Identification of lactic flora of fermented carrot using biochemical and molecular method and determination of their relationship with phylogenetic analysis

Introduction: Carrot products such as carrot juice and fermented carrot products possess high nutritional value and they are considered as a major source of β-carotene. Carotenoids because of containing conjugated double bonds, have antioxidant properties and provide the natural yellow, orange and red colors in fruits and vegetables. Due to the outbreak of some problems such as lactose-intolerance and high blood cholesterol especially in dairy products’ consumption, great attention has been drawn toward fermented vegetable products. Lactic acid bacteria (LAB) including important genera: leuconostocs, lactobacilli, streptococci and pediococci are wide-spread and have been divided according to morphological features and fermentation pathway, which utilize glucose. Current knowledge regarding involved microorganisms in vegetable fermentation is still dependent on biochemical and classical data. Nowadays, application of molecular methods in the field of microbial identification has been provided better understanding from fermented foods ecology. Since local starter cultures are considered as precious genetic resources in each country and also they play an important role in production and creation of organoleptic characteristics in fermented products, therefore, the objective of present study was the isolation and identification of lactic flora from fermented carrot with the help of conventional (biochemical) and molecular methods and determination of phylogenetic relationships. Materials and methods: Following the production of fermented carrot samples, they were packed in plastic container and stored at ambient temperatures (25-27°C). In the next step, total LAB count was performed according to Iranian standard of 5484. Isolation and selection of LAB was done during 32 days with the intervals of 0, 4, 8, 16, 24 and 32. For initial identification of LAB, isolated were subjected to gram staining and catalase tests. Also biochemical tests including growth at 15 and 45C, at NaCl 6.5% and 18%, pH=4.4 and 9.6, were done in order to identify   and classify at genus level. Carbohydrate fermentation profiles were obtained for isolates with the aid of 10 sugars. Molecular identification was done with DNA extraction followed by amplification of 16S gene with universal primers (27 F and 1492 R). For sequencing of resulted PCR-products, they were sent to Macrogen Company, South Korea. Phylogenetic tree was plotted with Clustal Omega and Fig. Tree soft wares. Results and discussion: In the first step, 144 gram positive, catalase negative isolates were screened and selected as presumptive LAB according to gram staining and catalase test and morphological characteristics. Among them, 48 representative isolates were chosen and identified up to genus level according to biochemical tests. Five distinct genera were identified as Pediococci (4.08%), homofermentative lactobacilli (34.69%), hetero fermentative lactobacilli (36.74%), Leuoconostocs (20.41%) and enterococci (4.08%). Carbohydrate fermentation profiles revealed Lactobacilli constitute the highest percent among other genera and also some species like Lb. kimchi and Lb. parakefiri were detected. Growth of lactic acid bacteria experienced increasing trend up to day-16 but thereafter showed decline trend until the end of storage time (day-32). 26 out of 48 isolates were subjected to molecular analysis. Results of sequencing revealed following species: Lb.plantarum (9), Lb. brevis (8), Leu. mesenteroides (4), Lb. casei (1), Lb. paracasei (1), and Lb, pantheris (1). Changes and variation of lactic flora during fermentation stages revealed that at initial stages of fermentation  (0- day-8) Leuconostocs sp. were predominant species but disappeared then. In the next stages of fermentation Leuconostocs sp. were replaced by homo-fermentative strains such as Lb. plantarum which was present from the first day up to day-24 but constituted the majority of species on day-16. In the final stage, Lb. brevis dominated the others due to better survival and resistance of this bacterium at the increased acidity level. Phylogenetic tree results revealed three clusters including cluster I (composed of three sub-clusters), cluster II (three sub-clusters) and cluster III (two sub-clusters). Cluster I included two genera: Leuconostocs sp. (mesenteroides) and Lactobacillus (pantheris, casei and paracasei). Cluster II included Lb. brevis and finally cluster III composed of Lb. plantarum.