Contamination routes and control of Listeria monocytogenes in Food Production

Listeria monocytogenes is the causative agent of the severe foodborne infection listeriosis. The number of listeriosis cases in recent years has increased in many European countries, including Finland. Contamination of the pathogen needs to be minimized and growth to high numbers in foods prevented in order to reduce the incidence of human cases. The aim of this study was to evaluate contamination routes of L. monocytogenes in the food chain and to investigate methods for control of the pathogen in food processing. L. monocytogenes was commonly found in wild birds, the pig production chain and in pork production plants. It was found most frequently in birds feeding at landfill site, organic farms, tonsil samples, and sites associated with brining. L. monococytogenes in birds, farms, food processing plant or foods did not form distinct genetic groups, but populations overlapped. The majority of genotypes recovered from birds were also detected in foods, food processing environments and other animal species and birds may disseminate L. monocytogenes into food chain. Similar genotypes were found in different pigs on the same farm, as well as in pigs on farms and later in the slaughterhouse. L. monocytogenes contamination spreads at farm level and may be a contamination source into slaughterhouses and further into meat. Incoming raw pork in the processing plant was frequently contaminated with L. monocytogenes and genotypes in raw meat were also found in processing environment and in RTE products. Thus, raw material seems to be a considerable source of contamination into processing facilities. In the pork processing plant, the prevalence of L. monocytogenes increased in the brining area, showing that the brining was an important contamination site. Recovery of the inoculated L. monocytogenes strains showed that there were strain-specific differences in the ability to survive in lettuce and dry sausage. The ability of some L. monocytogenes strains to survive well in food production raises a challenge for industry, because these strains can be especially difficult to remove from the products and raises a need to use an appropriate hurdle concept to control most resistant strains. Control of L. monocytogenes can be implemented throughout the food chain. Farm-specific factors affected the prevalence of L. monocytogenes and good farm-level practices can therefore be utilized to reduce the prevalence of this pathogen on the farm and possibly further in the food chain. Well separated areas in a pork production plant had low prevalences of L. monocytogenes, thus showing that compartmentalization controls the pathogen in the processing line. The food processing plant, especially the brining area, should be subjected to disassembling, extensive cleaning and disinfection to eliminate persistent contamination by L. monocytogenes, and replacing brining with dry-salting should be considered. All of the evaluated washing solutions decreased the populations of L. monocytogenes on precut lettuce, but did not eliminate the pathogen. Thus, the safety of fresh-cut produce cannot rely on washing with disinfectants, and high-quality raw material and good manufacturing practices remain important. L. monocytogenes was detected in higher levels in sausages without the protective culture than in sausages with this protective strain, although numbers of L. monocytogenes by the end of the ripening decreased to the level of < 100 MPN/g in all sausages. Protective starter cultures provide an appealing hurdle in dry sausage processing and assist in the control of L. monocytogenes.

Everyday beliefs about food and health

The series of studies addresses several everyday beliefs about food and health from the perspective of everyday thinking and paranormal beliefs. They are "you are what you eat" beliefs, attitudes towards genetically modified and organic foods, and belief in alternative medicine. The survey studies included from 239 to 3261 Finnish participants. It was found that food consumption can have far-stretching consequences for the impressions of the eater in a "you are what you eat" manner. The results also demonstrated that belief in alternative medicine was related to belief in the paranormal, as were to a lesser degree attitudes towards genetically modified and organic foods. The study also addressed paranormal beliefs and belief in alternative medicine from the perspective of category observance. Paranormal believers as well as believers in alternative medicine were much more liberal than skeptics in violating categorical boundaries and attributed, for example, intentionality (mental) to body growth (biological) and life (biological) to energy (physical). In addition, the study addressed the relation of these attitudes and beliefs with preferred thinking style. The results demonstrated that belief in alternative medicine was especially appealing to intuitive thinkers, while rational thinking was unrelated to it. The same pattern was demonstrated for negative attitudes towards genetically modified food and positive attitudes towards organic food. In addition, it was demonstrated, however, that such unscientific notions may exist not instead of but parallel with "better knowledge". In sum, the present thesis contributes to the understanding of superstitious elements in various everyday attitudes and beliefs, and investigates their relationship with general inclinations towards belief in the paranormal. It appears that some very common everyday beliefs and attitudes about food and health contain elements of a superstitious nature. Involving conceptual enmeshment they go beyond mere associations, and can coincide with scientifically valid views on the same topic.

