Tracking cereulide producing Bacillus cereus in foods, papermaking and biowaste management

Bacillus cereus is a ubiquitous food poisoning bacterium, but only producers of the emetic toxin, cereulide can be life threatening. Therefore a fast and reliable method is needed for identifying strains that produce the toxin. In this thesis the previously developed sperm bioassay was refined into a tool for the detection of microbially produced mitochondria toxic substances. This refined tool, Sperm Combi Assay effectively detected not only cereulide, but also other mitochondrial toxic substances and yielded data for classifying the substances into 3 different classes. The cereulide class (includes also valinomycin and enniatin) was characterised by a spotted mitochondrial sheath when stained with the membrane potential responsive dye JC-1. The gramicidin class (includes nigericin, calcimycin, monensin, narasin, salinomycin and antimycin A) was characterised by gradual fading of the dye during loss of the membrane potential. The ionomycin type (includes staurosporine, oligomycin) was characterised by loss of sperm motility with no changes of the mitochondrial membrane potential. The novel Sperm Combi Assay was put to the test to clarify the cause of illness connected to consumption of food supplements (seaweed capsules). The Sperm Combi Assay revealed highly toxic contaminants in the capsules. The test revealed that the poisonous substance was in the seaweed inside the capsule, not involving the capsule shell. The currently available official methods had failed to disclose this information. The test further helped to identify bacteria responsible for producing the toxic substances in the seaweed. The effects shown by the Sperm Combi Assay were similar to the gramicidin type of response. Next the Sperm Combi Assay was used to track potential sources of cereulide producers in food. We found cereulide producing B. cereus from potato tuber originating from the consumer market and had not undergone industrial treatment. We describe in this thesis how the initial plate count procedure can be improved to recognise the cereulide producing B. cereus already on a plate. Cereulide producers have been reported in industrial foods and rice by many researchers. Our report appears to be the first one where potato crops were screened for cereulide producers by methods that were effective for detecting cereulide producing B. cereus. This development is important because the potato is a major food crop in the European diet. Not only potatoes but many other plants such as trees in the forest contain cereulide producing B. cereus as an endophyte. The forest industry makes food packaging material from wood pulp. To trace the behaviour cereulide producing B. cereus contained in wood, we used the same protocol which was useful for finding the cereulide producing B. cereus in potato. We found that, of the spores of cereulide producing B. cereus, ca. 5 % may be retained in the paper product. The Sperm Combi Assay was used to track the distribution of cereulide, potentially released into the pulp slurry by cereulide producing B. cereus. We found that ca. 10 % present in the pulp slurry was recovered in the paper product. We measured if cereulide contained in paper could migrate from the paper product into packaged food and found almost all cereulide leached into simulated fatty foods, but less than the detection limit into warm or cold drinks. Food waste contains valuable nutrients and should be recycled into agricultural production. The question arises about the fate of cereulide producing B. cereus contained in food. We studied the behaviour of cereulide producing B. cereus spores throughout four seasons in an industrial scale biowaste processing plant that involved a heating step of 24 min at 137 °C followed by anaerobic digestion. We traced