Natamycin is a natural antimycotic, produced by fermentation of the actinomycete bacterium Streptomyces natalensis or other related species such as Streptomyces Gilvosporeus in a carbohydrate-based medium. Natamycin is also known as pimaricin and it is used as a food preservative in a variety of foods and beverages worldwide. It is a white/cream-colored powder with no taste and little odor .
Natamycin has the empirical formula C33H47NO13 and a molecular weight of 665,7 g/mol. It has a crystalline form and belongs to the group of polyene macrolide antifungals, or more specifically the tetraenes . The structure (fig. 6), which possesses a macrocyclic ring of carbon atoms closed by lactonization, is closely related to other antimycotics such as nystatin, rimodicin and amphotericin .
Figure 6. The structure of Natamycin [*].
The molecule is amphoteric with one acid and one basic group. Natamycin is poorly soluble in water (30 - 100 ppm at room temperature) and almost insoluble in non-polar solvents. However, it shows good solubility in strongly polar organic solvents, e.g. glycerol (15000 ppm). The poor solubility in water is, most of the time, not a problem because of the relatively low concentrations required for natamycin to be effective. On the contrary, the low solubility can be an advantage because the preservative remains effective on the surface of the food for longer periods. When first applied, only 30 - 50 ppm will be present on the surface, the remainder will be present in the more stable crystal formation. A gradual dissolvation then insures a slow release and prolonged effectiveness . Natamycin is most effective at pH-values between 5 and 7. Below pH 4, 5 and above pH 9 its effectiveness may drop by as much as 30 %. Aqueous solutions/suspensions of natamycin at neutral pH remains stable for 24 hours at 50 Â°C but longer periods of exposure to this temperature will cause a reduction in effectiveness due to hydrolysis of the ring structure .
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Exposure to high temperatures 100 Â°C for shorter periods shows little reduction in activity. Exposure to ultraviolet light for longer periods reduce the activity of natamycin and contact with oxidizing agents and heavy metals should also be avoided. Heavy metals reduce the stability of dilute solutions and chemical oxidation leads to reduced effectiveness of the antimycotic. Therefore, exposure to direct sunlight should be avoided and glass, plastic or stainless steel containers should be used. Natamycin in solution has an ultraviolet absorption spectrum with minima at 250, 295.5, and 311 nm, and maxima at 220, 290, 303, and 318 nm .
Natamycin is effective against moulds and yeasts but not against bacteria, viruses or other microorganisms such as protozoa. This is because natamycin acts by combining with ergosterol and other sterols, e.g. 24- and 28-dehydroergosterol and cholesterol, which are present in the cell membranes of moulds and yeasts but not in bacteria, with few exceptions. The binding of natamycin to the sterols disrupts the cell membrane leading to increased permeability and leakage of essential cellular material. This in turn leads to a rapid drop in intracellular pH and possibly cell lysis. Natamycin also inhibits the glycolysis and respiration .
There exist no naturally natamycin resistant strains in the environment. This is because ergosterol is an essential component of the cell membrane, present in all yeasts and fungi, and the fact that natamycin is present as micelles in solution. An organism that comes into contact with the antimycotic encounters a high and lethal concentration of the compound .
Natamycin is an approved food additive in over 40 countries. However, the regulations regarding its use differ from country to country. In the EU, natamycin has the additive number E235 and is permitted for use as surface treatment of specified cheeses and sausages. .
Natamycin is usually effective against most moulds and yeasts at concentration levels between <5 - 20 ppm. Yeasts are generally more sensitive than moulds to the preservative. The minimum inhibitory concentration (MIC) of A. niger is 1,0 - 1,8 Î¼g/ml and for S. cerevisiae the MIC is 0,15 Î¼g/ml .
Toxicology studies on natamycin have been done using mice, rats, rabbits and guinea pigs. Natamycin was proven to be least toxic when administrated orally (LD50 = 1500 mg/kg in mice and rats) or subcutaneously (LD50 = 5000 mg/kg in rats) and most toxic when administrated intravenously (LD50 = 5 - 10 mg/kg). It has also been shown that no natamycin was absorbed from the human intestinal system after an intake of up to 500 mg/day under a period of seven days . Handling natamycin in bulk, as with any dry powder, can result in skin and eye irritation .
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 Thomas, L. V., Delves-Broughton, J. Danisco Innovation, Beaminster, Dorset, UK. Natamycin (Technical Paper, TP 21-1e). 2005, Elsevier Science Ltd.
