سطـوح مختلـف اسانس‌های میخـک و مـرزه بـر فراسنجه‌های تخمیری در تکنیک تولید گاز و تاثیـرات ضد‌میکروبی آنها بر باکتری پپتواستـرپتوکـوکوس انئـروبیوس(Peptostreptococcus anaerobius) جداسازی شده از شکمبه

نوع مقاله : علمی پژوهشی - تغذیه نشخوارکنندگان

نویسندگان

1 گروه علوم دامی، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران.

2 دپارتمان کشاورزی، دانشکده فنی و کشاورزی شهریار، دانشگاه فنی و حرفه ای استان تهران، ایران

3 گروه علوم کشاورزی ، واحد مشهد، دانشگاه آزاد اسلامی، مشهد، ایران

4 پژوهشکده کشاورزی هسته ای، پژوهشگاه علوم و فنون هسته ای، کرج، ایران

چکیده

به­منظور بررسی اثر مقادیر مختلف اسانس­های میخک و مرزه بر فراسنجه ­های تخمیری در شرایط برون­تنی، دو آزمایش تولید گاز و کشت میکروبی طراحی­گردید. تکنیک تولید گاز در قالب آزمایش فاکتوریل 3×3 شامل اسانس­های مرزه و میخک هرکدام 3 سطح (0، 250 و 500 میلی­گرم) طراحی­ شد. مقدار تولید گاز در طی ساعات 2، 4، 6، 8، 16، 24، 48، 72، 96 و 120 اندازه‌­گیری­شد. سطوح بالای اسانس میخک در ساعات ابتدایی تخمیر باعث افزایش معنی دار گاز تولیدی گردید. در مقابل اسانس میخک در ساعات 24-8 تولید گاز را کاهش­داد. در آزمایش میکروبی، سطوح مختلف اسانس­های مرزه و میخک (20، 35 و 40 میکرولیتر برای هر اسانس بصورت جداگانه) و همچنین سطوح ترکیبی اسانس­های مزبور ( 9 تیمار حاوی سطوح ترکیبی) و یک تیمار شاهد (استرپتومایسین) مورد مقایسه قرار گرفتند. بعد ازانجام کشت باکتری پپتواستـرپتوکـوکوس انئـروبیوس در محیط کشتو تقابل آن با اسانس­های مرزه و میخک مشاهده گردید تمامی تیمارهای حاوی اسانس (ساده و ترکیبی) نسبت به شاهد، تیمارهای حاوی اسانس مرزه نسبت به شاهد و سطوح ترکیبی اسانس­های مزبور نسبت به سطوح ساده اثر بازدارندگی بیشتری بر رشد این باکتری داشته­است. به طور کلی نتایج مرحله اول آزمایش نشان داد که استفاده از اسانس میخک باعث کاهش تولید گاز و فراسنجه‌های تخمیری شد، اما اسانس مرزه بر مقدار تولید گاز، قابلیت هضم ماده آلی، انرژی قابل متابولیسم و مقدار اسیدهای چرب فرار تأثیر نداشت. نتایج حاصل از آزمایشات کشت میکروبی نیز نشان داد اسانسهای مرزه و میخک اثر ممانعت کنندگی بر رشد باکتری پپتواستـرپتوکـوکوس انئـروبیوس داشته و در نتیجه تولید آمونیاک توسط این باکتری کاهش می یابد.

کلیدواژه‌ها


عنوان مقاله [English]

Different levels of clove and savory essences on fermentative parameters in gas production technique and their antimicrobial effects on Peptostreptococcusanaerobius bacteria isolated from rumen

نویسندگان [English]

  • Hojat Imani Moghadam 1
  • Samaneh ghasemi 2
  • saeid sobhanirad 3
  • Mehdi Behgar 4
1 Department of Agricultural Science, Mashhad Branch, Islamic Azad University, Mashhad, Iran
2 Department of Agriculture, Faculty of Shahryar, Tehran branch, Technical and Vocational University, Tehran
3 Department of Agricultural Science, Mashhad Branch, Islamic Azad University, Mashhad, Iran
4 Nuclear science &technology research institute
چکیده [English]

