4. RESULTS AND DISCUSSIONS
4.1. C HITOSAN ’ S IN VITRO ANTIMICROBIAL ACTIVITY — A CRITICAL LOOK
4.1.11. Discussion
ATCC 12472, which possesses a considerable chitosan hydrolytic activity; on the other hand the MIC for B. subtilis 165, also a chitosan‐hydrolyzing strain, was below 1 µg/ml, indicating the absence of a direct correlation between hydrolysis and susceptibility to chitosan. These findings debunk the theory of Beausejour et al. 13, who claimed that the presence of a chitosan‐hydrolyzing activity is virtually synonymous with immunity towards chitosan, and therefore suggested its use together with a biocontrol strain exhibiting chitosanolytic activity as a promising biocontrol tool.
Although previous studies have investigated the chitosanolytic properties of a number of organisms, and their possible use for chitosan‐degradation, we are not aware of any reports studying the relation between chitosanolytic activity on one hand, and bacterial susceptibility to chitosan in an in vitro setting.
Whereas both S. simulans 22 and S. aureus SG511 have no chitosan‐degrading activity, B. subtilis 168 demonstrated an adequate activity in chitosanase‐detection medium. The highest chitosan‐hydrolyzing activity of B. subtilis 168 was observed in cultures grown in rich media, such as TSB and BHI broth, followed by NI broth, and a residual activity in CAMHB; the activities being detected both in the culture, as well as in the cell‐free supernatant.
In an in vitro setting, the potency of an antimicrobial agent is governed on the one hand by its properties and on the other hand by the specific environmental context, including the type of growth medium, the presence of extraneous matter and the experimental setting used to assess the potency. Underestimation of any of these factors would inevitably lead to false conclusions.
The MIC is usually taken as a measure of the susceptibility of a bacterial strain towards a specific antimicrobial substance. In this study we present evidence that a number of factors may account for large variations in the reported MIC values of chitosan in the available literature.
The discrepancies between data may result from the different degrees of deacetylation and molecular weights of chitosan. The evaluation of the dependence of the antimicrobial activity of chitosan on each of these characteristics requires keeping all other variables constant, for example by using chitosans of a wide MW range with the same DD. This is however almost impossible to achieve, since chitosan is a natural polymer; there would always be variations within product batches. Therefore, it would be rather difficult to determine the optimal MW for the maximum antimicrobial activity.
Liu et al. 182 studied the antimicrobial activity of chitosan against E. coli, and came to the conclusion that its activity increased with increasing MW, up to a MW of 91.6 kDa;
above that value, there was an inverse relationship between both. We were not able to reach a similar conclusion based on our findings; however, we discovered that oligosaccharides lack the antimicrobial activity that is exhibited by large chains of the polymer. This view is shared by Uchida et al. 323, who observed that chitosan oligomers possessed weak or no antibacterial activity at levels as high as 0.5 –1.0%. In addition, Young et al. 361 demonstrated in their study that chitosan increases the membrane permeability of plant cells, a property shared by basic polymers, such as poly‐L‐lysine, histone, DEAE‐dextran, protamine sulfate, and glycol chitosan; in contrast, monomeric D‐glucosamine and L‐lysine showed no effect at concentrations up to 500 µg/ml. On the other hand, Rhoades and Roller 255 theorized that a mild degradion of chitosan enhances
action. Therefore, it seems reasonable to suggest that there seems to be a minimum degree of polymerization required for antimicrobial activity, above which the activity remains more or less constant.
Noteworthy of mentioning is that the reported MIC values of chitosan are usually much higher than those encountered in our current study. Apart from differences in chitosan characteristics (including the use of plain or derivatized chitosans, oligomers or high molecular weight polymers), we can conclusively demonstrate that this could be ascribed to differences in experimental settings, including both the culture medium used, as well as the method used for susceptibility assessment, which play critical roles in the antimicrobial potency of chitosan.
For instance, we chose to use the broth microdilution method for susceptibility testings, based on preliminary results that demonstrated the inferior activity of chitosan in agar media. Nonetheless, some researchers used agar‐based methods (agar dilution
215 and agar diffusion 128) to assess the antimicrobial potency of chitosan.
Moreover, the antibacterial effects of chitosan and its oligosaccharides were often studied in nutrient broth 182,335, tryptic soy broth 144,145 and phosphate buffer 318, where the activity of chitosan is only minimal, resulting in considerably higher MIC values. In addition, some of these researchers tracked the inhibitory effects of chitosan spectrophotometrically 144,182. Based on the results of flocculation assays, we deem such a method inappropriate, due to large fluctuations in optical density measurements in presence of chitosan. Therefore, it seems that several studies ostensibly demonstrating the antimicrobial efficiency of chitosan were based on unsuitable approaches.
Among other criteria that need to be taken into consideration when evaluating the efficiency of chitosan for use as a preservative is its concentration, which is a major factor in antimicrobial activity. Most chitosan formulations contain high concentrations of chitosan to achieve an optimal, broad spectrum activity. Here, we would like to point
out that the concentrations of chitosan used in this study are far below those used in chitosan formulations or in other studies. For instance, Bae et al. used a 1.0% chitosan solution in a clinical trial to study the effect of chitosan on plaque formation 10, while Roller and Covill studied the antimicrobial properties of chitosan glutamate in mayonnaise and mayonnaise‐based shrimp salad 259 as well as in laboratory media and apple juice 258, at a level of 3 g/l and 0.1 ‐ 5 g/l, respectively.
In sum, our findings uniformly indicate that, while there is little doubt that chitosan could indeed be included as a preservative in certain systems; there is still much to be learned about its antimicrobial potential. However, a stage has being reached at which it is becoming possible to present a general account of the main criteria that should be closely observed while developing antimicrobial systems to be implemented into industrial applications. Therefore, at this point we would like to reiterate that applications relying solely upon in vitro results should be treated with caution, since results may differ considerably in a food or pharmaceutical formulation, where such factors as the complexity of the medium, the presence of organic matter, the pH of the formulation as well as the presence of other active agents, would all play an important role in the final efficiency of the formulation.