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Probiotic Bacteria from Pollen: Selection, Characterization and in Vitro Inhibition of Helicobacter Pylori

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Probiotic Bacteria from Pollen: Selection, Characterization and in Vitro Inhibition of Helicobacter Pylori الجمهورية الجزائرية الديمقراطية الشعبية وزارة التعليم العالي و البحث العلمي University of Ferhat Abbas Sétif 1 1 جامعة فرحات عباس، سطيف كلية علوم الطبيعة و الحياة Faculty of Natural and Life Sciences DEPARTMENT OF MICROBIOLOGY N°………………………………………….…………..…….……/SNV/2014 Thesis Presented by BELHADJ Hani For the fulfillment of the requirements for the degree of Doctorate of Sciences In Biology Option: Microbiology Topic Probiotic Bacteria from Pollen: Selection, Characterization and in vitro Inhibition of Helicobacter pylori Presented publically in 29 / 10 /2014 Jury President Guechi Abdelhadi Pr. U. Ferhat Abbas Sétif 1 Supervisor Harzallah Daoud Pr. U. Ferhat Abbas Sétif 1 Examiners Maamache Bakir Pr. U. Batna Cheriguene Abderrahim Pr. U. Mostaganem Laboratory of Applied Microbiology مــلـــخــــص بكتيريا بروبيوتيك من حبوب الطلع: اﻻنتقاء و التشخيص والتثبيط المخبري للبكتيريا Helicobacter pylori في هذا الـبحث، تم التحقـق من الجودة الميكروبيولوجية لعـينات من طـلع الـنحل. تراوح تعـداد الفـلورا الكلية الهـوائية بين 3 و 5 لغ وم م / غ، أما الفـطريات فـقدرت بيـن 3.3 و حوالي 7 لغ وم م /غ. كما لوحظت كثافـة عالية للمكورات العنقودية الذهـبـية )وصلت إلى 2.33 لغ وم م / غ(. فـي حـيـن تأرجح تـعـداد بكتـيـريا القـولون من 3 إلى أكـثـر من 2 لغ وم م / غ. باﻹضافة إلى ذلك، تم الكـشـف عن اﻷمعائـيات الممرضة و عزلت الفـطريات المنتجة للسموم. انـتــشـلت 567 عـزلـة من بكـتـيـريا حـمـض الــلبـن و اعــتمادا علي الــنشاطـية العــدائـية تم تحيــيـد 55 بكـتـريا حـمض الـلبن من بـيـن 316 عــزلة ذات نــشاطــية حــيوية. حددت سبعة مجموعات مع عضوين آخرين على مستوى التشابه 77 ٪ علي أساس الصفات الـمـظهــرية. كما بين التسلسل الجزيئي لمورثات الرنا الريبوزي 16S، أن عشر سﻻﻻت من البكتيريا اللبنية تنتمي إلى سبعة أنواع : ,Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis Lactobacillus ingluviei, Pediococcus pentosaceus, Lactobacillus acidipiscis , Weissella cibaria . شخصت بعد ذلك عشر سﻻﻻت من العصيات اللبنية )Lactobacillus( علي أساس الميزات المظهرية و الوراثية و التي اختيرت لمزيد من تقييم خصائص التعزيز الحيوي. لم يتمكن من انتشال إﻻ سﻻلتين فقط )Lactobacillus sp. BH1398 وBH1480( بعد ثﻻث ساعات من التجربة في درجة الحموضة 3. ثبت أن اﻷنواع .Lb. fermentum, Lb.acidophyllus, Lb Lb. plantarum, pentosus هي اﻷكثر مقاومة للظروف القاسية. يـبـدو أن، 3.3٪ و 3.5٪ من اﻷمﻻح الصفراوية ﻻ تؤثر بشكل كبير على بقاء معظم السﻻﻻت ، باستثناء Lactobacillus sp.BH1398. أبدت كل السﻻﻻت مقاومة للكوليستين، الكليندامايسين، الكلورامفينيكول، وسيبروفلوكساسين، لكن معظم السﻻﻻت حساسة للمضادات الحيوية، أوكساسيلين، أوكسيتيتراسيكلين، وأموكسيسيلين. تم التحقــق في اﻷخير من أمكانية تأثير Lactobacillus fermentum BH1509 الكابح ضد سﻻلتين من سﻻﻻت Helicobacter pylori. قيس الفعل القاتل للمحلول الطافي لمستنبت البكتيريا اللبنية. و تم التأكد من إنتاج السﻻلة BH1509 لمبيد ميكروبي )بكتيريوسين( منخفض الوزن الجزيئي، سمي BAC-F15. بعد تنقية جزئية بالترسيب بسلفات اﻷمونيوم و كروماتوغرافيا Sephadex G-50، قدرالوزن الجزيئي التقريبي لـ BAC-F15 بعد الهجرة الكهربائية بين 5133 و 6533 دالتن. يبدي هذا البكتيريوسين نشاطية تثبيطية واسعة ضد بكتيريا