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.

Cow’s milk related gastrointestinal symptoms in adults

The purpose of this study was to evaluate subjective food-related gastrointestinal symptoms and their relation to cow’s milk by determining the genotype of adult-type hypolactasia, measuring antibodies against milk protein, and screening the most common cause for secondary hypolactasia, namely coeliac disease. The whole study group comprised 1900 adults who gave a blood sample for the study when they attended a health care centre laboratory for various reasons. Of these 1885 (99%) completed a questionnaire on food-related gastrointestinal symptoms. Study No. I evaluated the prevalence of adult-type hypolactasia and its correlation to self-reported milk induced gastrointestinal symptoms. The testing for hypolactasia was done by determination of the C/T-13910 genotypes of the study subjects. The results show that patients with the C/C-13910 genotype associated with adult type hypolactasia consume less milk than those with C/T-13910 and T/T-13910 genotypes. Study No. II evaluated the prevalence and clinical characteristics of undiagnosed coeliac disease in the whole study population with transglutaminase and endomysium antibodies and their correlation with gastrointestinal symptoms. The prevalence of coeliac disease was 2 %, which is surprisingly high. Serum transglutaminase and endomysium antibodies are valuable tools for recognising an undiagnosed coeliac disease in outpatient clinics. In the study No. III the evaluation of milk protein IgE related hypersensitivity was carried out by stratifying all 756 study subjects with milk related problems and randomly choosing 100 age and sex matched controls with no such symptoms from the rest of the original study group. In the study No. IV 400 serum samples were randomly selected for analyzing milk protein related IgA and IgG antibodies and their correlation to milk related GI-symptoms. The measurement of milk protein IgA, IgE or IgG (studies No. III and IV) did not correlate clearly to milk induced symptoms and gave no clinically significant information; hence their measurement is not encouraged in outpatient clinics. In conclusion, adult type hypolactasia is often considered the reason for gastrointestinal symptoms in adults and determination of the C/T-13910 genotypes is a practical way of diagnosing adult type hypolactasia in an outpatient setting. Undiagnosed coeliac disease, should be actively screened and diagnosed in order to apply a gluten free diet and avoid the GI-symptoms and nutritional deficiencies. Cow’s milk hypersensitivity in the adult population is difficult to diagnose since the mechanism in which it is mediated is still unclear. Measuring of cow’s milk protein specific antibodies IgE, IgA or IgG do not correlate with subjective milk-related GI-symptoms.

