Influence of essential oils on selected enterobacteria strains biofilms formed on the vegetable leaf surfaces

Pojava i učestalost epidemija izazvanih konzumiranjem hrane beleži stalni rast. Tome su doprinele promene u ishrani i težnja savremenih potrošača za konzumiranjem minimalno obrađene hrane koja u najvećoj meri obuhvata sveže voće i lisnato povrće. Kao jedan od čestih izvora kontaminacije navodi se zelena salata koja se pre konzumiranja podvrgava samo pranju. S obzirom na način gajenja, putevi kontaminacije ovog povrća patogenim bakterijama kao što su Escherichia coli - E. coli i Salmonella Typhimurium - S. Typhimurium su mnogobrojni. Dodatni problem predstavlja sposobnost ovih patogena da formiraju biofilmove u kojima su zaštićeni od nepovoljnih uslova spoljašnje sredine, a koje je nemoguće potpuno ukloniti pranjem vodom. Činjenica da mikroorganizmi u biofilm formaciji pokazuju znatno veću otpornost prema dejstvu antibiotika i raznih dezinficijenasa u odnosu na planktonski oblik, doprinela je izboru etarskih ulja (EU) kao prirodnih jedinjenja velikog i do sada nepotpuno iztraženog antimikrobnog i antibiofilm potencijala. Kulinarske i začinske biljke, bogate EU se od davnina koriste u kulinarstvu i farmaciji, te svako novo saznanje vezano za njihov antimikrobni potencijal može doprineti iznalaženju novog biološkog rešenja za eliminisanje patogena i njihovih biofilmova u hrani, posebno onoj koja se konzumira sveža, kao što je lisnato povrće. Imajući u vidu navedene činjenice, cilj rada je bio da se ispita antibiofilm potencijal EU prema biofilmovima E. coli i S. Typhimurium ATCC 14028 formiranim u in vitro uslovima i na površini lista zelene salate. S tim u vezi prvo je ispitivana sposobnost izolata E. coli različitog porekla da formiraju biofilmove in vitro uz prethodno određivanje morfotipa kolonija na Kongo crvenom agaru (KCA) na dve različite temperature, 25 oC i 37 oC. Zatim je izvršeno ispitivanje sposobnosti preživljavanja odabranog izolata E. coli (koji je prethodnim testiranjem svrstan u jake biofilm producere) i laboratorijskog tipskog soja S. Typhimurium u zemljištu pri konstantnim i promenljivim uslovima vlage i temperature tokom 120 dana inkubiranja. Ispitana je sposobnost adherencije i formiranje biofilma ova dva mikroorganizma na površini lista zelene salate na temperaturi od 25 oC i 37 oC. Na kraju je određen uticaj odabranih EU na adherenciju i formirane biofilmove E. coli i S. Typhimurium u in vitro uslovima i na listu zelene salate. Vizuelizacija biofilma formiranog na površini lista zelene salate izvedena je primenom skening elektronske mikroskopije (SEM). Određivanjem morfotipa kolonija na KCA na temperaturi inkubiranja od 25 oC utvrđeno je da su testirani izolati E. coli formirali kolonije morfotipa RDAR (eng. red, dry and rough), BDAR (eng. brown, dry and rough), RAS (eng. red and smooth) i SAW (eng. smooth and white), dok su na temperaturi inkubiranja od 37 oC formirali kolonije morfotipa, RDAR, BDAR i SAW. Laboratorijski tipski soj S. Typhimurium formirao je kolonije morfotipa RDAR na temperaturi inkubiranja od 25 oC, odnosno SAW na temperaturi inkubiranja od 37 oC. Ispitivanjem sposobnosti formiranja biofilma in vitro uz kvantifikaciju ukupne mase biofilma primenom kristal violet testa na obe temperature uočen je jak uticaj temperature u smislu većeg broja izolata koji su svrstani u jake biofilm producere na nižoj temperaturi inkubiranja. Uporednom analizom rezultata dobijenih za određivanje morfotipa i sposobnosti formiranja biofilma na obe temperature uočena je pozitivna korelacija između broja izolata E. coli koji su formirali RDAR morfotip i broja izolata koji su svrstani u jake biofilm producere. Ispitivanjem sposobnosti preživljavanja odabranog izolata E. coli i S. Typhimurium u zemljištu, nakon 120 dana inkubiranja, ustanovljeno je prisustvo vijabilnih ćelija oba mikroorganizma pri konstantnim i pri promenljivim uslovima vlažnosti i temperature. Stopa preživljavanja S. Typhimurium bila je veća i pri konstantnim i pri promenljivim uslovima temperature i vlažnosti i iznosila je 0,60% odnosno 0,27%. Stepen adherencije na površinu lista zelene salate tokom 3 h na temperaturi od 25 oC iznosio je 6,75 log cfu/cm2 lista za S. Typhimurium, odnosno 6,64 log cfu/cm2 lista za E. coli. Viša temperatura inkubiranja (37 oC) različito je uticala na stepen adherencije ispitivanih mikroorganizama na list zelene salate, generalno ga redukujući na nižu vrednost, pri čemu je kod S. Typhimurium redukcija iznosila 50%, a kod E. coli 10%. Primenom SEM, na mikrografijama manjeg uvećanja uočeno je formiranje ćelijskih agregata E. coli i S. Typhimurium na površini lista zelene salate, koji su sastavni deo biofilma dok je na mikrografijama većeg uvećanja uočena interkonekcija između ćelija i formirani biofilm. Drugi deo ispitivanja sproveden je u cilju određivanja uticaja EU začinskih biljaka bosiljka, mirođije, nane i vreska na inicijalnu ćelijsku adheziju i prethodno formirani biofilm. Pre ispitivanja antimikrobnog i antibiofilm potencijala, analiziran je hemijski sastav EU, koji je pokazao da sva četiri EU sadrže visok % monoterpena. Najzastupljenije identifikovano jedinjenje EU bosiljka bilo je linalool (69,2%), EU mirođije karvon (66,6%) i