فساد اﻷغذية و الممرضة، بما في ذلك البكتيريا إيجابية الجرام مثل Micrococcus luteus, Staphylococcus aureus, Listeria innocua وسالبة الجرام، Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Stenotrophomonas Campylobacter jejuni ، maltophilia, Helicobacter pylori . لم يتأثر نشاط السائل الطافي المضاد للبكتيريا بالكاتاﻻز أو الليباز، لكن ألغي من قبل اﻹنزيمات المحللة للبروتين مثل بروتيناز K، التربسين و بروناز BAC-F15 .E هو بروتين مقاوم للحرارة. و أظهر نشاطا مثبطا على نطاق واسع من درجة الحموضة )5.3-2.3(. و أبدى فعﻻ قاتﻻ للميكروبات و اتضح دلك جليا من نمط تأثيره على سﻻﻻت Helicobacter pylori. إن المبيد الميكروبي والبكتيريا المنتجة له، Lactobacillus fermentum BHB1509، قد تكون مضادات ميكروبية مفيدة للسيطرة على اﻻلتهابات المعوية التي تحدثها .Helicobacter pylori كلمات مفتاحية: طلع النحل، مراقبة الجودة النوعية، اﻷغذية الوظيفية، الملوية البوابية، المعززات الحيوية، اﻻلتهابات المعوية و المعدية، البكتيريوسين، العصيات اللبنية، البكتيريا اللبنية. ii ABSTRACT Probiotic Bacteria from pollen: Selection, Characterization and in vitro Inhibition of Helicobacter pylori In this research, microbiological quality control of pollen samples was investigated. TAMC ranged from 3.00 to 5.80 Log CFU/g. TMYC was between 2.3 and about 7 Log CFU/g. However, Staphylococcus aureus was recovered with high density (up to 8.32 Log CFU/g). Enterobactericeae count ranges from 3.00 to more than 8 Log CFU/g. In addition, samples contain pathogenic members of Enterobacteriaceae and potent toxinogenic molds Furthermore, 567 isolates of lactic acid bacteria were recovered from raw bee pollen grains. Based on antagonistic activity, neutralized supernatants of 54 lactic acid bacteria (LAB) cultures from 216 active isolates inhibited the growth of indicator bacteria. They were phenotypically characterized and seven clusters with other two members were defined at the 79% similarity level. Partial sequencing of the 16S rRNA gene of representative isolates, 10 strains were assigned to seven species: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Lactobacillus ingluviei, Pediococcus pentosaceus, Lactobacillus acidipiscis and Weissella cibaria. After that, 10 Lactobacillus isolates identified, by phenotypic and genotypic methods, were selected for further evaluation of their probiotic properties. At pH 2, only tow strains (Lactobacillus sp. BH1398 and BH1480) that could not recovered after three hours. It seems that, 0.3% and 0.5% of bile salts does not affect greatly the survival of most strains, excluding Lactobacillus sp. BH1398. In contrast, in the presence of 1.0% bile salts, survival of five strains was decreased by more than 50 %. In addition, all strains were resistant to colistine, clindamycine, chloramphenicol, and ciprofloxacine, but most of the strains were susceptible to peniciline, oxacillin, Oxytetracyclin, and amoxicillin. Finally, Lactobacillus fermentum BH1509 was investigated for its inhibitory effect against two Helicobacter pylori strains. The bactericidal activity of spent culture supernatants (SCS) was measured. The strain BH1509 produced a small bacteriocin, designated Bac-F15. After partial purification by ammonium sulfate precipitation and Sephadex G-50 chromatography, the approximate molecular weight of Bac-F15 was estimated by SDS-PAGE between 4100 and 6500 Da. It displayed a wide inhibitory spectrum against food-spoiling bacteria and food-borne pathogens. