Karakterizacija i determinacija bakteriocina autohtonih laktokoka

Lactic acid bacteria (LAB), synthesized a number of different antimicrobial substances among wich bacteriocins are the most important. Bacteriocins of LAB, with regard to the GRAS (Generaly Recognized As Safe) status of LAB, can be used as food preservatives, but also in pharmaceutical industry as an alternative or supplement to antibiotic therapy. A large number of species of the genus Lactococcus sp., not only to play a key role in the production of fermented food where they contribute to specific aroma and structure of fermented products, but also they have ability to synthesize different bacteriocins. Bacteriocins are small, ribosomaly synthesized peptides or proteins with relatively narrow spectrum of antimicrobial activity which is limited to closely related species, and producer has specific mechanism of self-protection. The aim of this thesis was to select a natural isolates of lactococci which have ability to produce bacteriocins. Then, to characterize the selected bacteriocins, to determine their antimicrobial potential and analyze potential applications in the food industry. The strains Lactoccus lactis ssp. lactis BGBM50, LMG2081 and BGBU1-4, whose bacteriocins were analyzed in this paper, were determined using conventional microbiological-biochemical tests and molecular methods of determination (sequencing of gene for 16S rRNA). Testing ability of lactococci to produce bacteriocins, was performed using bacteriocin activity assay in which as sensitive strains were used lactococci, lactobacilli and pathogenic bacteria. Determinations of bacteriocins were performed using cross-resistance test between bacteriocin producer strains and by using PCR method with specific primers for the knowing genes coding for different bacteriocins. It was found that the strain L. lactis ssp. lactis BGBM50 synthesizes bacteriocin named BacBM50. Plasmid curing experiments showed that genes for bacteriocin production are located on the big plasmid of 145,5 kb...Bakterije mlečne kiseline (BMK), sintetišu veliki broj antimikrobnih jedinjenja, među kojima značajno mesto zauzimaju bakteriocini. Bakteriocini, s obzirom na GRAS (Generalno Prepoznati Kao Sigurni) status koje imaju BMK, mogu se primenjivati kao konzervansi hrane, ali i u farmaceutskoj industriji kao alternativa ili dodatak antibiotskoj terapiji. Veliki broj vrsta roda Lactococcus sp., pored toga što zauzima ključnu ulogu u proizvodnji fermentisane hrane gde doprinose specifičnoj aromi i strukturi fermentisanih proizvoda, ima i sposobnost sinteze različitih bakteriocina. Bakteriocini su mali, ribozomalno sintetisani peptidi ili proteini koji imaju relativno uzak spektar antimikrobne aktivnosti koja je ograničena na blisko srodne vrste i prema kojoj soj proizvođač ima mehanizam specifične samozaštite. Cilj ove teze, bio je da se odaberu prirodni izolati laktokoka koji imaju sposobnost produkcije bakteriocina. Zatim, da se odabrani bakteriocini okarakterišu, da se utvrdi njihov antimikrobni potencijal i analizira potencijalna primena u prehrambenoj industriji. Sojevi L. lactis ssp. lactis BGBM50, LMG2081 i BGBU1-4, čiji su bakteriocini analizirani u ovom radu, determinisani su korišćenjem klasičnih mikrobiološko-biohemijskih metoda, kao i metoda molekularne determinacije (sekvenciranjem gena za 16S rRNK). Testiranje laktokoka na sposobnost produkcije bakteriocina, urađeno je primenom bakteriocinskih testova sa senzitivnim sojem laktokoka, laktobacila i patogenih bakterija kao indikatorima. Determinacija bakteriocina je određena testovima unakrsne inhibicije između sojeva bakteriocin proizvođača, korišćenjem PCR metoda sa specifičnim prajmerima za gene koji kodiraju različite bakteriocine i analizom sekvence genoma..