Introduction Since the legislation of European Union has prohibited the use of growth-promoting antibiotics such as monensin, scientists have been interested in alternatives to manipulate rumen fermentation. The use of growth-promoting antibiotics in animal feeds is banned in Europe due to potential risks such as the spread of antibiotic resistance genes or the contamination of milk or meat with antibiotic residues. Recently, essential oils have been increasingly evaluated to replace or facilitate reductions in the use of antibiotics. The most effects of plant essential oils, especially cloves and savory oils, are their antioxidant effects and their effects on the metabolism of ruminal microbes. The antiprotozoal effects of clove extract have been proven in the studies in vitro by gas production technique. Few studies have been done on the effects of clove oil, especially the savory oil, on the digestive properties in the country. Also, no study was found on the interactions of these essential oils in the experiments in vitro. Thus, the aim of this study was investigated to evaluate the effects of clove and savory oils on gas production and in vitro fermentation process and estimation of gas production parameters of feedstuffs (alfalfa hay and barley grain).
Materials and Methods Experimental treatments were included control (basal feeds without additive), basal feeds supplemented with three levels of clove oil (0, 250, 500 mg) and three levels of savory oil (0, 250, 500 mg) per kg of DM in a rumen culture. Ruminal fluid was collected from two fistulated sheep (49.5±2.5 kg). All samples isolated from the rumen were withdrawn 2 h after the morning ration had been consumed. Collected rumen contents were strained through four layers of cheesecloth and brought immediately to the laboratory. Gas production technique was used to detect the fermentation parameters of the treatments. About 200 mg of basal diet (alfalfa hay and barley grain, 1:1, with clove and savory oils) were incubated in 100ml glass syringes and 30ml of incubation liquid were added and were incubated in 39 C° water bath. The gas production was measured in 2, 4, 6, 8, 16, 24, 48, 72, 96, 120h. Three parallel syringes of each treatment were prepared in this experiment in a completely randomized design in a factorial arrangement. They were used to measure the gas production parameters (fermentable fraction (b) and rate (c) of gas production) cumulative gas production, organic digestibility and metabolizable energy of treatments until 120 h. In the present study, digestible organic matter, metabolizable energy, and production of volatile fatty acids were estimated based on the presented equations.In the second experiment, Peptostreptococcus anaerobic was isolated from the ruminal fluid, cultured in the medium of BAAA) Bile EsculinAzideAgar(, and evaluated by different levels of cloves and fennel essences in a randomized complete design with sixteen treatments and three replicates using Duncan test at level 0.05. Experimental levels in this experiment including: 20, 35 and 40 µl of cloves essence, 20, 35 and 40 µl of fennel essence, 20 µl of fennel essence + 20 µl of cloves essence, 20 µl of fennel essence + 35 µl of cloves essence, 20 µl of fennel essence + 40 µl of cloves essence, 35 µl of fennel essence + 20 µl of cloves essence, 35 µl of fennel essence + 350 µl of cloves essence, 35 µl of fennel essence + 40 µl of cloves essence, 40 µl of fennel essence + 20 µl of cloves essence, 40 µl of fennel essence + 35 µl of cloves essence, 40 µl of fennel essence + 40 µl of cloves essence, control.Statistical analysis of data was performed by SAS statistical software (9.1 version). Duncan's multiple test range was conducted in level 5%.
Results and Discussion High levels of savory oil were increased gas production in the first hours (p<0.05), but clove oil was reduced gas production in 8-24 hours (p<0.05). Although, few data have been published on the interaction effects of the use of the essential oils, as well as on the effects of savory oil on fermentation parameters. The amount of gas production (b) tend to be decreased (P=0.06) due to the simultaneous use of savory and clove oils. Different levels of savory oil had no effect on estimated organic matter digestibility, metabolizable energy, and volatile fatty acids. But, with increasing the levels of clove oil, the amount of estimated organic matter digestibility, metabolizable energy tend to be decreased (P=0.09). In the microbial experiment, after incubation of peptostreptococcus anaerobic in medium of Bile AesculinAzideAgar and adding different levels of cloves and fennel essences was observed that all levels of treatments were significant (P<0.05); the highest growth of bacteria was related to treatment 11 (35 µl of fennel essence + 350 µl of cloves essence). This result showed ammonia-producing bacteria known peptostreptococcus anaerobic is inhibiting by different levels of clove and savory essences and finally resulting in the decrease of rumen fluid Ammonia nitrogen.
Conclusion According to our results we can conclude thatusing savory oil improves ruminal fermentation in vitro and with increasing the levels of savory oil, the amount of gas production, the organic matter digestibility, the metabolizable energy and volatile fatty acidsconcentration were decreased. Also in this study, were shown the co-effects of simultaneous use of savory and clove oils on the reduction of gas production. Therefore the effects of essential oils should be considered. For further studies, it is suggested that the effect of essential oils along with various sources of energy, such as pectin, starch, and various protein sources, should be studied in vitro and in vivo. Also, consideration of other changes in fermentation products such as ammonia nitrogen, methane and the composition of the microbial population in these experiments can also be of particular importance.
 

