Efficacy of a Probiotic Formulation in Adults With Lactose Intolerance and Methanogenic Intestinal Overgrowth (LAC L-134/33)

Lactose intolerance (LI) is a syndrome associated with specific symptoms, including abdominal bloating and pain, nausea, flatulence, borborygmi, and diarrhea following the consumption of foods containing lactose, a disaccharide that is hydrolyzed in the intestine by the enzyme lactase (β-D-galactosidase). However, when there is enzymatic inefficiency or deficiency, malabsorption of the carbohydrate occurs; once present in the large intestine, it is fermented by microorganisms that mediate the production of hydrogen gas (H₂), carbon dioxide (CO₂), methane (CH₄), and short-chain fatty acids. Epidemiological data indicate a global prevalence of LI ranging from 65% to 70%; consequently, a significant percentage of people have difficulty consuming dairy products and must follow a treatment regimen that involves reducing or avoiding their intake. Consequently, the search for tools to manage LI has sparked interest in effective probiotic species capable of acting as an enzymatic source of β-galactosidase in the gastrointestinal tract. In particular, certain strains with β-galactosidase activity can contribute to lactose hydrolysis and symptom relief, such as: Streptococcus thermophilus, Lactobacillus bulgaricus, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium, and Saccharomyces boulardii. Previous clinical trials have noted its use in treating gastrointestinal symptoms and the amount of exhaled H₂ measured by the Hydrogen Breath Test (HBT) before and after probiotic interventions.

However, given that the gut microbiota comprises bacterial communities, viruses, fungi, archaea, and protozoa, it is important to consider its influence on the pathogenesis of certain conditions; in particular, intestinal methanogenic overgrowth (IMO) may have clinical relevance as it is associated with functional gastrointestinal disorders where symptoms may overlap with those of other conditions. It has been suggested that IL may be related to the presence of Small Intestinal Bacterial Overgrowth (SIBO) and share symptoms, since SIBO reduces lactose absorption due to abnormal villus function and the destruction of enzymes in the intestinal mucosa, leading to positive IL cases being secondary to SIBO. Additionally, it has been reported that approximately 30% of SIBO cases present with concomitant IMO. However, the therapeutic targets for IMO include antibiotic administration, dietary modifications, and, it is speculated, the use of probiotics, for which there is limited evidence. Therefore, while it is possible to consider the role of probiotics in modulating the gastrointestinal symptoms associated with IL and IMO, the clinical results of probiotic supplementation are inconclusive. For this reason, we conducted a preliminary pilot efficacy study using the strains Limosilactobacillus fermentum L-33 and Lacticaseibacillus rhamnosus L-134, both isolated from a gastric biopsy and possessing high lactase-producing capacity.

Therefore, the objective of this pilot study was to analyze the efficacy of a capsule formulation containing both probiotics and its effect on gastrointestinal symptoms in adults with lactose intolerance and intestinal methanogenic overgrowth.

Research hypothesis: daily administration, for 12 weeks, of a probiotic formulation with the strains Lacticaseibacillus rhamnosus L-134 and Limosilactobacillus fermentum L-33 with β-galactosidase activity decreases the quantity of exhaled H2 measured through the intervention of HBT and the presentation of gastrointestinal symptoms in adults.

Material and methods: The group of participants was made up of 22 adults of both genders (13 women and 9 men). The group underwent an HBT at the beginning of the intervention to confirm the diagnosis of LI and perform the initial H2 measurement. 12 participants (6 women and 6 men) received a diagnosis of LI. 2 participants tested positive for LI and IMO (2 men) and 8 participants were diagnosed with IMO (7 women and 1 man).

The HBT is the most used diagnostic method for determining LI, is not invasive, is relatively economical and has an optimal sensitivity (> 73.2%) and a specificity of 85.6%. On the other hand, if the golden standard for the diagnosis of SIBO is the aspiration of yeyunal liquid, followed by cultivation and detection of bacterial communities, this intervention stands out for being invasive and expensive, and if it is well known that BT with lactulose or glucose is postulated for the diagnosis of SIBO, in the present pilot study it was possible to identify some subjects positive for IMO from the initial screen specifically targeted at LI (BT with lactose). HBT was applied before and after the intervention to all participants, initially between 2 and 3 weeks before the start of the intervention, indicating the necessary preparation that included a carbohydrate-free diet 48 hours before the test, avoiding the use of antibiotics, laxatives and prokinetic drugs for four weeks beforehand, suspending the habit tobacco the night before and last at least 12 hours.

The specific gastrointestinal symptomatic presentation associated with LI (diarrhea or need to go to the bathroom, vomiting or nausea, cramp or abdominal pain, abdominal cramping or audible intestinal noises and flatulence or abdominal distention) was evaluated through the use of a Questionnaire on symptoms of Lactose Intolerance (QSLT) type visual analogue scale (VAS) which ranges from 0 to 10 with a total score of 50. The form has a sensitivity of 0.75 and a specificity of 0.67 and allows the identification of lactose malabsorption with a cutoff point of 6.5. This questionnaire was applied during the performance of the respective HBT, before and after the intervention. Randomization and masking: The randomized, double-blind and placebo-controlled pilot study was carried out in the facilities of the University of Concepción, Faculty of Pharmacy and/or Biological Sciences during the year 2024. An independent researcher carried out the randomization using a list with the names of participants generated in one Microsoft Office Excel® spreadsheet, which was safeguarded by it to maintain the anonymity and masking of participants throughout the study.

The group of 22 participants was classified into groups: those with LI and those with IMO (positive results for LI + IMO were considered within the LI group). Accordingly, each group was randomized separately and formed into 2 subgroups for each group, having the intervention groups (which received capsules with two probiotic strains) and the placebo groups, over a period of 12 weeks. Liva Company® (Santiago, Chile) provided bottles containing capsules with the biceps probiotic formulation (Lacticaseibacillus rhamnosus L-134 and Limosilactobacillus fermentum L-33) at a concentration of three thousand million CFU of each strain per capsule and placebo capsules were made up of maltodextrin 10-14 dimerco. Both capsules are coated with vegetable cellulose (hydroxypropylmethylcellulose), which are indistinct in appearance. Individuals must administer one capsule daily, in water and in fast with cold water according to the manufacturer's instructions, for 12 weeks. To evaluate self-administration compliance, participants returned the 3 delivered bottles containing non-self-administered capsules.

Nutritional assessment: Includes the measurement of anthropometric and dietary variables, through which body composition, nutritional status and the association between the intake of dairy products and lactose and expressed symptomatology and the result of the intervention are globally analyzed.

Statistical analysis: The size of the sample was defined based on García et al recommendation for pilot studies. The statistical treatment and data tabulation were carried out using the Microsoft Office Excel® version 16.72 program, while the same process and analysis were carried out using the SPSS-24.0® and R 4.4.3 packages. The numerical variables were presented by its mean and standard deviation (D.E), the categorical variables by the frequency and percentage of each one of its levels. Due to the size of the samples, the student test is applied to paired groups. For the statistical analysis, a significance level of 0.05 was used, so every time the value was less than 0.05, the result was considered statistically significant.