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High-intensity-exercise-induced intestinal damage is protected by fermented milk supplemented with whey protein, probiotic and pomegranate (Punica granatum L.)

  • Fernanda M. Chaves (a1), Igor L. Baptista (a1), Fernando M. Simabuco (a1), Paula G. F. Quaresma (a2), Fabiola L. Pena (a1), Rosangela M. N. Bezerra (a1), Jose R. Pauli (a1), Diogo T. da Cunha (a1), Patricia L. Campos-Ferraz (a1) and Adriane E. C. Antunes (a1)...

Here we evaluated the effect of fermented milk supplemented with whey protein (approximately 80 % protein), probiotic (Bifidobacterium animalis subsp. lactis BB12) and pomegranate juice (Punica granatum L.) on the physical performance, intestinal motility and villi structure, inflammatory markers and intestinal microbiota of rats under high-intensity acute exercise. In all, twenty-four Wistar rats were separated into groups: control (Ctrl), supplemented (Supp), exercised (Exe) and exercised and supplemented (Exe+Supp). Rats in the Supp groups received fermented milk during 6 weeks by oral administration. At the end of the supplementation period, the Exe groups were submitted to high-intensity acute exercise on a treadmill. We found that intense acute exercise caused changes in the intestinal villi interspace, changes in the proportion of Lactobacillus species and an increase in Clostridium species, as well as a decrease in intestinal motility. Supplementation increased intestinal motility, and maintained the intestinal villi interspace and the natural microbiota proportions of the exercised rats. Physical performance was not improved by fermented milk supplementation. We conclude that the fermented milk containing whey protein, B. animalis (BB12) and pomegranate juice can re-establish intestinal microbiota and protect the animals from the undesirable effects of intense acute exercise.

Corresponding author
* Corresponding author: A. E. C. Antunes, fax +55 19 3701 6680, email
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1. Cruzat, VF, Krause, M & Newsholme, P (2014) Amino acid supplementation and impact on immune function in the context of exercise. J Int Soc Sports Nutr 11, 61.
2. Newsholme, EA & Parry-Billings, M (1990) Properties of glutamine release from muscle and its importance for the immune system. JPEN J Parenter Enteral Nutr 14, 63S67S.
3. Newsholme, P (2001) Why is L-glutamine metabolism important to cells of the immune system in health, postinjury, surgery or infection? J Nutr 131, 2515s2522s.
4. Newsholme, EA & Calder, PC (1997) The proposed role of glutamine in some cells of the immune system and speculative consequences for the whole animal. Nutrition 13, 728730.
5. Rohde, T, MacLean, DA & Pedersen, BK (1998) Effect of glutamine supplementation on changes in the immune system induced by repeated exercise. Med Sci Sports Exerc 30, 856862.
6. Wang, B, Wu, G, Zhou, Z, et al. (2015) Glutamine and intestinal barrier function. Amino Acids 47, 21432154.
7. Clark, A & Mach, N (2016) Exercise-induced stress behavior, gut-microbiota-brain axis and diet: a systematic review for athletes. J Int Soc Sports Nutr 13, 43.
8. Nieman, DC (2008) Immunonutrition support for athletes. Nutr Rev 66, 310320.
9. van Wijck, K, Lenaerts, K, van Loon, LJ, et al. (2011) Exercise-induced splanchnic hypoperfusion results in gut dysfunction in healthy men. PLoS ONE 6, e22366.
10. Lamprecht, M & Frauwallner, A (2012) Exercise, intestinal barrier dysfunction and probiotic supplementation. Med Sport Sci 59, 4756.
11. Garcia-Hernandez, V, Quiros, M & Nusrat, A (2017) Intestinal epithelial claudins: expression and regulation in homeostasis and inflammation. Ann N Y Acad Sci 1397, 6679.
12. Fanning, AS, Mitic, LL & Anderson, JM (1999) Transmembrane proteins in the tight junction barrier. J Am Soc Nephrol 10, 13371345.
13. Gunzel, D & Yu, AS (2013) Claudins and the modulation of tight junction permeability. Physiol Rev 93, 525569.
14. Pyne, DB, West, NP, Cox, AJ, et al. (2015) Probiotics supplementation for athletes – clinical and physiological effects. Eur J Sport Sci 15, 6372.