 URL: http://dailymed.nlm.nih.gov/dailymed/image.cfm?id=2300&type=img&name=natacyn-01.jpg Â© The United States National Library of Medicine (NLM)
Last consulted: 2007-06-03
 Introduction to NatamaxÂ® Natural Antimicrobial (Technical Memorandum, TM 35-6e). Danisco A/S.
* http://en.wikipedia.org/wiki/File:Natamycin.svg 2009/12/29
The term 'fermentation' was obtained from the Latin verb 'fervere', which describes the action of yeast or malt on sugar or fruit extracts and grain. The 'boiling' is due to the production of carbon dioxide bubbles from the aqueous phase under the anaerobic catabolism of carbohydrates in the fermentation media. The art of fermentation is defined as the chemical transformation of organic compounds with the aid of enzymes. The ability of yeast to make alcohol was known to the Babylonians and Sumerians before 6000 BC.
The major steps in the commercial production of penicillin are:
(1) Preparation of inoculum.
(2) Preparation and sterilization of the medium.
(3) Inoculation of the medium in the fermentor.
(4) Forced aeration with sterile air during incubation.
(5) Removal of mould mycelium after fermentation.
(6) Extraction and purification of the penicillin.
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1. In a process for preparing natamycin including the steps of (a) obtaining an inoculum by propogating a spore suspension containing a natamycin-producing Streptomyces species in an inoculum medium; (b) introducing the inoculum to a fermentation medium and providing a fermentation broth comprising said fermentation medium and inoculum; (c) producing natamycin by a fermentation in said fermentation broth; and (d) recovering natamycin produced by said fermentation, an improvement comprising:
in (c) using a fermentation with a cell propogation stage followed by major natamycin production stage, adding a basic pH control agent at a rate sufficient to maintain the fermentation broth at a pH of from 5.0 to 6.5 during said major natamycin production stage, and continuing the fermentation to provide a fermentation broth containing at least about 5 g/l natamycin.
United state patent: Patent 5231014
Figure 4 The morphology of the mutant LK-119 at 8h, 12h, 16h, 24h, and 36h cultivated in liquid medium at 28oC
2.1.1 Aspects of Submerged Culture
Fungi and bacteria are commonly used in submerged fermentation to produce a wide
range of primary and secondary metabolites. Many other plant, insect and
mammalian cell lines are also used to produce secondary metabolites, but these
systems usually are not as effective as bacteria and fungi.
Three distinct phases of a micro-organism's growth can normally be distinguished in
submerged culture fermentations: the lag, the linear (also known as log or
exponential), and stationary phases (Fig. 2.1). The autolytic, or death phase, is not
usually classed as a growth phase. The length of the lag phase, where cells adapt to
the new environment and begin to grow depends on the micro-organism, initial cell
concentration, environmental conditions and growth medium. In the linear phase
(sometimes called the tropophase), there is rapid growth and the stationary phase
(sometimes called the idiophase), there is no further net growth. Secondary
metabolites such as antibiotics are usually produced in the stationary phase (Pelczar
& Reid 1972; Giancarlo & Rolando 1993).
Figure 2.1 Typical growth phases for micro-organisms in submerged culture
Any fermentation involving biocatalysts, such as micro-organisms, plant cells, animal cells or enzymes, will result in transformation and production of biochemical substances. Fermenter or bioreactors should provide optimal growth conditions for micro-organisms to achieve conversion and/or production of biological products. Generally, only one specific form of life has to grow and produce the desired product. To prevent contamination by other micro-organisms, sterility of the reactors and media are an indispensable requirement. Cleaning is important to make sure that impurities will not affect the bioconversion or spoil the final product. Homogeneous conditions for temperature, pH, dissolved oxygen, substrate, and product concentration have to be maintained in the reactors to ensure consistent processes and products. The bioprocess needs to be controlled and all available measurements should be logged to enable quality assurance. Safety regulations also have to be followed to prevent accidents and release of toxic products. Therefore, fermentations take time and consequently any experiments are going take time to get the results.
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Screening applications such as media optimization, searching for new bioactive molecules or characterizing recombinant/mutated micro-organisms, always involves analysing many medium compositions or cultures (Minas et al. 2000). Several different experimental designs may be used for a single investigation. For example, screening the main factors affecting the process may be done using the Plackett-Burman method initially, followed by optimization of critical medium components using the response surface methodology. Thus, a medium design campaign can involve testing hundreds of different media.
Another difficult aspect of the medium design process is recording and analysing the data produced. In reality, information generated from design experiments is often difficult to assess because of its sheer volume. After about 20 experiments with five variables, the researcher may find it very difficult to remember the trends in medium components, especially when more than one variable has been changed at a time. Therefore, data capture and data mining techniques are crucial (Kennedy & Krouse 1999).