Applications of a Functional Dairy Product in Diet Therapy

Determining the Possibility of Using a Functional Dairy Product in the Dietary Therapy of Irritable Bowel Syndrome and Metabolic Syndrome

The aim of the study will be to evaluate the effectiveness of dietary therapy using a functional dairy product, using fermented milk with selected probiotic cultures, in individuals with irritable bowel syndrome and metabolic syndrome.

The technology for preparing fermented milk from probiotic bacteria and assessing its physicochemical and sensory characteristics was developed and conducted as part of the SUPRIM research project at the Department of Gastronomic Technology and Food Hygiene, Institute of Human Nutrition, Warsaw University of Life Sciences (SGGW).

Compliance with dietary recommendations will be monitored by qualified dietitians, and will be monitored by a physician. Nutritional status, selected metabolic parameters, quality of life, and the occurrence of gastrointestinal symptoms will be assessed before and after the program. Patients' dietary habits will be monitored throughout the study.

Recruitment of participants for the study in individuals with irritable bowel syndrome:

Purposeful selection based on established inclusion and exclusion criteria (20 participants in each group: intervention, in which a functional milk drink will be included in the diet, and control, in which individuals with IBS will consume a natural, unadulterated, acidified milk drink), conducted by a physician.

Survey methodology:

• Food Frequency Questionnaire: FFQ6;
• Food consumption diary - the questionnaire will be completed by study participants before and during the intervention and given to the dietitian during a follow-up meeting every 7 days to monitor compliance with the recommendations.
• Short Form Health Survey SP-36
• Quality of Life Questionnaire for IBS patients: Quality of Life Questionnaire and IBS symptom severity: Birmingham IBS Symptom Questionnaire
• Stool quality questionnaire based on the Bristol Stool Formation Scale
• Physical activity questionnaire: International Physical Activity Questionnaire

Nutritional intervention regimen:

8 weeks of 2 servings per day (2 x 100 ml/day) of a functional milk drink and a milk drink without probiotics in individuals with Irritable bowel syndrome or metabolic syndrome

Assessment of the following in the qualified individuals:

• Dietary habits
• Anthropometric indicators (body weight and height, body composition using bioimpedance and the Accuniq BC-750 device, waist circumference)
• Quality of life
• Severity of gastrointestinal symptoms and self-assessment of stool quality
• Energy expenditure (resting energy expenditure and RQ index using indirect respirometry, Metalyzer 3B device from Cortex)
• Blood pressure measurement under standard conditions (Accuniq BP-250 device)
• Indicators of carbohydrate and lipid metabolism (blood concentrations of glucose, insulin, total cholesterol, LDL, HDL, and triglycerides)
• Indicators of inflammation severity (blood concentrations of C-reactive protein and a cytokine panel (using the Bio-Plex 200 system for immunofluorescence assays)
• Indicators of liver and pancreas function (blood concentrations of enzymes) liver function, gamma-glutamyltranspeptidase activity, and amylase and lipase activity)
• Evaluation of the colonic microbiota genotype (Real Time PCR)
• Random selection into the intervention group (n=20) and control group (n=20) consuming 200 ml/day of a functional, probiotic milk drink or 200 ml/day of a control milk drink (in two 100 ml portions) for 8 weeks. All patients will receive recommendations for a standard diet for this group of patients.
• Delivery every 8 days of 4,000 ml of milk drink (8 200 ml plastic packages intended for contact with this type of food), with or without probiotics, packed in packaging ensuring appropriate shelf life. Each portion will be stored at a refrigerated temperature (no higher than 4°C) and marked with a best-before date (no longer than 2 days after opening the package and 10 days without opening it),
• 8 weeks of dietary care, monitoring compliance with recommendations (meetings every 8 days),
• Completion of the program and re-evaluation of the above parameters;

Anthropometric, body composition and respirometric tests will be conducted at the Nutrition and Dietetics Clinic of the Department of Dietetics, Institute of Human Nutrition Sciences, Warsaw University of Life Sciences (SGGW). Blood cytokine panel testing using the immunofluorescence method will be performed at the Department of Dietetics, Institute of Nutrition and Food Sciences, Warsaw University of Life Sciences (SGGW). Real-time PCR characterization of the colonic microbiome will be performed at the Department of Gastronomic Technology and Food Hygiene, Institute of Nutrition and Food Sciences, Warsaw University of Life Sciences (SGGW).

