3 Biotech

, 9:294 | Cite as

An in vitro chicken gut model for the assessment of phytase producing bacteria

  • Paul Priyodip
  • Seetharaman BalajiEmail author
Original Article


An in vitro simulated chicken gut model was proposed for studying the phytase activity of selected bacteria such as Streptococcus thermophilus, Sporosarcina pasteurii, Sporosarcina globispora, and Sporosarcina psychrophila using known probiotic bacterium, Lactobacillus helveticus as a control. The selected bacteria were viable in the intestinal lumen and produced extracellular phytase at optimal phytate concentration of 6.25 mM when compared to 3.125 mM and 12.5 mM. These bacteria demonstrated significantly higher (p < 0.05) phosphate liberation (up to 387 µM) due to better phytase activity in the production medium, when compared to the growth medium (339 µM). The phytase activity showed a steady increase in phosphate liberation up to 150 min after which it became constant. This trend is observed for the selected bacteria at pH 5, 6 and 7. However, the liberation of phosphates showed no significant difference (p > 0.05) at the tested pH. Among the analyzed bacteria, the members of the genus Sporosarcina showed better phytate degradation when compared to S. thermophilus. The proposed model can be extended to analyze any extracellular enzymes produced by gut microbes.


Bacteria Chicken-gut Phosphate Phytase Phytate Probiotic 



The authors acknowledge the funding received from the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India (sanction order no. BT/IN/Indo-US/Foldscope/39/2015) for carrying out the project. The first author is the junior research fellow and the corresponding author is the principal investigator of the same project.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

13205_2019_1825_MOESM1_ESM.doc (266 kb)
Table S1. Simulated phosphate release for S. thermophilus; in the presence of phytate (a) 3.125 mM, (b) 6.25 mM and (c) 12.50 mM. Table S2. Simulated phosphate release for S. pasteurii. Table S3. Simulated phosphate release for S. globispora. Table S4. Simulated phosphate release for S. psychrophila; Bacteria cultured in NB-nutrient broth medium, PSM-phytase screening medium, MRS-deMan Rogosa Sharpe broth medium; ‘±’ indicates standard deviations for triplicate experiments (DOC 265 kb)