cereulide producing B. cereus in the digested biowaste with the same culture methods and the Sperm Combi Assay which proved operative in potato and with non culture based methods (PCR). We showed that the liquid biofertilizer contained ca. 1000 total B. cereus per ml of which 10 % were cesB (indicator of cereulide synthase) positive. This persistence of cereulide producing B. cereus under harsh conditions demonstrates that it is not possible to eradicate cereulide producers from the food cycle. Instead, we have to learn to design our food processing protocols so as to not give cereulide producers a chance to accumulate the cereulide toxin in foods. In this study we used porcine sperm cells as a tool to detect cereulide (their sensitivity is high), with an EC100 (equal to lethal dose) 0.3 ng ml-1. The question is if sperm sensitivity to cereulide reflects the sensitivity of other kinds of cells and tissues, for instance in the human body. We assessed this question by comparing the sensitivity of primary cells, cell lines and pancreatic islets towards cereulide. We found that the EC100 values for loss of mitochondrial membrane potential (24 h exposure) to human peripheral blood mononuclear cells and keratinocytes, porcine kidney epithelial cells and murine fibroblasts and pancreatic islets (containing the beta cells, which produce insulin) ranged from 0.4 to 2 ng of cereulide per ml. The results show that different kinds of cells were similarly vulnerable to mitochondrial toxicity by cereulide. These results strongly indicate that the Sperm Combi Assay is useful not only for tracking cereulide in foods and environmental samples, but can also be useful in assessing human health risk by materials contaminated by cereulide. We also showed that the external concentration of potassium (300µM increased to 850-950 µM) influences the speed of the potassium efflux, measured in HaCaT and PBMC cells. We introduced this Sperm Combi Assay to deal with a bacterium and/or toxin that can hardly be eliminated in foods. This assay is a fast and reliable method to detect mitochondria toxic substances as well as the reliable plating method for screening the producing organisms. Because it is not possible to eliminate cereulide producing B. cereus, it should be kept under control by avoiding conditions permissive for cereulide production.Kereulidi-toksiinia tuottavien Bacillus cereus -bakteerien tunnistaminen elintarvikkeista sekä paperintuotannon ja biojätteiden prosessointinäytteistä Bacillus cereus on hyvin yleinen bakteeri ympäristössä, erityisesti kasveissa ja maaperässä. Elintarvikkeissa lisääntyessään sen toksineeja tuottavat kannat aiheuttavat ruokamyrkytyksiä. Sen monista toksiineista eli myrkkyaineista kereulidi, jota sanotaan myös oksetustoksiiniksi, on erityisen vaarallinen. Se tunnetaan jopa kuolemaan johtaneiden, vakavien myrkytysten aiheuttajana. Siksi tätä toksiinia tuottamaan kykenevien B. cereus -kantojen tunnistaminen on tärkeää. Tässä työssä kehitettiin kuumennusta kestävien toksiinien mittausmenetelmä, Sperm Combi Assay (SCA). Menetelmässä hyödynnetään sikataloudessa tuotettavaa siemennestettä. Eri toksiinit aiheuttavat sian siittiöissä erityyppisiä soluvammoja. Mikroskooppisesti todennettavien, kullekin toksiinityypille ominaisten soluvaurioiden avulla toksiinit voidaan tunnistaa suoraan elintarvikkeista ja mikrobinäytteistä. Siittiöreaktiivisia mikrobitoksiineja löytyi kolmenlaisia: 1. kereulidi ja sen kaltaisesti vaikuttavat, kuten kymmen- tai satakertaisesti vähemmän myrkylliset, valinomysiini ja enniatiini. Näille myrkyille on tunnusomaista siittiön hännän värjäytyminen pilkulliseksi JC-1 väriaineella, joka vaihtaa väriä solujen kalvopotentiaalin muuttuessa; 2. gramisidiinin kaltaiset myrkkyaineet nigerisiini, kalsimysiini, monensiini, narasiini, salinomysiini ja antimysiini A, joille tyypillistä oli JC-1 väriaineen