Autohtoni sojevi roda Lactobacillus izolirani iz Istarskog sira kao potencijalne starter kulture

Istrian ewe’s milk cheese is an autochthonous product that is manufactured for generations on small family farms in the Croatian peninsula Istria. Traditional Istrian cheese is made from unpasteurized ewe’s milk, without the addition of starter cultures. Consequently, the specific flavour and texture of the Istrian cheese is owed to metabolic processes of indigenous microflora of which Lactobacillus species play pivotal role. Characterisation and selection of indigenous lactobacilli may result in the potential use of selected strains as starter, bioprotective or even probiotic cultures. This study focuses on potential use of Lactobacillus plantarum and Lactobacillus casei isolated from traditional Istrian cheese as starter cultures, by using methods that determine their proteolytic, lipolytic, antimicrobial and haemolytic potential, as well as their ability of acidification, autoaggregation and survival in simulated gastrointestinal conditions. Our results indicated that from 12 representative strains most revealed a low or moderate proteolytic activity as well as absence of lipolytic and haemolytic activities. From 12 strains, 5 of them showed a medium to strong acidification ability and lowered the pH of milk below 5.00 after 24 hours of incubation. Furthermore, almost all isolates exhibited antimicrobial activity against Serratia marcescens, and lowest number of isolates showed antimicrobial activity against Staphylococcus aureus and Listeria innocua. The studied Lactobacillus strains revealed high survival rate in a simulated oral cavity and duodenum conditions, while the survival ability in a simulated gastric conditions was much lower. Ability to aggregate was low for all tested strains, after 3 hours and after 5 hours of incubation.Tradicionalni Istarski sir proizvodi se od nepasteriziranog ovčjeg mlijeka, bez dodatka starter kultura. Prema tome, specifična aroma i tekstura Istarskog sira pripisuje se, uz ostale faktore, metaboličkim procesima autohtone mikroflore od kojih vrste roda Lactobacillus imaju ključnu ulogu. Karakterizacija i izbor autohtonih sojeva laktobacila može dovesti do potencijalnog korištenje odabranih sojeva kao starter, zaštitnih ili probiotičkih kultura. U ovom istraživanju željeli smo istražiti tehnološki i probiotički potencijal sojeva Lactobacillus plantarum i Lactobacillus casei izoliranih iz tradicionalnog Istarskog sira pomoću metoda koje određuju njihov proteolitički, kazeinolitički, lipolitički, antimikrobni i hemolitički potencijal, kao i njihovu sposobnost zakiseljavanja, autoagregacije i preživljavanja u simuliranim gastrointestinalnim uvjetima. Svih 12 reprezentativnih sojeva pokazuju nisku do umjerenu proteolitičku aktivnost te odsutnost lipolitičke i hemolitičke aktivnosti. Od 12 sojeva, 5 je pokazalo jaku sposobnost zakiseljavanja, spuštajući pH mlijeka ispod 5,00 nakon 24 sati inkubacije. Nadalje, gotovo svi izolati pokazuju antimikrobno djelovanje u odnosu na vrstu Serratia marcescens te slabo antimikrobno djelovanje u odnosu na Staphylococcus aureus i Listeria innocua. Istraživani sojevi pokazali su visoku prosječnu stopu preživljavanja u simuliranim uvjetima usne šupljine i dvanaesnika, dok je prosječna stopa preživljavanja u simuliranim želučanim uvjetima bila znatno niža. Nakon 5 sati inkubacije, autoagregacijska sposobnost svih istraživanih sojeva bila je niska

Utjecaj inkubacijske temperature i koncentracije aflatoksina M1 u mlijeku na larve račića Artemia salina

Temperature-dose relationships with aflatoxin M1 (AFM1) were studied using the brine shrimp Artemia salina larvae as an biological indicator in the temperature range from 20 °C to 40 °C. Increase in the incubation temperature resulted in sensitivity increase by the brine shrimp to AFM1. Optimum sensitivity occured at 30 °C. Positive results were obtained at 0.18 μg AFM1 x L-1 of whole pasteurized milk with a mortality of over 15%. Greater than 90 % mortality occurred at dose levels of 0.9 μg AFM1 x L-1 and above. The test can be conducted during 30-60 hours.Proučavani su utjecaji inkubacijske temperature i koncentracije aflatoksina M1 (AFM1) na larve račića Artemia salina kao biologijskog indikatora u temperaturnom rasponu od 20 °C do 40 °C. Povišenje temperature inkubacije uzrokovalo je povećanu osjetljivost račića na AFM1. Optimalna osjetljivost očitovala se pri 30 °C. Pozitivni rezultati dobiveni su pri koncentraciji AFM1 0,18 μ g x L-1 pasteriziranog mlijeka uz izraženu smrtnost višu od 15 %. Smrtnost viša od 90 % očitovala se pri koncentracijama AFM1 0,9 μ g x L-1 i višim. Test se može provesti u tijeku 30-60 sati

