The antifungal properties of chitosan in laboratory media and apple juice

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Abstract

The antimicrobial properties of chitosan glutamate, a derivative of chitin, were investigated in laboratory media and apple juice against 15 yeasts and moulds associated with food spoilage in order to assess the potential for using chitosan as a natural food preservative. Of the seven strains of filamentous fungi studied, chitosan reduced the growth rate of Mucor racemosus at 1 g/l whilst concentrations of 5 g/l were required to completely prevent growth of three strains of Byssochlamys spp. on agar plates incubated at 25°C for 3 weeks. Three strains of filamentous fungi were resistant to the antifungal effects of chitosan at 10 g/l. The presence of chitosan in apple juice (pH 3.4) at levels ranging from 0.1 to 5 g/l inhibited growth at 25°C of all eight spoilage yeasts examined in this study. The initial effect of chitosan in apple juice was biocidal with viable numbers reduced by up to 3 log cycles. Following an extended lag phase, some strains recovered and resumed growth to levels similar to those observed in unsupplemented apple juice. The most sensitive strain was an isolate of Zygosaccharomyces bailii obtained from a spoiled carbonated beverage; this yeast was completely inactivated by chitosan at 0.1 and 0.4 g/l for 32 days of storage at 25°C. The most resistant strain was Saccharomycodes ludwigii, an isolate from spoiled cider: a level of addition of 5 g/l of chitosan was required to inactivate this strain and to maintain yeast-free conditions in apple juice for 14 days at 25°C. Growth inhibition and inactivation of filamentous moulds and yeasts, respectively, was concentration-, pH- and temperature-dependent. It was concluded that chitosan was worthy of further study as a natural preservative for foods prone to fungal spoilage.

Introduction

Chitin, a β-(1,4)-D-linked polymer of N-acetylglucosamine, is a common constituent of crustacean and arthropod cell walls and is extracted commercially from shellfish wastes (Skjak-Braek et al., 1989, Goosen, 1997). Chitin makes up to 45% of the cell wall of Aspergillus niger and Mucor rouxii and 20% of the cell wall of Penicillium notatum; however, it is present at only around 3% in Saccharomyces cerevisiae (Arcidiacono and Kaplan, 1992, Knorr, 1984). It has been estimated that chitin is synthesised in nature at a level of up to 109–1010 tonnes per year (Peter, 1997).

Chitosan, a deacetylated derivative of chitin, is found in the cell walls of some fungi (Basidiomycetes spp.) and is produced from chitin by alkali treatment. Most commercial chitosans have a degree of deacetylation that is greater than 70% and a molecular weight ranging between 100 000 and 1.2 million Da (Li et al., 1997, Onsoyen and Skaugrud, 1990). Chitosan is polycationic at pH<6 and interacts readily with negatively charged substances such as proteins, anionic polysaccharides (alginate, carrgeenan), fatty acids, bile acids and phospholipids due to the high density of amino groups present in the polymer (Knorr, 1984, Muzzarelli, 1996). Perhaps less predictably, chitosan also selectively chelates metal ions such as iron, copper, cadmium and magnesium. Chitosan has attracted much research attention in the last 20 years as a potentially important renewable resource that is both non-toxic and biodegradable (Goosen, 1997, Muzzarelli et al., 1997, Skjak-Braek et al., 1989).

One of the earliest applications of chitosan has been in chelation of harmful metal ions such as lead, mercury and uranium out of industrial wastewaters (reviewed by Li et al., 1997, Onsoyen and Skaugrud, 1990) and in the removal of suspended solids from food processing wastes (reviewed by Knorr, 1984). The coagulating ability of chitosan has been harnessed to remove solids, unwanted dyes and acid substances from fruit juices effectively (Li et al., 1997). Chitosan and its depolymerised derivatives are reportedly used in personal care products such as hair conditioners and facial creams to impart moisturising properties (Hirano, 1989, Muzzarelli and De Vincenzi, 1997). Recently, the hypocholesteraemic properties of chitosan in the diet have been demonstrated in laboratory rats and in a limited number of human volunteers in short-term trials (Muzzarelli, 1996, Muzzarelli and De Vincenzi, 1997).

Much of the interest in the antimicrobial properties of chitosan has focused on its possible role in plant protection (Gooday, 1991, Gooday, 1994, Gooday, 1997). Minimum Inhibitory Concentrations (MICs) as low as 0.075 g/l for particulate chitosan and 0.018 g/l for soluble chitosan against some plant pathogenic fungi in liquid growth media have been reported (Allan and Hadwiger, 1979, Kendra and Hadwiger, 1989). However, other authors have found that levels as high as 10 g/l were necessary to inhibit growth of some fungal strains (Stossel and Leuba, 1984). Chitosan at a concentration of 1 g/l has been reported to reduce growth by up to 50% on agar plates at 25°C of several phytopathogenic fungi important in post-harvest spoilage of fruit and vegetables, including Botrytis cinerea (Hirano, 1997). The use of acidic chitosan solutions (containing 0.1% Tween as wetting agent) as disinfecting dips for fresh strawberries inoculated with B. cinerea conidia and stored at 13°C has been reported to reduce spoilage to a similar extent as treatment with the conventional chemical fungicide iprodione (Ghaouth et al., 1991).

