PCR provjera identifikacija fenotipskog sistema s mikropločama za BMK iz tradicionalnih sireva od sirovog mlijeka sa Zapadnog Balkana

Fermentation and ripening specificity of traditional cheeses are predominantly directed by the natural microbial community present in milk selected by the cheese-making environment and technology. Therefore the traditional cheeses are unique products with specific microbiota biodiversity. There are several approaches for the identification of microbial population, however all of them have certain advantages and disadvantages. In this study the eligibility and performance of the Biolog phenotypic identification system (Biolog, Inc.) with GEN III microplates was tested. Parallel to this method, polymerase chain reaction with genus- and species-specific primers was performed. One hundred sixty-five isolates from nine types of artisan cheeses were isolated and analysed. Cheeses were produced from raw ewe’s milk in Slovenia, Bosnia and Herzegovina, Croatia and Serbia. The Biolog phenotypic identification system identified 90 isolates, but only 55 identifications acquired by the Biolog system were supported by polymerase chain reaction at a genus level and 28 at a species level. The obtained results showed that the reliability of commercial phenotypic identification systems was inadequate when analysing lactic acid bacteria isolates from natural, spontaneous fermentations and needs to be additionally corroborated by genotypic identification methods.Fermentacija i specifično zrenje sira najvećim je dijelom uvjetovano prirodnom mikrobnom zajednicom prisutnom u mlijeku, a koja se selekcionira uz pomoć sirarskog okoliša i tehnologije. Stoga su tradicionalni sirevi unikatni proizvodi sa specifičnom mikrobnom bioraznolikošću. Postoje različite mogućnosti identifikacije mikrobne populacije, međutim svi pristupi imaju svoje prednosti i nedostatke. U ovom istraživanju testirana je prikladnost i učinkovitost fenotipskog identifikacijskog sustava Biolog (Biolog, Inc.) s GEN III mikropločama. Usporedno je izvođena lančana reakcija polimeraze s rodom i vrstom specifičnih početnica. U ovom je istraživanju izolirano i analizirano ukupno 165 izolata porijeklom od devet vrsta tradicionalnih sireva. Sirevi su bili proizvedeni od sirovog ovčjeg mlijeka u Sloveniji, Bosni i Hercegovini, Hrvatskoj i Srbiji. Fenotipski identifikacijski sustav Biolog identificirao je 90 izolata. 55 identifikacija bilo je potvrđeno metodom lančane reakcije polimeraze na razini roda, a 28 na razini vrsta. Rezultati su pokazali da je pouzdanost komercijalnog fenotipskog identifikacijskog sistema neadekvatna kada se analiziraju izolati mliječno kiselinskih bakterija prirodnih i spontanih fermentacija. Taj identifikacijski sistem potrebno je dodatno potvrditi genotipskim identifikacijskim metodama

