The aim of this study is to investigate whether a fruit snack containing probiotic ''Lactobacillus rhamnosus'' can survive and establish itself more effectively in the gut when it is protected inside a natural plant-protein coating and delivered in a fruit snack.

Lactobacillus rhamnosus is a beneficial bacterium that may support gut health and influence communication between the gut and the brain. In this study, we will examine how the probiotic affects the gut microbiome (the community of bacteria that naturally live in the gut).

We are recruiting 25 healthy men and women aged 25-65 years with a body mass index (BMI) between 18.5 and 31.9 kg/m². Participants will be randomly assigned to receive either:

A) Fruit snack containing encapsulated Lactobacillus rhamnosus probiotics, or a similar fruit snack that does not contain probiotics (placebo).

Neither participants nor researchers (blinded) will know which snack is being provided during the study.

Participants will attend four study visits over approximately 10 weeks (70 days): at the baseline visit Day 0 and again at Days 28, Day 42, and Day 70. A blood and stool samples will be collected at the beginning of the study, and samples will be used to assess changes in the gut microbiome over time.

The findings from this study will help us better understand how targeted delivery of probiotics to the gut may influence the gut microbiome and support digestive health.

The gut microbiota plays an important role in human health through interactions with the gastrointestinal, immune, endocrine, and nervous systems. Increasing evidence suggests that the gut microbiota influences gastrointestinal and brain function through the microbiota-gut-brain axis, a bidirectional communication network linking the gastrointestinal tract and the central nervous system. Alterations in gut microbial composition have been associated with aging-related physiological changes, gastrointestinal function, inflammation, cognition, and mood.

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. However, the effectiveness of probiotic supplementation depends on the ability of viable microorganisms to survive food processing, storage, gastric acidity, and gastrointestinal transit before reaching their target site in the intestine. Consequently, strategies that improve probiotic survival and targeted delivery may enhance intestinal colonisation and biological activity within the gastrointestinal tract.

Microencapsulation is a promising approach for protecting probiotic bacteria during manufacturing, storage, and passage through the upper gastrointestinal tract. Encapsulation within a protective plant protein matrix may increase delivery of viable probiotic cells to the intestine by reducing exposure to gastric acid and digestive enzymes. The investigational product used in this study contains Lactobacillus rhamnosus microencapsulated using a plant protein-based delivery system and incorporated into a fruit snack.

The primary objective of this study is to determine whether daily consumption of a fruit snack containing microencapsulated Lactobacillus rhamnosus increases intestinal colonisation compared with a matched fruit snack without probiotics. Colonisation will be assessed by measuring the abundance of Lactobacillus rhamnosus in stool samples.

Secondary objectives include evaluating the effects of the intervention on gut microbiome composition and diversity, and exploring changes in markers related to gastrointestinal function and microbiota-gut-brain axis physiology.

This study is a randomised, double-blind, placebo-controlled crossover trial. Participants will complete two 28-day intervention periods separated by a 14-day washout period. Participants will receive both study interventions in a random order: (1) a fruit snack containing microencapsulated Lactobacillus rhamnosus and (2) a matched fruit snack without probiotics.

Participants will consume one 28-g serving of the assigned fruit snack each day during each intervention period. The probiotic snack contains Lactobacillus rhamnosus at a concentration of 2 × 10⁹ colony-forming units (CFU) per gram, providing a total daily dose of approximately 5.6 × 10¹⁰ CFU (56 billion CFU/day). Stool samples will be collected at predefined study visits to assess probiotic colonisation and changes in gut microbiome composition.

This study will provide evidence regarding the effectiveness of plant protein-based microencapsulation technology for probiotic delivery and improve understanding of the impact of targeted probiotic supplementation on intestinal colonisation and the human gut microbiome.

Inclusion Criteria:

• Signed informed consent prior to any study-related procedure Age 25-65 years-old
• 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:

• Smoking
• Substance abuse
• Pregnancy and lactaction
• 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)
• People with intellectual disabilities.
• History of complicated gastrointestinal surgery
• Diagnosed 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 principle 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.

Justification: it is necessary to exclude participation from individuals with smoking or substance abuse as that may compromise the physiological gut response.