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.

Understanding the organic chain : The framework of the interaction between the actors in organic chains in relation to ecological modernisation of food production

The sustainability of food production has increasingly attracted the attention of consumers, farmers, food and retailing companies, and politicians. One manifestation of such attention is the growing interest in organic foods. Organic agriculture has the potential to enhance the ecological modernisation of food production by implementing the organic method as a preventative innovation that simultaneously produces environmental and economic benefits. However, in addition to the challenges to organic farming, the small market share of organic products in many countries today and Finland in particular risks undermining the achievement of such benefits. The problems identified as hindrances to the increased consumption of organic food are the poor availability, limited variety and high prices of organic products, the complicated buying decisions and the difficulties in delivering the intangible value of organic foods. Small volumes and sporadic markets, high costs, lack of market information, as well as poor supply reliability are obstacles to increasing the volume of organic production and processing. These problems shift the focus from a single actor to the entire supply chain and require solutions that involve more interaction among the actors within the organic chain. As an entity, the organic food chain has received very little scholarly attention. Researchers have mainly approached the organic chain from the perspective of a single actor, or they have described its structure rather than the interaction between the actors. Consequently, interaction among the primary actors in organic chains, i.e. farmers, manufacturers, retailers and consumers, has largely gone unexamined. The purpose of this study is to shed light on the interaction of the primary actors within a whole organic chain in relation to the ecological modernisation of food production. This information is organised into a conceptual framework to help illuminate this complex field. This thesis integrates the theories and concepts of three approaches: food system studies, supply chain management and ecological modernisation. Through a case study, a conceptual system framework will be developed and applied to a real life-situation. The thesis is supported by research published in four articles. All examine the same organic chains through case studies, but each approaches the problem from a different, complementary perspective. The findings indicated that regardless of the coherent values emphasising responsibility, the organic chains were loosely integrated to operate as a system. The focus was on product flow, leaving other aspects of value creation largely aside. Communication with consumers was rare, and none of the actors had taken a leading role in enhancing the market for organic products. Such a situation presents unsuitable conditions for ecological modernisation of food production through organic food and calls for contributions from stakeholders other than those directly involved in the product chain. The findings inspired a revision of the original conceptual framework. The revised framework, the three-layer framework , distinguishes the different layers of interaction. By gradually enlarging the chain orientation the different but interrelated layers become visible. A framework is thus provided for further research and for understanding practical implications of the performance of organic food chains. The revised framework provides both an ideal model for organic chains in relation to ecological modernisation and demonstrates a situation consistent with the empirical evidence.