Cultivable Bacteria from Milk from Slovenian Breastfeeding Mothers

Majčino mlijeko ima važnu ulogu u razvoju crijevne mikrobiote, te u zaštiti dojenčadi od patogenih mikroorganizama. Svrha je ovoga rada bila istražiti mikrobni sastav majčinog mlijeka, uzorkovanog iz lijeve (L) i desne (R) dojke 47 dojilja, tridesetog dana nakon poroda. Kvantificirane su neke glavne skupine bakterija u majčinom mlijeku, uspoređene kultivabilne bakterije iz lijeve i desne dojke, te je identificirana raznolikost sojeva laktobacila. Rezultati pokazuju da majčino mlijeko sadržava bakterije mliječne kiseline, bifidobakterije i mezofilne aerobne bakterije, od kojih je posljednja skupina najviše zastupljena. Iako je mikrobiološki sastav majčinog mlijeka u uzorcima L i R bio usporediv, koncentracija se bakterija u ta dva uzorka od iste majke mogla razlikovati, pa uzorak mlijeka uzet samo iz jedne dojke ne odražava prosječni mikrobiološki sastav. Na osnovi rezultata dobivenih metodom RAPD (engl. random amplified polymorphic DNA), 86 je bakterijskih sojeva za koje se pretpostavljalo da pripadaju rodu Lactobacillus razvrstano u jedanaest skupina, te identificirano sekvenciranjem 16S rDNA. Od jedanaest analiziranih skupina izolata, za njih je četiri (21 % svih sojeva) ustanovljeno da pripadaju vrsti Lactobacillus gasseri. Utvrđeno je da 48 % izolata najzastupljenijeg profila RAPD pripada vrsti Lactobacillus fermentum, a u preostalim su skupinama izolata identificirane vrste L. salivarius, L. reuteri, Enterococcus faecium, Staphylococcus epidermidis i Bifidobacterium breve.The human milk microbiota plays an important role in the development of infant´s intestinal microbiota and in the protection of infants against pathogenic microorganisms. The aim of this study is to investigate the microbial composition of human milk from 47 breastfeeding mothers, sampled separately from the left (L) and the right (R) breast, on the 30th day after giving birth. We quantified some major bacterial groups in human milk, compared the cultivable bacteria from the left and the right breast and identified strain diversity of lactobacilli. The results revealed that human milk contains lactic acid bacteria, bifidobacteria and mesophilic aerobic bacteria, of which the last were the most abundant group. Although the microbial composition of human milk in L and R breast samples was comparable, the concentration of bacteria in the two samples from the same mother might vary, therefore milk sample taken from one breast only does not reflect the average microbial composition. Using random amplified polymorphic DNA (RAPD), 86 presumptive isolates of lactobacilli from representative samples of human milk from 11 mothers were classified into 11 groups. Moreover, representatives of different RAPD groups were identified using 16S rDNA sequencing. Out of 11 RAPD groups, 4 groups (21 % of all isolates) belonged to the species Lactobacillus gasseri. The most representative RAPD profile (48 % of isolates) was found to belong to the species Lactobacillus fermentum. Other RAPD groups were associated with L. salivarius, L. reuteri, Enterococcus faecium, Staphylococcus epidermidis and Bifidobacterium breve species

How Can We Advance Integrative Biology Research in Animal Science in 21st Century?:Experience at University of Ljubljana from 2002 to 2022

In this perspective analysis, we strive to answer the following question: how can we advance integrative biology research in the 21st century with lessons from animal science? At the University of Ljubljana, Biotechnical Faculty, Department of Animal Science, we share here our three lessons learned in the two decades from 2002 to 2022 that we believe could inform integrative biology, systems science, and animal science scholarship in other countries and geographies. Cultivating multiomics knowledge through a conceptual lens of integrative biology is crucial for life sciences research that can stand the test of diverse biological, clinical, and ecological contexts. Moreover, in an era of the current COVID-19 pandemic, animal nutrition and animal science, and the study of their interactions with human health (and vice versa) through integrative biology approaches hold enormous prospects and significance for systems medicine and ecosystem health

Mechanisms of innate immunity activation in intestinal epithelial cells with selected probiotic bacterial strains

