The Effect of Probiotic Feed Use on Intestinal Microbiota in Experimental Animals

Öz

Laboratory animals are frequently used in scientific studies and are of great importance. To clearly demonstrate the effectiveness of the researched in in-vivo studies, many conditions such as the breed, species and age of the animals used in the experiment, as well as environmental conditions, should be uniform in the control and test groups. However, in these experiments it is often overlooked that the intestinal microbiota, which plays an important role in the function and integrity of the gastrointestinal tract, protection of immune homeostasis and host energy metabolism, may differ in the experimental animals used, and this may adversely affect the results of the study. In this study, it was aimed to produce probiotic-containing feeds which have the potential to synchronize intestinal microbiota of laboratory animals (rat and mouse) to be used in the adaptation period before scientific studies and to investigate the effectiveness of feeds on the intestinal microbiota. According to the analysis, at the end of the storage period of the produced liquid feeds, the total number of general aerobic organisms and the number of lactic acid bacteria (L. acidophilus and L. plantarum) reached to 109 cfu/ml, however, coliform bacteria, yeast-mold and other pathogenic microorganisms were not detected. As a result of microbiota analysis, it was revealed that 10-day probiotic feed application was effective on intestinal microbiota composition in both mice and rats compared to control groups and 0. day test groups. In the literature reviews carried out to date, it has been determined that there is no study in this scope in Turkey. In this context, it was concluded that feeding experimental animals with probiotic-containing feeds that produced within the scope of this study before in-vivo studies will contribute to the improvement of the intestinal microbiota of the laboratory animals.

Anahtar Kelimeler

• Laboratory animals
• L. acidophilus
• L. plantarum
• microbiota
• synchronization

Deney Hayvanlarında Probiyotikli Yem Kullanımının Bağırsak Mikrobiyotasına Etkisi

Öz

Bilimsel çalışmalarda laboratuvar hayvanı kullanımı büyük önem taşımakta ve önemli bir yer tutmaktadır. In-vivo çalışmalarda araştırılanın kesin olarak etkinliğini ortaya koyabilmek için deneyde kullanılan hayvanların ırkı, türü ve yaşının yanı sıra çevre koşulları gibi pek çok koşul kontrol ve test gruplarında bir örnek hale getirilmektedir. Ancak bu deneylerde gastrointestinal sistemin işlevi ve bütünlüğünde, bağışıklık homeostazının korunmasında ve konak enerji metabolizmasında önemli bir rol oynayan bağırsak mikrobiyotasının kullanılan deney hayvanlarında farklılıklar gösterebileceği bu durumun ise çalışma sonuçlarını olumsuz etkileyebileceği çoğu zaman gözden kaçmaktadır. Bu çalışmada, bilimsel çalışmalar öncesi adaptasyon periyodunda kullanılmak üzere laboratuvar hayvanlarının (rat ve fare) bağırsak mikrobiyotalarının senkronizasyonunda kullanım potansiyeli olan probiyotik içerikli yemlerin üretimi ve bağırsak mikrobiyotası üzerine etkinliğinin araştırılması amaçlanmıştır. Analizler neticesinde üretilen sıvı yemlerin muhafaza süresi sonunda toplam aerob genel canlı sayısı ve laktik asit bakteri sayısının (L. acidophilus ve L. plantarum) 109 kob/ml ulaştığı buna karşın koliform bakteri, maya-küf ile diğer patojen mikroorganizmalara rastlanmamıştır. Mikrobiyota analizleri neticesinde ise hem fare hem de ratlarda 10 günlük probiyotikli yem uygulamasının kontrol grupları ile 0. gündeki test gruplarına göre bağırsak mikrobiyotası kompozisyonu üzerine etkili olduğu ortaya konmuştur. Günümüze kadar yapılan literatür incelemelerinde Türkiye’de bu kapsamda bir çalışma olmadığı belirlenmiştir. Bu bağlamda in-vivo çalışmalar öncesinde deney hayvanlarının bu çalışma kapsamında üretilen probiyotik içerikli yemler ile beslenmesinin hayvanların bağırsak mikrobiyotasının geliştirilmesine katkı sunacağı sonucuna varılmıştır.