Characterization and determination of bacteriocins produced by autochthonous Lactococci

Bakterije mlečne kiseline (BMK), sintetišu veliki broj antimikrobnih jedinjenja, među kojima značajno mesto zauzimaju bakteriocini. Bakteriocini, s obzirom na GRAS (Generalno Prepoznati Kao Sigurni) status koje imaju BMK, mogu se primenjivati kao konzervansi hrane, ali i u farmaceutskoj industriji kao alternativa ili dodatak antibiotskoj terapiji. Veliki broj vrsta roda Lactococcus sp., pored toga što zauzima ključnu ulogu u proizvodnji fermentisane hrane gde doprinose specifičnoj aromi i strukturi fermentisanih proizvoda, ima i sposobnost sinteze različitih bakteriocina. Bakteriocini su mali, ribozomalno sintetisani peptidi ili proteini koji imaju relativno uzak spektar antimikrobne aktivnosti koja je ograničena na blisko srodne vrste i prema kojoj soj proizvođač ima mehanizam specifične samozaštite. Cilj ove teze, bio je da se odaberu prirodni izolati laktokoka koji imaju sposobnost produkcije bakteriocina. Zatim, da se odabrani bakteriocini okarakterišu, da se utvrdi njihov antimikrobni potencijal i analizira potencijalna primena u prehrambenoj industriji. Sojevi L. lactis ssp. lactis BGBM50, LMG2081 i BGBU1-4, čiji su bakteriocini analizirani u ovom radu, determinisani su korišćenjem klasičnih mikrobiološko-biohemijskih metoda, kao i metoda molekularne determinacije (sekvenciranjem gena za 16S rRNK). Testiranje laktokoka na sposobnost produkcije bakteriocina, urađeno je primenom bakteriocinskih testova sa senzitivnim sojem laktokoka, laktobacila i patogenih bakterija kao indikatorima. Determinacija bakteriocina je određena testovima unakrsne inhibicije između sojeva bakteriocin proizvođača, korišćenjem PCR metoda sa specifičnim prajmerima za gene koji kodiraju različite bakteriocine i analizom sekvence genoma...Lactic acid bacteria (LAB), synthesized a number of different antimicrobial substances among wich bacteriocins are the most important. Bacteriocins of LAB, with regard to the GRAS (Generaly Recognized As Safe) status of LAB, can be used as food preservatives, but also in pharmaceutical industry as an alternative or supplement to antibiotic therapy. A large number of species of the genus Lactococcus sp., not only to play a key role in the production of fermented food where they contribute to specific aroma and structure of fermented products, but also they have ability to synthesize different bacteriocins. Bacteriocins are small, ribosomaly synthesized peptides or proteins with relatively narrow spectrum of antimicrobial activity which is limited to closely related species, and producer has specific mechanism of self-protection. The aim of this thesis was to select a natural isolates of lactococci which have ability to produce bacteriocins. Then, to characterize the selected bacteriocins, to determine their antimicrobial potential and analyze potential applications in the food industry. The strains Lactoccus lactis ssp. lactis BGBM50, LMG2081 and BGBU1-4, whose bacteriocins were analyzed in this paper, were determined using conventional microbiological-biochemical tests and molecular methods of determination (sequencing of gene for 16S rRNA). Testing ability of lactococci to produce bacteriocins, was performed using bacteriocin activity assay in which as sensitive strains were used lactococci, lactobacilli and pathogenic bacteria. Determinations of bacteriocins were performed using cross-resistance test between bacteriocin producer strains and by using PCR method with specific primers for the knowing genes coding for different bacteriocins. It was found that the strain L. lactis ssp. lactis BGBM50 synthesizes bacteriocin named BacBM50. Plasmid curing experiments showed that genes for bacteriocin production are located on the big plasmid of 145,5 kb..

Fracture toughness of zirconia ceramic crowns made by feather-edge tooth preparation design

