كليدواژه :
نعناع فلفلي , لاكتو باسيلوس , اسانس , شكمبه
چكيده فارسي :
زمينه مطالعاتي: اسانس برخي گياهان فعاليت ضد ميكروارگانيزمي دارد. هدف: اين تحقيق به منظور بررسي اثر اسانس گياه نعناع فلفلي بر لاكتوباسيلهاي جداسازي شده از شكمبه گاو به منظور مطالعه امكان كنترل اسيدوز از طريق كاهش اين ميكروارگانيزم‌ها انجام شد. روش كار: نمونه مايع شكمبه از شكمبه گاوهايي كه با جيره حاوي 65%كنستانتره و 35% علوفه تغذيه شده بودند جمع‌آوري و صاف گرديد. به‌منظور جداسازي لاكتوباسيلها در سطح جنس از محيط اختصاصي MRS استفاده گرديد و انتخاب كلني‌ها بر اساس اندازه و مورفولوژي و تست‌هاي كاتالاز و اكسيداز و رنگ‌آميزي گرم انجام گرفت. سپس تست‌هاي بيوشيميايي تخمير قندها و هيدروليز آرژنين و توليد CO2 از گلوكز انجام شد. به منظور بررسي تأثير اسانس نعناع فلفلي بر لاكتوباسيلهاي جداسازي شده و لاكتوباسيلوس پلنتارم ابتدا اسانس گياه بوسيله دستگاه كلونجر استخراج شد و سپس اثر آن به كمك روش ايجاد حلقه در آگار و اندازه‌گيري قطر هاله عدم رشد انجام گرفت. آزمايش در قالب طرح كاملاً تصادفي با سه تكرار انجام شد. تيمارهاي آزمايش شامل 16 گونه لاكتوباسيلوس بود كه در معرض غلظت ثابتي از اسانس خالص نعناع فلفلي وآب مقطر (شاهد) قرار گرفتند. نتايج: تعداد 36 كلني مربوط به جنس لاكتوباسيلها جداسازي و شناسايي گرديد كه در15گروه در سه طبقه تقسيم بندي شدند. لاكتوباسيلهاي هتروفرمنتيتيو با 58% فراواني بيشتري نسبت به لاكتو باسيل‌هاي هموفرمنتيتيو با 42% داشتند. اسانس نعناع بر تمام باكتري‌هاي آزمايش شده اثر مهاري داشت. نتيجه گيري نهايي: اسانس گياه نعناع فلفلي اثر معني‌داري (p≤0/05) بر كاهش رشد لاكتوباسيلها در محيط آزمايشگاه داشت.
چكيده لاتين :
Introduction: Plants produce a wide variety of organic compounds as metabolites derived from the secondary metabolism. Some plants are sources of several types of essential oil. Essential oils often have antimicrobial activity against a wide range of bacteria, yeasts and molds. Carvacrol, a terpene found in oregano oil, inhibits the growth of several bacteria strains including Escherichia coli and Bacillus cereus. Another example of essential oils is thymol, isomeric with carvacrol and found in oil of the common spice thyme. Thymol is part of a naturally occurring class of compounds know as biocides. An essential oil is a concentrated hydrophobic liquid containing volatile aroma compounds from plants. Essential oils are also known as volatile oils. Essential oils are usually lipophilic compounds that usually are not miscible with water. They can be diluted in solvents like pure ethanol and polyethylene glycol. Lactobacillus is a genus of Gram-positive, facultatively anaerobic or microaerophilic, rod-shaped, non-spore forming bacteria. They are a major part of the lactic acid bacteria group that convert sugars to lactic acid. Lactobacillus has many species. Many lactobacilli operate using homofermentative metabolism (they produce only lactic acid from sugars), and some species use heterofermentative metabolism (they can produce either alcohol or lactic acid from sugars). Lactobacilli are catalase negative lactic acid bacteria. Strains of lactobacilli are isolated from the rumen of cattle, sheep and other ruminating animals. In order to evaluate the effect of Mentha piperita essential oil on the isolated Lactobacillus engaged with rumen acidosis, ruminal fluid samples were collected from the rumen of cows equipped with permanent fistula. Material and methods: The cows were fed by a diet containing 65% concentrate and 35% forage. The Menthapiperita essential oil was extracted by the clevenger apparatus. Bacterial isolation was done according to the standard methods. Bacterial culture was done on the special media of MRS and colonies were selected and distinguished based on their size and morphology as well as catalase, oxidase and Gram staining tests. Catalase test was performed in two ways, tube metod and slide method. Tube method poured 1-2 ml of hydrogen peroxide solution into a test tube and used a sterile wooden stick or a glass rod, taked several colonies of 18-24 hours test organism and immerse in the hydrogen peroxide solution and observed for immediate bubbling. Slid method used a loop or sterile wooden stick to transfer a small amount of colony growth in the surface of a clean, dry glass slide and placed a drop of 3% H2O2 in the glass slide and observed for the evolution of oxygen bubbles. The oxidase test is used to identify bacteria that produce cytochorome oxidase, an enzyme of the bacterial electron transport chain. All bacteria that are oxidase positive are aerobic, and can use oxygen as a terminal electron acceptor in respiration. This does not mean that they are strict aerobes. Oxidase test was performed with this method. The amount of 2-3 drops of oxidase reagent (kovacs) was poured onto filter paper and with the loop, some bacterial colonies were tested on the filter paper. After 10 seconds, the dark blue color indicates that the test is positive. Complementary test experiments including biochemical, fermentation of carbohydrates, arginine hydrolysis, study of bacterial growth in different conditions with regards to environmental temperature and pH, sodium chloride concentration and CO2 production from glucose were performed. The antibacterial effects of M.piperita essential oil was evaluated by using well diffusion agar method and measuring the inhibitory zone diameter in comparison with the control samples (distilled water) and t-test statistic.
Results and discussion: Thirty-six colonies of Lactobacillus genus were identified according to their characteristics. It was confirmed that heterofermentative Lactobacillus with a propertion of 58% were more than the homofermentative lactobacillus with 42% following running the purification and biochemical tests of arginine hydrolysis and fermentation of carbohydrates and Co2 production from glucose. Based on the biochemical similarity and growth characteristics, the 36 isolated Lactobacillus were classified into 15 groups and 3 stories. Fifteen species of Lactobacilli were diagnosed following the commentary experimental tests in comparison with Bergey's Manual of Systematic Bacteriology. By using Levene's test, the equality of variances between 2 groups didn't reject, hence the variations between these 2 groups were equal with the 95% level of significance. In all cases the number was less than 0.05 (P<0.05) or the average of inhibitory zone diameter in all treated Lactobacilli were significantly higher than the control. Lactobacillus were sensitive to the peppermint essential oil.
Conclusion: All the 15 isolated groups of Lactobacillus and Lactobacillus plantarum were sensitive to the peppermint essential oil based on colony characteristics. Nearly all species of isolated Lactobacilli species were sensitive to the essential oils. More in vivo experiments are required for reaching to the practical application of M.piperita essential oil utilization against the Lactobacillus engaged with rumen acidosis in ruminants.