کلیدواژه‌ها [English]

  • clove oil
  • peptostreptococcus anaerobic
  • savory oil
  • Gas production
  • Volatile Fatty Acid
  1. Akhtar, M. S., B. Degaga, and T. Azam. 2014. Antimicrobial activity of essential oils extracted from medicinal plants against the pathogenic microorganisms: a review, Biological Sciences and Pharmaceutical Research, 2 (1): 1–7.
  2. Benchaar, C., S. Calsamiglia, A. V. Chaves, G. R. Fraser, D. Colombatto, T.A. McAllister, and K.A. Beauchemin. 2008. A review of plant-derived essential oils in ruminant nutrition and production. Animal Feed Science and Technology, 145:209-228.
  3. Bromand, M. A., M. Sharifi, Gh. Irajan, F. shahcheraghi, B. Valizadeh, M. Rahbar, F. Fllah, F. RashedMarandi, and M. Sarmi. 2012. Performance Standard for Antimicrobial Susceptibility Testing. Seda, Tehran, Iran (In Persian).
  4. Burt, S. 2004. Essential oils: Their antibacterial properties and potential applications in foods—a review. International Journal of Food Microbiology, 94: 223–253.
  5. Busquet, M., S. Calsamiglia, A. Ferret, and C. Kamel. 2006. Plant extracts affect in vitro rumen microbial fermentation. Journal of Dairy Science, 89: 761-771.
  6. Calsamiglia, S., L. Castillejos, and M. Busquet. 2006. Alternatives to antimicrobial growth promoters in cattle.Pages 129–167 in Recent Advances in Animal Nutrition. P. C. Garnsworthy, and J. Wiseman, ed. Nottingham University Press, Nottingham, UK.
  7. Calsamiglia, S., M. Busquet, P. W Cardozo, L. Castillejos, and A. Ferret. 2007. Invited Review: Essential Oils as Modifiers of Rumen Microbial Fermentation. Journal of Dairy Science, 90:2580–2595.
  8. Cardozo, P.W., S. Calsamiglia , A. Ferret, and C. Kamel. 2005. Screening for the effects of natural plant extracts at different pH on in vitro rumen microbial fermentation of a high-concentrate diet for beef cattle. Journal of Animal Science, 83:2572–2579.
  9. Castillejos, L., S. Calsamiglia, J. Martín-Tereso, and H. TerWijlen. 2008. In vitroevaluation of effects of ten essential oils at three doses on ruminal fermentation of high concentrate feedlot-type diets. Animal Feed Science and Technology, 145:259-270.
  10. Chandrasekharaiah, M., A. Thulasi, and L. Jose. 2015. Effect of Supplementation of Different Essential Oils on in-Vitro Methanogenesis, Fermentation and Digestibility of Finger millet straw based Diet in Rumen Liquor of Crossbred Cattle. Research Journal of Pharmaceutical, Biological and Chemical Sciences, 6 (2): 480-486.
  11. Chen, G., C. J. Sniffen, and J. B. Russell. 1987. Concentration and estimated flow of peptides from the rumen of dairy cattle: Effects of protein quantity, protein solubility, and feeding frequency. Journal of Dairy Science. 70:983–992.
  12. Dorman, H J. D., and S. G. Deans. 2000. Antimicrobial agents from plants: antibacterial activity of plant volatile oils. Journal of Applied Microbiology, 88: 308-316.
  13. Dehority, B.A. 2003. Rumen microbiology. Nottingham University Press, Nottingham, UK.