Manipulacija črevesne mikrobiote s koristnimi mikroorganizmi predstavlja obetavno alternativo in podporo za zdravljenje črevesnega vnetja in motenj v delovanju črevesne epitelne bariere. Probiotiki lahko namreč stimulirajo tako pridobljeni kot tudi naravni imunski odziv. Namen raziskave je bil zato pojasniti signalne poti, ki jih v črevesnih epitelnih celicah sprožijo probiotični bakterijski sevi Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) in Lactobacillus plantarum WCFS1 (WCFS1). Izhodišče raziskave je bila in vivo študija, v kateri so na miših s povzročenim kolitisom proučevali zaščitni vpliv sevov L930BB in IM386. Rezultati analize transkriptoma mišjega kolona so pokazali spremembe v izražanju genov, ki so povezani z aktivacijo proti-apoptostskih poti preko fosfatidil inozitol 3-kinaze (PI3K)/Akt in aktivacijo poti, ki vodijo v regulacijo aktinskega citoskeleta in tesnih stikov preko protein-kinaze C (PKC). Tako reorganizacija aktinskega citoskeleta, kot tudi zmanjšana apoptoza, prispevata k obnavljanju monosloja črevesnih epitelnih celic. V in vitro poskusih smo dokazali, da so aktivirane signalne poti posledica signalizacije preko receptorja naravne imunosti, Toll-u podobnega receptorja 2 (TLR2). Z uporabo pretočne citometrije smo potrdili, da so probiotični sevi in ligand za TLR2 sposobni zmanjšati s citokini povzročeno celično smrt. S pregledovanjem črevesne epitelne celične linije Caco-2 pod konfokalnim mikroskopom pa smo opazili, da probiotični sevi, ob simulaciji vnetnih razmer s H2O2, zmanjšajo internalizacijo proteina tesnih stikov ZO-1 in tako stabilizirajo medcelične povezave. V stanju vnetja se je ob dodatku probiotičnih sevov, zmanjšala tudi permeabilnost celičnega monosloja, kar dodatno dokazuje zaščitni vpliv izbranih sevov in njihovo zmožnost podpore vzdrževanja črevesne epitelne bariere.Manipulation of the intestinal microbiota with beneficial microorganisms represents a promising alternative and support to the treatment of intestinal inflammation and disorders in intestinal epithelial barrier function. Probiotics can stimulate both the adaptive and innate immune responses. The aim of the study was therefore to clarify the signalling pathways in intestinal epithelial cells triggered by probiotic bacterial strains Lactobacillus gasseri K7 (K7), Lactobacillus fermentum L930BB (L930BB), Bifidobacterium animalis subsp. animalis IM386 (IM386) and Lactobacillus plantarum WCFS1 (WCFS1). The starting point of the research was an in vivo study in which the protective effect of strains L930BB and IM386 was examined in a mouse model with induced colitis. Results of mice colon transcriptome analysis revealed changes in gene expression, involved in activation of anti-apoptotic pathways through phosphatidylinositol 3-kinase (PI3K)/Akt and activation of pathways that lead to regulation of actin cytoskeleton and tight junctions through protein kinase C (PKC). Reorganisation of actin cytoskeleton and decreased apoptosis are both helpful in intestinal epithelial cell monolayer reconstitution. We proved in in vitro experiments that activated signalling pathways are a consequence of signalization through the innate immune receptor Toll-like receptor 2 (TLR2). With the use of flow cytometry we confirmed that probiotic strains and TLR2 ligand are able to reduce cytokine induced cell death. By examining the intestinal epithelial cell line Caco-2 under the confocal microscope, we observed that probiotic strains, in addition to the simulation of inflammatory conditions with H2O2, reduce the internalization of the tight junction protein ZO-1 and thus stabilize intercellular connections. In inflammatory conditions, the addition of probiotic strains also reduced the permeability of the cell monolayer, further proving the protective effect of selected strains and their ability to support the maintenance of the intestinal epithelial barrier

Correlations of goat milk coagulation properties between dams and daughters

Goat milk coagulation properties (gMCP) are important trait for the cheesemaking industry but are rarely included in national breeding programs. In the present study, we monitored goat milk quality traits and gMCP of Slovenian Alpine goats within a single flock (N = 77), reared in the same common environment over three-year period. In addition, using pedigree data, we looked for correlations among dams, daughters (N = 32 pairs) and gMCP. Traditional gMCP measurements were introduced into a 4-parameter curd firmness over time (CFt) model. Several gMCP parameters were associated with parity and stage of lactation. Most notably, estimated rennet coagulation time (RCTeq), curd firmness at 15 (a15eq) and 20 min (a20eq) after rennet addition were strongly correlated between dams and their daughters. These results support further research of genetic parameters for gMCP and the inclusion of breeding value predictions for gMCP in national breeding programs

Characterisation of lactoferrin isolated from acid whey using pilot-scale monolithic ion-exchange chromatography

The aim of this study was to characterize the properties of lactoferrin (LF) obtained in a process developed for its isolation from acid whey derived from the production of fresh curd cheese, using a unique technology of ion-exchange chromatography on CIM^® monolithic columns. The freeze-dried lactoferrin samples produced on the pilot plant (capacity 1 m$^3$) were examined for the purity, iron-binding capacity, antibacterial activity, and pH- and temperature-stability. Apo-LF inhibited several tested strains (enterobacteria, Staphylococcus, Streptococcus salivarius) except clostridia, lactic acid bacteria, and bifidobacteria. Sample of LF intentionally saturated with Fe$^{3+}$ lost its antibacterial activity, indicating the involvement of mechanisms based on depriving bacteria of an iron source. All samples, regardless of the iron-saturation level, exhibited stability in pH range 4.0 to 11.0. LF with higher iron content (A-value = 41.9%) showed better thermal stability. Heat treatment up to 72 °C/3 s did not reduce antimicrobial activity against E. coli O157: H7 tox-. Higher purity (above 91%), higher iron-binding capacity and higher inhibitory activity against E. coli O157: H7 tox- compared to some similar products from the market was observed. These results demonstrate a high potential of monolithic ion-exchange chromatography for industrial processing of acid whey as a source of LF that can be used in new products with high-added value. The upscaling of the process is ongoing on a demonstration plant (10–30 m$^3$/day capacity)