Spoilage microorganisms in milk and dairy products

Mikroorganizmi - uzročnici kvarenja svojim prisustvom u mlijeku i mliječnim proizvodima mijenjaju njihova primarna svojstva i osobine. Nastale promjene, koje se događaju prije i nakon tehnološke obrade, uvjetovane su brojem i vrstom mikrobnih uzročnika. Najčešće se te promjene odnose na samo jednu pogrešku okusa, mirisa, arome ili konzistencije mliječnog proizvoda. Međutim, u slučajevima većih mikrobnih kontaminacija, sve te pogreške mogu se dogoditi istovremeno. Osim toga, svaka i najmanja promjena, uvjetovana prisutnošću mikrobnih uzročnika kvarenja, umanjuje prehrambenu vrijednost proizvoda. Usprkos značenju koje ima za ukupnu kvalitetu, kontrola mikroorganizama uzročnika kvarenja za mljekarsku industriju nije obvezna, te ih u proizvodima kontrolira zanemariv broj proizvođača. U radu je opisan neželjeni učinak mikroorganizama uzročnika kvarenja na kvalitetu sirovog, pasteriziranog i steriliziranog mlijeka, fermentiranih mlijeka, vrhnja, maslaca i sira s namjerom da se potakne važnost i značenje njihove kontrole u mljekarskoj industriji.Spoilage microorganisms cause changes of primary characteristics and properties of milk and dairy products. The product defects depends on the specific species and number of microorganisms involved in pre- and post- technological processing. Most often, these changes are related to single undesirable sensory characteristic, smell, flavour or conistency. However, in the case of heavier microbial contamination all these undesirable characteristics can occur simultaneously. Besides, even small changes caused by presence of spoilage microorganisms lead to decreased quality of milk and various dairy products. Despite of the importance for the overall quality, the control of spoilage microorganisms for dairy industry is not obligated and therefore, only a few producers control them. Therefore, the present study describes the undesirable effect of spoilage microorganisms on quality of raw, pasteurized and sterilized milk, fermented milk, butter, sour cream and cheeses with the intention to emphasize the importance and significance of their control in the dairy industry

Microbial Communities and Biofilms

Sve do godine 1960. mnoga stajališta okolišne mikrobiologije bila su postupci na osnovi promatranja. U novije doba bilo je izvrsnih prosudbi za razumijevanje ekonomski važnih neobičnih pojava, poput simbiotskih odnosa između bakterija iz roda Rhizobium i leguminoznih biljaka, dok je osnova bila temeljno razumijevanje odnosa između mikrobnoga rasta i fizičkih parametara, poput temperature i različitih tipova međudjelovanja između različitih mikroba u danome okolišu. Sveukupno, međutim, velik broj opažanja ostao je slabo objašnjen i po mnoge mikrobiologe činilo se da su različiti primjeri upotrijebljeni radi objašnjenja i interpretacije ponašanja mikroba u prirodnome okolišu bili manjkavi. U protekla tri desetljeća situacija se bitno promijenila i svo vrijeme, na sreću okolišnih mikrobiologa, mnogi slabo razumljivi zaostaci, sada ponovo egzistiraju kao poredak razumljiv koji je iznimno proširio razumijevanje odnosa između mikroba i njihova okoliša. Dva primjera za to jesu razumijevanje tvorbe biofilma i njegova okolišna važnost i strategija preživljavanja mikroba koji ne tvore endospore.Until the 1960s many aspects of environmental microbiology were dealt with on an observational basis. Certainly there was a good measure of understanding of economically important phenomena, such as the symbiotic relationship between Rhizobium and leguminous plants, while there was a basic understanding of the relationship between microbial growth and physical parameters, such as temperature and of the various types of interaction between different microorganisms in the same environment. Overall, however, many observations remained poorly explained and, to many microbiologists, it appeared that the various paradigms used to explain and interpret the behaviour of microorganisms in natural environments were defi cient. Over the past two decades, the situation has changed and while, happily for environmental microbiologists, much remains less than fully understood, there now exists a framework of knowledge which has greatly expanded the understanding of the relationship between microorganisms and their environments. Two examples are an understanding of biofi lm formation and its environmental importance and of the survival strategies of non-endospore forming microorganisms