  14. Eschenlauer, S.C. P., N. Mckain, N. D. Walker, N. R. McEwan, C. J. Newbold, and R. J. Wallace . 2002. Ammonia production by ruminal microorganisms and enumeration, isolation and characterization of bacteria capable of growth on peptides and amino acids from the sheep rumen .Applied Environmental Microbiology, 68: 4925-4931.
  15. Gershenzon, J., and R. Croteau. 1991. Terpenoids. Pages 165–219 in Herbivores: Their interactions with secondary plant metabolites. Vol. 1.G. A. Rosenthal, and M. R. Berenbaum, ed. Academic Press, San Diego, CA.
  16. Getachew, G., H. P. S. Makkar, and K. Becker. 2002. Tropical browses: contents of phenolic compounds, in vitro gas production and stoichiometric relationship between short chain fatty acid and in vitro gas production. Journal of Agricultural Science, 139(3): 341-352.
  17. Griffin, S.G., S.G. Wyllie, J.L. Markham, and D.N. Leach. 1999.The role of structure and molecular properties of terpernoids in determining their antimicrobial activity. Flavour and Fragrance Journal, 14: 322-332.
  18. Günal, M., B. Pinski, and A. A. AbuGhazaleh. 2017. Evaluating the effects of essential oils on methane production and fermentation under in vitro conditions. Italian Journal of Animal Science, 16(3): 500-506.
  19. Halimi Shabestari, A., R. Salamat doustnobar, and N. Maheri-Sis. 2011. Evaluation Effects of Clove Methanol Extract on Methane Production in the in vitro Condition. Pakistan Journal of Nutrition, 10 (12): 1154-1157.
  20. Hart, K. J., D. R. Yanez-Ruiz, S. M. Duval, N. R. McEwan, and C. J. Newbold. 2008. Plant extracts to manipulate rumen fermentation. Animal Feed Science and Technology, 147: 8-35.
  21. Jahani-Azizabadi, H., M. DaneshMesgaran, A. R. Vakili, and K. Rezayazdi. 2014. Effect of some plant essential oils on in vitro ruminal methane production and on fermentation characteristics of a mid-forage diet. Journal of Agricultural Science and Technology, 16: 1543-1554.
  22. Lee, S. S., J. K. Ha, and K. J. Cheng. 2000. Relative contributions of bacteria, protozoa, and fungi to in vitro degradation of orchard grass cell walls and their interactions. Applied and Environmental Microbiology, 66: 3807 – 3813.
  23. Macheboeuf, D., D.P.Morgavi, Y.Papon, J.L.Mousset, M.Arturo-Schaan. Dose-response effects of essential oils on in vitro fermentation activity of the rumen microbial population. Animal Feed Science and Technollogy, 145: 335–350.
  24. Mateos, I., M. J. Ranilla, M. L. Tejido, C. Saro, C. Kemal, and M. D. Carro. 2013. The influence of diet type (dairy versus intensive fattening) on the effectiveness of garlic oil and cinnamaldehyde to manipulate in vitro ruminal fermentation and methane production.Animal Production Science, 53: 299-307.
  25. McIntosh, F. M., P. Williams, R. Losa, R. J. Wallace, D. A. Beever, and C. J. Newbold. 2003. Effects of essential oils on ruminal microorganisms and their protein metabolism. Applied and Environmental Microbiology, 69:5011–5014.
  26. Menke, K. H., and H. Steingass. 1988. Estimation of the energetic feed value obtained from chemical analyses and gas production using rumen fluid. Animal Research Development, 28(3): 7-55.
