Christiaan Michiels

Biofilm formation and the food industry, a focus on the bacterial outer surface.

The ability of many bacteria to adhere to surfaces and to form biofilms has major implications in a variety of industries including the food industry, where biofilms create a persistent source of contamination. The formation of a biofilm is determined not only by the nature of the attachment surface, but also by the characteristics of the bacterial cell and by environmental factors. This review focuses on the features of the bacterial cell surface such as flagella, surface appendages and polysaccharides that play a role in this process, in particular for bacteria linked to food‐processing environments. In addition, some aspects of the attachment surface, biofilm control and eradication will be highlighted.

Lysozymes in the animal kingdom.

Lysozymes (EC 3.2.1.17) are hydrolytic enzymes, characterized by their ability to cleave the β-(1,4)-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine in peptidoglycan, the major bacterial cell wall polymer. In the animal kingdom, three major distinct lysozyme types have been identified — the c-type (chicken or conventional type), the g-type (goose-type) and the i-type (invertebrate type) lysozyme. Examination of the phylogenetic distribution of these lysozymes reveals that c-type lysozymes are predominantly present in the phylum of the Chordata and in different classes of the Arthropoda. Moreover, g-type lysozymes (or at least their corresponding genes) are found in members of the Chordata, as well as in some bivalve mollusks belonging to the invertebrates. In general, the latter animals are known to produce i-type lysozymes. Although the homology in primary structure for representatives of these three lysozyme types is limited, their three-dimensional structures show striking similarities. Nevertheless, some variation exists in their catalytic mechanisms and the genomic organization of their genes. Regarding their biological role, the widely recognized function of lysozymes is their contribution to antibacterial defence but, additionally, some lysozymes (belonging to different types) are known to function as digestive enzymes.

Antimicrobial Properties of Lysozyme in Relation to Foodborne Vegetative Bacteria

The purpose of this review is to describe the antibacterial properties and mode of action of lysozyme against Gram-positive and Gram-negative bacteria, and to provide insight in the underlying causes of bacterial resistance or sensitivity to lysozyme. Such insight improves our understanding of the role of this ubiquitous enzyme in antibacterial defense strategies in nature and provides a basis for the development and improvement of applications of this enzyme as an antibacterial agent. The bactericidal properties of lysozyme are primarily ascribed to its N-acetylmuramoylhydrolase enzymic activity, resulting in peptidoglycan hydrolysis and cell lysis. However, an increasing body of evidence supports the existence of a nonenzymic and/or nonlytic mode of action. Because Gram-negative bacteria, including some major foodborne pathogens, are normally insensitive to lysozyme by virtue of their outer membrane that acts as a physical barrier preventing access of the enzyme, several strategies have been developed to extend the working spectrum of lysozyme to Gram-negative bacteria. These include denaturation of lysozyme, modification of lysozyme by covalent attachment of polysaccharides, fatty acids and other compounds, attachment of C-terminal hydrophobic peptides to lysozyme by genetic modification, and the use of outer membrane permeabilizing agents such as EDTA or polycations or permeabilizing treatments such as high hydrostatic pressure treatment.

Comparison of sublethal injury induced in Salmonella enterica serovar Typhimurium by heat and by different nonthermal treatments

We have studied sublethal injury in Salmonella enterica serovar Typhimurium caused by mild heat and by different emerging nonthermal food preservation treatments, i.e., high-pressure homogenization, high hydrostatic pressure, pulsed white light, and pulsed electric field. Sublethal injury was determined by plating on different selective media, i.e., tryptic soy agar (TSA) plus 3% NaCl, TSA adjusted to pH 5.5, and violet red bile glucose agar. For each inactivation technique, at least five treatments using different doses were applied in order to cover an inactivation range of 0 to 5 log units. For all of the treatments performed with a technique, the logarithm of the viability reductions measured on each of the selective plating media was plotted against the logarithm of the viability reduction on TSA as a nonselective medium, and these points were fined by a straight line. Sublethal injury between different techniques was then compared by the slope and the y intercept of these regression lines. The highest levels of sublethal injury were observed for the heat and high hydrostatic pressure treatments. Sublethal injury after those treatments was observed on all selective plating media. For the heat treatment, but not for the high-pressure treatment, sublethal injury occurred at low doses, which were not yet lethal. The other nonthermal techniques resulted in sublethal injury on only some of the selective plating media, and the levels of injury were much lower. The different manifestations of sublethal injury were attributed to different inactivation mechanisms by each of the techniques, and a mechanistic model is proposed to explain these differences.