Relatively little work has been reported on the antagonistic properties of chitosan against microorganisms important in foods. Furthermore, many researchers have targeted bacteria rather than fungi as target organisms for chitosan. For example, Sudarshan et al. (1992) tested the sensitivity of nine bacteria, including Salmonella typhimurium, to chitosan glutamate and chitosan lactate (both at 2 g/l) in phosphate buffer (pH 5.8) at 32°C and reported inactivation ranges of between one and five log cycles (depending on the organism) within 1 h of exposure. Both chitosan salts were similarly bactericidal against Gram-positive and Gram-negative organisms, indicating non-specific action. Papineau et al. (1991) have reported greater sensitivity of S. cerevisiae compared with E. coli or Staph. aureus in the presence of 1 g/l chitosan lactate in distilled water at 37°C. However, the biocidal properties of chitosan in relatively ‘clean’ systems such as distilled water and buffers are a poor indication of likely performance in complex food systems where interactions with other components may modulate the activity of chitosan, as well as of other food preservatives that may be present.

Very few attempts have been made to date to assess the antimicrobial properties of chitosan in real foods. Darmadji and Izumimoto (1994) investigated the effect of chitosan (type of salt not specified by authors) on the development of spoilage in minced beef patties stored at 30°C for 2 days and at 4°C for 10 days. At the higher storage temperature, a reduction of one to two log cycles of total bacteria, pseudomonads, Staphylococci, coliforms, Gram-negative bacteria and Micrococci was observed in the presence of 1% chitosan; at the lower storage temperature, similar reductions in spoilage flora were reported after 10 days. Fang et al. (1994) have investigated the use of chitosan as an antimicrobial agent against mould spoilage in candied kumquat. The authors reported that a concentration of 6 g/l of chitosan was required to maintain a mould-free shelf life of 65 days when the sugar concentration in the syrup was reduced from the traditional 65° Brix to 61.9° Brix at pH 4 (Fang et al., 1994). Chitosan is reportedly used as a preservative in solid foods in Japan in products such as kamaboko, noodles, soy sauce, Chinese cabbage and sardines; however, many of these reports are lacking in detail so that the conditions/formulations would be difficult to replicate and verify (Li et al., 1997, Hirano, 1997).

The objective of this study was to investigate the antifungal properties of chitosan against 15 food-associated yeasts and moulds in laboratory media and an acidic beverage in order to assess the potential for using chitosan as a natural preservative in foods prone to spoilage by yeasts and moulds.

Section snippets

Materials

Chitosan glutamate (trade name Seacure 110) was obtained from Pronova (Drammen, Norway). This chitosan preparation contained 42% glutamate and had a deacetylation range of 75–85% (manufacturer’s data). All growth media and diluents were obtained from Oxoid (Basingstoke, UK). All other chemicals were from Sigma Chemical Co. unless otherwise indicated. Clear, UHT-treated, shelf-stable apple juice containing no added preservatives and packed in laminated, 1 litre cartons was purchased from a local

Antimicrobial activity of chitosan against filamentous fungi

Of the seven strains of filamentous fungi studied, three strains (A. flavus, C. cladosporioides and P. aurantiogriseum) showed no discernible sensitivity (in terms of germination time and number of days to complete growth) when exposed to chitosan-supplemented agar (up to 10 g/l) at 25°C for up to 3 weeks. By contrast, M. racemosus and 3 strains of Byssochlamys spp inoculated on agar plates containing 5 and 10 g/l of chitosan glutamate failed to grow at 25°C within 3 weeks at which point the

The effect of chitosan on filamentous fungi

Of the seven strains of filamentous fungi tested in this study, M. racemosus, a rapidly-growing Zygomycete, and three strains of Byssochlamys spp. (belonging to the Ascomycetes group) were inhibited by chitosan glutamate at levels near or just above 1 g/l. By contrast, the fungi in the Deuteromycete group, A. flavus, C. cladosporioides and P. aurantiogriseum were resistant to chitosan at the maximum concentrations tested in this study (10 g/l). Following a study on the sensitivity of 46 fungal

Acknowledgements

The authors would like to thank Mr Jonathan Rhoades for assistance in the preparation of the figures. In addition, the following organisations are gratefully acknowledged for providing funding for this work: European Commission (Contracts FLAIR AGRF 0048 and FAIR CT96-1066), Aplin and Barrett Ltd. (UK), CPC International (UK), Gervais Danone (France), Gist-brocades (Netherlands), Meat and Livestock Commission (UK), Nestec Ltd. (Switzerland), Pepsico International (USA) and Unilever Research

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