  27. Menke, K. H., L. Raab, A. Salewski, H. Steingass, D. Fritz, and W. Schneider. 1979. The estimation of the digestibility and metabolizable energy content of ruminant feedstuffs from the gas production when they are incubated with rumen liquorin vitro.Journal of Agricultural Science, 92: 217–222.
  28. Momeni, T. K., and N. Shahrokhi. 1998. Essential oils and their therapeutic actions. Tehran University Press (In Persian).
  29. Moree, L.V., J. L. Johnson, and W. E. C. Moree.1986. Genus Peptococcus Kluyver and Van Neil 1936, 400AL , pages 1082-1083, and  Genus Peptostreptococcus Kluyver and Van Neil , pages 1083-1092 in Bergey,s manual of systematic bacteriology. P. H. Sneath, N. S. Mair, E. M. Sharpe, and J G. Holt, ed.  The Williams & Williams CO., Baltiore.
  30. Paster, B. J., J. B. Russell, C. M. J. Yang, J. M. Chow, C. R. Woese, and R. Tanner. 1993. Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcusanaerobius, Clostridium sticklandii and Clostridiumaminophilumsp. International Journal Systematic Bacteriology, 43:107–110.
  31. Patra, A. K. 2011.Effects of essential oils on rumen fermentation, microbial ecology and ruminant production. Asian Journal of Animal and Veterinary Advances, 6(5): 416-428.
  32. Patra, A.K., and Z. Yu. 2012. Effects of essential oils on methane production, fermentation, abundance and diversity of rumen microbial populations. Applied Environmental Microbiology, 78: 4271–4280.
  33. Righi, F., M. Simoni, A. Foskolos, V. Beretti, A. Sabbioni, and A. Quarantelli. 2017. In vitro ruminal dry matter and neutral detergent fiber digestibility of common feedstuffs as affected by the addition of essential oils and their active compounds. Journal of Animal and Feed Sciences, 26: 204–212.
  34. Rofiq, M. N., S. Martono, M. Görgülü, and M. Boga. 2012. Combination Effect of Clove and Cinnamon Oil on in Vitro Rumen Gas and Methane Production. Proceeding of the 2nd International Seminar on Animal Industry, Jakarta, 431-437.
  35. Roy, D., S. K. Tomar, S. K. Sirohi, V. Kumar, and M. Kumar. 2014. Efficacy of different essential oils in modulating rumen fermentation in vitro using buffalo rumen liquor. Veterinary World, 7: 213-218.
  36. Russell, J. B., H. J. Strobel, and G. Chen. 1988. Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production. Applied and Environmental Microbiology, 54: 872-877.
  37. SAS User’s Guide: Statistics, Version 9.0 Edition. SAS Inst. Inc., Cary, NC.
  38. Talatapeh, A., P. Farhoomand, Y. A. Alijoo, M. Ghaderzadeh, and E. Norouzi. 2013. Effects of Summer Savory essential oil with two types of diets on performance, rumen fermentation and blood parameters of West Azerbaijan native kids. Animal Sciences Journal (Pajouhesh & Sazandegi), 102: 71-80 (In Persian).
  39. Wallace, R. J., N. R. McEwan, F. M. McIntosh, B. Teferedegne, and C. J. Newbold. 2002. Natural products as manipulators of rumen fermentation. Asian-Australasian Journal of Animal Sciences, 10: 1458–1468.
  40. Zhang, Y., W. Gao, and Q. Meng . Fermentation of plant cell walls by ruminal bacteria, protozoa and fungi and their interaction with fiber particle size. Archives of Animal Nutrition, 61(2): 114–125.