ilmaiseman korkean kalvopotentiaalin vähittäinen sammuminen; 3. jonomysiinin kaltaiset myrkkyaineet, joille tyypillistä oli siittiöiden lakkaaminen uimasta ilman, että niiden kalvopotentiaalissa näkyy muutosta. Uutta mittausmenelmää testattiin erään ravintolisäkapselien käyttöön liittyneen epidemian yhteydessä (merileväkapseleita). SCA-mittaus paljasti kapselien sisällön vahvan myrkyllisyyden, jota ei tavanomaisilla, viranomaiskäytössä olevilla menetelmillä voitu todeta. Myrkkyvaikutus liittyi kapselin sisältöön, ei kuoreen. Testillä selvitettiin, että myrkyn tuottajia olivat bakteerit, jotka olivat kapselin sisällössä epäpuhtauksina. Niiden tuottama myrkkyaine oli gramisidiini-tyyppinen. SCA-testiä sovellettiin myös kereulidia tuottavien B. cereus -bakteerien kulkureittien paljastamiseen elintarviketuotantoketjussa. Havaitsimme, että perunassa on luontaisesti kereulidia tuottavia bakteereja jo ennen perunan elintarvikkeeksi jalostamista. Tässä väitöskirjassa kerrotaan, miten kereulidin tuottajat voidaan tunnistaa tätä tuottamattomien B. cereus -bakteerityyppien joukosta viljelymaljalla. Koska kereulidin tuottajia esiintyy monissa tuotantokasveissa, kuten riisissä, ja niiden itiöt kestävät poikkeuksellisen hyvin kuumuutta, niitä löytyy myös teollisesti tuotetuissa valmisteissa ja puolivalmisteissa. Tämä asia on tärkeä ymmärtää, jotta kereulidin tuottajien läsnäolo osataan ottaa huomioon ja pystytään minimoimaan niiden lisääntyminen tuotantoprosessin aikana. Kereulidia tuottavia B. cereus -bakteereja esiintyy luultavasti lähes kaikissa kasveissa, koska niitä löytyi kuusien (Picea abies) sisältäkin. Kuusihiokkeesta valmistetun sulpun sisältämistä kereulidin tuottajista n. 5 % kulkeutui valmiiseen paperiin saakka. Tutkimme simulointitestien avulla, siirtyykö kereulidi pahvista elintarvikkeeseen. Havaitsimme, että kulkeutumista tapahtui pahvista rasvaiseen elintarvikkeeseen mutta ei kuumaan tai kylmään juomaan. Osa elintarvikkeista päätyy yhteiskunnassa biojätteeksi. Koska biojätteen ravinteet pitäisi saada kiertoon, peltojen lannoitteeksi, selvitimme kereulidia tuottavien B. cereus -bakteerien käyttäytymistä biojätteen kuumennus- (24 min, 137˚C) ja mädätysprosesseissa. Mittausmenetelminä oli SCA-, viljely- ja PCR-tekniikka (DNA tunnistus). Totesimme, että n. 10 % prosessin läpi selvinneistä B. cereus -bakteereista oli kereulidi-geenin suhteen positiivisia eli todennäköisiä tuottajia. Tulokset, jotka saatiin täysimittaisella biojätteen teollisen käsittelyn laitteistolla, eri työvaiheineen osoittivat, että kereulidia tuottavat B. cereus -bakteerit ovat osa luontoa, eivätkä ne ole tekniikan keinoin hävitettävissä elintarvikkeista. Koska siittiöt osoittautuivat tehokkaiksi ilmaisemaan kereulidin solumyrkyllisyyttä, halusimme selvittää, mikä on elimistön muiden solutyyppien kereulidi-herkkyys. Käytimme testisoluina ihmisen, sian ja hiiren primaarisoluja ja solulinjoja. Mikroskoopilla ja ionielektrodeilla havaittiin, että solut vammautuivat kun ne altistuivat 24 tuntia kereulidi-pitoisuudelle 0.4 2 ng ml-1. Tulokset osoittivat, että nisäkkään, kuten ihminen, monet eri kudos- ja solutyypit ovat herkkiä kereulidin myrkkyvaikutuksille. Tärkein soluvammojen syntyreitti oli ehkä kalium-ionien massiivinen vuoto ulos soluista, jonka kereulidi aiheutti ihmisen veren yksitumaisissa valkosoluissa ja ihon keratinosyytteissä jo muutaman minuutin altistuksen seurauksena. Tässä työssä kehitetyllä SCA-menetelmällä voidaan tutkia vaikeasti tuhottavia bakteereja ja niiden toksiineja elintarvikkeissa. Menetelmällä havaitaan mitokondrioille myrkylliset aineet nopeasti ja luotettavasti. Lisäksi käytettiin maljausmenetelmää, jolla voidaan löytää kereulidin tuottajat. Koska kereulidia tuottavien B. cereus -bakteerien tuhoaminen elintarvikkeesta on mahdotonta, on tärkeää pitää tuotteiden säilytysolosuhteet sellaisina, että kereulidin tuotto estyy