limonen (25,3%), EU nane mentol (30,8%) i menton (28,8%) i EU vreska p-cimen (33,8%) i karvakrol (32,9%). Najbolji antimikrobni potencijal prema bujonskim kulturama E. coli i S. Typhimurium pokazalo je EU mirođije, sledi EU bosiljka i vreska, dok je EU nane pokazalo najmanji antimikrobni potencijal, koji je vrednovan dobijenim vrednostima za minimalnu inhibitornu koncentraciju (eng. minimal inhibitory concentration, MIC) i minimalnu baktericidnu koncentraciju (eng. minimal bactericidal concentration, MBC). Etarska ulja pokazala su dozno-zavistan uticaj na inicijalnu ćelijsku adheziju i posledično na formiranje biofilma u in vitro uslovima, prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane (odnosi se na koncentracije EU od 2 MIC). Ispitivanjem uticaja EU na ukupnu masu biofilma in vitro dokazano je da EU u primenjenim koncentracijama od 0,5 MIC, MIC i 2 MIC ispoljavaju uticaj na redukciju ukupne mase formiranog biofilma na dozno-zavistan način u funkciji vremena, tako da je najveći % eradikacije biofilma E. coli i S. Typhimurium postignut nakon 60 minuta ekspozicije primenom EU koncentracije 2 MIC prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane. Uticaj EU na prethodno formirani biofilm E. coli i S. Typhimurium na površini lista zelene salate takođe je zavisio od koncentracije. Posmatrajući njihov uticaj sa aspekta broja vijabilnih ćelija biofilma, najefikasnije je bilo EU mirođije koncentracije 2 MIC, a najmanje efikasno EU nane koncentracije MIC. Najveći % redukcije ukupne mase biofilma i E. coli i S. Typhimurium postignut je primenom EU koncentracije 2 MIC prema sledećem redosledu: EU mirođije > EU bosiljka > EU vreska > EU nane. Poređenjem rezultata redukcije ukupne mase biofilma E. coli i S. Typhimurium, formiranog u in vitro uslovima i na površini lista zelene salate, veći procenat redukcije zabeležen je kod biofilmova formiranih u in vitro uslovima, što se može objasniti hrapavom strukturom površine lista zelene salate koja je ograničila kontakt EU i biofilma ispitivanih mikroorganizama. Na osnovu izvedenih ispitivanja, u skladu sa ciljevima ove doktorske disertacije i postignutih rezultata, može se zaključiti da je EU mirođije pokazalo najbolji antimikrobni i antibiofilm potencijal u in vitro uslovima i na kuponima lista zelene salate prema oba ispitivana mikroorganizma. Sledi EU bosiljka, vreska i nane. Dobijeni rezultati upućuju da EU ovih biljaka na osnovu dokazanog antimikrobnog i antibiofilm potencijala koji poseduju mogu naći primenu kao sastojci salatnih preliva, mogu da se inkorporiraju u materijale za pakovanje ili u atmosferu pakovanja doprinoseći time zdravstvenoj bezbednosti zelene salate.The frequency of foodborne outbreaks is constantly increasing. This trend has been contributed to by changes in diet and the desire of modern consumers to consume minimally processed foods, which primarily include fresh fruits and leafy vegetables. Lettuce is one of the frequent sources of contamination, consumed after washing only. Due to the growing techniques, the contamination pathways of this vegetable with pathogenic bacteria, such as Escherichia coli - E. coli and Salmonella Typhimurium - S. Typhimurium, are numerous. An additional problem is the ability of these pathogens to form a biofilm. In biofilms, pathogens are protected from adverse environmental conditions and cannot be entirely removed by water washing. The fact that microorganisms in biofilm formation show significantly higher resistance to antibiotics and various disinfectants compared to planktonic form, contributed to the choice of essential oils (EOs). EOs as natural compounds of great and until now incompletely investigated antimicrobial and antibiofilm potential. Culinary and spice plants rich in EOs have long been used in cooking and pharmacy, and any new knowledge about their antimicrobial potential can contribute to finding a new biological solution to eliminate pathogens and their biofilms in food, especially those consumed fresh as leafy vegetables. Having in mind this fact, this study aimed to examine the effect EOs on the removal of biofilms of E. coli and S. Typhimurium ATCC 14028 formed in vitro and on lettuce leaves. In this regard, the ability of E. coli isolates of different origins to form biofilms in vitro was examined. Previous, the morphotype of the colonies on the Congo Red Agar (CRA) at two different temperatures, 25 °C and 37 °C, was determined, as well as the viability of the selected E. coli isolates, previously classified as a strong biofilm producer. Furthermore, the viability of the laboratory-type strain S. Typhimurium in soil under constant and variable humidity and temperature during 120 days of incubation was investigated, along with these two microorganisms’ ability to adhere and form a biofilm on the lettuce leaves surface at temperatures of 25 °C and 37 °C. Finally, the influence of selected EO on adherence and formed biofilms of E. coli and S. Typhimurium in vitro and on lettuce leaf was examined. Obtained biofilm visualization was performed using scanning electron microscopy (SEM). By determining the colony morphotypes on CRA at an incubation temperature of 25 °C, the tested isolates of E. coli formed colony morphotypes RDAR (red, dry