Cow's milk allergy and the development of tolerance

Aims: To gain insight on the immunological processes behind cow’s milk allergy (CMA) and the development of oral tolerance. To furthermore investigate the associations of HLA II and filaggrin genotypes with humoral responses to early oral antigens. Methods: The study population was from a cohort of 6209 healthy, full-term infants who in a double-blind randomized trial received supplementary feeding at maternity hospitals (mean duration 4 days): cow’s milk (CM) formula, extensively hydrolyzed whey formula or donor breast milk. Infants who developed CM associated symptoms that subsided during elimination diet (n=223) underwent an open oral CM challenge (at mean age 7 months). The challenge was negative in 112, and in 111 it confirmed CMA, which was IgE-mediated in 83. Patients with CMA were followed until recovery, and 94 of them participated in a follow-up study at age 8-9 years. We investigated serum samples at diagnosis (mean age 7 months, n=111), one year later (19 months, n=101) and at follow-up (8.6 years, n=85). At follow-up, also 76 children randomly selected from the original cohort and without CM associated symptoms were included. We measured CM specific IgE levels with UniCAP (Phadia, Uppsala, Sweden), and β-lactoglobulin, α-casein and ovalbumin specific IgA, IgG1, IgG4 and IgG levels with enzyme-linked immunosorbent assay in sera. We applied a microarray based immunoassay to measure the binding of IgE, IgG4 and IgA serum antibodies to sequential epitopes derived from five major CM proteins at the three time points in 11 patients with active IgE-mediated CMA at age 8-9 years and in 12 patients who had recovered from IgE-mediated CMA by age 3 years. We used bioinformatic methods to analyze the microarray data. We studied T cell expression profile in peripheral blood mononuclear cell (PBMC) samples from 57 children aged 5-12 years (median 8.3): 16 with active CMA, 20 who had recovered from CMA by age 3 years, 21 non-atopic control subjects. Following in vitro β-lactoglobulin stimulation, we measured the mRNA expression in PBMCs of 12 T-cell markers (T-bet, GATA-3, IFN-γ, CTLA4, IL-10, IL-16, TGF-β, FOXP3, Nfat-C2, TIM3, TIM4, STIM-1) with quantitative real time polymerase chain reaction, and the protein expression of CD4, CD25, CD127, FoxP3 with flow cytometry. To optimally distinguish the three study groups, we performed artificial neural networks with exhaustive search for all marker combinations. For genetic associations with specific humoral responses, we analyzed 14 HLA class II haplotypes, the PTPN22 1858 SNP (R620W allele) and 5 known filaggrin null mutations from blood samples of 87 patients with CMA and 76 control subjects (age 8.0-9.3 years). Results: High IgG and IgG4 levels to β-lactoglobulin and α-casein were associated with the HLA (DR15)-DQB1*0602 haplotype in patients with CMA, but not in control subjects. Conversely, (DR1/10)-DQB1*0501 was associated with lower IgG and IgG4 levels to these CM antigens, and to ovalbumin, most significantly among control subjects. Infants with IgE-mediated CMA had lower β -lactoglobulin and α-casein specific IgG1, IgG4 and IgG levels (p<0.05) at diagnosis than infants with non-IgE-mediated CMA or control subjects. When CMA persisted beyond age 8 years, CM specific IgE levels were higher at all three time points investigated and IgE epitope binding pattern remained stable (p<0.001) compared with recovery from CMA by age 3 years. Patients with persisting CMA at 8-9 years had lower serum IgA levels to β-lactoglobulin at diagnosis (p=0.01), and lower IgG4 levels to β-lactoglobulin (p=0.04) and α-casein (p=0.05) at follow-up compared with patients who recovered by age 3 years. In early recovery, signal of IgG4 epitope binding increased while that of IgE decreased over time, and binding patterns of IgE and IgG4 overlapped. In T cell expression profile in response to β –lactoglobulin, the combination of markers FoxP3, Nfat-C2, IL-16, GATA-3 distinguished patients with persisting CMA most accurately from patients who had become tolerant and from non-atopic subjects. FoxP3 expression at both RNA and protein level was higher in children with CMA compared with non-atopic children. Conclusions: Genetic factors (the HLA II genotype) are associated with humoral responses to early food allergens. High CM specific IgE levels predict persistence of CMA. Development of tolerance is associated with higher specific IgA and IgG4 levels and lower specific IgE levels, with decreased CM epitope binding by IgE and concurrent increase in corresponding epitope binding by IgG4. Both Th2 and Treg pathways are activated upon CM antigen stimulation in patients with CMA. In the clinical management of CMA, HLA II or filaggrin genotyping are not applicable, whereas the measurement of CM specific antibodies may assist in estimating the prognosis.

Characterisation of antibiotic-resistant psychrotrophic bacteria in raw milk

Dynamics of raw milk associated bacteria during cold storage of raw milk and their antibiotic resistance was reviewed, with focus on psychrotrophic bacteria. This study aimed to investigate the significance of cold storage of raw milk on antibiotic-resistant bacterial population and analyse the antibiotic resistance of the Gram-negative antibiotic-resistant psychrotrophic bacteria isolated from the cold-stored raw milk samples. Twenty-four raw milk samples, six at a time, were obtained from lorries that collected milk from Finnish farms and were stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Antibiotics representing four classes of antibiotics (gentamicin, ceftazidime, levofloxacin and trimethoprim-sulfamethoxazole) were used to determine the antibiotic resistance of mesophilic and psychrotrophic bacteria during the storage period. A representative number of antibiotic-resistant Gram-negative isolates retrieved from the cold-stored raw milk samples were identified by the phenotypic API 20 NE system and a few isolates by the 16S rDNA gene sequencing. Some of the isolates were further evaluated for their antibiotic resistance by the ATB PSE 5 and HiComb system. The initial average mesophilic counts were found below 105 CFU/mL, suggesting that the raw milk samples were of good quality. However, the mesophilic and psychrotrophic population increased when stored at 4°C/4 d, 6°C/3 d and 6°C/4 d. Gentamicin- and levofloxacin-resistant bacteria increased moderately (P < 0.05) while there was a considerable rise (P < 0.05) of ceftazidime- and trimethoprim-sulfamethoxazole-resistant population during the cold storage. Of the 50.9 % (28) of resistant isolates (total 55) identified by API 20 NE, the majority were Sphingomonas paucimobilis (8), Pseudomonas putida (5), Sphingobacterium spiritivorum (3) and Acinetobacter baumanii (2). The analysis by ATB PSE 5 system suggested that 57.1% of the isolates (total 49) were multiresistant. This study showed that the dairy environment harbours multidrug-resistant Gramnegative psychrotrophic bacteria and the cold chain of raw milk storage amplifies the antibioticresistant psychrotrophic bacterial population.

