Identification of acetic acid bacteria during industrial submerged apple cider vinegar based on integenic 16S-23S rDNA region

Zavedanje ljudi o pomembnosti uživanja zdrave hrane postaja vedno močnejše, zato se v zadnjih letih povečuje trend uživanja živil, pridobljenih iz surovin, ki so čim manj izpostavljene kemikalijam, npr. s škropljenjem. Tudi kis, kot eden izmed pogosto uporabljenih dodatkov k hrani, se lahko pridobiva iz jabolk, ki so pridelana na konvencionalen način, s škropljenjem, ali pa brez škropljenja, s čimer pridobimo bio-jabolčni kis. V okviru te magistrske naloge smo primerjali mikrobioto iz industrijskih submerznih bioreaktorjev za pridobivanje konvencionalnega in bio-jabolčnega kisa. V ta namen smo na gojiščih RAE v aerobni atmosferi pridobili 41 izolatov ocetnokislinskih bakterij in kvasovk, od tega 21 iz bioreaktorja za pridobivanje konvencionalnega jabolčnega kisa. Pri vsakem od obeh bioprocesov smo vzorce odvzeli iz različnih stopenj oksidacije etanola v ocetno kislino med enim dobro delujočim oksidacijskim ciklusom pridobivanja kisa. Izolate smo najprej fenotipsko okarakterizirali in jih nato s pomočjo molekulskih metod identificirali. Na osnovi primerjav nukleotidnega zaporedja medgenske regije 16S-23S rDNA med izoliranimi sevi in referenčnimi sevi ocetnokislinskih bakterij smo v konvencionalnem jabolčnem kisu identificirali dve vrsti ocetnokislinskih bakterij: Komagataeibacter oboediens in Acetobacter pasteurianus. Poleg tega smo identificirali še vrsto Komagataeibacter maltaceti, ki pa je živela v simbiozi s kvasovko in nam je od nje ni uspelo izolirati. V bio-jabolčnem kisu smo identificirali štiri vrste ocetnokislinskih bakterij: A. pasteurianus, Acetobacter ghanensis, K. oboediens in Komagataeibacter saccharivorans. Iz obeh vrst kisa smo izolirali tudi kvasovke in jih identificirali na osnovi nukleotidnega zaporedja regije ITS15.8S rDNA‒ITS2. Kvasovki Candida ethanolica in Saccharomycodes ludwigii sta bili prisotni v obeh vrstah kisa, medtem ko smo iz bio-jabolčnega kisa izolirali še vrsto Pichia membranifaciens. Pri predstavnikih posameznih vrst, pridobljenih v tem delu, in izbranih referenčnih sevih ocetnokislinskih bakterij smo ugotavljali sposobnost rasti pri temperaturah 30 °C in 37 °C. Kljub temu da je za ocetnokislinske bakterije 30 °C opisanih kot optimalna temperatura namnoževanja, smo ugotovili, da se vsi sevi dobro namnožujejo tudi pri 37 °C, za nekatere seve pa je bila ta temperatura celo ugodnejša. Pri kvasovkah smo ugotavljali tudi sposobnost rasti ob prisotnosti različnih koncentracij ocetne kisline v gojišču RAE. Ugotovili smo, da vsi izolati rastejo v prisotnosti 3 % maksimalne koncentracije ocetne kisline, sev Candida ethanolica T4K1 pa je bil odporen tudi proti 4 % ocetni kislini.People are more and more aware of importance of consuming healthy food. In recent years, a consumption of food produced from raw materials that have been as little as possible exposed to chemicals is increasingly growing. Also vinegar as one of the commonly used food supplements may be produced from apples which are grown in a conventional way with sprinkling or without sprinkling for production of organic apple cider vinegar. In this work, we compared acetic acid bacterial microbiota for production of conventional apple cider vinegar and organic apple cider vinegar during industrial submerged processes. For this purpose we isolated 41 microorganisms on RAE medium incubated in aerobic atmosphere of which 21 were from bioreactor for conventional apple cider vinegar production and the others from bioreactor for production of organic apple cider vinegar production. Samples were taken from each of the bioprocesses at different stages of the oxidation of ethanol to acetic acid during one cycle of smoothly running vinegar producing process. At the beginning we performed basic phenotypic analyses of the isolates, then we proceeded with molecular methods for their identification. Based on a comparison of 16S-23S rRNA gene internal transcribed spacer region of isolated strains with reference strains of acetic acid bacteria, we determined the following species in conventional apple cider vinegar: Komagataeibacter oboediens and Acetobacter pasteurianus. Besides, we isolated Komagataeibacter maltaceti which lived in symbiosis with yeast from which we could not separate it so far. The acetic acid bacterial microbiota for organic apple cider vinegar contained four species of acetic acid bacteria: A. pasteurianus, Acetobacter ghanensis, K. oboediens and Komagataeibacter saccharivorans. We also isolated yeasts and further identified them by sequence analysis of the ITS15.8S rDNA‒ITS2 region. Candida ethanolica and Saccharomycodes ludwigii were present in both apple cider vinegars while Pichia membranifaciens was isolated only from organic apple cider vinegar. We compared the ability of the representatives of individual species of acetic acid bacteria isolated in this work and the reference strains of acetic acid bacteria for their ability to grow at 30°C and 37°C. Despite the fact that 30°C is described as an optimal temperature for multiplication of acetic acid bacteria, our isolates all grew well at 37°C and for some strains this temperature was even more favorable. The yeasts isolated in this work were also tested for their ability to grow in the presence of different concentrations of acetic acid in RAE medium. We found out that all isolates grew in the presence of 3 % of the maximum concentration of acetic acid, while the strain Candida ethanolica T4K1 was resistant against 4 % acetic acid