Genetic Diversity of Indigenous Saccharomyces sensu stricto Yeasts Isolated from Southern Croatia

Alcoholic fermentation is a polimicrobial process involving a large number of genera and species of yeast and bacteria. The major yeast genera involved in this process belongs to the Saccharomyces spp. The aim of this study was the isolation, identification and determination of genetic diversity of indigenous Saccharomyces cerevisiae natural population taken from cv. Plavac mali from secluded wine growing areas of Southern Croatia. Must samples were taken during the spontaneous alcoholic fermentation followed by yeast isolation. A total of 40 isolates that were physiologically confirmed to belong to the Saccharomyces sensu stricto group were furthermore differentiated by molecular methods. PCR-RFLP analyses of the internal transcribed spacer (ITS1) region of the 18S ribosomal DNA identified 37 of the isolates as S. cerevisiae and two of the isolates as S. bayanus/pastorianus. All isolates were further analyzed by RAPD fingerprinting. The results of this study showed that in some cases the RAPD assay may be useful to separate species within the Saccharomyces sensu stricto group. The molecular analysis confirmed genetic diversity of S. cerevisiae indigenous population and additionally the involvement of indigenous S. paradoxus and S. bayanus was determined. The population structure of Saccharomyces cerevisiae has indicated that each vineyard is characterized with particular S. cerevisiae microflora. It is an important step towards the preservation and exploitation of yeast biodiversity in Southern Croatia

Aflatoxin Accumulation During the Growth of Mould Aspergillus flavus ATCC 26949 on Corn in Pure and Mixed Culture as Related to Various Temperature and Moisture Content

The relationship between mould biomass and the biosynthesis of afl atoxin B1 and G1 on solid substrates (whole and crushed maize grain) at temperatures from 15-40oC and a water content in the substrate of 20-38% has been investigated. The experiments have been carried out with the mould Aspergillus flavus ATCC 26949 in pure culture and in mixed culture respectively, the latter with the mould Trichothecium roseum ZMPBF 1226. The biomass growth during cultivation was measured by the chitin content, and the concentration of aflatoxins was determined by means of a ELISA. It has been established that the biosynthesis of the examined afl atoxins and their ratio primarily depend on the temperature of cultivation, rather than on the growth of the mycelium. The biomass of the mixed culture of A. flavus and T. roseum after 35 days of cultivation reduces the amount of aflatoxin B1 by 19-40%, and the amount of aflatoxin G1 by 30-45%. The decrease of concentration of both toxins is more pronounced in the substrate with a higher initial water content and at a higher temperature of cultivation

Early results of intra-articular micro-fragmented lipoaspirate treatment in patients with late stages knee osteoarthritis: a prospective study

Aim To analyze clinical and functional effects of intra-articular injection of autologous micro-fragmented lipoaspirate (MLA) in patients with late stage knee osteoarthritis (KOA). Secondary aims included classifying cell types contributing to the treatment effect, performing detailed MRIbased classification of KOA, and elucidating the predictors for functional outcomes. Methods This prospective, non-randomized study was conducted from June 2016 to February 2018 and enrolled 20 patients with late stage symptomatic KOA (Kellgren Lawrence grade III, n = 4; and IV, n = 16) who received an intra-articular injection of autologous MLA in the index knee joint. At baseline radiological KOA grade and MRI were assessed in order to classify the morphology of KOA changes. Stromal vascular fraction cells obtained from MLA samples were stained with antibodies specific for cell surface markers. Patients were evaluated at baseline and 12-months after treatment with visual analog scale (VAS), Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and Knee Injury and Osteoarthritis Outcome Score (KOOS).Results Three patients (15%) received a total knee replacement and were not followed up completely. Seventeen patients (85%) showed a substantial pattern of KOOS and WOMAC improvement, significant in all accounts. KOOS score improved from 46 to 176% when compared with baseline, WOMAC decreased from 40 to 45%, while VAS rating decreased from 54% to 82% (all P values were <0.001). MLA contained endothelial progenitor cells, pericytes, and supra-adventitial adipose stromal cells as most abundant cell phenotypes. Conclusion This study is among the first to show a positive effect of MLA on patients with late stages KOA