Anahtar Kelimeler

• Laboratuvar hayvanları
• L. acidophilus
• L. plantarum
• mikrobiyota
• senkronizasyon

Kaynakça

1. Anonim (2006) Food and Agriculture Organization of the United Nations, World Health Organization, Joint FAO/ WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, and Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food (2006)
2. Probiotics in food: health and nutritional properties and guidelines for evaluation. In Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, Cordoba, Argentina, 1–4 October 2001 [and] Report of a Joint FAO/WHO Working Group on Drafting Guidelines for the Evaluation of Probiotics in Food, London, ON, Canada, 30 April –1 May 2002. Rome: Food and Agriculture Organization of the United Nations, World Health Organization.
3. Anonim. (2010) Directive 2010/63/EU of the European Parliament anf of the Council of 22 September 2010 on the protection of animals used for scientific purposes. 20.10.2010 EN L 276/33.
4. Anonim. (2019) European Union Comission Report. 2019 report on the statistics on the use of animals for scientific purposes in the Member States of the European Union in 2015-2017 {SWD(2020) 10 final, Brussels, 5.2.2020 COM(2020) 16 final.
5. Anonim. (2020a) American Association for Laboratory Animal Science. AALAS, Erişim adresi: https://www.aalas.org/
6. Anonim. (2020b) International Council for Laboratory Animal Science. ICLAS, Erişim adresi: https://iclas.org/
7. Anonim. (2021) HADMEK (Hayvan Deneyleri Merkez Etik Kurulu). 2018 – 2020 Yılları Hayvan Deneyleri Merkez Etik Kurulu Faaliyet Raporu, Ankara 2021.
8. Anonim. (2023) Tarım ve Orman Bakanlığı. Çalışma izni verilen deney hayvanı üretici, kullanıcı ve tedarikçi kuruluşlar. Erişim adresi: https://www.tarimorman.gov.tr/Konu/1007/Calisma-Izni-Verilen-Deney-Hayvani-Uretici-Kullanici-ve-Tedarikci-Kuruluslar. Erişim tarihi: 26.07.2023.