15. Fuster-Munoz, E, Roche, E, Funes, L, et al. (2016) Effects of pomegranate juice in circulating parameters, cytokines, and oxidative stress markers in endurance-based athletes: A randomized controlled trial. Nutrition 32, 539545.
16. Gracious Ross, R, Selvasubramanian, S & Jayasundar, S (2001) Immunomodulatory activity of Punica granatum in rabbits – a preliminary study. J Ethnopharmacol 78, 8587.
17. Varghese, S, Joseph, MM, RA, S, et al. (2017) The inhibitory effect of anti- tumor polysaccharide from Punica granatum on metastasis. Int J Biol Macromol 103, 10001010.
18. Sharma, P, McClees, SF & Afaq, F (2017) Pomegranate for prevention and treatment of cancer: an update. Molecules 22, E177.
19. Drochioiu, G, Ciobanu, CI, Bancila, S, et al. (2016) Ultrasound-based protein determination in maize seeds. Ultrason Sonochem 29, 93103.
20. Musci, M & Yao, S (2017) Optimization and validation of Folin-Ciocalteu method for the determination of total polyphenol content of Pu-erh tea. Int J Food Sci Nutr 68, 19.
21. Benzie, IF & Strain, JJ (1996) The ferric reducing ability of plasma (FRAP) as a measure of ‘antioxidant power’: the FRAP assay. Anal Biochem 239, 7076.
22. Witard, OC, Jackman, SR, Breen, L, et al. (2014) Myofibrillar muscle protein synthesis rates subsequent to a meal in response to increasing doses of whey protein at rest and after resistance exercise. Am J Clin Nutr 99, 8695.
23. Ferreira, JC, Rolim, NP, Bartholomeu, JB, et al. (2007) Maximal lactate steady state in running mice: effect of exercise training. Clin Exp Pharmacol Physiol 34, 760765.
24. Wang, Y & Qian, PY (2009) Conservative fragments in bacterial 16S rRNA genes and primer design for 16S ribosomal DNA amplicons in metagenomic studies. PLoS ONE 4, e7401.
25. Caporaso, JG, Lauber, CL, Walters, WA, et al. (2011) Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci U S A 108, Suppl. 1, 45164522.
26. Takahashi, S, Tomita, J, Nishioka, K, et al. (2014) Development of a prokaryotic universal primer for simultaneous analysis of Bacteria and Archaea using next-generation sequencing. PLOS ONE 9, e105592.
27. Chen, YM, Wei, L, Chiu, YS, et al. (2016) Lactobacillus plantarum TWK10 supplementation improves exercise performance and increases muscle mass in mice. Nutrients 8, 205.
28. Games, PA, Keselman, HJ & Rogan, JC (1983) A review of simultaneous pairwise multiple comparisons. Statistica Neerlandica 37, 5358.
29. Hill, C, Guarner, F, Reid, G, et al. (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 Gastroenterol Hepatol 11, 506514.
30. Bertazzoni, E, Donelli, G, Midtvedt, T, et al. (2013) Probiotics and clinical effects: is the number what counts? J Chemother 25, 193212.
31. BRASIL. Ministerio da Saude (BRAZIL. Health Ministry) (2016) Alimentos com alegacoes de propriedades funcionais e ou de saude (Foods With Claims of Functional and/or Health Properties). (accessed January 2018).
32. Lollo, PC, Cruz, AG, Morato, PN, et al. (2012) Probiotic cheese attenuates exercise-induced immune suppression in Wistar rats. J Dairy Sci 95, 35493558.
33. Cox, AJ, Pyne, DB, Saunders, PU, et al. (2010) Oral administration of the probiotic Lactobacillus fermentum VRI-003 and mucosal immunity in endurance athletes. Br J Sports Med 44, 222226.
34. West, NP, Horn, PL, Pyne, DB, et al. (2014) Probiotic supplementation for respiratory and gastrointestinal illness symptoms in healthy physically active individuals. Clin Nutr 33, 581587.
35. West, NP, Pyne, DB, Cripps, AW, et al. (2011) Lactobacillus fermentum (PCC(R)) supplementation and gastrointestinal and respiratory-tract illness symptoms: a randomised control trial in athletes. Nutr J 10, 30.
36. Gleeson, M, Bishop, NC, Oliveira, M, et al. (2012) Effects of a Lactobacillus salivarius probiotic intervention on infection, cold symptom duration and severity, and mucosal immunity in endurance athletes. Int J Sport Nutr Exerc Metab 22, 235242.