and rough), BDAR (brown, dry and rough), RAS (red and smooth) and SAW (smooth and white), while at an incubation temperature of 37 °C they formed colony morphotypes, RDAR, BDAR and SAW. The laboratory-type strain S. Typhimurium formed the RDAR and SAW morphotype at incubation temperatures of 25 °C and 37 °C, respectively. Examination of the ability to form biofilms in vitro with quantification of total biofilm mass using crystal violet test at both temperatures showed a strong influence of temperature in terms of a higher number of isolates classified as strong biofilm producers at lower incubation temperature. A comparative analysis of the results obtained to determine morphotype and biofilm formation ability at both temperatures showed a positive correlation between the number of E. coli isolates that formed the RDAR morphotype and the number of isolates classified as strong biofilm producers. By examining the viability of the selected isolate of E. coli and S. Typhimurium in soil, after 120 days of incubation, the presence of viable cells of both microorganisms was detected under both constant and variable conditions of humidity and temperature. The survival rate of S. Typhimurium was higher under both constant and variable temperature and humidity conditions and was 0.60% and 0.27%, respectively. The adhesion degree to the surface of lettuce leaves for 3 h at a temperature of 25 °C was 6.75 log cfu/cm2 leaves for S. Typhimurium and 6.64 log cfu/cm2 leaves for E. coli. Higher incubation temperature (37 °C) differently affected the degree of adhesion of the tested microorganisms to a lettuce leaf, generally reducing it to a lower value, with a reduction of 50% in S. Typhimurium and 10% in E. coli. Using SEM, low magnification micrographs showed the formation of E. coli and S. Typhimurium cell aggregates on the surface of lettuce leaves, which are an integral part of the biofilm, while high magnification micrographs showed interconnection between cells and the formed biofilm. Therefore, the second part of the research within this doctoral dissertation aimed to examine the influence of EOs of spice plants of basil, dill, mint, and heather on the initial cell adhesion and preformed biofilm. Before testing the antimicrobial and antibiofilm potentials, the chemical composition of the EOs was analyzed, this showed that all four EOs contain a high share of monoterpenes. The major compounds of basil EO were linalool (69.2%), dill EO carvone (66.6%) and limonene (25.3%), mint EO menthol (30.8%) and menthone (28.8 %) and heather EO p- cymene (33.8%) and carvacrol (32.9%). The best antimicrobial potential against the broth cultures of E. coli and S. Typhimurium showed dill EO, followed by EOs of basil and heather, while mint EO showed the lowest antimicrobial potential. The antimicrobial potential was evaluated based on the obtained values for minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Tested EOs showed a dose-dependent effect on initial cell adhesion and consequently on biofilm formation in vitro according to the following order: dill EO > basil EO > heather EO > mint EO (refers to EO concentrations of 2 MIC). By examining the influence of the EO on the total mass of biofilm in vitro, it was proved that EOs in applied concentrations of 0.5 MIC, MIC, and 2 MIC have an effect on reducing the total mass of preformed biofilm in a dose-dependent manner as a function of time. Eradication of preformed biofilms of E. coli and S. Typhimurium were achieved after 60 minutes of exposure to EOs in concentration of 2 MIC in the following order: EO dill > EO basil > EO heather > EO mint. The influence of the EO on the preformed biofilm of E. coli and S. Typhimurium on the surface of lettuce leaves showed also a dose-dependent manner as a function of time. Observing their influence regarding to the number of viable biofilm cells, the most effective was EO dill in a concentration of 2 MIC, and the least efficient was EO dill in a concentration of MIC. The highest % reduction of the total biofilm mass of both E. coli and S. Typhimurium was determined by applying the EO concentration of 2 MIC in the following order: EO dill > EO basil > EO heather > EO mint. By comparing the results of reducing the total mass of E.coli and S. Typhimurium biofilms formed in vitro and on the surface of lettuce leaves, a higher percent reduction was observed in biofilms formed in vitro. This result can be explained by the rough surface structure of lettuce leaves, which limited the contact between the EO and the biofilm of the tested microorganisms. Based on the performed tests following the goals of this doctoral dissertation and the achieved results, it can be concluded that dill EO showed the best antimicrobial and antibiofilm potential in vitro and on lettuce coupons against both tested microorganisms. It is followed by basil, heather and mint EOs. Obtained results indicated that investigated EOs next to be used as ingredients in salad dressings also can be incorporated into packaging materials as well as packaging atmosphere. Based on their proven antimicrobial and antibiofilm potential their application can contribute to food safety and lead to improving health safety in general

