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.

Mannans as film formers and emulsion stabilizers

Mannans are abundant plant polysaccharides found in the endosperm of certain leguminous seeds (guar gum galactomannan, GG; locust bean gum galactomannan, LBG), in the tuber of the konjac plant (konjac glucomannan, KGM), and in softwoods (galactoglucomannan, GGM). This study focused on the effects of the chemical structure of mannans on their film-forming and emulsion-stabilizing properties. Special focus was on spruce GGM, which is an interesting new product from forest biorefineries. A plasticizer was needed for the formation of films from mannans other than KGM and the optimal proportion was 40% (w/w of polymers) glycerol or sorbitol. Galactomannans with lower galactose content (LBG, modified GG) produced films with higher elongation at break and tensile strength. The mechanical properties of GG-based films were improved by decreasing the degree of polymerization of the polysaccharide with moderate mannanase treatments. The improvement of mechanical properties of GGM-based films was sought by blending GGM with each of poly(vinyl alcohol) (PVOH), corn arabinoxylan (cAX), and KGM. Adding other polymers increased the elongation at break of GGM blend films. The tensile strength of films increased with increasing amounts of PVOH and KGM, but the effect of cAX was the opposite. Dynamic mechanical analysis showed two separate loss modulus peaks for blends of GGM and PVOH, but a single peak for all other films. Optical and scanning electron microscopy confirmed good miscibility of GGM with cAX and KGM. In contrast, films blended from GGM and PVOH showed phase separation. GGM and KGM were mixed with cellulose nanowhiskers (CNW) to form composite films. Addition of CNW to KGM-based films induced the formation of fiberlike structures with lengths of several millimeters. In GGM-based films, rodlike structures with lengths of tens of micrometers were formed. Interestingly, the notable differences in the film structure did not appear to be related to the mechanical and thermal properties of the films. Permeability properties of GGM-based films were compared to those of films from commercial mannans KGM, GG, and LBG. GGM-based films had the lowest water vapor permeability when compared to films from other mannans. The oxygen permeability of GGM films was of the same magnitude as that of commercial polyethylene / ethylene vinyl alcohol / polyethylene laminate film. The aroma permeability of GGM films was low. All films were transparent in the visible region, but GGM films blocked the light transmission in the ultraviolet region of the spectra. The stabilizing effect of GGM on a model beverage emulsion system was studied and compared to that of GG, LBG, KGM, and cAX. In addition, GG was enzymatically modified in order to examine the effect of the degree of polymerization and the degree of substitution of galactomannans on emulsion stability. Use of GGM increased the turbidity of emulsions both immediately after preparation and after storage of up to 14 days at room temperature. GGM emulsions had higher turbidity than the emulsions containing other mannans. Increasing the storage temperature to +45 ºC led to rapid emulsion breakdown, but a decrease in storage temperature increased emulsion stability after 14 days. A low degree of polymerization and a high degree of substitution of the modified galactomannans were associated with a decrease in emulsion turbidity.

Glycoalkaloid Content and Starch Structure in Solanum Species and Interspecific Somatic Potato Hybrids

When genome sections of wild Solanum species are bred into the cultivated potato (S. tuberosum L.) to obtain improved potato cultivars, the new cultivars must be evaluated for their beneficial and undesirable traits. Glycoalkaloids present in Solanum species are known for their toxic as well as for beneficial effects on mammals. On the other hand, glycoalkaloids in potato leaves provide natural protection against pests. Due to breeding, glycoalkaloid profile of the plant is affected. In addition, the starch properties in potato tubers can be affected as a result of breeding, because the crystalline properties are determined by the botanical source of the starch. Starch content and composition affect the texture of cooked and processed potatoes. In order to determine glycoalkaloid contents in Solanum species, simultaneous separation of glycoalkaloids and aglycones using reversed-phase high-performance liquid chromatography (HPLC) was developed. Clean-up of foliage samples was improved using a silica-based strong cation exchanger instead of octadecyl phases in solid-phase extraction. Glycoalkaloids alpha-solanine and alpha-chaconine were detected in potato tubers of cvs. Satu and Sini. The total glycoalkaloid concentration of non-peeled and immature tubers was at an acceptable level (under 20 mg/100 g of FW) in the cv. Satu, whereas concentration in cv. Sini was 23 mg/100 g FW. Solanum species (S. tuberosum, S. brevidens, S. acaule, and S. commersonii) and interspecific somatic hybrids (brd + tbr, acl + tbr, cmm + tbr) were analyzed for their glycoalkaloid contents using liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). The concentrations in the tubers of the brd + tbr and acl + tbr hybrids remained under 20 mg/100 g FW. Glycoalkaloid concentration in the foliage of the Solanum species was between 110 mg and 890 mg/100 g FW. However, the concentration in the foliage of S. acaule was as low as 26 mg/100 g FW. The total concentrations of brd + tbr, acl + tbr, and cmm + tbr hybrid foliages were 88 mg, 180 mg, and 685 mg/100 g FW, respectively. Glycoalkaloids of both parental plants as well as new combinations of aglycones and saccharides were detected in somatic hybrids. The hybrids contained mainly spirosolanes, and glycoalkaloid structures having no 5,6-double bond in the aglycone. Based on these results, the glycoalkaloid profiles of the hybrids may represent a safer and more beneficial spectrum of glycoalkaloids than that found in currently cultivated varieties. Starch nanostructure of three different cultivars (Satu, Saturna, and Lady Rosetta), a wild species S. acaule, and interspecific somatic hybrids were examined by wide-angle and small-angle X-ray scattering (WAXS, SAXS). For the first time, the measurements were conducted on fresh potato tuber samples. Crystallinity of starch, average crystallite size, and lamellar distance were determined from the X-ray patterns. No differences in the starch nanostructure between the three different cultivars were detected. However, tuber immaturity was detected by X-ray scattering methods when large numbers of immature and mature samples were measured and the results were compared. The present study shows that no significant changes occurred in the nanostructures of starches resulting from hybridizations of potato cultivars.