Pregled higijenskih i fizikalno-kemijskih parametara istarskog sira

Istrian cheese is the traditional hard cheese produced exclusively from the raw milk of the autochthonous Istrian sheep. The aim of this study was to determine the bacteriological quality, physicochemical properties of the sheep milk and Istrian cheese as well as proteolysis of cheese produced on six family farms during the cheese ripening. The hygienic microbial indicators of the sheep milk were poor in comparison to the other East Adriatic regions. However, the number of bacterial indicators rapidly declined during the ripening and they were under the detection limit for “ready to consume” cheese and therefore indicated their good bacteriological quality. Regression function of the effects of the ripening time on physicochemical properties of Istrian cheese during ripening showed a substantial increase in total solids, in salt, in moisture and in salt content as well as in the lactic acid in the total solids. However, these changes were not significant due to the high variability of the Istrian cheese production. The farm cheese making procedure affected the electrophoretic profile of the primary proteolysis in the cheese samples. Significant (P5 %). Equal degradation of as1-casein and ß-casein during ripening of Istrian cheese occurred. A low percentage of nitrogen fractions soluble in water and in 12 % trichloroacetic acid were determined.Istarski sir je tradicionalni tvrdi sir koji se proizvodi prvenstveno od sirovog mlijeka autohtone istarske ovce. Cilj je ovog rada bio utvrditi bakteriološku kvalitetu i fizikalno-kemijske parametre ovčjeg mlijeka te istarskog sira kao i proteolizu sira proizvedenog na šest obiteljskih gospodarstava tijekom zrenja. Mikrobiološki indikatori higijene ovčjeg mlijeka bili su loši u usporedbi s drugim regijama istočnog Jadrana. Međutim, tijekom zrenja sira broj bakterijskih indikatora higijene drastično je pao, te je njihov broj bio ispod detekcijskog limita u siru “spremnom za konzumaciju” što je bio pokazatelj njegove dobre bakteriološke kvalitete. Regresijska funkcija utjecaja trajanja zrenja na fizikalno-kemijske osobine istarskog sira tijekom zrenja pokazala je osjetno povećanje sadržaja suhe tvari, soli u vodenoj fazi sira i soli u siru kao i sadržaja mliječne kiseline u suhoj tvari sira. Međutim, te promjene nisu bile značajne zbog velike varijabilnosti u proizvodnji istarskog sira. Način proizvodnje sira na gospodarstvima utjecao je na elektroforetski profil primarne proteolize u uzorcima sira. Utvrđeno je signifikantno (P5 %). Tijekom zrenja istarskog sira utvrđena je jednaka razgradnja as1-kazeina i β-kazeina te niski postotak dušičnih frakcija topljivih u vodi i 12 %-tnoj trikloroctenoj kiselini

Fenotipska i genotipska karakterizacija autohtone zajednice laktobacila izolirane iz tradicionalnog istarskog ovčjeg sira

