion complexes was studied in an acid-based beverage matrix. Finally, the stability of the complexes stored in PET containers was compared to the stability of the same complexes stored in glass containers. Twenty three of the compounds evaluated are components of plant essential oils and the remaining five compounds are alkyl esters of para-hydroxybenzoic acid. The test compounds were evaluated for aqueous solubility as well as their solubility in an acid-based beverage mixture. The compounds were found to be practically insoluble (less than100 mg/L), very slightly soluble (100 mg/L - 1,000 mg/L) or slightly soluble (1,000 mg/L to 10,000 mg/L). -Methoxycinnamaldehyde, trans,trans-2,4-decadienal, cinnamic acid, and citronellol were complexed with alpha- and beta- cyclodextrin and evaluated through phase solubility analyses. The complexes formed showed improved aqueous solubility for all compounds. Complexation with alpha-CD resulted in an increase of aqueous solubility of omega-methoxycinnamaldehyde by 10-fold, trans,trans-2,4-decadienal by 3.2-fold, cinnamic acid by 6.3-fold, and citronellol by 8-fold. In addition, complexation with beta-CD resulted in an increase of aqueous solubility of omega-methoxycinnamaldehyde by 1.6-fold, trans,trans-2,4-decadienal by 3.1-fold, cinnamic acid by 1.7-fold, and citronellol by 1.6- fold. The storage stability of the alpha-CD complexes of omega-methoxycinnamaldehyde trans,trans-2,4-decadienal and citronellol were evaluated for 7 days in an acid-based beverage solution by SPME GC-MS. The complexes exhibited varying levels of degradation throughout the duration of the study all. The concentration of omega-methoxycinnamaldehyde detected by SPME GC-MS decreased by 61.7 percent. Similarly, the concentration of trans,trans-2,4-decadienal and that of citronellol decreased by 62.7 and 43 percent respectively. Additionally, a comparison by UV/Vis of the storage stability of the complexes stored in glass and PET containers was performed. The storage stability comparison proved that absorption into the PET polymer membrane did not occur.

Publications

  • Samperio, Cristian. 2009. Formation, Characterization and Stability of Natural Antimicrobial-Cyclodextrin Complexes. MS Thesis. Virginia Polytechnic Institute and State University.
  • McKinney, J. Boyer, R. and Marcy, J. 2009. Antimicrobial potential of thirty-two natural compounds against common juice spoilage microorganisms (Saccharomyces cerevisiae, Zygosaccharomyces bailli, Z. bisporus) IAFP Annual Meeting Abstract book. P. 142.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Increased surface area of the shredded cheese allows mold to have more surfaces on which to grow allowing the cheese to spoil quicker. Manufacturers have tried to limit the growth of mold by modifying the atmosphere within the package. Oxygen is required for mold growth and replacing the atmosphere of a package with nitrogen and other inert gases prior to package sealing, mold growth is inhibited. Changing the atmosphere of the package protects the cheese while on the grocer's shelves but once purchased by the consumer, further protection is needed. When the consumer first opens the package of shredded cheese, the modified atmosphere packaging is lost allowing oxygen to enter the package giving mold the oxygen needed for growth. To help further protect the cheese, an antimycotic such as sorbic acid or natamycin is added. Natamycin kills germinating mold spores before the consumer can see visible mold growth. Without the added protection of natamycin, the cheese is able to spoil quicker in the consumer's refrigerator resulting in returned product. With shredded cheese accounting for nearly a quarter of all the cheese sold in the United States, cheese processors are investigating ways to increase shelf life after the consumer has released the modified atmosphere while still keeping a high quality product. 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. Polymer packages containing a UV absorber allow significantly less UV-associated degradation of natamycin than those packages that lacked a UV protectant. Further work with cyclodextrin compound has been investigated. In response to a need for a natural antimicrobial to replace sodium benzoate, cinnamic acid was chosen. Due to cinnamic acid's solubility issues, alpha-cyclodextrin was used as a host molecule to form an inclusion complex with the cinnamic acid molecule. The cinnamic acid: alpha-cyclodextrin inclusion complex was then characterized using phase solubility analysis, proton nuclear magnetic resonance (H-NMR), and solid inclusion. Phase solubility analysis verified the maximum amount of cinnamic acid that alpha-cyclodextrin was able to host. H-NMR was used to determine the complex association constant, determine the chemical shifts of available protons, and yield a stoichiometry for the complex. The solid inclusion complex allowed for a physical formation of the complex, yielding further information in support of the complex stoichiometry. Microbiological tests were also performed to quantify the antimicrobial abilities of the complex, the guest, and the host against the yeast Saccharomyces cerevisiae and mold Paecilomyces variotii. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This research showed the current IDFA method of natamycin detection to be inadequate. An HPLC method of natmycin detection has been shown to be highly effective in measuring both the active and light oxidizied (inactive) form of natamycin. Other natural antimicrobial compounds were complexed with cyclodextrins to test their effectiveness. Cinnamic acid was complexed with cyclodextrins to make them more water soluble.

