Characterization of the Microbiota of Naturally Fermented Pirot “Ironed” Sausage During Maturation and the Finished Product

Pirotska „peglana“ kobasica je tradicionalna, fermentisana kobasica, koja se pravi od različitih vrsta mesa, soli i začina, a bez dodatka starter kultura. Testirani su uzorci kobasica šest proizvođača od kojih su neki koristili standardnu a neki nestandardnu recepturu. Ukupni diverzitet bakterija je ispitivan u gotovom proizvodu i tokom zrenja u različitim vremenskim tačkama korišćenjem odgajivačkog i neodgajivačkog (sekvenciranje gena za 16S rRNK) pristupa. Genetički polimorfizam predstavnika različitih grupa bakterijskih izolata određivan je korišćenjem rep-PCR i RAPD-PCR tehnika sa specijalizovanim prajmerima. Rezultati identifikacije sekvenciranjem gena za 16S rRNK ukazuju na prisustvo samo dve vrste bakterija mlečne kiseline, Lactobacillus sakei (76%) i Leuconostoc mesenteroides (24%). U poređenju sa drugim prajmerima, najveću diskriminatornu moć pokazao je prajmer GTG5, kod većine testiranih izolata. Sekvenciranjem naredne generacije utvrđena je dominacija predstavnika iz razdela Proteobacteria i Firmicutes u svim proučavanim uzorcima. Tokom procesa zrenja uočen je trend povećanja zastupljenosti predstavnika Firmicutes (sa 33,5% na 63,5%) i smanjenje udela Proteobacteria (sa 65,4% na 22,3%). Metabarkoding analiza je pokazala da su dominantni bakterijski rodovi tokom procesa zrenja kobasice bili: Lactobacillus, Photobacterium, Leuconostoc, Weissella i Lactococcus, dok su detektovani i Carnobacterium, Brochothrix i Acinetobacter. Odgajivačkim pristupom je utvrđeno prisustvo vrsta Lactobacillus sakei i Leuconostoc mesenteoides u svim fazama zrenja. Upoređen je sastav bakterijskih zajednica u gotovom proizvodu svakog testiranog proizvođača. U uzorcima PK1 i PK2 pokazano je najveće bogatstvo bakterijskih vrsta. Predstavnici razdela Firmicutes su bili dominantni u uzorcima PK3, PK4, PK5 i PK6, dok su predstavnici Proteobacteria bili najzastupljeniji u uzorcima PK1 i PK2. U zavisnosti od uzorka, dominiraju vrste roda Lactobacillus, L. sakei i L. algidus, dok je rod Leuconostoc nađen u svim uzorcima. U gotovom proizvodu i tokom zrenja u različitim vremenskim tačkama određivane su i fizičko-hemijske osobine kobasice. Primećeno je smanjenje pH vrednosti, aktivnosti vode i sadržaja vlage, i povećanje sadržaja proteina i masti. Različite recepture i različito vreme zrenja pirotske “peglane” kobasice nisu imale značajan uticaj na profil masnih kiselina, ali su imale značajan efekat na instrumentalne parametre boje. Takođe, zabeležene su i statistički značajne razlike ispitivanih parametara teksture između kobasica proizvedenih po različitim recepturama. Različiti udeli upotrebljenog mesa prouzrokovali su značajne razlike u pogledu senzornih karakteristika.Pirot "ironed" sausage is a traditional fermented sausage made from various meats, salt, and spices, without the addition of starter cultures. Sausage samples from six producers were studied, some of which used standard and some non-standard recipes. Total bacterial diversity was studied in the finished product and during ripening at different time points using cultivating and non-cultivating methods. Genetic polymorphism of representatives of different groups of bacterial isolates was determined using rep-PCR and RAPD-PCR techniques with specific primers. The results of 16S rRNA gene sequencing indicated the presence of only two species of lactic acid bacteria, Lactobacillus sakei (76%) and Leuconostoc mesenteroides (24%). Compared to other primers, the GTG5 primer showed the highest discriminatory power for most of the isolates tested. Next generation sequencing showed that representatives of Proteobacteria and Firmicutes dominated in all samples tested. During maturation, a trend of increasing the proportion of Firmicutes representatives (from 33.5% to 63.5%) and decreasing the proportion of Proteobacteria (from 65.4% to 22.3%) was observed. Metabarcoding analysis showed that the dominant bacterial genera during the sausage ripening process were the following: Lactobacillus, Photobacterium, Leuconostoc, Weissella and Lactococcus, while Carnobacterium, Brochothrix and Acinetobacter were also detected. The presence of the species Lactobacillus sakei and Leuconostoc mesenteoides at all stages of maturation was detected in the cultivation approach. The composition of bacterial communities in the final product of each tested producer was compared. Samples PK1 and PK2 showed the highest abundance of bacterial species. Representatives of the Firmicutes dominated in samples PK3, PK4, PK5 and PK6, while representatives of Proteobacteria were most abundant in samples PK1 and PK2. Depending on the sample, species of the genus Lactobacillus, L. sakei and L. algidus dominated, while the genus Leuconostoc was found in all samples. The physical and chemical properties of the sausage were also determined in the finished product and during ripening at different times. Decreases in pH, water activity, and moisture content were found, as well as increases in protein and fat content. Different recipes and different ripening times of Pirot "ironed" sausage had no significant effect on the fatty acid profile, but a significant effect on the instrumental color parameters. Statistically significant differences were also found in the investigated texture parameters between sausages produced according to different recipes. Different proportions of meat used resulted in significant differences in sensory properties