37. Valimaki, IA, Vuorimaa, T, Ahotupa, M, et al. (2012) Decreased training volume and increased carbohydrate intake increases oxidized LDL levels. Int J Sports Med 33, 291296.
38. Shing, CM, Peake, JM, Lim, CL, et al. (2014) Effects of probiotics supplementation on gastrointestinal permeability, inflammation and exercise performance in the heat. Eur J Appl Physiol 114, 93103.
39. Crum, EM, Che Muhamed, AM, Barnes, M, et al. (2017) The effect of acute pomegranate extract supplementation on oxygen uptake in highly-trained cyclists during high-intensity exercise in a high altitude environment. J Int Soc Sports Nutr 14, 14.
40. Rolfe, RD (2000) The role of probiotic cultures in the control of gastrointestinal health. J Nutr 130, 396S402S.
41. Gil, MI, Tomas-Barberan, FA, Hess-Pierce, B, et al. (2000) Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48, 45814589.
42. Simren, M (2002) Physical activity and the gastrointestinal tract. Eur J Gastroenterol Hepatol 14, 10531056.
43. van Nieuwenhoven, MA, Brouns, F & Brummer, RJ (2004) Gastrointestinal profile of symptomatic athletes at rest and during physical exercise. Eur J Appl Physiol 91, 429434.
44. Lu, Z, Ding, L, Lu, Q, et al. (2013) Claudins in intestines: distribution and functional significance in health and diseases. Tissue Barriers 1, e24978.
45. Tokes, AM, Kulka, J, Paku, S, et al. (2005) Claudin-1, -3 and -4 proteins and mRNA expression in benign and malignant breast lesions: a research study. Breast Cancer Res 7, R296R305.
46. Li, Q, Zhang, Q, Wang, C, et al. (2009) Altered distribution of tight junction proteins after intestinal ischaemia/reperfusion injury in rats. J Cell Mol Med 13, 40614076.
47. Overgaard, CE, Daugherty, BL, Mitchell, LA, et al. (2011) Claudins: control of barrier function and regulation in response to oxidant stress. Antioxid Redox Signal 15, 11791193.
48. Bron, PA, Kleerebezem, M, Brummer, RJ, et al. (2017) Can probiotics modulate human disease by impacting intestinal barrier function? Br J Nutr 117, 93107.
49. Karczewski, J, Troost, FJ, Konings, I, et al. (2010) Regulation of human epithelial tight junction proteins by Lactobacillus plantarum in vivo and protective effects on the epithelial barrier. Am J Physiol Gastrointest Liver Physiol 298, G851G859.
50. Macpherson, AJ & Harris, NL (2004) Interactions between commensal intestinal bacteria and the immune system. Nat Rev Immunol 4, 478485.
51. Keita, AV & Soderholm, JD (2010) The intestinal barrier and its regulation by neuroimmune factors. Neurogastroenterol Motil 22, 718733.
52. Patel, RM, Myers, LS, Kurundkar, AR, et al. (2012) Probiotic bacteria induce maturation of intestinal claudin 3 expression and barrier function. Am J Pathol 180, 626635.
53. Castell, LM & Newsholme, EA (1997) The effects of oral glutamine supplementation on athletes after prolonged, exhaustive exercise. Nutrition 13, 738742.
54. Li, N & Neu, J (2009) Glutamine deprivation alters intestinal tight junctions via a PI3-K/Akt mediated pathway in Caco-2 cells. J Nutr 139, 710714.
55. Cannon, JG (2000) Inflammatory cytokines in nonpathological states. News Physiol Sci 15, 298303.
56. Nieman, DC & Pedersen, BK (1999) Exercise and immune function. Recent developments. Sports Med 27, 7380.
57. Drenth, JP, Van Uum, SH, Van Deuren, M, et al. (1995) Endurance run increases circulating IL-6 and IL-1ra but downregulates ex vivo TNF-alpha and IL-1 beta production. J Appl Physiol (1985) 79, 14971503.
58. Ostrowski, K, Rohde, T, Asp, S, et al. (1999) Pro- and anti-inflammatory cytokine balance in strenuous exercise in humans. J Physiol 515, 287291.