Social dynamics for sustainable food systems. Actors’ orientations towards sustainability in primary production and public consumption

Abstract The modern food system and sustainable development form a conceptual combination that suggests sustainability deficits in the ways we deal with food consumption and production - in terms of economic relations, environmental impacts and nutritional status of western population. This study explores actors’ orientations towards sustainability by taking into account actors’ embedded positions within structures of the food system, actors’ economic relations and views about sustainability as well as their possibilities for progressive activities. The study looks particularly at social dynamics for sustainability within primary production and public consumption. If actors within these two worlds were to express converging orientations for sustainability, the system dynamics of the market would enable more sustainable growth in terms of production dictated by consumption. The study is based on a constructivist research approach with qualitative text analyses. The data consisted of three text corpora, the ‘local food corpus’, the ‘catering corpus’ and the ‘mixed corpus’. The local food actors were interviewed about their economic exchange relations. The caterers’ interviews dealt with their professional identity for sustainability. Finally, the mixed corpus assembled a dialogue as a participatory research approach, which was applied in order to enable researcher and caterer learning about the use of organic milk in public catering. The data were analysed for theoretically conceptualised relations, expressing behavioural patterns in actors’ everyday work as interpreted by the researcher. The findings were corroborated by the internal and external communities of food system actors. The interpretations have some validity, although they only present abstractions of everyday life and its rich, even opaque, fabric of meanings and aims. The key findings included primary producers’ social skilfulness, which enabled networking with other actors in very different paths of life, learning in order to promote one’s trade, and trusting reflectively in partners in order to extend business. These activities expanded the supply chain in a spiral fashion by horizontal and vertical forward integration, until large retailers were met for negotiations on a more equal or ‘other regarding’ basis. This kind of chain level coordination, typically building around the core of social and partnership relations, was coined as a socially overlaid network. It supported market access of local farmers, rooted in their farms, who were able to draw on local capital and labour in promotion of competitive business; the growth was endogenous. These kinds of chains – one conventional and one organic – were different from the strategic chain, which was more profit based and while highly competitive, presented exogenous growth as it depended on imported capital and local employees. However, the strategic chain offered learning opportunities and support for the local economy. The caterers exhibited more or less committed professional identity for sustainability within their reach. The facilitating and balanced approaches for professional identities dealt successfully with local and organic food in addition to domestic food, and also imported food. The co-operation with supply chains created innovative solutions and savings for the business parties to be shared. The rule-abiding approach for sustainability only made choices among organic supply chains without extending into co-operation with actors. There were also more complicated and troubled identities as juggling, critical and delimited approaches for sustainability, with less productive efforts due to restrictions such as absence of organisational sustainability strategy, weak presence of local and organic suppliers, limited understanding about sustainability and no organisational resources to develop changes towards a sustainable food system. Learning in the workplace about food system reality in terms of supply chain co-operation may prove to be a change engine that leads to advanced network operations and a more sustainable food system. The convergence between primary producers and caterers existed to an extent allowing suggestion that increased clarity about sustainable consumption and production by actors could be approached using advanced tools. The study looks for introduction of more profound environmental and socio-economic knowledge through participatory research with supply chain actors in order to promote more sustainable food systems. Summary of original publications and the authors’ contribution I Mikkola, M. & Seppänen, L. 2006. Farmers’ new participation in food chains: making horizontal and vertical progress by networking. In: Langeveld, H. & Röling N. (Eds.). Changing European farming systems for a better future. New visions for rural areas. Wageningen, The Netherlands. Wageningen Academic Publishers: 267–271. II Mikkola, M. 2008. Coordinative structures and development of food supply chains. British Food Journal 110 (2): 189–205. III Mikkola, M. 2009. Shaping professional identity for sustainability. Evidence in Finnish public catering. Appetite 53 (1): 56–65. IV Mikkola, M. 2009. Catering for sustainability: building a dialogue on organic milk. Agronomy Research 7 (Special issue 2): 668–676. Minna Mikkola has been responsible for developing the generic research frame, particular research questions, the planning and collection of the data, their qualitative analysis and writing the articles I, II, III and IV. Dr Laura Seppänen has contributed to the development of the generic research frame and article I by introducing the author to the basic concepts of economic sociology and by supporting the writing of article II with her critical comments. Articles are printed with permission from the publishers.

