Encapsulated Probiotics & Gut Microbiota

The investigators plan to assess the gut microbiome and changes in probiotic diversity at the end of a course of a beverage containing encapsulated Lacticaseibacillus rhamnosus, compared to those at the end of a course of the same beverage without probiotics.

This is a randomised, double-blinded, placebo-controlled crossover study. Twenty-five healthy men and women with a body mass index (BMI) between 18.5-31.9 kg/m2, aged 25-65 years, will be recruited and randomised to a commercial fermented (yoghurt) drink containing a) encapsulated probiotics consisting of Lacticaseibacillus rhamnosus, or b) a non-probiotic fermented (yoghurt) dairy drink (control). The number of participants includes a 10% drop-out rate.

Each participant will be studied on four occasions in random order following a crossover design. Each will receive a 200 ml beverage containing yoghurt per day for 28 days, either a) fortified with 1 billion probiotics contained in pea protein capsules or b) the control beverage (same yoghurt without probiotic). Faecal samples will be collected on four occasions: at baseline, and at 28, 42 and 70 days after baseline.

The current study will provide important information on the physiological function of probiotics in metabolic and inflammatory biochemical parameters, as well as in their capacity to colonise the gastrointestinal tract.

• probiotic containing L.rhamnosus R0011 strain (Biological): Twenty-five participants will be studied on four occasions in random order and in a crossover design. Subjects will receive a daily dose of 200 ml on two different occasions for 28 days, a commercial dairy drink fortified with 1Billion probiotics in the pea protein capsules. Feacal samples will be collected on four occasions: at baseline, 28 days after baseline, 42 days and 70 days after baseline.
• 200 ml yogurt drink (Dietary supplement): Twenty-five participants will be studied on four occasions in random order and in a crossover design. Subjects will receive a daily dose 200 ml on two different occasions for 28 days, a commercial dairy drink (no probiotics). Feacal samples will be collected on four occasions: at baseline, 28 days after baseline, 42 days and 70 days after baseline.

Primary outcomes

• Colonisation [28 days]: Faecal abundance of Lactobacillus rhamnosus after probiotic consumption measured through shotgun metagenomics -libraries with Illumina Nextera XT, sequenced 150 bp paired-end on NextSeq 2000. Negative + positive controls will be processed alongside study samples with around 15M paired reads/sample after quality controls.

Sex: All, ages: 25 Years to 65 Years, accepts healthy volunteers

Inclusion Criteria:

* Capacity to obtain and sign informed consent before any study-related procedure
* BMI range 18.5-31.9 kg/m2
* Willing to abstain from regular consumption of probiotic supplements or food products containing probiotic bacteria (including fermented food and beverages).
* Willing to abstain from regular consumption of supplements and medications known to alter gastrointestinal function or inflammatory status during the study.

Exclusion Criteria:

Participants who meet any of the following criteria will be excluded:

* Smoking
* Substance abuse
* Pregnancy
* Diagnosis of type 1 and/or type 2 diabetes
* Current (or within the last 4 weeks prior to study start) use of probiotic supplementation.
* Immobile (defined as the inability to participate in all study-related procedures)
* History of complicated gastrointestinal surgery
* Diagnosed with inflammatory bowel disease (IBD)
* Current diagnosis of psychiatric disease/s or syndromes
* Current diagnosis of neurodegenerative disease
* Systemic use of antibiotics and/or steroid medication in the last 4 months prior to inclusion
* Use of any non-steroidal anti-inflammatory drug (NSAID) more than 3 times a week for the last 2 months
* Consumption of any NSAID within 7 days of study start
* Any condition which could substantially interfere with intestinal barrier function (e.g. gluten sensitivity, lactose intolerance, celiac disease, IBS, IBD) or in any other way with the outcome of the study, as decided by the principal investigator's discretion
* Regular smoking, use of snuff, nicotine, cannabidiol narcotics/supplements, or e-cigarette use
* Drinking more than 9 standard cups of alcohol per week and/or more than 3 standard cups of alcohol per occasion
* Regular use, for more than three times a week for the last 2 months and/or 7 days prior to inclusion, of medications which, according to the principal investigator, can have an anti-inflammatory effect or affect in any way the intestinal barrier function or have an impact on the study analysis (such as laxatives, anti-diarrheal, anti-cholinergic, etc.)
* After being included in the study, starting any medication or treatment that could potentially influence the study participation and/or study analysis.

