Browsing by Subject "Lactobacillus"

Aflatoxins are harmful compounds found in food and feed. The literature review of this thesis looked at aflatoxin M1 (AFM1) found in milk and its occurrence, significance and prevention methods using lactic acid bacteria (LAB). Furthermore, the assay methods of aflatoxins and the factors affecting the analysis were discussed. The aim of the experimental work was 1) to investigate the ability of five LAB strains to bind AFM1 in vitro and 2) to investigate how different matrixes affect the assay of AFM1 and to evaluate the feasibility of the selected ELISA kit for the study. In the first phase of the study the AFM1 concentrations of three different matrixes were analyzed using ELISA when known concentrations of the AFM1-standardsolution were added (0, 20 ja 40 ppt). In the second phase of the study the abilities of five LAB strains to bind AFM1 in UHT skimmed milk and MRS-broth were investigated. The bacterial suspensions were incubated at +32 °C for one hour, and the AFM1 concentration in the matrixes were 50, 15 and 10 ppt. The unbound AFM1 concentrations of the supernatants were analyzed from the samples using ELISA. The unbound AFM1 concentrations were converted to the proportional portion of the bound AFM1. In this study, statistically significant differences were observed in the abilities of the LAB to bind AFM1. The viable cells of strain B2 27 (Lb. plantarum-, pentosus- or paraplantarum) were the best binders of AFM1. They removed 43.7 % AFM1 in UHT skimmed milk, where the AFM1 concentration was 15 ppt. Unlike in previous studies viable cells bound AFM1 better than the heat-killed cells. The used ELISA kit was a sensitive method for analyzing low concentrations of AFM1, but at higher concentrations the assay results were inaccurate. The nonspecific interaction due to the components of the matrixes had to be taken into account when the results were reviewed. In the future, it may be possible to utilize the LAB strains, such as B2 27 for reducing AFM1 the concentration in milk and probably in other foodstuffs. There is a need to develop a practical application which can be used in the binding of AFM1 using lactic acid bacteria and thus reduce the bioavailability of AFM1.

The literature review of the study focused on intestinal microbiota and the connection between its imbalance and Inflammatory Bowel Disease (IBD). Specific focus was on communication between microbes and human host through congenital immune defense. The purpose of the experimental phase was to research in vitro, the adherence of the human Lactobacillus rhamnosus GG, Bifidobacterium bifidum strain DSM20456 and the Lactobacillus acidophilus strain LAB20 of canine and the EPS mutant strain LAB20 to Caco-2 and HT-29 cell lines and mucus. The adhesion method was based on bacterial cells that were marked with tritium. The next experiment was whether the bacteria could reduce inflammatory response in the LPS-induced HT-29 cell line. HT-29 cells produced inflammatory mediator IL-8, and its concentration was measured by collecting the supernatant above the cells and measuring the IL-8 concentration with the ELISA-method. In conclusion, the effect of adhesin proteins SpaC and BopA as anti-inflammatory components was tested. L. rhamnosus GG, B. bifidum DSM20456 and L. acidophilus LAB20 adhered to the HT-29, Caro-2 cells of epithelial cell lines and to mucus. LAB20 EPS mutants did not adhere to mucus at all, so the EPS-construction of the strain LAB20 would appear to be relevant to the bacteria’s adherence. L. rhamnosus GG, B. bifidum DSM20456 and L. acidophilus LAB20 strains showed anti-inflammatory properties. They significantly reduced IL-8 yield in the LPS-induced inflammatory response in the HT-29 cell line. L. acidophilus LAB20 significantly reduced the yield of IL-8 in one test, and therefore the result of this study is indicative. LAB20 EPS mutants did not cut the IL-8 yield, so EPS structures may be responsible for the anti-inflammatory feature of the strain LAB20. Bacteria SpaC- and BopA-adhesin proteins showed pro-inflammatory properties, i.e. an inflammatory response in the HT-29 cell line. The results showed that L. rhamnosus GG, B. bifidum DSM20456 and L. acidophilus LAB20 strains have adhesion and anti-inflammatory properties. The LAB20 is a new and potential probiotic for canines. B. bifidum also showed anti-inflammatory properties, so it could also act as a palliative for IBD. The SpaC and BopA adhesin proteins did not show any anti-inflammatory effects, but they still proved to be stimulating host's immune defense, which plays an important role in the host's immune system regulation. EPS structures may convey the LAB20 adhesion to mucus and anti-inflammatory properties.