Folates in rye: Determination and enhancement by food processing

The average daily intake of folate, one of the B vitamins, falls below recommendations among the Finnish population. Bread and cereals are the main sources of folate, rye being the most significant single source. Processing is a prerequisite for the consumption of whole grain rye; however, little is known about the effect of processing on folates. Moreover, data on the bioavailability of endogenous cereal folates are scarce. The aim of this study was to examine the variation in as well as the effect of fermentation, germination, and thermal processes on folate contents in rye. Bioavailability of endogenous rye folates was investigated in a four-week human intervention study. One of the objectives throughout the work was to optimise and evaluate analytical methods for determining folate contents in cereals. Affinity chromatographic purification followed by high-performance liquid chromatography (HPLC) was a suitable method for analysing cereal products for folate vitamers, and microbiological assay with Lactobacillus rhamnosus reliably quantified the total folate. However, HPLC gave approximately 30% lower results than the microbiological assay. The folate content of rye was high and could be further increased by targeted processing. The vitamer distribution of whole grain rye was characterised by a large proportion of formylated vitamers followed by 5-methyltetrahydrofolate. In sourdough fermentation of rye, the studied yeasts synthesized and lactic acid bacteria mainly depleted folate. Two endogenous bacteria isolated from rye flour were found to produce folate during fermentation. Inclusion of baker s yeast in sourdough fermentation raised the folate level so that the bread could contain more folate than the flour it was made of. Germination markedly increased the folate content of rye, with particularly high folate concentrations in hypocotylar roots. Thermal treatments caused significant folate losses but the preceding germination compensated well for the losses. In the bioavailability study, moderate amounts of endogenous folates in the form of different rye products and orange juice incorporated in the diet improved the folate status among healthy adults. Endogenous folates from rye and orange juice showed similar bioavailability to folic acid from fortified white bread. In brief, it was shown that the folate content of rye can be enhanced manifold by optimising and combining food processing techniques. This offers some practical means to increase the daily intake of folate in a bioavailable form.

Bacillus cereus spores and cereulide in food-borne illness

B. cereus is a gram-positive bacterium that possesses two different forms of life:the large, rod-shaped cells (ca. 0.002 mm by 0.004 mm) that are able to propagate and the small (0.001 mm), oval shaped spores. The spores can survive in almost any environment for up to centuries without nourishment or water. They are insensitive towards most agents that normally kill bacteria: heating up to several hours at 90 ºC, radiation, disinfectants and extreme alkaline (≥ pH 13) and acid (≤ pH 1) environment. The spores are highly hydrophobic and therefore make them tend to stick to all kinds of surfaces, steel, plastics and live cells. In favorable conditions the spores of B. cereus may germinate into vegetative cells capable of producing food poisoning toxins. The toxins can be heat-labile protein formed after ingestion of the contaminated food, inside the gastrointestinal tract (diarrhoeal toxins), or heat stable peptides formed in the food (emesis causing toxin, cereulide). Cereulide cannot be inactivated in foods by cooking or any other procedure applicable on food. Cereulide in consumed food causes serious illness in human, even fatalities. In this thesis, B. cereus strains originating from different kinds of foods and environments and 8 different countries were inspected for their capability of forming cereulide. Of the 1041 isolates from soil, animal feed, water, air, used bedding, grass, dung and equipment only 1.2 % were capable of producing cereulide, whereas of the 144 isolates originating from foods 24 % were cereulide producers. Cereulide was detected by two methods: by its toxicity towards mammalian cells (sperm assay) and by its peculiar chemical structure using liquid-chromatograph-mass spectrometry equipment. B. cereus is known as one of the most frequent bacteria occurring in food. Most foods contain more than one kind of B. cereus. When randomly selected 100 isolates of B. cereus from commercial infant foods (dry formulas) were tested, 11% of these produced cereulide. Considering a frequent content of 103 to 104 cfu (colony forming units) of B. cereus per gram of infant food formula (dry), it appears likely that most servings (200 ml, 30 g of the powder reconstituted with water) may contain cereulide producers. When a reconstituted infant formula was inoculated with >105 cfu of cereulide producing B. cereus per ml and left at room temperature, cereulide accumulated to food poisoning levels (> 0.1 mg of cereulide per serving) within 24 hours. Paradoxically, the amount of cereulide (per g of food) increased 10 to 50 fold when the food was diluted 4 - 15 fold with water. The amount of the produced cereulide strongly depended on the composition of the formula: most toxin was formed in formulas with cereals mixed with milk, and least toxin in formulas based on milk only. In spite of the aggressive cleaning practices executed by the modern dairy industry, certain genotypes of B. cereus appear to colonise the silos tanks. In this thesis four strategies to explain their survival of their spores in dairy silos were identified. First, high survival (log 15 min kill ≤ 1.5) in the hot alkaline (pH >13) wash liquid, used at the dairies for cleaning-in-place. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterized by slow germination in rich medium and well preserved viability when exposed to heating at 90 ºC. Fourth, spores capable of germinating at 8 ºC and possessing the psychrotolerance gene, cspA. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus. In this thesis it was shown that cereulide producing B. cereus was capable of inhibiting the growth of cereulide non-producing B. cereus occurring in the same food. This phenomenon, called antagonism, has long been known to exist between B. cereus and other microbial species, e.g. various species of Bacillus, gram-negative bacteria and plant pathogenic fungi. In this thesis intra-species antagonism of B. cereus was shown for the first time. This brother-killing did not depend on the cereulide molecule, also some of the cereulide non-producers were potent antagonists. Interestingly, the antagonistic clades were most frequently found in isolates from food implicated with human illness. The antagonistic property was therefore proposed in this thesis as a novel virulence factor that increases the human morbidity of the species B. cereus, in particular of the cereulide producers.

