Identification of lactic acid bacteria in probiotic products (tablets) using amplification methods

Laktobacily hrají důležitou roli ve fermentačních procesech již po tisíciletí. Byly prostudovány jejich probiotické vlastnosti a dnes jsou používány při výrobě fermentovaných potravinových výrobků a doplňků stravy včetně farmakologických preparátů. V této práci byla pozornost zaměřena na zástupce rodu Lactobacillus v probiotických preparátech (tabletách). Pro identifikaci laktobacilů v sedmi probiotických preparátech byla použita metoda polymerázové řetězové reakce (PCR), založené na amplifikaci sekvence DNA. Jako DNA matrice byla do PCR směsí použita celková DNA izolovaná z tablet pomocí magnetických nosičů (P(HEMA-co¬-GMA) pokrytých karboxylovými skupinami. Rodově specifické produkty (250 bp) byly detekovány pomocí gelové elektroforézy na agaróse. Přítomnost DNA rodu Lactobacillus byla prokázána ve všech výrobcích kromě jednoho. Na etiketě tohoto výrobku byla uvedena pouze přítomnost probiotických bakterií. Pravděpodobně se jednalo o zástupce jiného rodu než Lactobacillus.Lactobacillus have been playing a crucial role in the fermentation processes for millenia. Their probiotic effects have been studied deeply and nowadays they are used in the production of fermented products and food adjunkt including probiotic preparations. This thesis was focused on bakteria of genus Lactobacillus in probiotic preparations (tablets). Polymerase chain reaction method (PCR) based on amplification of DNA was used to identify lactobacillus in seven probiotic preparations. As DNA matrices whole DNA recovered from tablets with the help of magnetic microparticles (P(HEMA-co¬-GMA) coated by carboxylic groups was used into a PCR mixture. Genus specific PCR products (250 bp) were detected with the help of gel electrophoresis on agaróse. DNA of genus Lactobacillus was proved in all but one product. In this product only probiotic bacteria were declared presence without family identification was proved. It contained different bakteria then bakteria of Genus Lactobacillus probably.

Fysiske og psykiske mure

Noter i margine

Izolation and identification of DNA from probiotic bacteria in complex matrices

V dnešní době jsou hojně využívány v potravinářském průmyslu probiotické bakterie mléčného kvašení (BMK) a bifidobakterie. Tvoří mikroflóru gastrointestinálního traktu. Mohou být do organismu dodávané jako potravinové doplňky. Nejnovější metody identifikace bakterií mléčného kvašení a bifidobakterií jsou založeny na analýze DNA s využitím amplifikačních metod. Nejvíce se využívá polymerázová řetězová reakce (PCR). Cílem diplomové práce bylo využití rodově a druhově specifických PCR pro identifikaci kmenů rodu Lactobacillus a Bifidobacterium v komplexních matricích (6 potravinových doplňků: Zenflo, Linex Forte, Probian, Nutra Bona, GS Laktobacily Forte, Pangamin Bifi Plus). Do PCR směsi byla použita celková DNA izolovaná z tablet pomocí magnetických nosičů pokrytých karboxylovými skupinami. DNA byla izolována z hrubých lyzátů buněk. Při přípravě hrubých lyzátů bylo testováno množství lysozymu (3 mg/ml, 10 mg/ml) v lyzačním roztoku, délka inkubace s lysozymem při laboratorní teplotě (1,5 hod, 3 hod) a s proteinázou K a SDS při 55 °C (1 hod, 3 hod, přes celou noc). Izolovaná DNA byla detekována pomocí agarósové gelové elektroforézy a spektrofotometricky. Přítomnost bakteriální DNA byla ověřena v PCR .K identifikaci byly použity specifické primery pro rod Lactobacillus, Bifidobacterium a druhy Lb. acidophilus, Lb. casei/paracasei, Lb.rRhamnosus, Lb. Plantarum, B. animalis, B. bifidum, B. infantis, B. longum. U třech probiotických preparátů (Zenflo, Linex Forte a Pangamin Bifi Plus) byla prokázána shoda PCR produktů s deklarovanými údaji. U ostatních probiotických tablet (Probian, Nutra Bona, GS Laktobacily Forte) byla shoda jen na úrovni rodů.Nowadays, probiotic lactic acid bacteria (LAB) and bifidobacteria are plentifully exploited in food processing industry. LAB and bifidobacteria are important part of microflora of gastro intestinal tract (GIT). Probiotics (most often just lactobacilli and bifidobakteria) can be supplied to the GIT of the organism like food complements. Species identification is therefore very important. New methods of identification of LAB and bifidobacteria are based on analysis of DNA. Mostly exploited method is polymerase chain reaction (PCR). In my diploma work, genus and species specific PCRs were used for identification of different species of bacteria of genus Lactobacillus and Bifidobacterium in complex matrices of six food supplements (Zenflo, Linex Forte, Probian, Nutra Bona, GS lactobacillus Forte, Pangamin Bifi plus). Total DNA was isolated from crude lysates of cells present in tablets by magnetic particles coated by carboxyl groups . The preparation of cell lysates was optimalised. Different amounts of lysozyme (3 mg/ml, 10 mg/ml), time of incubation at laboratory temperature (1,5 hour, 3 hour) and time of incubation with SDS and proteinase K at 55 °C (1 hour, 3 hour, over all night) were tested. Isolated DNA was quantified and checked in PCR. Primers specific for genus Lactobacillus and Bifidobacterium and for species Lb. acidophilus, Lb. casei, Lb. rhamnosus and Lb. plantarum and B. animalis, B. bifidum, B. infantis, B. longum were used, respectively. All identified bacteria were in accord with the data declared by producer in 3 food supplements (Zenflo, Linex Forte and Pangamin Bifi Plus). The genus indentification was in accord with declaration of producer in other food products only (Probian, Nutra Bona, GS Laktobacily Forte).