Fermented Lingonberry Juice Inhibits Oral Tongue Squamous Cell Carcinoma Invasion In Vitro Similarly to Curcumin

Background: Oral tongue squamous cell carcinoma (OTSCC) cells are highly proliferative and invasive. Lingonberry contains several polyphenolic compounds similar to curcumin. We hypothesize that fermented lingonberry juice (FLJ) has an anti-invasive and anti-proliferative effect on OTSCC cells similarly to curcumin, which is known to be anti-carcinogenic. Materials and Methods: FLJ, curcumin dissolved in ethanol, or curcumin loaded in Candida extracellular vesicles (EVs) were added to more (HSC-3) and less aggressive (SCC-25) OTSCC cells. Cell proliferation was measured with a 5-bromo-2'-deoxyuridine kit and invasion in the three-dimensional Myogel spheroid assay. Statistical analyses were completed with one-way ANOVA and Bonferroni post-hoc testing. Results: Both FLJ and curcumin significantly reduced the proliferation and invasion of HSC-3 and SCC-25 cells. The effects of curcumin were not improved when cells were treated with curcumin loaded within EVs. Conclusion: Our results suggest that FLJ, like curcumin, has an anti-carcinogenic effect on aggressive OTSCC cells in vitro.Peer reviewe

Chemical and microbiological hazards associated with recycling of anaerobic digested residue intended for agricultural use

In the present study, three full-scale biogas plants (BGP) were investigated for the concentration of heavy metals, organic pollutants, pesticides and the pathogenic bacteria Bacillus cereus and Escherichia coli in the anaerobically digested residues (ADR). The BGPs mainly utilize source-separated organic wastes and industrial food waste as energy sources and separate the ADR into an ADR-liquid and an ADR-solid fraction by centrifugation at the BGP. According to the Norwegian standard for organic fertilizers, the ADR were classified as quality 1 mainly because of high zinc (132–422 mg kg−1 DM) and copper (23–93 mg kg−1 DM) concentrations, but also because of high cadmium (0.21–0.60 mg kg−1 DM) concentrations in the liquid-ADR. In the screening of organic pollutants, only DEHP (9.7–62.1 mg kg−1) and ∑ PAH 16 (0.2–1.98 mg kg−1 DM) were detected in high concentrations according to international regulations. Of the 250 pesticides analyzed, 11 were detected, but only imazalil (<0.30–5.77 mg kg−1 DM) and thiabendazol (<0.14–0.73 mg kg−1 DM) were frequently detected in the ADR-fiber. Concentrations of imazalil and thiabendazol were highest during the winter months, due to a high consumption of citrus fruits in Norway in this period. Ten percent of the ADR-liquid samples contained cereulide-producing B. cereus, whereas no verotoxigenic E. coli was detected. The authors conclude that the risk of chemical and bacterial contamination of the food chain or the environment from agricultural use of ADR seems low

Fermented lingonberry juice inhibits oral tongue squamous cell carcinoma invasion in vitro similarly to curcumin

Abstract Background: Oral tongue squamous cell carcinoma (OTSCC) cells are highly proliferative and invasive. Lingonberry contains several polyphenolic compounds similar to curcumin. We hypothesize that fermented lingonberry juice (FLJ) has an anti-invasive and anti-proliferative effect on OTSCC cells similarly to curcumin, which is known to be anti-carcinogenic. Materials and methods: FLJ, curcumin dissolved in ethanol, or curcumin loaded in Candida extracellular vesicles (EVs) were added to more (HSC-3) and less aggressive (SCC-25) OTSCC cells. Cell proliferation was measured with a 5-bromo-2’-deoxyuridine kit and invasion in the three-dimensional Myogel spheroid assay. Statistical analyses were completed with one-way ANOVA and Bonferroni post-hoc testing. Results: Both FLJ and curcumin significantly reduced the proliferation and invasion of HSC-3 and SCC-25 cells. The effects of curcumin were not improved when cells were treated with curcumin loaded within EVs. Conclusions: Our results suggest that FLJ, like curcumin, has an anti-carcinogenic effect on aggressive OTSCC cells in vitro

Safety assessment of food-contact paper and board using a battery of short-term toxicity tests: European union BIOSAFEPAPER project

International audienceAn European Union (EU)-funded project QLK1-CT-2001-00930 (BIOSAFEPAPER) involves the development, validation and intercalibration of a short-term battery of toxicological tests for the safety assessment of food-contact paper and board. Dissemination of the results to industry, legislators (e.g. DG Consumer Protection, DG Enterprises, DG Research), standardization bodies such as CEN, and consumers will create an agreed risk evaluation procedure. The project involves pre-normative research in order to establish a set of in-vitro cytotoxicity and genotoxicity tests that will be easily adaptable to food-contact fibre-based materials and have endpoints relevant to consumer safety, including sub-lethal cellular events. These tests will be performed on samples representing actual migration conditions from food-contact paper and board with respect to different foodstuffs, and should form an experimental basis for scientifically sound recommendations for a harmonized system of risk evaluation and product testing