Dietary aspects of cow's milk allergy in young children

Cow s milk allergy (CMA) affects about 2-6% of infants and young children. Environmental factors during early life are suggested to play a role in the development of allergic diseases. One of these factors is likely to be maternal diet during pregnancy and lactation. The association between maternal diet and development of CMA in offspring is not well known, but diet could contain factors that facilitate development of tolerance. After an established food allergy, another issue is gaining tolerance towards an antigen that causes symptoms. The strictness of the elimination depends on the individual level of tolerance. This study aimed at validating a questionnaire used to inquire about food allergies in children, at researching associations between maternal diet during pregnancy and lactation and subsequent development of cow s milk allergy in the offspring, and at evaluating the degree of adherence to a therapeutic elimination diet of children with CMA and factors associated with the adherence and age of recovery. These research questions were addressed in a prospective birth cohort born between 1997 and 2004 at the Tampere and Oulu University Hospitals. Altogether 6753 children of the Diabetes Prediction and Prevention (DIPP) Nutrition cohort were investigated. Questionnaires regarding allergic diseases are often used in studies without validation. High-quality valid tools are therefore needed. Two validation studies were conducted here: one by comparing parentally reported food allergies with information gathered from patient records of 1122 children, and the other one by comparing parentally reported CMA with information in the reimbursement records of special infant formulae in the registers of the Social Insurance Institution for 6753 children. Both of these studies showed that the questionnaire works well and is a valid tool for measuring food allergies in children. In the first validation study, Cohen s kappa values were within 0.71-0.88 for CMA, 0.74-0.82 for cereal allergy, and 0.66-0.86 for any reported food allergy. In the second validation study, the kappa value was 0.79, sensitivity 0.958, and specificity 0.965 for reported and diagnosed CMA. To investigate the associations between maternal diet during pregnancy and lactation and CMA in offspring, 6288 children were studied. Maternal diet during pregnancy (8th month) and lactation (3rd month) was assessed by a validated, 181-item semi-quantitative food frequency questionnaire (FFQ), and as an endpoint register-based information on diagnosed CMA was obtained from the Social Insurance Institution and complemented with parental reports of CMA in their children. The associations between maternal food consumption and CMA in offspring were analyzed by logistic regression comparing the highest and lowest quarters with two middle quarters of consumption and adjusted for several potential confounding factors. High maternal intake of milk products (OR 0.56, 95% CI 0.37-0.86 p = 0.002) was associated with a lower risk of CMA in offspring. When stratified according to maternal allergic rhinitis or asthma, a protective association of high use of milk products with CMA was seen in children of allergy-free mothers (OR 0.30, 95% CI 0.13 - 0.69, p < 0.001), but not in children of allergic mothers. Moreover, low maternal consumption of fish during pregnancy was associated with a higher risk of CMA in children of mothers with allergic rhinitis or asthma (OR 1.47, 95% CI 0.96 - 2.27 for the lowest quarter, p = 0.043). In children of nonallergic mothers, this association was not seen. Maternal diet during lactation was not associated with CMA in offspring, apart from an inverse association between citrus and kiwi fruit consumption and CMA. These results imply that maternal diet during pregnancy may contain factors protective against CMA in offspring, more so than maternal diet during lactation. These results need to be confirmed in other studies before giving recommendations to the public. To evaluate the degree of adherence to a therapeutic elimination diet in children with diagnosed CMA, food records of 267 children were studied. Subsequent food records were examined to assess the age at reintroduction of milk products to the child s diet. Nine of ten families adhered to the elimination diet of the child with extreme accuracy. Older and monosensitized children had more often small amounts of cow s milk protein in their diet (p < 0.001 for both). Adherence to the diet was not related to any other sociodemographic factor studied or to the age at reintroduction of milk products to the diet. Low intakes of vitamin D, calcium, and riboflavin are of concern in children following a cow s milk-free diet. In summary, we found that the questionnaires used in the DIPP study are valid in investigating CMA in young children; that there are associations between maternal diet during pregnancy and lactation and the development of CMA in offspring; and that the therapeutic elimination diet in children with diagnosed CMA is rigorously adhered to.

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.

Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Milk casein-derived tripeptides : A special focus on blood pressure and vascular function

Increased consumption of low-fat milk products is inversely associated with the risk of hypertension. The beneficial effect of milk on blood pressure is attributed to high calcium and potassium content but also to specific peptide sequences, which are cleaved from milk protein during gastrointestinal digestion, fermentation of milk with proteolytic starter cultures or enzymatic hydrolysis. Milk products fermented with Lactobacillus helveticus contain casein-derived tripeptides isoleucine-proline-proline (Ile-Pro-Pro) and valine-proline-proline (Val-Pro-Pro), which have been shown to possess antihypertensive effects in humans and in experimental animals. The aim of the present series of studies was to investigate the effects of tripeptides Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them on vascular function and blood pressure and to elucidate the mechanisms behind them by using different experimental models of hypertension. Another aim was to characterize the acute effects of tripeptides on blood pressure and arterial stiffness in mildly hypertensive humans. Ile-Pro-Pro and Val-Pro-Pro or fermented milk products containing them attenuated the development of hypertension in two experimental models of hypertension, spontaneously hypertensive rat (SHR) and type 2 diabetic Goto-Kakizaki (GK) rat fed with high-salt diet. Significant differences in systolic blood pressure (SBP) were seen after 8 weeks treatment with tripeptide-containing products compared to control product. Plant sterols did not enhance this effect. Two differently produced tripeptide powders produced a similar attenuating effect on SBP in SHR. In mildly hypertensive subjects, a single administration of tripeptide- and plant sterol-containing fermented milk product decreased both SBP and diastolic blood pressure (DBP) over a period of 8 hours. Protective effect of tripeptides Ile-Pro-Pro and Val-Pro-Pro and fermented milk products containing them on vascular function was demonstrated in in vitro studies and long-term experimental studies. The effect was shown to be endothelium-dependent and possibly involving endothelium-derived hyperpolarizing factor (EDHF). In the clinical study, single administration of tripeptide-containing fermented milk product did not affect measures of arterial stiffness. Long-term treatment with fermented milk product containing Ile-Pro-Pro and Val-Pro-Pro inhibited angiotensin-converting enzyme (ACE) and decreased aldosterone levels thus showing beneficial effects on the renin-angiotensin system (RAS) in SHR and GK. No changes in the components of RAS were observed by the single administration of the same product in mildly hypertensive subjects. Increased levels of cGMP, NOx and citrulline suggest increased nitric oxide (NO) production by the tripeptides. Taken together, Ile-Pro-Pro and Val-Pro-Pro -containing products attenuate the development of hypertension after long-term treatment in experimental models of hypertension and possess an acute antihypertensive effect in mildly hypertensive subjects. In addition, these tripeptides show endothelium-mediated beneficial effects on vascular function. Attenuation of blood pressure increase by the tripeptides in experimental animals involves RAS, but its role in the antihypertensive effect in humans remains to be elucidated.