High-Pressure Homogenization as a Non-Thermal Technique for the Inactivation of Microorganisms

In the pharmaceutical, cosmetic, chemical, and food industries high-pressure homogenization is used for the preparation or stabilization of emulsions and suspensions, or for creating physical changes, such as viscosity changes, in products. Another well-known application is cell disruption of yeasts or bacteria in order to release intracellular products such as recombinant proteins. The development over the last few years of homogenizing equipment that operates at increasingly higher pressures has also stimulated research into the possible application of high-pressure homogenization as a unit process for microbial load reduction of liquid products. Several studies have indicated that gram-negative bacteria are more sensitive to high-pressure homogenization than gram-positive bacteria supporting the widely held belief that high-pressure homogenization kills vegetative bacteria mainly through mechanical disruption. However, controversy exists in the literature regarding the exact cause(s) of cell disruption by high-pressure homogenization. The causes that have been proposed include spatial pressure and velocity gradients, turbulence, cavitation, impact with solid surfaces, and extensional stress. The purpose of this review is to give an overview of the existing literature about microbial inactivation by high-pressure homogenization. Particular attention will be devoted to the different proposed microbial inactivation mechanisms. Further, the different parameters that influence the microbial inactivation by high-pressure homogenization will be scrutinized.

Bacterial interactions in biofilms

It is generally acknowledged that biofilms are the dominant lifestyle of bacteria, both in the natural environment as on manmade settings such as industrial and medical devices. This attached form of cell growth consists of slime matrix embedded bacteria of either a single, but mostly of multiple microbial species that form an interdependent structured community, capable of coordinated and collective behavior. Although research on multispecies biofilms is still in its infancy, this review will focus on these complex communities where cooperation and antagonism are keys to increase the fitness of the different species and where intercellular interactions and communication are means to achieve this goal.

High pressure increases bactericidal activity and spectrum of lactoferrin, lactoferricin and nisin.

We have studied the inactivation of a panel of eight test bacteria (two Escherichia coli strains, Salmonella enteritidis, Salmonella typhimurium, Shigella sonnei, Shigella flexneri, Pseudomonas fluorescens and Staphylococcus aureus) by high pressure in the presence of bovine lactoferrin (500 microg/ml), pepsin hydrolysate of lactoferrin (500 microg/ml), lactoferricin (20 microg/ml) and nisin (100 IU/ml). None of these compounds, at the indicated dosage, were bactericidal when applied at atmospheric pressure, except nisin, which caused a low level of inactivation of the bacteria. Under high pressure, lactoferrin, lactoferrin hydrolysate and lactoferricin displayed bactericidal activity against some of the test bacteria, however, the former had a narrower bactericidal spectrum than the two latter compounds. The bactericidal efficiency and spectrum of nisin were also enhanced under high pressure. The sensitisation of the test bacteria to these antimicrobials under pressure was transient, since no bactericidal activity was observed when bacteria were pressure treated before exposure to the compounds. We propose a mechanism of pressure-promoted uptake of these antimicrobial proteins and peptides in gram-negative bacteria to explain this sensitisation.

Using survival analysis to investigate the effect of UV-C and heat treatment on storage rot of strawberry and sweet cherry.

