Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum

https://doi.org/10.1016/j.ijfoodmicro.2004.01.017Get rights and content

Abstract

Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets. Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol (∼8% w/w) were required for complete inhibition of all strains of C. botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone. However, only low levels of mastic oil (∼0.3% v/v) were required for inhibition of proteolytic strains of C. botulinum. Both studies showed a strain specific inhibition, with C. botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains. However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media. While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C. botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C. botulinum in English-style crumpets.

Introduction

Modified atmosphere packaging (MAP), involving gas flushing and interactive oxygen absorbent sachets, is being increasingly used by the bakery industry worldwide to extend the shelf-life of high-moisture bakery products. However, there are regulatory concerns about the safety of such products with extended shelf-life at ambient temperature, particularly with respect to the growth of Clostridium botulinum. While there have been no botulism outbreaks associated with commercially produced MAP bakery products, several challenge studies have shown that bakery products are a suitable substrate for the growth of C. botulinum (Daifas et al., 1999a). Daifas et al. (1999b) have shown also that MAP crumpets, a high-moisture minimally processed product, could support the growth of proteolytic strains of C. botulinum yet remain sensorially acceptable at the time of toxigenesis—a highly dangerous scenario. As a result of the potential of bakery products to support the growth of C. botulinum, it has been recommended that additional barriers, such as aw and pH reduction, be incorporated into MAP bakery products to ensure their continued safety at ambient storage. However, reformulation of high-moisture products to lower aw and pH levels is not always practical due to textural and sensory constraints. More recently, Daifas et al. (2000) investigated the potential of ethanol vapor as an additional barrier to enhance the safety of MAP crumpets. However, while this approach enhanced the safety of crumpets, their sensory shelf-life was compromised due to absorption of ethanol from the package headspace (Daifas et al., 2000). It is well known that two or more barriers, in conjunction with one another, can be far more effective than using each barrier separately. This “hurdle” approach is widely used by the food industry to extend both the shelf-life and safety of foods. A novel potential barrier that could be used, in conjunction with ethanol vapor, is mastic resin or its essential oil. Derived from the shrub Pistacia lentiscus var. chia, mastic resin, and its essential oil, have been used as flavoring for breads, as well as for other foods and beverages (Wyllie et al., 1990), and have also been shown to have antioxidant properties (Abdel-Rahman and Soud, 1975). The antimicrobial activity of mastic against several foodborne pathogens, including Staphylococcus aureus, Salmonella enteritidis, Bacillus cereus, Escherichia coli and Helicobacter pylori, as well as many yeasts and molds has also been reported Alippi et al., 1996, Iuak et al., 1996, Tassou and Nychas, 1995, Huwez et al., 1998, Magiatis et al., 1999, Koukoutsis, 2002 with Gram-positive bacteria being generally more sensitive than Gram-negative Hussain and Tabji, 1997, Ali-Shtayeh et al., 1998. However, the effect of mastic on C. botulinum has never been reported.

Therefore, the objective of this study was to determine the effect of mastic resin and mastic oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of C. botulinum in media and on neurotoxin production in challenge studies with English-style crumpets.

Section snippets

Preparation of inocula

Spores of proteolytic C. botulinum (A6, 17A, 62A, CK2A, MRB, 13983IIB, and 368B) were used in these studies. Spore crops of each strain were prepared separately, enumerated as described by Hauschild and Hilsheimer (1977), and stored at −80 °C. Spores and overnight cultures of individual strains were used in the “spot-on-the-lawn” and microtiter assays respectively, while a composite spore inoculum was used in the agar plate study and in the challenge study with crumpets. All spores and spore

Preliminary screening

The effects of mastic resin solutions (0–10% [w/w]) in 95% ethanol against five strains of proteolytic C. botulinum are shown in Table 1. While the mastic solutions separated into two distinct, concentric phases in MMT agar plates—the outer of which had an iridescent, oil-like sheen (Fig. 1)—no such separation occurred with the control solution (0% mastic in ethanol). Inhibition of C. botulinum was consistently greater in the outer oil-like phase (Table 1) while partial inhibition was observed

Conclusion

This preliminary study has clearly shown the potential of mastic resin and its essential oil as a novel anti-botulinal agent in media. Since mastic is used as a flavoring agent, it could have the additional advantage of being a “natural” preservative in bakery products. However, further studies are required to enhance the activity of both mastic resin and its essential oil against C. botulinum in bakery products. While the anti-botulinal activity of mastic appears to be greater in the vapor

Acknowledgements

The authors would like to thank the Natural Sciences and Engineering Research Council of Canada for financial assistance to complete this study. The authors would also like to thank Cryovac Liquid Sealed Air for supplies of high gas barrier films and the Mitsubishi Gas and Chemical Company for supplies of oxygen absorbents.

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