• Zhou JS, Shu Q, Rutherfurd KJ, Prasad J, Gopal PK, Gill HS. Acute oral toxicity and bacterial translocation studies on potentially probiotic strains of lactic acid bacteria. Food Chem Toxicol. 2000 Feb-Mar;38(2-3):153-61. doi: 10.1016/s0278-6915(99)00154-4. PMID 10717355
• Zhou JS, Shu Q, Rutherfurd KJ, Prasad J, Birtles MJ, Gopal PK, Gill HS. Safety assessment of potential probiotic lactic acid bacterial strains Lactobacillus rhamnosus HN001, Lb. acidophilus HN017, and Bifidobacterium lactis HN019 in BALB/c mice. Int J Food Microbiol. 2000 May 25;56(1):87-96. doi: 10.1016/s0168-1605(00)00219-1. PMID 10857928
• Zhou JS, Pillidge CJ, Gopal PK, Gill HS. Antibiotic susceptibility profiles of new probiotic Lactobacillus and Bifidobacterium strains. Int J Food Microbiol. 2005 Feb 1;98(2):211-7. doi: 10.1016/j.ijfoodmicro.2004.05.011. PMID 15681048
• Zheng Y, Yu Z, Zhang W, Sun T. Lactobacillus rhamnosus Probio-M9 Improves the Quality of Life in Stressed Adults by Gut Microbiota. Foods. 2021 Oct 8;10(10):2384. doi: 10.3390/foods10102384. PMID 34681433
• Zahr NM, Mayer D, Pfefferbaum A, Sullivan EV. Low striatal glutamate levels underlie cognitive decline in the elderly: evidence from in vivo molecular spectroscopy. Cereb Cortex. 2008 Oct;18(10):2241-50. doi: 10.1093/cercor/bhm250. Epub 2008 Jan 29. PMID 18234683
• Yao M, Xie J, Du H, McClements DJ, Xiao H, Li L. Progress in microencapsulation of probiotics: A review. Compr Rev Food Sci Food Saf. 2020 Mar;19(2):857-874. doi: 10.1111/1541-4337.12532. Epub 2020 Feb 11. PMID 33325164
• Wu D, Lewis ED, Pae M, Meydani SN. Nutritional Modulation of Immune Function: Analysis of Evidence, Mechanisms, and Clinical Relevance. Front Immunol. 2019 Jan 15;9:3160. doi: 10.3389/fimmu.2018.03160. eCollection 2018. PMID 30697214
• World Medical Association. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013 Nov 27;310(20):2191-4. doi: 10.1001/jama.2013.281053. No abstract available. PMID 24141714
• Wickens KL, Barthow CA, Murphy R, Abels PR, Maude RM, Stone PR, Mitchell EA, Stanley TV, Purdie GL, Kang JM, Hood FE, Rowden JL, Barnes PK, Fitzharris PF, Crane J. Early pregnancy probiotic supplementation with Lactobacillus rhamnosus HN001 may reduce the prevalence of gestational diabetes mellitus: a randomised controlled trial. Br J Nutr. 2017 Mar;117(6):804-813. doi: 10.1017/S0007114517000289. Epub 2017 Apr 3. PMID 28367765
• Rode J, Edebol Carlman HMT, Konig J, Repsilber D, Hutchinson AN, Thunberg P, Andersson P, Persson J, Kiselev A, Lathrop Stern L, Salomon B, Mohammed AA, Labus JS, Brummer RJ. Probiotic Mixture Containing Lactobacillus helveticus, Bifidobacterium longum and Lactiplantibacillus plantarum Affects Brain Responses Toward an Emotional Task in Healthy Subjects: A Randomized Clinical Trial. Front Nutr. 2022 Apr 29;9:827182. doi: 10.3389/fnut.2022.827182. eCollection 2022. PMID 35571902
• Moro-Garcia MA, Alonso-Arias R, Baltadjieva M, Fernandez Benitez C, Fernandez Barrial MA, Diaz Ruisanchez E, Alonso Santos R, Alvarez Sanchez M, Saavedra Mijan J, Lopez-Larrea C. Oral supplementation with Lactobacillus delbrueckii subsp. bulgaricus 8481 enhances systemic immunity in elderly subjects. Age (Dordr). 2013 Aug;35(4):1311-26. doi: 10.1007/s11357-012-9434-6. Epub 2012 May 30. PMID 22645023
• Hutchinson AN, Bergh C, Kruger K, Susserova M, Allen J, Ameen S, Tingo L. The Effect of Probiotics on Health Outcomes in the Elderly: A Systematic Review of Randomized, Placebo-Controlled Studies. Microorganisms. 2021 Jun 21;9(6):1344. doi: 10.3390/microorganisms9061344. PMID 34205818