Background/Aim. Fracture toughness determines functional crown strenght and prevents damages on ceramics during mastication. There is a lack of relevant literature data about fracture toughness of crowns made by feather-edge preparation. Mechanical testing of ceramic samples is supposed to show if feather-edge tooth preparation is a successful method for making ceramic crowns without any risk of reduction of their mechanical properties. This research was done to establish effects of feather-edge tooth preparation on fracture toughness of single zirconia ceramic crowns. Methods. The research was performed as an experimental study. Sixty (60) ceramic crowns were made on non-carious extracted human premolars. Thirty (30) crowns were made on the basis of feather-edge preparation (experimental group I). The group II included 30 crowns made on 1 mm rounded shoulder. Crowns fabrication was executed on a copy mill production system “Zirkonzahn” (Zirkonzahn GMBH, Gais, Germany). The spherical compression test was used to determine fracture toughness, using 6 mm diameter ceramic ball. Fracture load for damaging ceramic crown was recorded on a universal testing machine - Zwick, type 1464, with the speed of 0.05 mm/min. Results. The results of this research introduced significant differences between fracture toughness of ceramic samples in every examined group. However, fracture toughness of crowns from both group was above 2 000 N, what was double beyond a recommended value. The mean value of fracture toughness in the feather-edge group was 2 090 N, and in shoulder group it was 2 214 N. Conclusion. This research showed a high fracture toughness of zirconia crowns made on feather-edge preparation. The examined crowns showed a fracture resistance at a sufficient distance in relation to the minimum values of functional loads. Further research of functional loads of these crown is necessary, as well as research of marginal adaptation of cemented crowns and gingival inflammatory response

Lactococcin B Is Inactivated by Intrinsic Proteinase PrtP Digestion in Lactococcus lactis subsp. lactis BGMN1-501

In our previous study we demonstrated that proteinase PrtP is able to impair bacteriocin LcnB activity, despite being produced by the same organism and encoded by the same plasmid. However, precise mechanism of this action, i.e., the exact cleavage site within LcnB bacteriocin, as well as its effect on antimicrobial activity of the resulting peptide remained vague. Here we further explored the interplay between these two proteins and defined, using mass spectrometry, that this unusual hydrolysis indeed occurs in vivo, between the sixth and seventh amino acid on the N terminus of LcnB. To address whether the cleaved form of LcnB retains any level of activity, both recombinant and chemically synthesized variant of truncated LcnB were engineered and produced, but demonstrated no antimicrobial activity. When LcnB was recombinantly overexpressed and subjected to PrtP digestion, the change in its antimicrobial activity was monitored and the degradation products analyzed with reverse-phase high-pressure liquid chromatography. The results confirmed the inactivity of the truncated LcnB and additionally corroborated the PrtP cleavage site in LcnB bacteriocin. In addition, it was demonstrated that, once truncated, LcnB is not able to bind its receptor and is susceptible to additional hydrolysis. This is the first report on proteolytic inactivation of bacteriocins inside the same bacterial host

Srpski crni tartuf tuber aestivum: mikrobiota I uticaj različitih režima smrzavanja na isparljiiva aromatična jedinjenja tokom skladištenja

The use of truffles in food is based mainly on the addition of artificial flavors, aiming to achieve an intense aroma in the products. As truffle is a natural product with nutritional and functional properties, it is important to find an optimal method for truffle storage. As the microbiota contribute to truffle aroma, the bacterial and yeast compositions in the rhizosphere and fruiting body of the truffle and the impact of different freezing methods on the volatile profile of the truffle Tuber aestivum during 90 days of the storage were determined. Bacteria and yeasts isolates were identified using 16s rRNA and 18s rRNA. The effect of freezing truffles at -20°C and -80°C with and without previous dipping in liquid N2 on the volatile compounds was observed using GC/MS. The results demonstrated that the isolated bacteria belonged to the phylum Proteobacteria, Firmicutes and Actinobacteria, and the identified species mainly belonged to Firmicutes, genus Bacillus sp. Isolated yeasts were identified as Cryptococcus sp., Debaromyces hanseinii, Candida fermentati and Rhodotorula mucilaginosa. The GC/MS analysis revealed that frozen truffle samples were richer in the compounds 2-butanone, 2-methyl-butanal, methanethiol and 2-butanol after freezing or during storage. The content of DMS, acetaldehyde, 3-octanone, ethanol, and 2-methyl-1-propanol significantly decreased immediately after freezing. Overall, the gained results indicated that freezing of truffles as a preservation method had profound effects on the volatile compounds, while previous dipping in liquid N2 showed no significant impact on the volatile profile of truffle Tuber aestivum.Primena tartufa u hrani se uglavnom zasniva na dodavanju veštačkih aroma, sa ciljem postizanja intenzivne arome tartufa u proizvodima. S obzirom na to da su tartufi proizvodi sa nutritivnim i funkcionalnim karakteristikama, važno je pronaći optimalan način za skladištenje tartufa. Pošto mikrobiota doprinosi aromi tartufa, sastav bakterija i kvasaca u rizosferi i plodonosnom telu srpskog tartufa, kao i uticaj različitih metoda smrzavanja na isparljiva jedinjenja tartufa Tuber aestivum ispitivan je tokom 90 dana skladištenja. Bakterije i kvasci su izolovani iz svežeg tartufa i izolati su identifikovani korišćenjem 16s rRNK i 18s rRNK. Uticaj smrzavanja tartufa na -20°C i -80°C sa i bez prethodnog potapanja u tečni N2 na sadržaj isparljivih jedinjenja ispitivan je korišćenjem GC/MS. Rezultati ispitivanja su pokazali da izolovane bakterije pripadaju carstvu Proteobacteria, Firmicutes i Actinobacteria, pri čemu identifikovane vrste uglavnom pripadaju Firmicutes, rod Bacillus sp. Izolovani kvasci su identifikovani kao Cryptococcus sp., Debaromyces hanseinii, Candida fermentati i Rhodotorula mucilaginosa. Analiza GC/MS je ukazala na razlike u profilu isparljivih jedinjenja svežeg i smrznutog tartufa. Smrznuti uzorci su više sadržali komponente kao što su 2-butanon, 2-metil-butanal, metanetiol i 2-butanol nakon smrzavanja ili tokom skladištenja. Sadržaj DMS, acetaldehida, 3-octanona, etanola, 2-metil-1-propanola značajno se smanjio odmah nakon smrzavanja. Dobijeni rezultati ukazuju da smrzavanje tartufa kao metoda konzervacije ima značajan uticaj na isparljiva jedinjenja, pri čemu potapanje u tečni N2 nije pokazalo značajan uticaj na isparljiva jedinjenja tartufa Tuber aestivum