9. Barko PC, McMichael MA, Swanson KS, Williams DA. (2018) The gastrointestinal microbiome: a review. J Vet Intern Med. 32, 9-25.
10. Benson AK, Kelly SA, Legge R, Pomp D. (2010) Individuality in gut microbiota composition is a complex polygenic trait shaped by multiple environmental and host genetic factors. Proc Natl Acad Sci U S A. 107(44), 18933–18938.
11. Ericsson AC, Davis JW, Spollen W, Bivens N, Givan S, Hagan CE, McIntosh M, Franklin CL. (2015) Effects of vendor and genetic background on the composition of the fecal microbiota of inbred mice. PLoS One. 10(2), e0116704. doi: 10.1371/journal.pone.0116704.
12. Ericsson AC, Personett AR, Turner G, Dorfmeyer RA, Franklin CL. (2017) Variable colonization after reciprocal fecal microbiota transfer between mice with low and high richness microbiota. Front Microbiol. 8, 196. https://doi.org/10.3389/fmicb.2017.00196.
13. Farzi A, Frohlich EE, Holzer P. (2018) Gut microbiota and the neuroendocrine system. Neurotherapeutics 15, 5–22. doi: 10.1007/s13311-017- 0600-5.
14. Ferrario C, Taverniti V, Milani C, Fiore W, Laureati M, De Noni I, Stuknyte M, Chouaia B, Riso P, Guglielmetti S. (2014) Modulation of fecal Clostridiales bacteria and butyrate by probiotic intervention with Lactobacillus paracasei DG varies among healthy adults. J Nutr. 144, 1787–1796. doi: 10.3945/jn.114.197723
15. Fox JG. (2007) Helicobacter bilis: bacterial provocateur orchestrates host immune responses to commensal f lora in a model of inflammatory bowel disease. Gut. 56(7), 898–900.
16. Franklin CL, Ericsson AC. (2020) Complex microbiota in laboratory rodents: management considerations. ILAR J. 60(2), 289–297. doi: 10.1093/ilar/ilaa011.
17. Gargari G, Taverniti V, Balzaretti S, Ferrario C, Gardana C, Simonetti P, Guglielmetti S. (2016) Consumption of a Bifidobacterium bifidum strain for 4 weeks modulates dominant intestinal bacterial taxa and fecal butyrate in healthy adults. Appl Environ Microbiol. 82, 5850–5859. doi: 10.1128/aem.01753-16.
18. Grazul H, Kanda LL, Gondek D. (2016) Impact of probiotic supplements on microbiome diversity following antibiotic treatment of mice. Gut Microbes. 7(2), 101-114.
19. Hart ML, Ericsson AC, Franklin CL. (2017) Differing complex microbiota alter disease severity of the IL-10(−/−) mouse model of inflammatory bowel disease. Front Microbiol. 8,792. doi: 10.3389/fmicb.2017.00792.
20. Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. (2014) Expert consensus document. the international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev
21. Gastroenterol Hepatol. 11, 506–514. doi: 10.1038/nrgastro.2014.66. ISO (International Organization for Standardization). (2013) Microbiology of the food chain - Horizontal method for the enumeration of microorganisms - Part 2: Colony count at 30° C by the surface plating technique. ISO 4833-2:2013. ICS: 07.100.30 Food microbiology.
22. ISO (International Organization for Standardization). (1998) Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of mesophilic lactic acid bacteria — Colony-count technique at 30 degrees C. ISO 15214:1998. ICS: 07.100.30 Food microbiology.
23. ISO (International Organization for Standardization). (2006) Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms — Colony-count technique. ISO 4832:2006. ICS: 07.100.30 Food microbiology.
24. ISO (International Organization for Standardization). (2008) Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of yeasts and moulds — Part 2: Colony count technique in products with water activity less than or equal to 0,95. ISO 21527-2:2008. ICS: 07.100.30 Food microbiology.
25. ISO (International Organization for Standardization). (2017a) Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp. ISO 6579-1:2017. ICS: 07.100.30 Food microbiology.
26. ISO (International Organization for Standardization). (2017b) Microbiology of the food chain — Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. — Part 1: Detection method. ISO 11290-1:2017. ICS: 07.100.30 Food microbiology.
27. ISO (International Organization for Standardization). (2021) Microbiology of the food chain — Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species) — Part 1: Method using Baird-Parker agar medium. ISO 6888-1:2021. ICS: 07.100.30 Food microbiology.
28. Jergens AE, Wilson-Welder JH, Dorn A, Henderson A, Liu Z, Evans RB, Hostetter J, Wannemuehler MJ. (2007) Helicobacter bilis triggers persistent immune reactivity to antigens derived from the commensal bacteria in gnotobiotic C3H/HeN mice. Gut. 56(7), 934–940.
29. Khailova L, Mount Patrick SK, Arganbright KM, Halpern MD, Kinouchi T, Dvorak B. (2010) Bifidobacterium bifidum reduces apoptosis in the intestinal epithelium in necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol. 299, G1118–G1127.
30. Lavasani S, Dzhambazov B, Nouri M, Fak F, Buske S, Molin G, Thorlacius H, Alenfall J, Jeppsson B, Weström B. (2010) A novel probiotic mixture exerts a therapeutic effect on experimental autoimmune encephalomyelitis mediated by IL-10 producing regulatory T cells. PLoS One. 5, e9009.
31. Lee SM, Donaldson GP, Mikulski Z, Boyajian S, Ley K, Mazmanian SK. (2013) Bacterial colonization factors control specifity and stability of the gut microbiota. Nature. 501, 426-429.
32. McCoy KD, Geuking MB, Ronchi F. (2017) Gut microbiome standardization in control and experimental mice. Curr Protoc Immunol. 117, 23.1.1–23.1.13.
33. McNulty N, Yatsunenko T, Hsiao A, Faith J, Muegge B, Goodman A, Henrissat B, Oozeer R, Cools-Portier S, Gobert G, Chervaux C, Knights D, Lozupone CA, Knight R, Duncan AE, Bain JR, Muehlbauer MJ, Newgard CB, Heath AC,
34. Gordon JI. (2011) The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins. Sci Transl Med. 3, 106.
35. Nicklas W. (2008) International harmonization of health monitoring. ILAR J. 49, 338-346. Pflughoeft KJ, Versalovic J. (2012) Human microbiome in health and disease. Annu Rev Pathol. 7, 99-122. doi: 10.1146/annurev-pathol-011811-132421.
36. Rasmussen TS, de Vries L, Kot W, Hansen LH, Castro-Mejia JL, Vogensen FK, Hansen AK, Nielsen DS. (2019)
37. Mouse vendor inf luence on the bacterial and viral gut composition exceeds the effect of diet. Viruses. 11(5), 435. doi: 10.3390/v11050435.
38. Taverniti V, Cesari V, Gargari G, Rossi U, Biddau C, Lecchi C, Fiore W, Arioli S, Toschi I, Guglielmetti G. (2021)
39. Probiotics Modulate Mouse Gut Microbiota and Influence Intestinal Immune and Serotonergic Gene Expression in a Site-Specific Fashion. Front Microbiol. 12, 706135. doi: 10.3389/fmicb.2021.706135
40. Wang HT, Anvari S, Anagnostou K. (2019) The role of probiotics in preventing allergic disease. Children (Basel). 6, 24. doi: 10.3390/children6020024
41. Wilkins T, Sequoia J. (2017) Probiotics for gastrointestinal conditions: a summary of the evidence. Am Fam Phys. 96, 170–178.
42. Wood DE, Salzberg SL. (2014) Kraken: ultrafast metagenomic sequence classification using exact alignments. Genome Biol. 15(3), R46.