The objective of this study is to describe the diversity of indigenous cultivable community of the lactobacilli associated with the production of traditional Istrian cheese and to get a collection of well characterized strains. Raw milk and cheese samples were collected from three different farms in Istria during ripening. A total of 212 mesophilic and thermophilic Lactobacillus isolates as well as bulk colonies (consortia) were investigated using culture-dependent approach combining phenotyping and genotyping. Biochemical fingerprinting with PhenePlate-LB system preliminary grouped 212 isolates in 16 distinct PhP types. Only one representative isolate from each PhP cluster was further analyzed by genotyping for a reliable identification at the genus and species level by employing PCR techniques and sequencing of 16S rRNA genes. Sequence analysis of 16S rRNA revealed the presence of Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei and Lactobacillus rhamnosus. Lactobacilli were screened for possible resistance against seven selected antibiotics: ampicillin, tetracycline, penicillin, rifampin, clindamycin, erythromycin and vancomycin. Although there was no clear pattern of antimicrobial susceptibility to most tested antibiotics, all representative isolates were resistant to vancomycin. The analysis of bulk colonies by denaturing gradient gel electrophoresis (DGGE) identified Lactobacillus plantarum and Lactobacillus brevis as predominant members of Lactobacillus population. Pediococcus pentosaceus, Pediococcus acidilactici, Streptococcus sp. and Leuconostoc mesenteroides were also detected as part of the analysed consortia. The prevalence of identified species and community members of lactobacilli agrees with other studies of raw milk cheese and represents a useful base for further selection.Svrha je ovog istraživanja bila utvrditi raznolikost autohtone mikrobne zajednice laktobacila u istarskom siru te detaljno genetički definirati sojeve. Uzorkovanje mlijeka i svježeg sira provodilo se na tri obiteljska poljoprivredna gospodarstva na području Istre. Ukupno 212 izolata mezofilnih i termofilnih laktobacila, te ukupna zajednica (konzorcij) istraženi su pomoću fenotipskih i genotipskih metoda ovisno o izolaciji na selektivnim hranjivim podlogama. Primjenom biokemijskog testa PhenePlate-LB, 212 izolata preliminarno je svrstano u 16 skupina prema načinu korištenja supstrata. Po jedan reprezentativni izolat laktobacila iz svake PhP skupine genotipski je analiziran PCR analizom i sekvencioniranjem 16S rRNA gena te je određeno kojem rodu i vrsti pripadaju. Sekvencijskom su analizom određene ove vrste: Lactobacillus plantarum, Lactobacillus brevis, Lactobacillus casei, Lactobacillus paracasei i Lactobacillus rhamnosus. Dodatno je testirana osjetljivost svakog reprezentativnog izolata na sljedeće antibiotike: ampicilin, tetraciklin, penicilin, rifampicin, klindamicin, eritromicin i vankomicin. Kod svih izolata utvrđena je rezistencija na vankomicin, iako nije jasno utvrđen mehanizam osjetljivosti na ostale testirane antibiotike. PCR DGGE analizom ukupne zajednice laktobacila (konzorcija) utvrđeno je da prevladavaju vrste Lactobacillus plantarum i Lactobacillus brevis. Također su pronađene vrste Pediococcus pentosaceus, Pediococcus acidilactici, Streptococcus sp. i Leuconostoc mesenteroides. Prisutnost određenih vrsta u kompleksnoj mikrobnoj zajednici laktobacila istarskog sira potvrđuje rezultate brojnih studija o tradicijskim sirevima, što predstavlja važan autohtoni genetički potencijal za daljnju selekciju izolata

A Study of Antifungal and Antiafl atoxigenic Action of Newly Synthesized Analogues of Dehydroacetic Acid

Mycelial growth and afl atoxin B1 accumulation by toxigenous mould Aspergillus flavus were inhibited by dehydroacetic acid and its newly synthesized analogues, 4-hydroxy-3-(p-toluoyl)-6-(p-tolil)-2H-yrane-2-one (DHT) and 5-Bromo-4-hydroxy-3-(p-toluoyl)-6-(p-tolil)-2Hpyrane-2-one (BrDHT) during the growth of investigated mould on solid substrate (soy-beans). Experiments were carried out in a stationary culture at 20 0C and 30 0C, respectively, during 21 days. Mycelial growth (biomass dry weight) was determined by measuring the chitin content and concentration of afl atoxin B1 (AFB1) was quantitated by HPLC. Concentrations of DHA and DHT of 0.1 and 0.2 μg x g-1 of substrate were stimulated, but concentrations higher than 0.5 μg x g-1 of these compounds, mould growth and AFB1 accumulation partially inhibited. In experiments with 0.2 μg x g-1 of BrDHT mould growth and AFB1 accumulation were inhibited completely depending on the time and temperature of cultivation.Rast micelija i nakupljanje afl atoksina B1 pomoću toksikogene plijesni Aspergillus flavus inhibirani su pomoću dehidracetne kiseline i njenih novosintetiziranih analoga, 4-hydroxy-3-(p-toluoyl)-6-(p-olil)-2H-pyrane-2-one (DHT) i 5-Bromo-4-hydroxy-3-(p-toluoyl)-6-(p-tolil)-2H-pyrane-2-one (BrDHT) u tijeku rasta istraživane plijesni na čvrstom supstratu (zrnima soje). Eksperimenti su načinjeni u stacionarnom uzgoju pri 20 0C odnosno 30 0C, u tijeku 21 dan. Rast micelija (suha tvar biomase) određena je mjerenjem količine hitina, a koncentracija afl atoksina B1 (AFB1) određena je kvantitativno pomoću HPLC metode. Koncentracije DHA i DHT od 0.1 i 0.2 μg x g-1 supstrata su stimulirane, a koncentracije više od 0.5 μg x g-1 tih spojeva, samo su djelomično inhibirale rast plijesni i nakupljanje AFB1. U eksperimentima s 0.2 μg x g-1 BrDHT, rast plijesni i nakupljanje afl atoksina potpuno su inhibirani u ovisnosti o vremenu i temperaturi uzgoja