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or lipase but was abolished by the proteolytic enzymes proteinase K, trypsin and pronase E. Bac-F15 was heat stable (10 min at 100°C) and showed inhibitory activity over a wide pH range (from 4.0 to 8.0). It has a bactericidal action as evidenced by its action upon Helicobacter pylori strains. The bacteriocin and its producer, Lactobacillus fermentum BHB1509, may be useful antimicrobial materials to control gastrointestinal infections caused by Helicobacter pylori. Keywords: Pollen, quality control, functional foods, Helicobacter pylori, probiotics, gastrointestinal ulceration, bacteriocin, Lactobacillus, lactic acid bacteria. iii RESUME Bactéries Probiotiques de Pollen: Sélection, Caractérisation et L’Inhibition in vitro de L’Helicobacter pylori Dans cette recherche, la qualité microbiologique d'échantillons de pollen a été étudiée. La MTAM variait de 3.00 à 5.80 Log UFC/g. Le NTML était entre 2.3 et environ 7 Log UFC/g. Cependant, Staphylococcus aureus a été récupéré à haute densité (jusqu'à 8.32 log UFC/g). Le nombre d’entérobactéries varie de 3.00 à plus de 8 Log UFC / g. En outre, les échantillons analysés contiennent des membres pathogènes d'entérobactéries et les moisissures potentiellement toxinogènes, Ensuite, 567 isolats de bactéries lactiques ont été récupérés. Les Surnageants neutralisés des cultures de 54 bactéries lactiques (BAL) parmi 216 isolats actifs inhibent la croissance des bactéries indicatrices. Sur la base de la classification numérique, sept groupes avec deux autres membres ont été définis au niveau de la similitude de 79%. Le séquençage partiel du gène 16S ARNr des isolats représentatifs de chaque groupe, 10 souches ont été attribués à sept espèces: Lactobacillus plantarum, Lactobacillus fermentum, Lactococcus lactis, Lactobacillus ingluvies, Pediococcus pentosaceus, Lactobacillus piscis et Weissella cibaria. Après cela, 10 isolats de Lactobacillus sont identifiés par des méthodes phénotypiques et génotypiques. À pH 2, seulement deux souches (Lactobacillus sp. BH1398 et BH1480) qui ne pouvaient pas récupérer au bout de trois heures. Lb. plantarum, Lb. fermentum, Lb. acidophilus, et L. pentosus sont révélés être les souches les plus résistantes. Il semble que, 0.3% et 0.5% de sels biliaires n'affecte pas considérablement la survie de la plupart des souches, à l'exclusion de Lactobacillus sp. BH 1398. En outre, toutes les souches étaient résistantes à la colistine, la clindamycine, le chloramphénicol et la ciprofloxacine, mais la plupart des souches étaient sensibles à la peniciline, oxacilline, l'oxytétracycline, et l'amoxicilline. Enfin, Lactobacillus fermentum BH1509 a été étudié pour son effet inhibiteur contre deux souches d'Helicobacter pylori. La souche BH1509 produit un petit bactériocine, désigné Bac-F15. Après purification partielle par précipitation au sulfate d'ammonium et Chromatographie sur Sephadex G-50, le poids moléculaire approximatif de Bac-F15 a été estimé par SDS-PAGE, entre 4100 et 6500 Da. Il présente un large spectre d’action. L'activité antibactérienne du surnageant de la culture bactérienne
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