Assessment and control of Bacillus cereus emetic toxin in food

Despite of improving levels of hygiene, the incidence of registered food borne disease has been at the same level for many years: there were 40 to 90 epidemics in which 1000-9000 persons contracted food poisoning through food or drinking water in Finland. Until the year 2004 salmonella and campylobacter were the most common bacterial causes of food borne diseases, but in years 2005-2006 Bacillus cereus was the most common. Similar developement has been published i.e. in Germany already in the 1990´s. One reason for this can be Bacillus cereus and its emetic toxin, cereulide. Bacillus cereus is a common environmental bacterium that contaminates raw materials of food. Otherwise than salmonella and campylobacter, Bacillus cereus is a heat resistant bacterium, capable of surviving most cooking procedures due to the production of highly thermo resistant spores. The food involved has usually been heat treated and surviving spores are the source of the food poisoning. The heat treatment induces germination of the spore and the vegetative cells then produce toxins. This doctoral thesis research focuses on developing methods for assessing and eliminating risks to food safety by cereulide producing Bacillus cereus. The biochemistry and physiology of cereulide production was investigated and the results were targeted to offer tools for minimizing toxin risk in food during the production. I developed methods for the extraction and quantitative analysis of cereulide directly from food. A prerequisite for that is knowledge of the chemical and physical properties of the toxin. Because cereulide is practically insoluble in water, I used organic solvents; methanol, ethanol and pentane for the extraction. For extraction of bakery products I used high temperature (100C) and pressure (103.4 bars). Alternaties for effective extraction is to flood the plain food with ethanol, followed by stationary equilibration at room temperature. I used this protocol for extracting cereulide from potato puree and penne. Using this extraction method it is also possible also extract cereulide from liquid food, like milk. These extraction methods are important improvement steps for studying of Bacillus cereus emetic food poisonings. Prior my work, cereulide extraction was done using water. As the result, the yield was poor and variable. To investigate suspected food poisonings, it is important to show actual toxicity of the incriminated food. Many toxins, but not cereulide, inactivate during food processing like heating. The next step is to identify toxin by chemical methods. I developed with my colleague Maria Andesson a rapid assay for the detection of cereulide toxicity, within 5 to 15 minutes. By applying this test it is possible to rapidly detect which food was causing the food poisoning. The chemical identification of cereulide was achieved using mass spectrometry. I used cereulide specific molecular ions, m/z (+/-0.3) 1153.8 (M+H+), 1171.0 (M+NH4+), 1176.0 (M+Na+) and 1191.7 (M+K+) for reliable identification. I investigated foods to find out their amenability to accumulate cereulide. Cereulide was formed high amounts (0.3 to 5.5 microg/g wet wt) when of cereulide producing B. cereus strains were present in beans, rice, rice-pastry and meat-pastry, if stored at non refrigerated temperatures (21-23C). Rice and meat pastries are frequently consumed under conditions where no cooled storage is available e.g. picnics and outdoor events. Bacillus cereus is a ubiquitous spore former and is therefore difficult to eliminate from foods. It is therefore important to know which conditions will affect the formation of cereulide in foods. My research showed that the cereulide content was strongly (10 to 1000 fold differences in toxin content) affected by the growth environment of the bacterium. Storage of foods under nitrogen atmosphere (> 99.5 %) prevented the production of cereulide. But when also carbon dioxide was present, minimizing the oxygen contant (< 1%) did not protect the food from formation of cereulide in preliminary experiments. Also food supplements affected cereulide production at least in the laboratory. Adding free amino acids, leucine and valine, stimulated cereulide production 10 to 20 fold. In peptide bonded form these amino acids are natural constituents in all proteins. Interestingly, adding peptide bonded leucine and valine had no significant effect on cereulide production. Free amino acids leucine and valine are approved food supplements and widely used as flawour modifiers in food technology. My research showed that these food supplements may increase food poisoning risk even though they are not toxic themselves.