Implementation of technical infrastructure in the Brno metropolitan area

Tato bakalářská práce se zaobírá tématem hodnocení realizace technické infrastruktury v Brněnské metropolitní oblasti. V první části, se věnuje programovému období 2014–2020, dále pak popisu strukturálních fondů a operačních programů. Závěr teoretické části je zaměřený na popis územně integrovaných investic. V praktické části je provedeno hodnocení přípravy projektů realizace technické infrastruktury vybraných obcí Tišnovska a Brna.This bachelor thesis deals with the topic of evalution the implementation of technical structure in Brno metropolitan area. First part deals with the programming period 2014–2020, next part is about description of the Structural Funds and operational programs. The end of theoretical part is focused on describing territorial integrated investments. In practical part you can find evaluation of preparation of implementation of technical structure selected in municipalities of Tišnov and Brno.

DETERMINATION OF THE GEOMETRICAL AND VISCOELASTIC PROPERTIES OF SCAFFOLDS MADE BY ADDITIVE MANUFACTURING USING BIOPLOTTER

Additive Manufacturing (AM) is a name of a group of technologies that build 3D objects by adding layer-upon-layer of material. There are many technologies, including Rapid Prototyping (RP), Direct Digital Manufacturing (DDM), layered manufacturing and additive fabrication. Many types of materials can be used for AM technology. Biodegradable polymers such as polylactic acid (PLA) and polyhydroxybutyrate (PHB), are currently the subject of intensive research in the field of additive manufacturing and regenerative medicine. A number of biodegradable and bioresorbable materials, as well as scaffold designs, have been experimentally and clinically studied in many research facilities around the world. For effective using of bioprinting technologies in tissue and biomedical engineering, the knowledge of material and technological parameters in the process of printing is necessary. In this study the 3D printer Bioplotter EnvisionTEC (the printer with ability to print different materials from hydrogel to plastic materials) was used. Scaffolds for the purpose of the experiment were prepared via extrusion-based bioprinting. Experimental part of this study was focused on defining the influence of printing parameters and technological pre-processing of the material on quality and mechanical and geometrical properties of printed parts. Testing of printed samples showed high influence of pre-processing of material, mainly drying process, on mechanical and geometric quality of samples. Drying of material before printing process makes the material more stable and allows it to maintain defined material properties for a longer time than non-dried material. Time of heating of the material in printing cartridge has also high impact on material behaviour. Test results showed that if the time of heating of the material in the high temperature cartridge exceeds defined time limit, the material starts to degrade and is no more usable

THE INFLUENCE OF CONSTRUCTION OF NON-WOVEN TEXTILES ON AIR PERMEABILITY FOR THEIR APPLICATION IN THE COMMUNITY FACE MASKS

The article is aimed at evaluating the air permeability of various types of non-woven fabrics prepared by Spunbond, Spunbond-Meltblown-Spunbond technology and non-woven fabrics reinforced mechanically by needling and thermal calendering. These are commercially available non-woven fabrics with various mass per unit area and thickness made of 100 % polypropylene, without special surface finish. By combining individual types of non-woven fabrics, three-layer and four-layer textile materials were prepared, meeting the minimum requirement of technical standardization information TNI CWA 17553 for air permeability, intended for application in the community face masks. As part of the experimental works, the effect of increased humidity on the air permeability of three-layer non-woven fabrics was verified as well

Dimensional Accuracy and Final Density Measurement of One-, Three-, and Eight-Unit Fixed Dental Frameworks Based on Co-Cr, Manufactured by Using Conventional, Additive and Subtractive Technologies

For the aim of this study, 90 fixed metal frameworks based on Co-Cr were made by using conventional lost vax technique, additive 3D printing done by Selective Laser melting (SLM) technique and subtractive manufacturing using CNC milling. Constructions were produced by the Ceramill Motion 2 milling machine (Amann Girrbach) and Mlab Cusing R 3D printer (GE Additive). Samples were made as one-, three- and eight-unit frameworks based on existing clinical cases. Initial stl. model was built up on a scanned plaster model of three clinical cases by ZirconZahn Modellier software. Evaluation of dimensional accuracy was made by comparison of initial stl. model with a scan of manufactured framework and analyzed by measurement software.  Density measurements were made by helium (He) based gas pycnometry.  Gained data was statistically analyzed by using T-test and F-test for technologies comparison and non-parametric form of ANOVA: Kruskal Wallis was applied in density evaluation of all samples