Improved applicability of nisin in novel combinations with other food preservation factors

<h3>General discussion</h3>Modern consumers nowadays, have a preference for more natural, mildly preserved food products with a fresh appearance over traditionally preserved products. Mild preservation techniques applied singly are usually not sufficient to control microbial outgrowth and combinations of measures are needed to ensure complete safe products (16). Bacteriocins, produced by lactic acid bacteria have been successfully used as biopreservatives in a number of food products to inhibit the growth of pathogenic and spoilage organisms (27). Up till now, nisin is the only bacteriocin that has been approved by the WHO to be used as a food preservative. Due to its restricted inhibition spectrum and the decreased solubility and heat sensitivity at neutral pH, application is still limited (10). The study described in this thesis aimed to increase the practical application of nisin by combinations with other biopreservatives or mild preservation techniques.<h3>Nisin and essential oils</h3>Essential oils, derived from plants, are known for their flavor characteristics. Many of the compounds found in essential oils possess antimicrobial activity (4, 9, 14, 22), and therefore are suitable candidates for mild food preservation in combination with nisin. The essential oils dramatically enhance the bactericidal activity of nisin at concentrations, which alone do not affect the bacterial cell counts of the foodborne pathogens Listeria monocytogenes and Bacillus cereus (chapter 2). Adaptation of these cells to lower temperatures resulted in an increased sensitivity towards nisin, possibly due to an altered membrane composition leading to a change in membrane fluidity or to an increased electrostatic interaction of nisin with phospholipids in the membrane caused by an increase in negative charges (8, 18 - 21, 31). Alternatively a decrease in lipid II content as a result of changes in the membrane composition might explain the decreased activity of nisin (5). Lowering the temperature had a negative influence on the synergy between nisin and the essential oils, which might result from the lower sensitivity of the cells towards essential oils at lower temperatures (28).The exact mechanism underlying this synergy is not exactly known. Both nisin and carvacrol cause a dissipation of the proton motive force as well as depletion of the internal ATP pool (6, 12, 23, 26, 30, 32, chapter 3). In combination, carvacrol enhances the membrane potential dissipating effect of nisin, at concentrations which do not affect the viable count of B. cereus . Apparently cells are able to cope with low concentrations of nisin and carvacrol. When concentrations increase, cells are no longer able to compensate for loss of membrane integrity and a synergistic reduction of the pH gradient and depletion of the intracellular ATP pool were observed. The reduction in internal ATP is not proportional to the increase in external ATP and no additional increase in external ATP was observed upon simultaneous exposure to nisin and carvacrol. This observation excludes increased leakage of ATP as an explanation for the synergistic depletion of the intracellular ATP pool. Consequently, the underlying mechanism of the synergistic inactivation of B. cereus is most likely not the increased poreforming ability of nisin by carvacrol. Presumably, the rate of ATP hydrolysis is increased upon simultaneous addition of nisin and carvacrol or the internal ATP pool is exhausted in an attempt to reenergize the membrane (1, 23, 29). Alternatively, the disturbance of the membrane permeability by carvacrol and nisin might lead to impairment of membrane bound enzymes like ATPase, resulting in a decreased ATP synthesis (15, 26).<h3>Nisin and PEF treatment</h3>In addition to essential oils, Pulsed Electric Field treatment was also found to improve the antimicrobial action of nisin against B. cereus. Synergy was only found when PEF treatment was spread over a period of 10 minutes to match the relevant inactivation time scale of nisin's action. The additional stress imposed by PEF treatment possibly facilitates the incorporation of nisin into the cytoplasmic membrane resulting in more or larger pores or pores with a longer lifetime (chapter 4). Further reduction of the intensities of the treatments was achieved by adding carvacrol as a third hurdle to the combination of nisin and PEF treatment (chapter 5).The fact that synergy was found between the three treatments renders the combination very interesting for mild food preservation. However, extrapolation of the results from labscale experiments in buffer systems to food model matrices is usually difficult and the influence of food ingredients on the efficiency of preservation techniques are not fully understood. The efficiency of PEF treatment against vegetative cells of B. cereus is not affected by proteins in skimmed milk (20 %). However, the proteins do have a negative