59. Cruzat, VF, Rogero, MM & Tirapegui, J (2010) Effects of supplementation with free glutamine and the dipeptide alanyl-glutamine on parameters of muscle damage and inflammation in rats submitted to prolonged exercise. Cell Biochem Funct 28, 2430.
60. Pithon-Curi, TC, Trezena, AG, Tavares-Lima, W, et al. (2002) Evidence that glutamine is involved in neutrophil function. Cell Biochem Funct 20, 8186.
61. Niess, AM & Simon, P (2007) Response and adaptation of skeletal muscle to exercise--the role of reactive oxygen species. Front Biosci 12, 48264838.
62. Nicholson, JK, Holmes, E, Kinross, J, et al. (2012) Host-gut microbiota metabolic interactions. Science 336, 12621267.
63. de La Serre, CB, Ellis, CL, Lee, J, et al. (2010) Propensity to high-fat diet-induced obesity in rats is associated with changes in the gut microbiota and gut inflammation. Am J Physiol Gastrointest Liver Physiol 299, G440G448.
64. Mackos, AR, Eubank, TD, Parry, NM, et al. (2013) Probiotic Lactobacillus reuteri attenuates the stressor-enhanced severity of Citrobacter rodentium infection. Infect Immun 81, 32533263.
65. Antunes, AEC, Silva, ERA, Van Dender, AGF, et al. (2009) Probiotic buttermilk-like fermented milk product development in a semiindustrial scale: Physicochemical, microbiological and sensory acceptability. Int J Dairy Technol 62, 556563.
66. Shah, NP (2007) Functional cultures and health benefits. Int Dairy J 17, 12621277.
67. Everard, A, Belzer, C, Geurts, L, et al. (2013) Cross-talk between Akkermansia muciniphila and intestinal epithelium controls diet-induced obesity. Proc Natl Acad Sci U S A 110, 90669071.
68. Clarke, SF, Murphy, EF, O’Sullivan, O, et al. (2014) Exercise and associated dietary extremes impact on gut microbial diversity. Gut 63, 19131920.
69. Barton, W, Penney, NC, Cronin, O, et al. (2017) The microbiome of professional athletes differs from that of more sedentary subjects in composition and particularly at the functional metabolic level. Gut 67, 625633.
70. Mika, A, Van Treuren, W, Gonzalez, A, et al. (2015) Exercise is more effective at altering gut microbial composition and producing stable changes in lean mass in juvenile versus adult male F344 rats. PLOS ONE 10, e0125889.
71. Ley, RE, Turnbaugh, PJ, Klein, S, et al. (2006) Microbial ecology: human gut microbes associated with obesity. Nature 444, 10221023.
72. Turnbaugh, PJ, Hamady, M, Yatsunenko, T, et al. (2009) A core gut microbiome in obese and lean twins. Nature 457, 480484.
73. Batacan, RB, Fenning, AS, Dalbo, VJ, et al. (2017) A gut reaction: the combined influence of exercise and diet on gastrointestinal microbiota in rats. J Appl Microbiol 122, 16271638.
74. Hsu, CK, Liao, JW, Chung, YC, et al. (2004) Xylooligosaccharides and fructooligosaccharides affect the intestinal microbiota and precancerous colonic lesion development in rats. J Nutr 134, 15231528.
75. Nusrat, A, von Eichel-Streiber, C, Turner, JR, et al. (2001) Clostridium difficile toxins disrupt epithelial barrier function by altering membrane microdomain localization of tight junction proteins. Infect Immun 69, 13291336.
76. Sanders, ME (1993) Summary of conclusions from a consensus panel of experts on health attributes of lactic cultures: significance to fluid milk products containing cultures. J Dairy Sci 76, 18191828.
77. Choi, JJ, Eum, SY, Rampersaud, E, et al. (2013) Exercise attenuates PCB-induced changes in the mouse gut microbiome. Environ Health Perspect 121, 725730.
78. Queipo-Ortuno, MI, Seoane, LM, Murri, M, et al. (2013) Gut microbiota composition in male rat models under different nutritional status and physical activity and its association with serum leptin and ghrelin levels. PLOS ONE 8, e65465.
79. Hell, M, Bernhofer, C, Stalzer, P, et al. (2013) Probiotics in Clostridium difficile infection: reviewing the need for a multistrain probiotic. Benef Microbes 4, 3951.
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