Respirometric tests will be performed under standard conditions: mental and physical comfort (temperature of 21°C, constant room humidity), under fasting conditions, using a respirometric mask, which will be washed and disinfected before and after each measurement.

Blood samples for the planned analyses will be collected under fasting conditions at the Warsaw University of Life Sciences (SGGW) Outpatient Clinic, where analyses of lipid and carbohydrate metabolism parameters will also be performed. Stool samples for gut microbiota genotype assessment will be collected according to the protocol established in the previous study by participants eligible for the study (Ethics Committee approval no. 25/RKE/2024U).

The milk drinks will be prepared at the Wesoła Zagroda farm (Stary Bazanów/Ryki), based on technology developed as part of the SupRim project by the team of Dr. Dorota Zielińska, Professor at the Warsaw University of Life Sciences (Department of Gastronomic Technology and Food Hygiene, Institute of Food and Nutrition), and Prof. Dr. hab. Marcin Kurek (Department of Food Technology and Design, Institute of Food and Nutrition).

In the first stage, after standardization, the cow's milk will be pasteurized at 90°C for 20 minutes, then cooled to 30°C for approximately 30 minutes. It will then be inoculated with Levilactobacillus brevis B1 bacteria (2.5g of freeze-dried bacteria with a count of 1010 CFU/g per 100 liters of milk). After inoculation and mixing (30 minutes), the product will be packaged in individual containers and incubated (fermented) for 24 hours at 30°C. A control milk drink will be prepared in a similar manner, with the addition of a commercial blend of mesophilic lactic acid bacteria from Danisco, which do not possess probiotic characteristics. The Levilactobacillus brevis B1 strain comes from the collection of the Department of Gastronomic Technology and Food Hygiene at the Institute of Food and Agriculture, SGGW and will be pre-processed for further production stages at the Department of Fermentation Technology, Institute of Agricultural and Food Biotechnology, National Research Institute (by the team of Dr. Antoni Miecznikowski). This strain inhibits the growth of pathogenic bacteria of the species Listeria monocytogenes, Bacillus subtilis, E. coli, Enterococcus feacium and Salmonella enteritidis (Patent No.: P.426002/UP RP 2020: A new strain of Lactobacillus brevis bacteria and the use of a new strain of Lactobacillus brevis bacteria).

Individuals participating in the study will be informed of the purpose and scope of the study, as well as the conditions for preparing for the study, including refraining from strenuous activity the day before, avoiding caffeinated beverages and alcohol, and consuming their last meal after 6:00 PM. They will also be informed about data security, data anonymization, and personal data protection.

Each participant will also be informed of the possibility of withdrawing from the study at any stage.

The Contractors undertake that once a person has been qualified for the study, all their data will be secured in accordance with the Security Policy adopted at SGGW - Regulation 88/2013 and in accordance with the Act. All data relating to study participants will be encrypted and will not be used for purposes other than those specified above. Study participants will provide written consent to participate and will have the right to access their data, correct it, and request that its processing be discontinued.

Study participants have the right to terminate their participation in the study at any time if they deem it in their best interest.

Inclusion Criteria:

• BMI > 18.5 and < 25 kg/m²
• adults > 18 years of age,
• diagnosed with irritable bowel syndrome or at least one criterion of metabolic syndrome, in addition to abdominal obesity (hypertension and/or hyperlipidemia and/or glucose intolerance),
• not taking pro- and/or prebiotic supplements

Exclusion Criteria:

• Presence of chronic diseases such as cancer, kidney, liver, and thyroid disease, autoimmune diseases, and food allergies requiring the complete elimination of dairy products,
• Type 1 and type 2 diabetes treated with insulin
• Antibiotic therapy
• Consumption of a vegetarian diet or a low-energy diet (pharmacological treatment of obesity) and/or diets, e.g., high-fat, high-protein diets
• possession of electrostimulation and/or measurement devices that prevent measurement
• lack of informed consent to participate in the study