• Gbassi GK, Vandamme T. Probiotic encapsulation technology: from microencapsulation to release into the gut. Pharmaceutics. 2012 Feb 6;4(1):149-63. doi: 10.3390/pharmaceutics4010149. PMID 24300185
• Claesson MJ, Jeffery IB, Conde S, Power SE, O'Connor EM, Cusack S, Harris HM, Coakley M, Lakshminarayanan B, O'Sullivan O, Fitzgerald GF, Deane J, O'Connor M, Harnedy N, O'Connor K, O'Mahony D, van Sinderen D, Wallace M, Brennan L, Stanton C, Marchesi JR, Fitzgerald AP, Shanahan F, Hill C, Ross RP, O'Toole PW. Gut microbiota composition correlates with diet and health in the elderly. Nature. 2012 Aug 9;488(7410):178-84. doi: 10.1038/nature11319. PMID 22797518
• Hill C, Guarner F, Reid G, Gibson GR, Merenstein DJ, Pot B, Morelli L, Canani RB, Flint HJ, Salminen S, Calder PC, Sanders ME. 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. 2014 Aug;11(8):506-14. doi: 10.1038/nrgastro.2014.66. Epub 2014 Jun 10. PMID 24912386
• Halverson T, Alagiakrishnan K. Gut microbes in neurocognitive and mental health disorders. Ann Med. 2020 Dec;52(8):423-443. doi: 10.1080/07853890.2020.1808239. Epub 2020 Aug 31. PMID 32772900
• Gill HS, Rutherfurd KJ, Cross ML. Dietary probiotic supplementation enhances natural killer cell activity in the elderly: an investigation of age-related immunological changes. J Clin Immunol. 2001 Jul;21(4):264-71. doi: 10.1023/a:1010979225018. PMID 11506196
• Costabile A, Bergillos-Meca T, Rasinkangas P, Korpela K, de Vos WM, Gibson GR. Effects of Soluble Corn Fiber Alone or in Synbiotic Combination with Lactobacillus rhamnosus GG and the Pilus-Deficient Derivative GG-PB12 on Fecal Microbiota, Metabolism, and Markers of Immune Function: A Randomized, Double-Blind, Placebo-Controlled, Crossover Study in Healthy Elderly (Saimes Study). Front Immunol. 2017 Dec 12;8:1443. doi: 10.3389/fimmu.2017.01443. eCollection 2017. PMID 29312280
• Christensen SE, Moller E, Bonn SE, Ploner A, Wright A, Sjolander A, Balter O, Lissner L, Balter K. Two new meal- and web-based interactive food frequency questionnaires: validation of energy and macronutrient intake. J Med Internet Res. 2013 Jun 5;15(6):e109. doi: 10.2196/jmir.2458. PMID 23739995
• Anderson LA, Goodman RA, Holtzman D, Posner SF, Northridge ME. Aging in the United States: opportunities and challenges for public health. Am J Public Health. 2012 Mar;102(3):393-5. doi: 10.2105/AJPH.2011.300617. Epub 2012 Jan 19. No abstract available. PMID 22390500
• Bjelland I, Dahl AA, Haug TT, Neckelmann D. The validity of the Hospital Anxiety and Depression Scale. An updated literature review. J Psychosom Res. 2002 Feb;52(2):69-77. doi: 10.1016/s0022-3999(01)00296-3. PMID 11832252
• Betzel RF, Byrge L, He Y, Goni J, Zuo XN, Sporns O. Changes in structural and functional connectivity among resting-state networks across the human lifespan. Neuroimage. 2014 Nov 15;102 Pt 2:345-57. doi: 10.1016/j.neuroimage.2014.07.067. Epub 2014 Aug 7. PMID 25109530
• Bagga D, Aigner CS, Reichert JL, Cecchetto C, Fischmeister FPS, Holzer P, Moissl-Eichinger C, Schopf V. Influence of 4-week multi-strain probiotic administration on resting-state functional connectivity in healthy volunteers. Eur J Nutr. 2019 Aug;58(5):1821-1827. doi: 10.1007/s00394-018-1732-z. Epub 2018 May 30. PMID 29850990

• Carel Le Roux, PhD, Principal investigator, St Vincent's University Hospital, Ireland