Development of postbiotics’ compositions including the evaluation of its bioactivity against pathogenic bacteria and their produced biofilms in the dairy industry

The project's objectives focus on addressing the problem of biofilm formation in the dairy industry, where milk serves as an ideal medium for the growth of microorganisms and the formation of biofilms. Biofilms not only reduce the microbial quality and shelf life of products, but pathogenic microbial biofilms also pose a threat to consumer health and negatively impact the health of milking animals, leading to mastitis and promoting the spread of antibiotic-resistant bacteria. Mature biofilms are difficult to eliminate because they are resistant to traditional detergents and disinfectants. Furthermore, the use of these traditional agents is not only associated with the development of resistance but also with the inactivation of beneficial microbiota and a potential deterioration of the organoleptic properties and nutritional value of dairy products. Thus, various new, safe both for an environment and society methods and agents for limiting biofilm formation in the food production sector are currently being researched worldwide.

This project aims to focus on evaluating the antimicrobial effects of whole inactivated lactic acid bacteria and their metabolites on pathogenic bacteria and the biofilms they produce, developing one or more compositions with antibacterial activity. Recent studies on postbiotics have revealed that they are very stable, do not require a cold chain during storage and transport, and maintain high activity over a long period, thus offering a safer and more cost-effective alternative than live probiotics. Additionally, postbiotics do not induce chemical reactions in food products, do not alter the aroma and taste of the product, do not possess the ability to develop resistance or transfer resistance genes, are not alive, and therefore cannot cause sepsis, making them a safe alternative to probiotics for immunocompromised individuals and infants/newborn animals.

Microorganisms can produce biofilms on any surface, both organic and inorganic, creating threats ranging from contaminated implantable medical devices and cariogenic colonization on tooth enamel to food and water contamination, corrosion of metal surfaces, and equipment clogging, along with other significant health issues due to their wide prevalence and potential harm. Therefore, the application possibilities of postbiotics as a natural, immune-modulating, antibacterial biotechnological product in combating biofilms and planktonic pathogenic bacteria are extensive, and the technology developed in this project for postbiotic extraction and bioactivity evaluation, as well as the compositions created, may also be applicable to solving problems for other target groups.

It is planned that the project will utilize lactic acid bacteria as a source of postbiotics, which are isolated from the milk of healthy cows and/or from traditionally fermented products in Latvia. The research results will be patentable and potentially commercializable.

Project Achievements

Project updates by 30 September 2026

During the reporting period, in project activity WP3, in collaboration with veterinary medicine students Signe Vessere and Viktorija Krasnova, the collection of samples and microbiological examination was completed in two milk production and one milk processing company. Repeated sampling enabled assessment of the recurrence of biofilm-forming species in the equipment. Approximately 1 000 pure cultures of bacteria and 200 pure cultures of microscopic fungi were isolated and preserved. Bacteria and yeast identification was completed, and the selected isolates were assessed for susceptibility to antibacterial and antifungal agents, as appropriate, and for their ability to form biofilms.

Bacterial and yeast isolates were identified by MALDI-TOF mass spectrometry in collaboration with the Scientific Laboratory of the Latvian University of Life Sciences and Technologies. The postdoctoral researcher acquired practical proficiency in the method and independently analysed the isolates. In addition, researchers at the RSU Institute of Oncology and Molecular Genetics performed molecular analysis of samples from the internal surfaces of milking systems using the 16S rRNA gene sequencing.

Under project activity WP4, the antimicrobial and antibiofilm activity of eight commercial probiotic sources and the postbiotic preparations derived from them was characterised. Approximately 90 lactic acid bacterial isolates were also isolated from milking equipment. This collection will form the basis for further selection to obtain postbiotic preparations from promising isolates and to test their ability to inhibit the growth of pathogenic bacteria and biofilm formation.

An international mobility at the Anti-Infectives Research (AIR) group of the Faculty of Pharmacy, University of Helsinki, took place from 1 June to 31 July 2026. During the mobility, the postdoctoral researcher learned new methods for assessing biofilm formation and antimicrobial activity, including the use of the nematode Caenorhabditis elegans to evaluate the toxicity of supernatants derived from probiotic microorganisms and their protective effects against Staphylococcus aureus infection. New data on the antimicrobial activity of probiotic-derived preparations were obtained. A joint publication with AIR researchers is planned.

Research results were presented as poster presentations at two international conferences: “Research for Rural Development 2026”, held in Jelgava on 13–14 May 2026, and “The 2nd International Online Conference on Veterinary Sciences”, held online on 7–9 September 2026.

Two scientific articles have been submitted for publication. The article “Comparative antibacterial and antibiofilm activity of Lactobacillus helveticus cell-free and non-filtered heat-inactivated supernatants” has been accepted for publication in the LBTU conference proceedings “Research for Rural Development”. The manuscript “Comparative screening of probiotic-derived formulations for inhibition of biofilm formation by milking equipment isolates and reference strains” has been submitted to the journal Fermentation.

Two theses are in progress. The experimental work for one thesis has been completed, and its defence is scheduled in May or June 2027.

A one-year interruption in project implementation is planned.

Project updates by 31 March 2026

Within the first activity of the project (WP1), the establishment of the microbiology laboratory has been completed, and a study course on test systems using mammalian cell cultures has been successfully completed. Results have been obtained on the antagonistic effects of commercial probiotic bacteria and their derived postbiotics against pathogenic bacteria, as well as their impact on biofilm formation. Part of the results was presented at the conference “International Probiotics Association (IPA) World Congress + Probiota 2026” (Dublin, February 11–13). In addition, abstract have been submitted for participation in the conference “Research for Rural Development 2026”, which will take place in Jelgava in May this year, and a draft scientific publication has been prepared.
Within the second activity of the project (WP2), English language skills have been improved by completing two courses at the B2.1 and B2.2 levels.

Within the framework of project activity WP3, samples were collected from equipment in several dairy production and processing companies. Over a six-month period, biofilm-forming microorganisms coming into contact with raw cow’s milk intended for consumer use were analyzed dynamically. Microbiological examination of the samples was performed, identifying biofilm-forming microorganisms (bacteria and yeasts) to the species level using MALDI-TOF mass spectrometry, while molds were identified to the genus level. For a subset of isolates—particularly species characterized by high persistence in milking equipment—biofilm-forming ability, antimicrobial resistance, as well as lipolytic and proteolytic activity were determined. Two students have been involved in the research and have initiated the development of their diploma theses on the topic of microbial biofilms.

As part of the project, public outreach and educational activities were also carried out. Presentations were delivered to pupils of Svēte Primary School on the professions of researcher and veterinarian, as well as on beneficial bacteria and their role in the human body, while also introducing the project objectives and research results.

Project updates by 30 September 2025

Within the framework of the project’s first activity (WP1) – learning the research methodology and feasibility study – a data management plan has been prepared and a methodology has been developed for the propagation of lactic acid bacteria, cultivation of biofilms, and bacteriological examination of samples. In line with the planned tasks, the necessary materials and reagents have been purchased, and the microbiology and biotechnology laboratory has been set up, including equipment requests and training for working with the new instruments.

At present, several commercially available species of lactic acid bacteria are being cultivated, postbiotics are being extracted, and their antagonistic activity against pathogenic bacteria is being tested. In parallel, training in the cultivation of pathogenic bacteria and Lactobacillus biofilms is ongoing, while the obtained data are already being compiled and supplemented with new experiments in order to begin preparing the first publication soon. Studies on test systems with mammalian cell cultures have also been initiated.

At the same time, the project’s second activity (WP2) – English language training – is being implemented, with one course completed and the next-level course already underway.