Options for selecting dairy cattle for milk coagulation ability

Lypsylehmien maidon juoksettumiskyvyn jalostuskeinot Väitöskirjassa tutkittiin lypsylehmien maidon juustonvalmistuslaadun parantamista jalostusvalinnan avulla. Tutkimusaihe on tärkeä, sillä yhä suurempi osa maidosta käytetään juustonvalmistukseen. Tutkimuksen kohteena oli maidon juoksettumiskyky, sillä se on yksi keskeisistä juustomäärään vaikuttavista tekijöistä. Maidon juoksettumiskyky vaihteli huomattavasti lehmien, sonnien, karjojen, rotujen ja lypsykauden vaiheiden välillä. Vaikka tankkimaidon juoksettumiskyvyssä olikin suuria eroja karjoittain, karja selitti vain pienen osan juoksettumiskyvyn kokonaisvaihtelusta. Todennäköisesti perinnölliset erot lehmien välillä selittävät suurimman osan karjojen tankkimaitojen juoksettumiskyvyssä havaituista eroista. Hyvä hoito ja ruokinta vähensivät kuitenkin jossain määrin huonosti juoksettuvien tankkimaitojen osuutta karjoissa. Holstein-friisiläiset lehmät olivat juoksettumiskyvyltään ayrshire-rotuisia lehmiä parempia. Huono juoksettuminen ja juoksettumattomuus oli vain vähäinen ongelma holstein-friisiläisillä (10 %), kun taas kolmannes ayrshire-lehmistä tuotti huonosti juoksettuvaa tai juoksettumatonta maitoa. Maitoa sanotaan huonosti juoksettuvaksi silloin, kun juustomassa ei ole riittävän kiinteää leikattavaksi puolen tunnin kuluttua juoksetteen lisäyksestä. Juoksettumattomaksi määriteltävä maito ei saostu lainkaan puolen tunnin aikana ja on siksi erittäin huonoa raaka-ainetta juustomeijereille. Noin 40 % lehmien välisistä eroista maidon juoksettumiskyvyssä selittyi perinnöllisillä tekijöillä. Juoksettumiskykyä voikin sanoa hyvin periytyväksi ominaisuudeksi. Kolme mittauskertaa lehmää kohti riittää varsin hyvin lehmän maidon keskimääräisen juoksettumiskyvyn arvioimiseen. Tällä hetkellä juoksettumiskyvyn suoran jalostamisen ongelmana on kuitenkin automatisoidun, laajamittaiseen käyttöön soveltuvan mittalaitteen puute. Tämän takia väitöskirjassa tutkittiin mahdollisuuksia jalostaa maidon juoksettumiskykyä epäsuorasti, jonkin toisen ominaisuuden kautta. Tällaisen ominaisuuden pitää olla kyllin voimakkaasti perinnöllisesti kytkeytynyt juoksettumiskykyyn, jotta jalostus olisi mahdollista sen avulla. Tutkittavat ominaisuudet olivat sonnien kokonaisjalostusarvossa jo mukana olevat maitotuotos ja utareterveyteen liittyvät ominaisuudet sekä kokonaisjalostusarvoon kuulumattomat maidon valkuais- ja kaseiinipitoisuus sekä maidon pH. Väitöskirjassa tutkittiin myös mahdollisuuksia ns. merkkiavusteiseen valintaan tutkimalla maidon juoksettumattomuuden perinnöllisyyttä ja kartoittamalla siihen liittyvät kromosomialueet. Tutkimuksen tulosten perusteella lehmien utareterveyden jalostaminen parantaa jonkin verran myös maidon juoksettumiskykyä sekä vähentää juoksettumattomuutta ayrshire-rotuisilla lehmillä. Lehmien maitotuotos ja maidon juoksettumiskyky sekä juoksettumattomuus ovat sen sijaan perinnöllisesti toisistaan riippumattomia ominaisuuksia. Myöskin maidon valkuais- ja kaseiinipitoisuuden perinnöllinen yhteys juoksettumiskykyyn oli likimain nolla. Maidon pH:n ja juoksettumiskyvyn välillä oli melko voimakas perinnöllinen yhteys, joten maidon pH:n jalostaminen parantaisi myös maidon juoksettumiskykyä. Todennäköisesti sen jalostaminen ei kuitenkaan vähentäisi juoksettumatonta maitoa tuottavien lehmien määrää. Koska maidon juoksettumattomuus on niin yleinen ongelma suomalaisilla ayrshire-lehmillä, väitöksessä selvitettiin tarkemmin ilmiön taustoja. Kaikissa kolmessa tutkimusaineistoissa noin 10 % ayrshire-lehmistä tuotti juoksettumatonta maitoa. Kahden vuoden kuukausittaisen seurannan aikana osa lehmistä tuotti juoksettumatonta maitoa lähes joka mittauskerralla. Maidon juoksettumattomuus oli yhteydessä lypsykauden vaiheeseen, mutta mikään ympäristötekijöistä ei pystynyt täysin selittämään sitä. Sen sijaan viitteet sen periytyvyydestä vahvistuivat tutkimusten edetessä. Lopuksi tutkimusryhmä onnistui kartoittamaan juoksettumattomuutta aiheuttavat kromosomialueet kromosomeihin 2 ja 18, lähelle DNA-merkkejä BMS1126 ja BMS1355. Tulosten perusteella maidon juoksettumattomuus ei ole yhteydessä maidon juoksettumistapahtumassa keskeisiin kaseiinigeeneihin. Sen sijaan on mahdollista, että juoksettumattomuusongelman aiheuttavat kaseiinigeenien syntetisoinnin jälkeisessä muokkauksessa tapahtuvat virheet. Asia vaatii kuitenkin perusteellista tutkimista. Väitöksen tulosten perusteella maidon juoksettumattomuusgeeniä kantavien eläinten karsiminen jalostuseläinten joukosta olisi tehokkain tapa jalostaa maidon juoksettumiskykyä suomalaisessa lypsykarjapopulaatiossa.