Supplementary data for the article: Vatić, S.; Mirković, N.; Milošević, J. R.; Jovčić, B.; Polović, N. Đ. Broad Range of Substrate Specificities in Papain and Fig Latex Enzymes Preparations Improve Enumeration of Listeria Monocytogenes. International Journal of Food Microbiology 2020, 334, 108851. https://doi.org/10.1016/j.ijfoodmicro.2020.108851

Supplementary material for: [https://doi.org/10.1016/j.ijfoodmicro.2020.108851]Related to published version: [https://cherry.chem.bg.ac.rs/handle/123456789/4090

The Sensory Quality and Volatile Profile of Dark Chocolate Enriched with Encapsulated Probiotic Lactobacillus plantarum Bacteria

peer-reviewedCocoa and dark chocolate have a wide variety of powerful antioxidants and other nutrients that can positively affect human health. Probiotic dark chocolate has the potential to be a new product in the growing number of functional foods. In this study, encapsulated potential probiotic Lactobacillus plantarum 564 and commercial probiotic Lactobacillus plantarum 299v were added in the production of dark chocolate. The results show very good survival of probiotic bacteria after production and during storage, reaching 108 cfu/g in the first 60 days and over 106 cfu/g up to 180 days. No statistically significant difference (p > 0.05) in chemical composition and no major differences in the volatile profiles between control and experimental chocolate samples were observed, indicating no impact of probiotic bacteria on compositional and sensory characteristics of dark chocolate. The sensory evaluation of control and both probiotic dark chocolate samples showed excellent sensory quality after 60 and 180 days of storage, demonstrating that probiotics did not affect aroma, texture and appearance of chocolate. Due to a high viability of bacterial cells and acceptable sensory properties, it can be concluded that encapsulated probiotics Lb. plantarum 564 and Lb. plantarum 299v could be successfully used in the production of probiotic dark chocolate

Accuracy of optical scanning methods of the Cerec®3D system in the process of making ceramic inlays

