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Identification and Phylogenetic Studies of a New Probiotic ACTA SCIENTIFIC NUTRITIONAL HEALTH (ISSN:2582-1423) Volume 4 Issue 3 March 2020 Research Article Lactobacillus spp. Identification and Phylogenetic Studies of a New Probiotic Ahmed R Awd1, Khater A Khater1 and Egyptian Ahmed IsolateI Ismail 2,3Based* on 16s rRNA Gene 1Dairy Science and Technology Department, Faculty of Agricultural, Al-Azhar Received: University, Egypt Published: February 17, 2020 2 Ahmed I Ismail., Biotechnology Department, Faculty of Agriculture, University of Al-Azhar, Cairo, February 26, 2020 et al. Egypt © All rights are reserved by 3Public Health Department, College of Applied Medical Sciences, Majmaah University, Al-Majmaah, Saudi Arabia *Corresponding Author: Ahmed I Ismail, Biotechnology Department, Faculty of Agriculture, University of Al-Azhar, Cairo, Egypt and Public Health Department, College ofDOI: Applied Medical Sciences, Majmaah University, Al-Majmaah, Saudi Arabia. 10.31080/ASNH.2020.04.0654 Abstract Lactobacillus Lactobacillus represents one of the most important genera of human and animal intestinal tract. They are used in dairy and non- dairy probiotics foods to restore the intestinal microflora, which provides a health benefit. strains were phenotypically identified and described using biochemical features and kits such as the API 50 CH system. Conventional biochemical and physiologi- cal studies have certain limitations when discriminating against large numbers of isolates with similar physiological characteristics. They are generally unreliable, and consuming money, time, and effort. There are many studies have focused on the rapid identifica- tion of molecular biology techniques for the rapid identification and detection of lactobacilli. In this study, we examined five pairs L. casei, L. rhamnosus L. gasseri of primers on complex communities to facilitate the examination of complex micro-ecosystems. The two primers Lac1-Lac2 and Joh16SI-Joh16II showed positive results with , and . Their strains were isolated from raw milk and feces of breast-fed infants. The fragments obtained from DNA samples after PCR amplification were sequenced and submitted in Lactobacillus GenBank "NCBI" under accessions number "KY123805", "KY123789" and "KY123806". As the long-term goal of this research was to obtainKeywords patent: protection for sp. isolated fromLactobacillus Egyptian resources and using it in food and dairy industries. API 50 CH; GenBank; Intestinal Microflora; ; Molecular Identification; Phylogenetic; Probiotics; 16S rRNA Introduction Lactobacillus rately. Recently, the use of biochemical characteristics in addition to spp. are microorganisms with a long-documented Kits likes API 50 CHL system and molecular methods such as poly- history of use in dairy technology and as a natural preservative in merase chain reaction (PCR) provided a novel approach for bacte- . dairy products due to their ability to inhibit the growth of harm- rial identification [6]. ful bacteria and thus increasing the shelf life of the products [1] Lactobacillus Therefore, the purpose of the inquiry under way was to isolate In addition, The interest is enhanced by proven probiotic proper- and classify some spp.by classical (morphology and ties that give consumers a health benefit [2]. The genus of Lacto- biochemical features included API 50 CHL system) and molecular bacillus is one of the major members of lactic acid bacteria (LAB), Lactobacillus (PCR technique) methods. An additional aim was to registering gram-positive, catalase-negative, rods shaped, non-spore forming patent protection for spp. Strains are isolated locally bacteria and produce lactic acid as the main end product during from Egyptian resources to increase the additive value of the Egyp- carbohydrate fermentation [3]. tianMaterials microbial and diversity. Methods The members of this genus have been isolated from different Samples collection sources, e.g. milk, dairy products, sewage, plants, food products, and animal intestines. Currently, this genus contains more than Forty-Nine Samples were collected from different healthy moth- 180 species [4]. However, there is a large degree of heterogeneity er breast milk, fresh feces of breast-fed infants, raw milk and some within the members of this genus is challenging [5]. Lactobacillus dairy products. The infant ages varied from 1 to 8 months. Samples classification is traditional based on morphologi- were kept in sterile glass containers, transported within two hours cal and physiological criteria. These conventional criteria are often in the icebox to the laboratory and analyzed on the same day. time-consuming and sometimes fail to identify the bacterial accu- Citation: , et al. Lactobacillus Acta Scientific Nutritional Health Ahmed I Ismail. “Identification and Phylogenetic Studies of a New Probiotic spp. Egyptian Isolate Based on 16s rRNA Gene". 