Oxidative stability of phytosterols in food models and foods

An important safety aspect to be considered when foods are enriched with phytosterols and phytostanols is the oxidative stability of these lipid compounds, i.e. their resistance to oxidation and thus to the formation of oxidation products. This study concentrated on producing scientific data to support this safety evaluation process. In the absence of an official method for analyzing of phytosterol/stanol oxidation products, we first developed a new gas chromatographic - mass spectrometric (GC-MS) method. We then investigated factors affecting these compounds' oxidative stability in lipid-based food models in order to identify critical conditions under which significant oxidation reactions may occur. Finally, the oxidative stability of phytosterols and stanols in enriched foods during processing and storage was evaluated. Enriched foods covered a range of commercially available phytosterol/stanol ingredients, different heat treatments during food processing, and different multiphase food structures. The GC-MS method was a powerful tool for measuring the oxidative stability. Data obtained in food model studies revealed that the critical factors for the formation and distribution of the main secondary oxidation products were sterol structure, reaction temperature, reaction time, and lipid matrix composition. Under all conditions studied, phytostanols as saturated compounds were more stable than unsaturated phytosterols. In addition, esterification made phytosterols more reactive than free sterols at low temperatures, while at high temperatures the situation was the reverse. Generally, oxidation reactions were more significant at temperatures above 100°C. At lower temperatures, the significance of these reactions increased with increasing reaction time. The effect of lipid matrix composition was dependent on temperature; at temperatures above 140°C, phytosterols were more stable in an unsaturated lipid matrix, whereas below 140°C they were more stable in a saturated lipid matrix. At 140°C, phytosterols oxidized at the same rate in both matrices. Regardless of temperature, phytostanols oxidized more in an unsaturated lipid matrix. Generally, the distribution of oxidation products seemed to be associated with the phase of overall oxidation. 7-ketophytosterols accumulated when oxidation had not yet reached the dynamic state. Once this state was attained, the major products were 5,6-epoxyphytosterols and 7-hydroxyphytosterols. The changes observed in phytostanol oxidation products were not as informative since all stanol oxides quantified represented hydroxyl compounds. The formation of these secondary oxidation products did not account for all of the phytosterol/stanol losses observed during the heating experiments, indicating the presence of dimeric, oligomeric or other oxidation products, especially when free phytosterols and stanols were heated at high temperatures. Commercially available phytosterol/stanol ingredients were stable during such food processes as spray-drying and ultra high temperature (UHT)-type heating and subsequent long-term storage. Pan-frying, however, induced phytosterol oxidation and was classified as a rather deteriorative process. Overall, the findings indicated that although phytosterols and stanols are stable in normal food processing conditions, attention should be paid to their use in frying. Complex interactions between other food constituents also suggested that when new phytosterol-enriched foods are developed their oxidative stability must first be established. The results presented here will assist in choosing safe conditions for phytosterol/stanol enrichment.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.