Background/Aim. One of the results of many years of Cerec® 3D CAD/CAM system technological development is implementation of one intraoral and two extraoral optical scanning methods which, depending on the current indications, are applied in making fixed restorations. The aim of this study was to determine the degree of precision of optical scanning methods by the use of the Cerec®3D CAD/CAM system in the process of making ceramic inlays. Methods. The study was conducted in three experimental groups of inlays prepared using the procedure of three methods of scanning Cerec ®3D system. Ceramic inlays made by conventional methodology were the control group. The accuracy of optical scanning methods of the Cerec®3D system computer aided designcomputer aided manufacturing (CAD/CAM) was indirectly examined by measuring a marginal gap size between inlays and demarcation preparation by scanning electron microscope (SEM). Results. The results of the study showed a difference in the accuracy of the existing methods of scanning dental CAD/CAM systems. The highest level of accuracy was achieved by the extraoral optical superficial scanning technique. The value of marginal gap size inlays made with the technique of extraoral optical superficial scanning was 32.97 ± 13.17 μ. Techniques of intraoral optical superficial and extraoral point laser scanning showed a lower level of accuracy (40.29 ± 21.46 μ for inlays of intraoral optical superficial scanning and 99.67 ± 37.25 μ for inlays of extraoral point laser scanning). Conclusion. Optical scanning methods in dental CAM/CAM technologies are precise methods of digitizing the spatial models; application of extraoral optical scanning methods provides the hightest precision

Karakterizacija autohtonih sojeva Lactobacillus paracasei na potencijalne probiotičke sposobnosti

Lactic acid bacteria strains isolated from traditional made cheeses constitute a reservoir of unexplored potential in biotechnology. In this study four autochthonous lactobacilli strains, isolated from traditional white brined cheeses and identified as Lactobacillus paracasei (08, 564, 05 and 02), were investigated on potential probiotic ability. The investigation comprised sensitivity to simulated gastrointestinal tract conditions, antimicrobial activity against wide range of pathogens, antibiotic resistance as well as autoaggregation ability. Lactobacillus rhamnosus GG was used as referent strain. Three tested strains grew well in simulated gastrointestinal conditions, but their sensitivity was greater on bile acids and pancreatin compared with pepsin low pH 2.5. The examined strains had different sensitivity to antibiotics, but three strains showed very good antimicrobial activity to pathogens. All strains demonstrated very good autoaggregation ability. For three of four examined strains of Lb. paracasei probiotic potential was similar with referent strain Lb. rhamnosus GG, determined in vitro.Bakterije mliječne kiseline izolirane iz tradicionalnih sireva predstavljaju neiscrpan potencijal u biotehnologiji. U ovom radu 4 soja laktobacila, izolirana iz autohtonih bijelih sireva u salamuri i identificirana kao Lactobacillus paracasei (08, 564, 05 and 02), ispitivana su na potencijalne probiotičke sposobnosti. Ispitivanje je obuhvatilo osjetljivost na simulirane gastrointestinalne uvjete, antimikrobnu aktivnost u odnosu na patogene, antibiotsku rezistentnost i sposobnost autoagregacije. Lactobacillus rhamnosus GG upotrijebljen je kao referentni soj. Tri testirana soja dobro su rasla u simuliranim gastrointestinalnim uvjetima, ali je njihova osjetljivost bila veća u prisustvu žučne kiseline i pankreatina u usporedbi s pepsinom niskog pH 2,5. Ispitivani sojevi pokazali su različitu osjetljivost na antibiotike, a 3 soja su pokazala dobru antimikrobnu aktivnost u odnosu na patogene. Svi su sojevi pokazali visoku sposobnost autoagregacije. Od 4 ispitana soja Lb. paracsei, tri su pokazala dobar probiotički potencijal u usporedbi s referentnim sojem Lb. rhamnosus GG, in vitro uvjetima

Isolation and Characterisation of Bacteriocin and Aggregation-Promoting Factor Production in Lactococcus lactis ssp lactis BGBM50 Strain

Lactococcus locus ssp. lactis BGBM50, a producer of lactococcin G and aggregation-promoting factor, was isolated from selected lactic acid bacteria taken from semi-hard cheese traditionally produced in the village Zanjic, Montenegro. Strain BGBM50 harbours a number of plasmids of different sizes. Plasmid curing experiments showed that genes for bacteriocin production are located on pBM140, a plasmid 140 kb in length. PCR analysis with primers specific for lactococcin Q and G genes gave fragment of the expected size. In addition, after plasmid curing of strain BGBM50, different derivatives with altered phenotypes were obtained, among them BGBM50-34 strain, which retained bacteriocin synthesis but had enhanced aggregation ability