Influence of Cropping System Intensity on Dry Matter Yield and Nitrogen Concentration in Different Parts of Soybean Plant

Two-year investigations were conducted within the three-year maize-soybean-wheat crop rotation with the aim to assess the influence of two cropping systems, conditionally marked as high-input and reduced-input systems, on soybean dry matter yield and nitrogen concentration in its different plant parts. The high-input system was characterized by: ploughing at 30-32 cm, fertilization with 80 kg N and 130 P2O5 and K2O kg ha-1, weed control based on oxasulfuron and, if required, a corrective treatment with propachizafop and bentazone. The reduced-input system involved: ploughing at 20-22 cm, fertilization with a total of 40 kg N and 130 kg P2O5 and K2O kg ha-1, and only oxasulfuron-based weed control. Investigations involved two soybean genotypes: L 940596 and L 910631, vegetation group I. An identical monofactorial trial with four replications was set up in each cropping system. Cropping system intensity had a positive effect on leaf and stem dry matter mass in 2002, and on leaf and stem nitrogen concentration, whereas it had no effect on pod dry matter, seed yield and seed nitrogen concentrations in either year. The highest leaf dry matter was recorded in R4 development stage and that of stem and pod without seeds in R7 development stage. In both trial years, the highest nitrogen concentrations in leaf and stem were recorded in R1 development stage. In both trial years, higher pod nitrogen concentration was achieved in R4 development stage than in the R7 stage

Nature and transcendence of marital love. Theological-moral aspect in the light of the encyclic Amoris laetitia