Publications

  • Romano,D.L. 2008. Characterization of Alpha-Cyclodextrin Inclusion Complexes with Trans-Cinnamic Acid in an Acid-Based Beverage System. Virginia Polytechnic Institute and State University. Masters Thesis.


Progress 10/01/06 to 09/30/07

Outputs
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 scenario of a high initial mold count was preformed to see how well natamycin and each of its applications could prevent visible mold growth from occurring. Natamycin was complexed with different cyclodextrins to help better solubilize natamycin - beta-cyclodextrin, hydroxy-propyl beta-cyclodextrin and gamma-cyclodextrin. Using cyclodextrins to apply natamycin more uniformly onto shredded cheese did not significantly increase the consistency of distribution (p less than 0.05). 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. 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. 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, the addition of the UV absorber does not protect the natamycin fully. Variability was uniform throughout all treatments with the exception of HPBCD complex. In HPBCD, variability increased significantly. High levels of variability were seen throughout the study primarily due to the complex nature of cheddar cheese. The use of natamycin in any type of treatment can help to increase the shelf life of shredded cheese for up to 9 days longer. The use of hydroxy-propyl beta-cyclodextrin-natamycin complex can increase that shelf life even further, up to 17 days past cheese that has been untreated.When untreated with natamycin and an initial concentration of 10 -100 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, beta-cyclodextrin-natamycin complex, and gamma-cyclodextrin-natamycin complex. However, there was a difference with the use of hydroxy-propyl beta-cyclodextrin-natamycin complex. Hydroxy-propyl beta-cyclodextrin-natamycin complex allowed the shredded cheese to last for 41 days, 17 days longer than the control sample.

Impacts
Natamycin is a widely used antimycotic agent however application of natamycin has been difficult since it is not soluble. The use of cyclodextrin complexes resulted in improved mold inhibition without additional natamycin application. Cheese products will have less mold spoilage resulting in few consumer complaints and less wasted cheese products.

Publications

  • No publications reported this period


Progress 10/01/05 to 09/30/06

Outputs
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. Unfortunately, natamycin is degraded by ultraviolet (UV) light at wavelengths of 350 nm and below. Typical packaging applications do not provide adequate UV protection causing the natamycin to degrade readily. In addition to being UV sensitive, natamycin is not readily water soluble making it harder to distribute evenly over shredded cheese. 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. Furthermore, 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. 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 natamycin after UV light degradation because the method gives erroneously high results. Polymer packages containing a UV absorber allow significantly less UV-associated degradation of natamycin than those packages that lacked a UV protectant (p less than 0.05). Polymer packages containing a UV absorber (11.4 per cent light transmission at 350 nm) allowed significantly less UV-associated degradation of natamycin than those packages that lacked a UV protectant (90 per cent light transmission at 350 nm). Incorporation of 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.

Impacts
This research demonstrated the International Dairy Federation method which uses spectrophotometric analysis to be unsuitable for natamycin quantification if the product is exposed to light. Cheese processors using this method will be incorrectly led to believe that natamycin is present and active when in fact it is not. This erroneous measurement will lead to unexpected spoilage and loss of product at the retail level. The International Dairy Federation HPLC method was suitable for determining both the active and inactive forms of natamycin on cheese. This research also demonstrated the use of UV barrier in cheese packaging could slow the light deterioration of natamycin and extend the shelf life of shredded cheeses. Extended shelf life will result in few consumer complaints and less wasted cheese.

Publications

  • Teter, Vanessa. 2006. Ensuring the Stability of Natamycin on Shredded Cheese. M.S. Thesis. Virginia Tech.