Tuotantotekniikan ja säilytysajan vaikutus kasvihuonekurkkujen ja -tomaattien aistittavaan laatuun.

Finland has moved from growing vegetables by natural light to year-round greenhouse production using artificial lighting. Determination of sensory effects on greenhouse-grown vegetables is important as sensory evaluation provides information which chemical methods can not. It can tell us about the quality of samples which affects the consumers' behaviour. There are different opinions on how the quality of vegetables should be determined. The consumers are dissatisfied with the quality of vegetables and fruits, although the variety of products is larger than ever. The aim of this study was to find out how artificial lighting contributes to the sensory quality of greenhouse tomatoes and cucumbers compared to traditional natural lighting, and how storage affects the sensory attributes of the samples. In this study there were two sets of tomatoes and two sets of cucumbers, representing two different harvest seasons. Sensory evaluation involved two steps. The first step was to sort the samples and the second step was to generate a profile using descriptive analysis. Sorting was found to give some approximate information on differences between tomato and cucumber samples. MDS-maps dimensions were presented by age and lighting technique. The reliability of sorting results was quite good. The quality of the natural products was inconsistent. Production technology had more of an effect on cucumber samples than tomato samples. Natural light cucumbers were, for example sweeter and softer than artificial light cucumbers. Age had an especially large effect on cucumber appearance characteristics. There were less differences between tomato samples than cucumber samples. Production technology had less of an effect on tomato samples than age, e.g. hardness decreased during storage. In this study, it was found that artificial lighting has little effect on the sensory quality of Finnish greenhouse tomatoes compared with tomatoes grown under natural light.

Information, risk and trust in the food chain : Ex-ante valuation of consumer willingness to pay for beef quality information using the contingent valuation method

The safety of food has become an increasingly interesting issue to consumers and the media. It has also become a source of concern, as the amount of information on the risks related to food safety continues to expand. Today, risk and safety are permanent elements within the concept of food quality. Safety, in particular, is the attribute that consumers find very difficult to assess. The literature in this study consists of three main themes: traceability; consumer behaviour related to both quality and safety issues and perception of risk; and valuation methods. The empirical scope of the study was restricted to beef, because the beef labelling system enables reliable tracing of the origin of beef, as well as attributes related to safety, environmental friendliness and animal welfare. The purpose of this study was to examine what kind of information flows are required to ensure quality and safety in the food chain for beef, and who should produce that information. Studying the willingness to pay of consumers makes it possible to determine whether the consumers consider the quantity of information available on the safety and quality of beef sufficient. One of the main findings of this study was that the majority of Finnish consumers (73%) regard increased quality information as beneficial. These benefits were assessed using the contingent valuation method. The results showed that those who were willing to pay for increased information on the quality and safety of beef would accept an average price increase of 24% per kilogram. The results showed that certain risk factors impact consumer willingness to pay. If the respondents considered genetic modification of food or foodborne zoonotic diseases as harmful or extremely harmful risk factors in food, they were more likely to be willing to pay for quality information. The results produced by the models thus confirmed the premise that certain food-related risks affect willingness to pay for beef quality information. The results also showed that safety-related quality cues are significant to the consumers. In the first place, the consumers would like to receive information on the control of zoonotic diseases that are contagious to humans. Similarly, other process-control related information ranked high among the top responses. Information on any potential genetic modification was also considered important, even though genetic modification was not regarded as a high risk factor.