influence on the nisin activity, either as a result of a decreased bioavailability of nisin due to binding of the molecule to proteins or because of protection of the microorganisms by the proteins. As a consequence, the synergy between nisin and PEF treatment is less pronounced in skimmed milk (20 %).In sharp contrast to the improved bactericidal activity found in HEPES buffer, carvacrol is not able to enhance the synergy between nisin and PEF treatment in diluted milk (only in high concentrations (1.2 mM)). Possibly, carvacrol binds to the proteins, reducing the availability of the molecule. However, this is not consistent with the fact that carvacrol increases the antimicrobial activity of PEF treatment in milk. Therefore, the absence of synergy between nisin, PEF treatment and carvacrol is more likely explained by the decreased bioavailability of nisin, thereby decreasing the extent of synergy between nisin and carvacrol and consequently between all three treatments. The influence of PEF treatment on the behavior of proteins is not exactly known. Proteins can carry electric charges and might behave as dipoles when subjected to PEF treatment, which cause the macromolecules to reorient or deform (such as protein unfolding and denaturation), and possibly some breakdown of covalent bonds or casein micelles may occur (3). These PEF induced changes in the structure of proteins may play a role in the existence of synergy between carvacrol and PEF. Dilution of the milk to 5 % still provides enough proteins to stimulate synergy between carvacrol and PEF treatment (chapter 5).Before such novel techniques can replace currently used thermal processes, more insight into spore inactivation is needed (chapter 6). Nisin and PEF treatment do not directly inactivate or damage spores of B. cereus , however germinated spores can be inactivated by nisin or PEF treatment to a certain extent. The PEF resistance of the germinated spores is lost 50 minutes after the onset of germination. Nisin resistance was lost immediately in parallel to heat resistance, suggesting that loss of nisin resistance might be ascribed to changes in the dehydrated state of the core. Sulfhydryl groups in the membrane, not available in ungerminated spores, were suggested to be the natural target for nisin and therefore access to the membrane is a prerequisite for inactivation (17, 24, 25). In addition, the increase in availability of the membrane-anchored cell wall precursor Lipid II upon germination could also play a role in the loss of nisin resistance (5). Apparently, nisin has gained access to the membrane by penetrating the coat, which was made more permeable upon germination or alternatively, the protective coat was degraded by spore lytic enzymes, allowing nisin to reach the cytoplasmic membrane. The late loss of PEF resistance can be explained by its dependence on the degradation of the spore coat. To exert antimicrobial inactivation by PEF treatment, free migration of ions is needed to increase the transmembrane potential of the spores. Formation of pores occurs after compression of the membrane and reorientation of the phospholipids in the membrane. In spores the ions are immobilized by proteins or DPA, restricting their mobility (7, 13) and subsequently the build up of an increased transmembrane potential is prevented. Secondly, the spore core is surrounded by several rigid protecting layers limiting the compression and reorientation of the phospholipids (2).Combining nisin and PEF treatment did not result in additional inactivation of the germinating spores. Since loss of PEF resistance occurs only after 50 minutes of germination and loss of nisin resistance seems to be an early event in spore germination, synergy would therefore be less likely due to different time scales of action. Furthermore, the incomplete germination of the spores reduces the margins to observe synergy. Ideally, complete and synchronized germination is needed to quantify the inactivation by nisin or PEF treatment and determine precisely the onset of loss of nisin or PEF resistance.One of the main problems associated with the use of antimicrobial compounds is the development of tolerance or resistance to certain compounds. Adaptation of cells to carvacrol was correlated to a decrease in membrane fluidity as demonstrated by Ultee et al. (30). In addition, they observed a change in phospholipid composition of the membrane. Cells adapted to carvacrol exhibited an increased sensitivity towards nisin compared to control cells (chapter 6). A decrease in the membrane fluidity is not expected to increase nisin's action, but a change in the head group composition, with an increase in negatively charged lipids, might stimulate the electrostatic binding of nisin and in this way enhance nisin's action (8, 18 - 21, 31). Alternatively an increase in lipid II content in carvacrol-adapted cells as a result of changes in the membrane composition might explain the increased activity of nisin (5). A decrease in the