Tyhjiöpakkausmenetelmä on nykyään hyvin yleisesti käytössä suomalaisessa lihanjalostusteollisuudessa. Viime vuosina on Suomessa kuitenkin noussut esille uusi ongelma; tyhjiöpakattujen lihatuotteiden venyvä limaantuminen säilytyksen aikana. Ongelma ilmeni äkillisesti ja sitä on esiintynyt erilaisissa lihatuotteissa sekä eri valmistajilla. Kuluttajien kannalta ilmiö on vastenmielinen aiheuttaen huomattavia taloudellisia tappioita ja heikentäen kuluttajien luottamusta suomalaisen lihanjalostusteollisuuden tuotteisiin. Eräät bakteerilajit voivat tuottaa elintarvikkeisiin ekstrasellulaarista limaa. Limaantumista on todettu ainakin leivässä, kalajalosteissa, suolatuissa lihavalmisteissa sekä sokeriliemissä, mutta suomalaisissa tyhjiöpakatuissa lihatuotteissa esiintyvän limaantumisen kaltaista ilmiötä ei ole kirjallisuudessa kuvattu. Limaantumisongelmaa on tutkittu EKK:n elintarvike- ja ympäristöhygienianosastolla. Limaantuneista lihatuotteista on eristetty bakteerikantoja, joilla on pystytty kokeellisesti aiheuttamaan tyhjiöpakattujen makkaroiden limaantuminen. Eristetyt kannat ovat maitohappobakteereita ja kuuluvat sukuihin Lactobacillus sekä Leuconostoc. Näitä bakteereita on löydetty runsaasti lihanjalostuslaitosten tuotantotiloista. Raaka-aineet lienevät niiden tärkeä kontaminaatiolähde. Yksi tutkimustavoite on ollut eristys- ja tunnistusmenetelmien kehittäminen limaantumista aiheuttaville bakteereille. Tässä tutkielmassa selvitettiin serologisen menetelmän soveltuvuutta limabakteerien tunnistamiseen. Limabakteeri A 210-kantaa vastaan valmistettiin antiseerumi, jolla testattiin agglutinaatiokokein eri limabakteeri- ja vertailukantoja. Menetelmä tunnisti melko huonosti A 210-kantaa muistuttavat limabakteerikannat testin herkkyyden ollessa vain 68.4% ja spesifisyyden 69.6%. Käytetty menetelmä oli vielä huonompi venyvien kantojen tunnistamisessa yleensä. Tuloksiin on saattanut vaikuttaa huonontavasti mm. subjektiivisuus niiden tulkinnassa. Jotkut kannat agglutinoivat spontaanisti ollen siten tutkimuskelvottomia. Lisäksi on huomattava, että kirjallisuuden mukaan agglutinaatiomenetelmä sopii parhaiten "gram-negatiivisten bakteerilajien tunnistamiseen (esim. Salmonella), kun taas tämän tutkimuksen kohteena olivat gram-positiiviset lajit.

Lactobacillus rhamnosus GG (GG) is one of the most studied probiotics worldwide and it has proved to confer health benefits to human. However, the exact mechanism of its probiotic action is still not fully understood. Some proteins secreted by probiotics, such as p75 (Msp1) and p40 (Msp2) in GG, are reported to play an important role in gut epithelial homeostasis. The aim of this work was to develop a static biofilm plate model for analyzing exoproteomes (all secreted proteins) produced by L. rhamnosus GG biofilms. First, different culture volumes (7 mL and 10 mL), incubation times and protein precipitation methods were tested to optimize conditions in order to maximize the protein yield. Next, the impact of different sugars on the exoproteome composition of the GG cells during planktonic and biofilm mode of growth was explored. Finally, the planktonic and biofilm exoproteins were subjected to antigen profiling and protein identification. The 6-well Polystyrene Microtiter plate was used as the static biofilm model for inducing the biofilm formation of GG. Biofilms were formed for 24 h and 48 h and the protein secretion from each time point was assessed by precipitating the supernatant proteins using 10% trichloroacetic acid (TCA)/acetone or 2-D Clean Up kit (GE Healthcare). The purified proteins were subjected to one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (1-DE) and the proteins were visualized using Coomassie blue staining. 1-DE immunoblotting using antibodies raised against whole GG cells was used to analyze antigens produced by the GG biofilms under the optimized biofilm formation conditions. The antigens produced by the GG biofilm cells were compared to those produced by the GG cells during planktonic growth (from over- night cultures) and the cross-reacting proteins were visualized using an IR800-conjugated secondary antibody. The immunoblots were scanned using an Odyssey Infrared Imaging System (Licor). In addition, the impact of two different carbohydrate sources on the antigen profiles was also explored. Finally, in-liquid tryptic digestion coupled with Liquid Chromatography- tandem Mass Spectrometry analysis (LC-MS/MS) was used to identify and compare exoproteomes produced by the biofilm cells cultured in the presence of the tested carbohydrates. The results showed that the commercial 2-D Clean Up kit is better than 10% TCA protein precipitation/acetone washing, because it produced clear protein patterns with less background. However, the TCA/acetone–protocol resulted in detection of higher number of proteins in 1-DE gels. Comparative immunoblot analyses of the planktonic and biofilm exoproteins at 24 h and 48 h time points revealed a clear difference in antigen profiles between the two modes of growth. In addition, the utilized carbon source was found to have a great impact on the antigen abundances and/or export. Using LC-MS/MS, 36 exoproteins were identified from the GG biofilm cultures (identification score ≥ 40, p < 0.05). Most of the identified proteins were associated with cell wall/membrane biogenesis, peptide and sugar transporters and transcription.