  1. Akhtar A (2015) The flaws and human harms of animal experimentation. Camb Q Healthc Ethics 24:407–419. CrossRefPubMedPubMedCentralGoogle Scholar
  2. Apajalahti J, Vienola K (2016) Interaction between chicken intestinal microbiota and protein digestion. Anim Feed Sci Technol 221:323–330. CrossRefGoogle Scholar
  3. Awad WA, Hess C, Hess M (2017) Enteric pathogens and their toxin-induced disruption of the intestinal barrier through alteration of tight junctions in chickens. Toxins 9:60. CrossRefPubMedCentralGoogle Scholar
  4. Bae HD, Yanke LJ, Cheng KJ, Selinger LB (1999) A novel staining method for detecting phytase activity. J Microbiol Methods 39:17–22. CrossRefPubMedGoogle Scholar
  5. Balaban NP, Suleimanova AD, Valeeva AR, Chastukhina IB, Sharipova MR (2014) Inositol phosphates and their biological effects. Biomed Pharmacol J 7:433–437. CrossRefGoogle Scholar
  6. Bohn L, Meyer S, Rasmussen SK (2008) Phytate: impact on environment and human nutrition. A challenge for molecular breeding. J Zhejiang Univ Sci B 9:165–191. CrossRefPubMedPubMedCentralGoogle Scholar
  7. Card MC, Cawthraw SA, Nunez-Garcia JA, Ellis RJ, Kay G, Pallen MJ, Woodward MJ, Anjum MF (2017) An in vitro chicken gut model demonstrates transfer of a multidrug resistance plasmid from Salmonella to commensal Escherichia coli. mBio 8:e00777-17. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Cowieson AJ, Ruckebusch JP, Knap I, Guggenbuhl P, Fru-Nji F (2016) Phytate-free nutrition: a new paradigm in monogastric animal production. Anim Feed Sci Technol 222:180–189. CrossRefGoogle Scholar
  9. de Verdal H, Mignon-Grasteau S, Jeulin C, Le Bihan-Duval E, Leconte M, Mallet S, Martin C, Narcy A (2010) Digestive tract measurements and histological adaptation in broiler lines divergently selected for digestive efficiency. Poult Sci 89:1955–1961. CrossRefPubMedGoogle Scholar
  10. Demirkan E, Baygın E, Usta A (2014) Screening of phytate hydrolysis Bacillus sp. isolated from soil and optimization of the certain nutritional and physical parameters on the production of phytase. Turk J Biochem 39:206–214. CrossRefGoogle Scholar
  11. Dersjant-Li Y, Awati A, Schulze H, Partridge G (2015) Phytase in non-ruminant animal nutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. J Sci Food Agric 95:878–896. CrossRefPubMedGoogle Scholar
  12. González-Córdova AF, Beltrán-Barrientos LM, Santiago-López L, Garcia HS, Vallejo-Cordoba B, Hernandez-Mendoza A (2016) Phytate-degrading activity of probiotic bacteria exposed to simulated gastrointestinal fluids. LWT Food Sci Technol 73:67–73. CrossRefGoogle Scholar
  13. Greiner R, Carlsson NG, Alminger ML (2000) Stereospecificity of myo-inositol hexakisphosphate dephosphorylation by a phytate-degrading enzyme of Escherichia coli. J Biotechnol 84:53–62. CrossRefGoogle Scholar
  14. Khobondo JO, Ogore PB, Atela JA, Onjoro PS, Ondiek JO, Kahi KA (2015) The effects of dietary probiotics on natural IgM antibody titres of Kenyan indigenous chicken. Livest Res Rural Dev 27:230Google Scholar
  15. Kumar V, Sinha AK, Makkar HPS, Becker K (2010) Dietary roles of phytate and phytase in human nutrition: a review. Food Chem 120:945–959. CrossRefGoogle Scholar
  16. Kuttappan VA, Berghman LR, Vicuña EA, Latorre JD, Menconi A, Wolchok JD, Wolfenden AD, Faulkner OB, Tellez GI, Hargis BM, Bielke LR (2015) Poultry enteric inflammation model with dextran sodium sulfate mediated chemical induction and feed restriction in broilers. Poult Sci 94:1220–1226. CrossRefPubMedGoogle Scholar
  17. Laquieze L, Lorencini M, Granjeiro JM (2015) Alternative methods to animal testing and cosmetic safety: an update on regulations and ethical considerations in Brazil. Appl In Vitro Toxicol 1:243–253. CrossRefGoogle Scholar
  18. Parent E, Archambault M, Charlebois A, Bernier-Lachance J, Boulianne M (2017) A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks. Avian Pathol 6:138–149. CrossRefGoogle Scholar
  19. Priyodip P, Balaji S (2018) Microbial degradation of myo-inositol hexakisphosphate (IP6): specificity, kinetics, and simulation. 3 Biotech 8:268. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Priyodip P, Balaji S (2019) A preliminary study on probiotic characteristics of Sporosarcina spp. for poultry applications. Curr Microbiol 76:448–461. CrossRefPubMedGoogle Scholar
  21. Rocky-Salimi K, Hashemi M, Safari M, Mousivand M (2016) A novel phytase characterized by thermostability and high pH tolerance from rice phyllosphere isolated Bacillus subtilis B.S.46. J Adv Res 7:381–390. CrossRefPubMedPubMedCentralGoogle Scholar
  22. Shin S, Ha NC, Oh BC, Oh TK, Oh BH (2001) Enzyme mechanism and catalytic property of beta propeller phytase. Structure 9:851–858. CrossRefPubMedGoogle Scholar
  23. Theerawatanasirikul S, Koomkrong N, Kayan A, Boonkaewwan C (2017) Intestinal barrier and mucosal immunity in broilers, Thai Betong, and native Thai Praduhangdum chickens. Turk J Vet Anim Sci 41:357–364. CrossRefGoogle Scholar
  24. Zelenka J, Mrkvicová E, Šťastník O, Jarošová M, Vlčko T, Ohnoutková L (2017) Effect of low- and high phytase barley on phosphorus retention and bone mineral composition in broilers. Acta Univ Agric Silvic Mendel Brun Acta 65:577–581. CrossRefGoogle Scholar
  25. Zhang GQ, Wu YY, Ng TB, Chen QJ, Wang HX (2013) A phytase characterized by relatively high pH tolerance and thermostability from the shiitake mushroom Lentinus edodes. Biomed Res Int 2013:540239. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© King Abdulaziz City for Science and Technology 2019

Authors and Affiliations

  1. 1.Department of Biotechnology, Manipal Institute of TechnologyManipal Academy of Higher EducationManipalIndia

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