Rad obrađuje poimanje bračne ljubavi na temelju apostolske pobudnice Amoris laetita pape Franje. Pri tome se polazi od naravnosti bračne ljubavi, zajedničke svim ljudima, da bi se došlo do poimanja njezine nadnaravnosti. U prvom dijelu rada, služeći se biblijskim izvještajima o stvaranju, ukazuje se da je Bog na samom početku, stvorivši muško i žensko na svoju sliku, upisao bračnu ljubav u samu ljudsku narav. Bog koji je Ljubav stvara čovjeka iz ljubavi i za ljubav te je poziv na ljubav temeljni poziv svakog čovjeka. Nadalje, polazeći od antropologije ljudske spolnosti, očito je da je upravo ona usmjerena na bračnu ljubav, jer uzajamnim darivanjem njih dvoje postaju jedno tijelo. Potom se, na temelju enciklike Humanae vitae, donose temeljne oznake bračne ljubavi u naravnom redu, ističući da je ona prije svega ljudska, posvemašnja, vjerna i isključiva, ali i plodna. To nas dovodi do pitanja odgovornog roditeljstva i naravi bračnog čina. U drugom dijelu rada, služeći se evanđeljima i poslanicama, naglašava se da je bračna ljubav između muža i žene slika ljubavi kojom je Krist ljubio Crkvu. Tumačeći bračnu ljubav kroz crkvene dokumente, osobito pastoralnu konstituciju Gaudium et spes Drugog vatikanskog sabora, encikliku Humanae vitae papa Pavla VI., apostolsku pobudnicu Familiaris consortio pape Ivana Pavla II. te Amoris laetitia pape Franje, iznose se kršćanska obilježja nadnaravnosti bračne ljubavi: strpljivost, stav dobrostivosti, lijek protiv zavisti te se naglašava da je ona bez hvastanja i nadimanja, potiče ljubaznost i velikodušno darivanje, a protivna je nasilju, potiče opraštanje, raduje se s drugima, sve pokriva, sve vjeruje, svemu se nada i sve podnosi. U trećem i posljednjem dijelu nastoji se problematizirati stav kanonskog prava koje promatra samo objektivne svrhe ženidbe, osobito privolu, dok se bračna ljubav samo pretpostavlja kao razlog davanja privole što danas izaziva mnoge teološke rasprave.This paper interprets marital love based on pope Francis' encyclic Amoris laetitia. Thereby, it begins with natural marital love, that is common to all people, on his path to transcendental love. In the first part, using the bible reports of creation, it points out that the God in the early beginnings created male and female in his own image and implanted marital love into every human being. God, who is a Love himself, creates human from love and for love which is particularly why is the need for love a basic call for every human. Later on, starting with the anthropology of human sexuality, it is obvious that the sexuality is directed to marital love, because by mutual donation two of them become one body. Based on the encyclic Humanae vitae, mutual marital love is firstly human, utter, faithful, exclusive and fertile. That leads us to responsible parenting and the question of the marriage act. Second part, written from using Gospels and Epistles, highlights that marital love between husband and wife pictures Christs love for the Church. Interpreting marital love through church documents, especially the pastoral constitution Guaudium et spes the Second Vatican Council, pope Paul VI encyclic Humanae vitae, pope John Paul II encyclic Familiaris consortio and pope Francis’ Amoris laetitia, Christian features of transcendental marital love are expressed: patience, the act of kindness, envy treatment, without boasting and arrogance, generosity, generous separation, anti internal violence, remission, looking forward to the other person, it covers all, it believes, hopes and takes everything on itself. In the third and final part, the problem of the Canon law which observes only the objective purposes of marriage, especially the cajolement, is sought to be solved, while marital love is merely supposed to be the cause of the cajolement which today causes many theological discussions

Ordering properties of anisotropic hard bodies in one-dimensional channels

The phase behavior and structural properties of hard anisotropic particles (prisms and dumbbells) are examined in one-dimensional channels using the Parsons--Lee (PL) theory, and the transfer-matrix and neighbor-distribution methods. The particles are allowed to move freely along the channel, while their orientations are constrained such that one particle can occupy only two or three different lengths along the channel. In this confinement setting, hard prisms behave as an additive mixture, while hard dumbbells behave as a non-additive one. We prove that all methods provide exact results for the phase properties of hard prisms, while only the neighbor-distribution and transfer-matrix methods are exact for hard dumbbells. This shows that non-additive effects are incorrectly included into the PL theory, which is a successful theory of the isotropic-nematic phase transition of rod-like particles in higher dimensions. In the one-dimensional channel, the orientational ordering develops continuously with increasing density, i.e., the system is isotropic only at zero density, while it becomes perfectly ordered at the close-packing density. We show that there is no orientational correlation in the hard prism system, while the hard dumbbells are orientationally correlated with diverging correlation length at close packing. On the other hand, positional correlations are present for all the systems, the associated correlation length diverging at close packing.Comment: 14 pages, 6 figures; v2: minor change