4.3 (2020): 104-110. Identification and Phylogenetic Studies of a New Probiotic Lactobacillus spp. Egyptian Isolate Based on 16s rRNA Gene Isolation of lactobacilli culture 105 72°C for 90 s and final cycle 72°C for 5 minutes. and then kept at 4°C until further use. Serial dilution of each sample in citrate buffer was plated on • Amplified fragments visualization: MRS agar medium [7], Then incubated at 37°C for 48 hours un- Agarose gel electropho- der anaerobic conditions using GENBox anaerobic kit. Obtained resis 1.5% (wt/vol) was used to visualize the amplified DNA colony were randomly picked, streaking twice on MRS agar plates fragments, gels were stained with ethidium bromide (0.5 μg/ and pre-incubated under the same previous conditions, to ensure ml) and loaded directly onto the gel, DNA ladder was also Identificationcomplete purification. of isolated cultures loaded onto the gel for comparison of fragment size and vi- sualized under UV light (Dual – Intensity Trans Illuminator) About 90 isolates were tested for Gram-staining [8], catalase and quantified using spectrophotometer (GENWAY630). The activity [9], and cell morphology. In order to obtain a complete PCR product containing DNA fragments with the expected size physiological characterization of the isolates, the following2 prop- were purified using GeneJET™ PCR Purification Kit (Thermo cose erties were also examined: the formation of acid and Co from glu- #K0701), then subjected to sequencing. • Sequencing of 16S: [10], growth at 15 and 45°C, fermentation of glucose, indol The species specificity of the primers APIproduction 50 CHL and system gelatin liquefaction [11] Fragments of PCR products for Lac1 - Lac2 and Joh16SI-Joh- Lactobacillus 16SII primers were sent to the University of Potsdam, Insti- API 50 CHL medium, intended to classify the ge- tute of Biochemistry and Biology, (Potsdam, Germany) for nus and related genera. This medium allows 49 different carbohy- sequencing using an ABI 3730xl DNA sequencer. The nucleo- drates to be fermented on the API 50 CHL strip. A suspension is tide sequences for resulted fragments were deposited to the made with the micro-organism to be evaluated in the liquid (API Genbank Database in date 14 and 16 Nov 2016 and it has been 50 CHL medium), and each strip tube is then inoculated with the named as Strain AZ1 using Bankert tool. Bioinformatics workflow (dry lab) suspension. Through incubation, the carbohydrates are fermented • Primer selectivity based on in silico studies: into acids that cause a decrease in pH, as indicated by the indicator in silico color change. The results constitute the biochemical profile which Before pro- Molecularthe identification biology software studies uses (Wet to lab) classify the strain. ceeding to wet laboratory, the have been validated. • Genomic DNA isolation and quantification: Each primer's properties including melting temperature, percentage of GC content and suitability for PCR have been Seven isolates described in the report. It helps us assess potential PCR of total genomic DNA were extracted using (Gene JET Ge- L. primers from the list of primers selected from Previous re- nomic DNA Purification Protocol Kit#K0721-Thermo Fisher acidophilus, L. johnsonii, L. rhamnosus, L. reuteri. search for all the species being studied Lactobacillus sp. Scientific Inc.) and eight isolates were extracted using. (ZR NCBI blast Fungal/Bacterial DNA MiniPrep™ Catalog No D6005 –Zymo primer tool http://www.ncbi.nlm.nih.gov/tools/primer Research CORP.). The DNA extraction procedure was per- Lactoba- was used to test the primers as it has a wide range of pa- formed in compliance with manual of the manufacturer. B- cillus. rameters in this process include, nr database and Total DNA was visualized on 1% agarose gel to determine DNA quality. Smeared appearance samples were considered • Sequences submissions and accession numbers: as fragmented DNA and were excluded, DNA concentration The se- was determined using a UV Dual-Intensity Trans Illuminator quences of this study have been submitted to the NCBI us- • and quantified using a spectrophotometer (GENWAY630). ing the Bankit tool http://www.ncbi.nlm.nih.gov/BankIt/. election of Primers for Lactobacillus identification: • Sequences analysis: S Lac- A search was carried out using the tobacillus species and group-specific primers were selected online tool NCBI BLASTn http:/ncbi.nlm.nih.gov/BLAST/ based on the sequences of 16S rRNA [12-14] (Table 1). • PCR amplification for 16S: against the database for the collection of nucleotides (nr/ nt) to find Sequence Similarity. The default parameters of Amplification reactions were BLASTN have been used. prepared in total volumes of 50 μl containing
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