The Impact of Dietary Supplementation With Lactobacillus Helveticus and Bifidobacterium Longum on the Functioning of the Brain-gut Axis, in the Context of Mental Health, Eating Patterns and Nutritional Status of Young Adults

Introduction The number of people receiving psychiatric services has been increasing over the past few years. In Poland, the number of people with mental disorders in outpatient care has increased by 150,000 in recent years, reaching a total of more than 1.2 million patients. The number of people requiring psychological and psychiatric care is expected to grow in the coming decades. The psychopharmacotherapy used does not always produce the expected treatment results. As a result, more and more attention is being paid to non-pharmacological interactions. In recent years, there has been a growing body of research on the relationship between nutrition and its impact on the mental state of people with mental disorders. Food and nutrition science, psychodietetics and nutripsychiatry, thriving scientific fields, are gaining prominence. Research findings indicate that nutrition and stress-coping resources affect an individual's health. The role of the probiotic bacteria Lactobacillus Helveticus and Bifidobacterium longum as part of the human microbiome has also been studied in this sphere, and results from a growing number of papers confirm their wide range of applications and positive effects on various domains of health. Previous studies have shown that supplementation with Bifidobacterium longum or a composite of them with other bacterial strains positively affected, among other things, the bone health of postmenopausal women, alleviated gastrointestinal discomfort, reduced perceived stress, and improved depression, anxiety, and mood indicators in adults of various ages. On the other hand, supplementation with a composite of several bacterial strains, including Lactobacillus Helveticus and Bifidobacterium longum, affected reducing fat mass in obese and overweight individuals. Probiotic supplementation appears to have great potential for improving health and quality of life, including the prevention and treatment of depressive and anxiety disorders and the ability to cope with stress. Nevertheless, there is still a lack of research in this area, especially concerning Lactobacillus Helveticus alone and the combination of Lactobacillus Helveticus and Bifidobacterium longum strains.

With this in mind, a research project was planned to investigate the effects of both probiotic supplements and dietary quality on a wide range of psychological, microbiological, biochemical, and clinical outcomes. The study aimed to evaluate the effects of targeted supplementation with a new composition of probiotic strains Lactobacillus Helveticus and Bifidobacterium longum on gut microbiota composition and quality, stress-related neuroendocrine markers, mental health, perceived stress intensity, psychosomatic response and coping strategies as well as body composition and bone density in young women who differed in diet quality. We hypothesised that supplementation with probiotic bacteria at a daily dose of 3 x 109 CFU, irrespective of diet quality, would result in changes in the gut microbiota towards higher proportions of beneficial bacteria, reduce stress-related neuroendocrine markers and the intensity of perceived stress and its psychosomatic symptoms, and improve stress-coping strategies, mental health potential and have a beneficial effect on body composition components and bone density in healthy young women, compared with placebo.

Study design and population This randomized, placebo-controlled, 2-arm, parallel, double-blind intervention trial was conducted in a few waves from September 2023 to March 2024 2023 at the Department of Human Nutrition and Dietetics (Poznań Univrsity of Life Sciences). Each wave started during the fall months and was completed during the winter months. The sample size was calculated using GPower software (version 3.1.9.7, Universität Düsseldorf, Germany). Briefly, to detect a significant change in perceived stress level with a two-sided t-test, a significance level of 5%, and a statistical power of 80%, a sample size of 110 was determined, considering a 30% dropout rate. Initially, 120 women were enrolled in this study. These were only women in early adulthood. Eligibility criteria for the study included female sex, age 20-30 years, BMI < 30 kg/m2. Exclusion criteria were: diagnosed chronic and/or serious health disorders (e.g. diabetes, asthma, hypertension, renal diseases), pregnancy, taking any probiotics for 8 weeks prior to enrollment to the study protocol, recent and current antibiotic treatment, ore use of pharmaceutical antidepressant treatment. All participants were healthy volunteers who were fully informed about the study's objectives and provided informed consent for participation before entering the study. The study protocol was reviewed and approved by the Bioethics Committee of the Karol Marcinkowski University of Medical Sciences in Poznan, Poland (permission no. 244/23, issued on March 9, 2023). The study complies with the CONSORT Statement on randomized trials. All procedures followed the guidelines of the 2013 Declaration of Helsinki and Good Clinical Practice.

The enrolled volunteers were evenly, randomly, and blindly assigned to either the intervention (Lh+Bl) group, receiving probiotic bacteria, or the placebo (PLA) group for 12 weeks of supplementation. Randomization was conducted by the supplement manufacturer with computer-generated codes using Research Randomizer (www.randomizer.org). The entire study protocol included familiarization and two laboratory visits in at the Department of Human Nutrition and Dietetics at baseline and at the end of the study (T1, T2; before/after Lh+Bl supplementation [Lh+Bl PRE and Lh+Bl POST] and PLA supplementation [PLAPRE and PLAPOST]). The study's main protocol included an assessment of dietary habits at the T1 study visit. Measurements of weight, height, body composition, bone density, stress response and stress-coping strategies, mental health assessment as well as saliva and stool sampling were conducted at each of the T1 and T2 study visits. The primary outcome measures are microbiota composition, perceived stress intensity and stress-related neuroendocrine markers (salivary cortisol, salivary DHEA and salivary cortisol:DHEA ratio, faecal cortisol); the secondary outcome measures are stress-coping strategies, psychosomatic symptoms, sense of coherence, body composition and bone density). All testing was performed in the morning at the same time for the participant. Participants declared that they had not introduced any changes in their lifestyles, nutrition, or supplementation during the study protocol and were prepared for each visit in the same manner. In addition, telephone contact was maintained in case of non-compliance with the protocol or gastrointestinal symptoms or adverse events.

There were 20 dropouts from the study protocol. The main reasons for dropping out were antibiotic therapy during the protocol (n=10) and discontinuation of the study without a reason before the second visit (n=10). There were 11 dropouts in the Lh+Bl and 9 in the PLA groups. Finally, 100 women (23.5 ± 2.7 years; 61.6 ± 9.4 kg body mass; 1.67 ± 0.06 m height) completed the entire study protocol and were included in the analyses.