Voice Therapy For Transgender People

Transrodne osobe su osobe koje osjećaju nesklad između roda s kojim se poistovjećuju i spola u kojem su se rodili. Transrodnost je širok pojam i obuhvaća čitav spektar rodno nenormativnih identiteta. Dva glavna obrasca tranzicije prema drugom spolu, odnosno rodu su: Male-to-Female (MtF) i Female-to-Male (FtM). Transrodne osobe navode da ih njihov glas često “izdaje”, odnosno otkriva njihov biološki rod te predstavlja posljednju prepreku u potpuno uživljavanje u novu rodnu ulogu. Bez obzira na to, često nisu svjesni da se zbog glasa mogu obratiti logopedu. Cilj ovog preglednog rada je dati uvid u poremećaje glasa kod transrodnih osoba, odnosno opisati logopedsku procjenu te terapijske postupke, a sve u svrhu boljeg razumijevanja potreba ovih osoba.Transgender individuals are people who feel an incongruity between their self-identified gender and their birth gender. Transgenderism is a broad term and includes a spectrum of gender-nonconforming identities. The two main patterns of gender transition are Male-to-Female (MtF) and Female-to-Male (FtM).Transgender people often think their voice “betrays” them, i.e. it reveals their biological gender and represents the last obstacle to the individual’s full enjoyment of his/her new gender role. Regardless of this, many transgender individuals are not aware of the fact that they can go to speech and language pathologists (SLP) for therapy. The aim of this review article is to provide insight into the voice disorders of transgender people, i.e. to describe the assessment process and therapy protocols in hope of gaining a better understanding of the needs of this population

Kroatische und deutsche Antisprichwörter in der Sprache der Jugendlichen

The present paper discusses the results of a survey conducted among adolescents aged 11-20 in Croatia, Germany, and Austria. The survey is the extension of research conducted in Croatia in 2014 with 897 respondents aged 14-87, which showed that the adolescents aged 11-20 in 79.30% of cases filled the first part of proverbs with the wrong phrase, indicating that they might be more familiar with the particular anti-proverb than with the canonical form of a particular proverb (Aleksa Varga, Matovac 2016). The present research was conducted on a sample of 202 Croatian native speaker, and 144 German native speaker adolescents, by filling out a paper questionnaire and supplying the first part of the most frequently occurring proverbs in Croatian and German written corpora. All the “wrong” answers, that is the items that do not correspond to the zero variant have been collected in a special sub-corpus which has been analyzed in the course of the present paper, with the special aim of describing and analyzing the anti-proverbs adolescents have provided

Hrvatske poslovice u slavenskome okruženju: određivanje hrvatskoga paremiološkog minimuma i optimuma

The present paper discusses the notion of Croatian proverbs, more precisely the steps that have been taken in an attempt to determine the Croatian paremiological minimum and optimum in the course of a project that started in 2014 by developing a part-text presentation questionnaire and conducting a field study with 867 participants from all major Croatian regions who filled in the first part of the most frequent proverbs found in the three major Croatian corpora. A second study was conducted in 2018 by using the same proverbs and resulted in 718 questionnaires filled out by Croatian participants, but this time they had to supply the second part of the proverb. The aim of this paper therefore is, a part from presenting a portion of the statistical results of the two surveys, to placing the issues regarding Croatian paremiology and the problems of determining the Croatian paremiological minimum/optimum into a wider Slavic context thus presenting similar attempts, methods and solutions in the Czech, Slovak, Slovene and Russian languages. Prispevek se ukvarja s hrvaškimi pregovori oziroma natančneje s koraki, ki so bili potrebni za določitev hrvaškega paremiološkega minimuma in optimuma v okviru projekta, ki se je začel leta 2014 z izdelavo ankete z delnim prikazom paremioloških enot ter izvedbo sistematične raziskave z 867 anketiranci z vseh večjih hrvaških govornih območij, ki so dopolnili prvo polovico najbolj pogostih pregovorov, najdenih v treh glavnih hrvaških jezikovnih korpusih. V naslednji raziskavi, ki je bila leta 2018 izvedena z istimi pregovori in je prinesla 718 izpolnjenih anketnih pol, so hrvaški anketiranci morali dopolniti drugo polovico vsakega izmed prikazanih pregovorov. Poleg prikaza nekaterih statističnih rezultatov obeh raziskav je namen prispevka umestiti temo hrvaške paremiologije in problematike vzpostavitve hrvaškega minimuma ter optimuma v širši slovanski kontekst s primerjavo s podobnimi namerami, metodami in izvedbami v češkem, slovaškem, slovenskem in ruskem jeziku