شناسایی فلور لاکتیکی هویج تخمیری با استفاده از روش‌های بیوشیمیایی و مولکولی و بررسی روابط خویشاوندی به کمک آنالیز فیلوژنتیکی

هویج تخمیری نوعی شوری تهیه شده از هویج و نمک است که سرشار از ویتامین‌ها می‌باشد. هدف از این پژوهش، بررسی تنوع زیستی باکتری‌های اسید لاکتیک فرآورده طی دوره نگهداری و رسیدگی بود. بدین منظور پس از تولید هویج تخمیری، تنوع باکتری‌های اسید لاکتیک موجود در فرآورده در شش تناوب زمانی بررسی گردید. 144 جدایه گرم مثبت و کاتالاز منفی، جداسازی شده که از میان آنها، 48 جدایه گرم مثبت و کاتالاز منفی انتخاب شدند. آزمون‌های فیزیولوژیکی و بیوشیمیایی شامل رشد در دماهای 10 و 45 درجه سانتی گراد، در 6.5 درصد نمک طعام، در 4.4 =ph و 9.6 ph=، وتولید گاز دی‌اکسیدکربن از قند گلوکز انجام پذیرفت. در مرحله بعد، شناسایی بر اساس پروفایل تخمیر کربوهیدرات (ده قند)، منجر به شناسایی جنس‌های پدیوکوکوس (2 مورد)، لویکونوستوک (10 مورد)، لاکتوباسیلوس (33 مورد) و انتروکوکوس (2 مورد) و یک مورد هم شناسایی نگردید و در مجموع 19 گونه مختلف گردید. در پایان، 26 جدایه از مجموع 48 جدایه توسط تعیین توالی ژن 16s rdna تا سطح جنس و گونه شناسایی شدند. نتایج توالی‌یابی منجر به شناسایی گونه‌های ذیل گردید: lactobacillus plantarum (33.34%)، leuconostoc mesenteroides (14.85%) ،lactobacillus.brevis (29.63%)، lactobacillus.casei (3.7%)، lactobacillus.pantheris (3.7%)، lactobacillus paracasei (3.7%) و سه ایزوله تا مرحله جنس به‌عنوان لاکتوباسیلوس شناسایی شدند. لویکونوستوک مزنترویدس در مراحل اولیه تخمیر با توجه به نتایج توالی‌یابی و مولکولی غالب بوده که به تدریج در مراحل پایانی توسط لاکتوباسیلوس برویس جایگزین گردید. آنالیز فیلوژنتیک وجود سه خوشه را نشان داد، به‌طوریکه خوشه اول شامل دو جنس لوکونستوک و لاکتوباسیلوس، خوشه دوم شامل لاکتوباسیلوس برویس و خوشه سوم شامل لاکتوباسیلوس پلانتاروم بوده است.

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 βcarotene. 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 lactoseintolerance 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 widespread 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 (2527°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 PCRproducts, 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 day16 but thereafter showed decline trend until the end of storage time (day32). 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 day8) Leuconostocs sp. were predominant species but disappeared then. In the next stages of fermentation Leuconostocs sp. were replaced by homofermentative strains such as Lb. plantarum which was present from the first day up to day24 but constituted the majority of species on day16. 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 subclusters), cluster II (three subclusters) and cluster III (two subclusters). 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.