Ultraviolet light and heat treatment are proposed as alternative techniques for the use of chemicals to reduce the development of the spoilage fungi Botrytis cinerea and Monilinia fructigena on strawberry and sweet cherry, respectively, during storage. In order to investigate the effect of both physical techniques on microbial inactivation and on fruit quality, inoculated berries were subjected to different temperatures (40-48 degrees C) and UV-C doses (0.05-1.50 J/cm2). For each condition, 20 berries were used. After the treatment, fungal growth, visual damage (holes, stains) and fruit firmness were evaluated during a period of 10 days. The experimental data were analysed statistically using survival analysis techniques. Fungal growth on strawberries was significantly retarded using UV-C doses of 0.05 J/cm2 and higher. The same treatment had no significant effect when applied to cherries. The highest doses (1.00 and 1.50 J/cm2) had a negative effect on the calyx of the strawberry, causing browning and drying of the leaves. No beneficial effect of a low temperature treatment (40-48 degrees C) on the shelf life of strawberries was observed, but fungal development on cherries was retarded at temperatures of 45 and 48 degrees C. These temperatures caused severe damage on strawberries (soft stains, holes, decreased firmness), but had no influence on the quality of sweet cherries.

From Field Barley to Malt: Detection and Specification of Microbial Activity for Quality Aspects

Barley grain carries a numerous, variable, and complex microbial population that mainly consists of bacteria, yeasts, and filamentous fungi and that can partly be detected and quantified using plating methods and microscopic and molecular techniques. The extent and the activity of this microflora are determined by the altering state of the grain and the environmental conditions in the malt production chain. Three ecological systems can be distinguished: the growing cereal in the field, the dry barley grain under storage, and the germinating barley kernel during actual malting. Microorganisms interact with the malting process both by their presence and by their metabolic activity. In this respect, interference with the oxygen uptake by the barley grain and secretion of enzymes, hormones, toxins, and acids that may affect the plant physiological processes have been studied. As a result of the interaction, microorganisms can cause important losses and influence malt quality as measured by brewhouse performance and beer quality. Of particular concern is the occurrence of mycotoxins that may affect the safety of malt. The development of the microflora during malt production can to a certain extent be controlled by the selection of appropriate process conditions. Physical and chemical treatments to inactivate the microbial population on the barley grain are suggested. Recent developments, however, aim to control the microbial activity during malt production by promoting the growth of desirable microbial cultures, selected either as biocontrol agents inhibiting mycotoxin-producing molds or as starter cultures actively contributing to malt modification. Such techniques may offer natural opportunities to improve the quality and safety of malt.

Combinations of pulsed white light and UV-C or mild heat treatment to inactivate conidia of Botrytis cinerea and Monilia fructigena.

The use of pulses of intense white light to inactivate conidia of the fungi Botrytis cinerea and Monilia fructigena, responsible for important economical losses during postharvest storage and transport of strawberries and sweet cherries, was investigated in this study. In the first stage, a light treatment applying pulses of 30 micros at a frequency of 15 Hz was investigated, resulting in a treatment duration varying from 1 to 250 s. The conidia of both fungi showed similar behaviour to pulsed light, with a maximal inactivation of 3 and 4 log units for B. cinerea and M. fructigena, respectively. The inactivation of the conidia increased with increasing treatment intensity, but no complete inactivation was achieved. The sigmoidal inactivation pattern obtained by the pulsed light treatment was described using a modification of the model of Geeraerd et al. [Int. J. Food Microbiol. 59 (2000) 185]. Hereto, the shoulder length was incorporated explicitly and relative values for the microbial populations were used. In the second stage, combinations of light pulses and ultraviolet-C or heat were applied. The UV light used in the experiments is the short-wave band or UV-C, running from 180 to 280 nm with a peak at 254 nm (UV-B runs from 280 to 320 nm and UV-A from 320 to 380 nm). The UV-C doses were 0.025, 0.05 and 0.10 J/cm(2), and the temperatures for the thermal treatment ranged from 35 to 45 degrees C during 3-15 min. When combining UV-C and light pulses, there was an increase in inactivation for both B. cinerea and M. fructigena, and synergism was observed. There was no effect of the order of the treatments. For the heat-light pulses combination, there was a difference between both fungi. The order of the treatments was highly significant for B. cinerea, but not for M. fructigena. Combining heat and light treatments improved the inactivation, and synergism between both methods was again observed. Complete inactivation of M. fructigena conidia was obtained after, e.g., a 40-s pulsed light treatment and 15 min at 41 degrees C, or after an 80-s light treatment and 10 min at 41 degrees C.