Progress 10/01/04 to 09/30/05

Outputs
Natamycin is an antifungal agent employed by the dairy industry to prevent mold growth on shredded cheese. The extreme sensitivity of natamycin to ultraviolet light causes rapid degradation of the antifungal agent on shredded cheese products stored under fluorescent lighting in the retail dairy case. Our first research objective is to quantify the effect of the delivery system of natamycin as a dry powder, an aqueous suspension, and as an aqueous solution when complexed with cyclodextrin. Preliminary data suggests 100 days of shelf-life without visible mold when shredded cheese is stored without MAP. The commercial product Devlocid was supplied by DSM Food Specialties (Delft, The Netherlands). Multilayer bags with and without an ultraviolet (UV) light absorber were supplied by Cryovac Sealed Air Corporation (Duncan, SC) Preliminary research has been started to look at the degradation rates of natamycin with the UV protecting bags. HPLC analysis will determine the stability of natamycin and the natamycin complexes under fluorescent lighting in an aqueous suspension to serve as a reference for the cheese samples. Once the rates are determined, the natamycin and the different complexes will be applied to the cheese to determine the degradation rates using HPLC analyses. Differential Spectrophotometry will be preformed using a Shimadzu UV-2101PC UV-VIS Scanning Spectrophotometer (Shimadzu Scientific Instruments, Inc., Columbia, MD) to quantify pure natamycin in aqueous solution. The spectral scan will be between the wavelengths of 250-400 nm. Sidewall cutouts of the multilayer bags containing UV absorber will be prepared. Each sample of polymer will be placed in a UV cuvette and qualitatively analyzed by a spectral scan between 250-400 nm to determine the spectral overlay with natamycin. The samples will then be transferred into the polymer packaging each of which contains different UV absorber amounts. The samples will be exposed to continuous fluorescent lighting by cool white 40-watt T12 fluorescent bulbs (GE Lighting, Cleveland, OH) mounted 16 cm above the samples to receive an illumination intensity of 1000. All light intensity measurements will be preformed with a Foot Candle/Lux Meter (Extech Instruments, Waltham, MA). These fluorescent light samples will be stored at 4 C under ambient air atmosphere until the natamycin is undetectable. Analyses was preformed with an Agilent 1100 series Autosampler, containing a controller and a Photodiode Array Detector (PDA) (Palo Alto, CA). A 4.6 x 50 mm, 1.8 nm Eclipse XDB C8 reverse phase analytical column, equipped with a 4.6 x 10 mm, 5 nm Waters Spherisorb C8 guard column. The mobile phase system employed is a methanol-water-acetic acid, 60:40:5, v/v/v. The samples were eluded in isocratic mode in the mobile phase for 20 minutes. The wavelength range of the PDA was 260-360 nm. An external standard curve was constructed using USP Reference Standard Natamycin to quantify free natamycin content in all samples. Waters Millennium software version 3.20 was used for data management.

Impacts
The mold spoilage of cheeses that can not be vacuum packaged continues to be a major problem for the cheese industry. Previous research has shown that modified atmosphere packaging can prevent mold spoilage during distribution and retail sale, but is lost as soon as the consumer opens the package. The current practice of using the natural antimycotic, natamycin, has had some success but not as much as would have been suggested by laboratory studies. Our research has shown that the effectiveness of natamycin is being lost during the retail display because of UV light degradation. Use of UV absorbent packaging or use of UV free lighting would eliminate this problem and allow the natamycin to be effective after the MAP atmosphere has been released. Extension of shelf-life for the consumer once the package has been opened would be a significant improvement for the cheese industry. While UV barrier packaging is available it is not know what wavelengths must be blocked to preserve the natamycin and what the ultimate extension of shelf-life will be. Preliminary data has suggested up to 100 days without MAP atmosphere is possible.

Publications

  • van Aardt, M. S. E. Duncan, J. E. Marcy, T. E. Long, S. F. O Keefe, S. R. Nielsen-Sims. 2005. Aroma analysis of light-exposed milk stored with and without natural and synthetic antioxidants. J. Dairy Sci. 88:881-890.
  • Yaun, B.R., S.S. Sumner J.D. Eifert, J.E. Marcy. 2004. Inhibition of Pathogens on Fresh Produce by Ultraviolet Energy. Int. J. Food Micro 40: 1-8.
  • van Aardt, M., S. E. Duncan, T. E. Long, S. F. O Keefe, J. E. Marcy, and S. R. Sims. 2004. Effects of antioxidants on oxidative stability of edible oils and fats: Thermogravimetric analysis. J. Agric. Food Chem. 52:587-591.
  • van Aardt, M. S. E. Duncan, T. E. Long, S. F. OKeefe, J. E. Marcy, and S. R. Sims. 2004. Effect of antioxidants on oxidative stability of edible fats and oils: Thermogravimetric analysis. J. Ag. Food Chem. 52:587-591.
  • van Aardt, M., S. E. Duncan, J. E. Marcy, T. E. Long, S. F. OKeefe, and S. R. Nielsen-Sims. 2005. Effect of antioxidant (alpha-tocopherol and ascorbic acid) fortification on light-induced flavor of milk. J. Dairy Sci. 88:872-880.
  • Koontz, J.L. and J.E. Marcy. 2003. Formation of Natamycin:Cyclodextrin Inclusion Complexes and Their Characterization. J Ag Food Chem 51(24) 7106-7110.
  • Koontz, J.L., J.E. Marcy, W.E. Barbeau and S.E. Duncan. 2003. Stability of natamycin and its cyclodextrin inclusion complexes in aqueous solution. Ag Food Chem 51(24) 7111-7114.