membrane fluidity did not change the susceptibility towards a PEF treatment. A more rigid membrane is less likely to be compressed by accumulating charges as a result of applied field strength and the ordered state of the phospholipids in the membrane decreases the chance of reorientation, which would reasonably lead to a decreased inactivation by PEF treatment. Although the bactericidal activity of nisin was increased by adaptation to carvacrol, the synergy between nisin and PEF treatment was not influenced by a change in membrane fluidity and membrane composition. Attemps to change the membrane composition of spores by adaptation of vegetative cells to carvacrol prior to and during sporulation did not lead to inactivation of spores by either nisin or PEF treatment.<h3>Application</h3>Combinations of nisin with essential oils or PEF treatment have been successful in overcoming the restrictions in practical application of nisin. For instance, the inhibition spectrum of nisin can be widened by combination with other preservation technologies like PEF treatment. In addition, the limited activity of nisin at higher temperatures can be complemented by the increased synergy between nisin and essential oils.The application of multiple hurdles has great potential to be used as a mild food preservation technology. The occurrence of synergy between nisin and essential oils or PEF technology allows for a reduction in the intensities of the treatments demonstrating the suitability for mild preservation. Increasing the number of hurdles (lysozyme) improves the observed synergy and further increases the mildness of the preservation technology (chapter 1).Consumer's acceptation of these combination techniques in case of the essential oils is not expected to meet difficulties. This combination meets with present preference for more natural and mild preservation methods. Herbs and spices, of which essential oils are the active components, are already used for centuries as flavoring agents and in homeopathic products and medicines. Currently, carvacrol is Generally Recognized As Safe (GRAS) and has been approved by the Code of Federal Regulation (CFR) to be used as a flavoring agent (11). However, when the essential oils are used for their antimicrobial activity, they will be regarded as new food additives and subsequently require a non-toxicity report (27). To circumvent these problems, the original herbs and spices can be used as food flavoring agents, while at the same time advantage can be taken of their antimicrobial activity. However, the producer has to take into account the low concentration of the active compound in herbs and spices. Furthermore, the essential oils have a strong and specific flavor and can only be applied in products where this aroma is appreciated.Acceptance of PEF technology is expected to give more problems and introduction of this technology has to be handled carefully. Consumers might associate PEF treated foods with residual electromagnetic raditation, just like radiated foods are associated with radioactivity. Only when PEF technology is introduced carefully and the consumers are supplied with the right information, they will accept this technology as mild preservation.At the moment, not enough information is known about PEF technology and its mechanism of action. Evidently, more research needs to be done to verify the influence of other food ingredients including fat particles on the antimicrobial activity. Furthermore the influence of PEF treatment on the product quality needs to be investigated. The fresh-like appearance, color and the vitamin content are seemingly unaffected however, the influence of PEF treatment on proteins, polysaccharides macromolecules, or lipids is not exactly known.The development of tolerance or resistance to the PEF treatment or the combination treatments is not clear and should receive more attention, since microorganisms generally adapt to environmental stress factors. Increased tolerance towards nisin and carvacrol has been studied in more detail (8, 18 - 21, 31) however, no such research has been conducted concerning PEF technology. Combining preservation technologies in which the microorganism is attacked from different sides should reduce the development of tolerance to a minimum. Inactivation of spores is another challenge to be overcome before such combination technologies can be implemented in current preservation strategies.In conclusion, these combination techniques are a welcome alternative to currently used pasteurization methods. The current limitations in the application of nisin can be complemented by the inhibition spectrum of the combination treatment. In addition, the synergy observed between the different preservation techniques allows for a reduction of the used intensities increasing the suitability for mild preservation.<h3>References</h3><ol><li>Abee, T., F. M. Rombouts, J. Hugenholtz, G. Guihard, and L. Letellier. 1994. 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