Pracownie badawcze

Badania

Najważniejsze osiągnięcia badawcze

• We developed bacterial starter cultures for the production of wholemeal bread from rye, common wheat, and spelt flour.
• We determined the microbiological composition of complex ecosystems, such as kefir grains, Oscypek cheeses, and spontaneous sourdoughs, based on wholegrain rye, common wheat, and spelt flour.
• We found that two genes (chromosomal AJ89-07570 [big] and AJ89_14230 [prtP]), located on plasmid pIBB477b, are involved in adhesion of the Lactococcus lactis IBB477 strain to components of the gastrointestinal mucosa.

Opis badań

Studying biodiversity of complex ecosystems and bacterial environmental interactions is crucial to understand the ecology of microbial communities, and to rationally select potentially probiotic strains as well as strains of industrial importance or for biotechnological purposes. Culture dependent and independent approaches have been used to study the microbiology of complex food ecosystems, such as raw milk, kefir grains, traditional Polish cheese – Oscypek and different wholegrain sourdoughs made from rye, wheat or spelt flours. An added value of the projects was that many different LAB strains from various environments have been isolated, genetically characterized and deposited in the publicly accessible IBB PAS laboratory culture collection. Among them there are strains collected from raw milk samples by Żylińska (PhD Thesis, 2017), kefir grains (Kowalczyk et al. 2012), Oscypek (Alegría et al. 2012) and sourdoughs (Boreczek et al. 2020). Based on the biodiversity analysis of wholegrain sourdough samples and information on LAB metabolism we selected strains for specific starter cultures for functional bread production (Litwinek et al. 2022) and efficient exopolysaccharide (EPS) production hindering resistant starch formation, thus improving the properties of sourdough bread (Buksa et al. 2021). Developed starter cultures, sourdough and wholemeal bread technology assessed at the industrial scale were awarded four patents.

Apart from food ecosystems LAB are also associated with animals and humans. Optimal adaptation to niches that LAB stably inhabit or temporarily encounter in the host depends on factors that promote survival (stress resistance), adaptation (active metabolism adapted to the host environment), and colonization of the host (adherence to the intestinal mucosa and mucus). Adhesion of the IBB477 strain, which is the first L. lactis strain exhibiting adhesive and muco-adhesive properties to be sequenced, was analyzed both in vitro and in vivo and putative adherence factors of IBB477 were identified (Radziwill-Bienkowska et al. 2014, 2016, 2017). The strain IBB477 was proposed and selected as a model of Gram-positive transient in gastro intestinal tract food-borne bacteria to study interactions between exogenously applied bacteria and nano-sized titanium dioxide (TiO₂) (Radziwill-Bienkowska et al. 2018).

Understanding of the mechanisms of probiotic action can support rational selection of probiotic strains. The probiotic properties are strain-specific, therefore detailed identification and characterization of the strains is essential. The result of our expert work at the Standing Committee of the International Dairy Federation provided updated guidelines for the identification of probiotics at the strain level (Yao et al. 2021).

• Bibliography

1. Alegría et al. Appl Environ Microbiol. 2012. doi: 10.1128/AEM.06081-11
2. Boreczek et al. MicrobiologyOpen. 2020. doi: 10.1002/mbo3.1009
3. Buksa et al. Food Chemistry. 2021. doi: 10.1016/j.foodchem.2021.130221
4. Kowalczyk et al. J Dairy Res. 2012. doi: 10.1017/S0022029911000677
5. Litwinek et al. PLOS ONE. 2022. doi: 10.1371/journal.pone.0261677
6. Radziwill-Bienkowska et al. Acta Biochim Pol. 2014
7. Radziwill-Bienkowska et al. Appl Microbiol Biotechnol. 2016. doi: 10.1007/s00253-016-7813-0
8. Radziwill-Bienkowska et al. Appl Microbiol Biotechnol. 2017. doi: 10.1007/s00253-017-8334-1
9. Radziwill-Bienkowska et al. Frontiers Microbiol. 2018. doi: 10.3389/fmicb.2018.00794
10. Yao et al. Bulletin of the IDF 513. 2021.

Metodologia

In our laboratory, we are currently using a large set of classic biochemical  and molecular-based methods:  DNA cloning, polymerase chain reaction, DNA gel electrophoresis, protein purification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, pH/bile salt resistance assays, bacterial adhesion assays, microbiological growth studies using Bioscreen C (Labsystems Oy, Finland), bacteria cultivation using fermenters, microdilution antibiotic resistance assays, and freeze-drying of bacterial cells.

Some analyses are performed in collaboration with other laboratories: Genomic sequencing of LAB strains and metagenomics of food/environmental samples are performed in the DNA Sequencing and Synthesis Facility (IBB PAS). Proteomics and metabolomics of LAB strains or complex microbial ecosystems are or will be performed in the Mass Spectrometry Facility (IBB PAS). Cell cultures are also employed to study the effects of the studied LAB strains or their metabolites on the properties of living systems. Bioinformatics including probiogenomics are done by ourselves and in collaboration with the DNA Sequencing and Synthesis Facility. Some of our experiments are being performed in vivo in mice thanks to the external collaboration.

Several specialized methodologies were developed in our laboratory, such as:

• Method for the cultivation of bacterial strains under controlled environmental conditions using BIOSTAT Bplus bioreactors (Sartorius Stedim Biotech, Germany).
• Method for monitoring the acidification process using a computerized acidifying activity evaluation system (iCINAC, AMS Alliance, Italy).

List of unique equipment available in the laboratory:

• BIOSTAT Bplus Exclusive Flow|Double Wall (Sartorius Stedim Biotech, Germany): System for animal, plant, and insect cell cultivation and microbial fermentation.
• iCINAC (AMS Alliance, Italy): System for monitoring acidification activity, which simultaneously observes changes in pH, temperature, and redox potential of one or several samples.
• Mini-BeadBeater-8 (16) (BioSpec Products, USA): High-energy cell disrupter that disrupts microbial cells and plant and animal tissue by violently agitating up to eight (sixteen) 2 ml screw-cap microvials that contain small glass, ceramic, or steel beads and disruption buffer.
• Whitley A35 Workstation with strictly controlled conditions for the processing, incubation and examination of samples without exposure to atmospheric oxygen. Anaerobic chamber can be used for incubation of microorganisms in strict anaerobic conditions.

Wybrane publikacje

• Developing lactic acid bacteria starter cultures for wholemeal rye flour bread and their evaluation on an industrial scale. Litwinek D, Boreczek J, Gambuś H, Buksa K, Berski W^#, Kowalczyk M^#. (^#corresponding authors) PLOS ONE. 2022. doi: 10.1371/journal.pone.0261677.
• Identification of probiotics at strain level - Guidance document - Update of the Bulletin of the IDF N°462/2013. Yao S^#, Bourdichon F, Kowalczyk M, Knudsen G, Bassett S, von Ah U, Dubois-Lozier A, Dekker J, Gerten B, Muto M. Bulletin of the IDF 513/2021. 2021.
• Bacterial community dynamics in spontaneous sourdoughs made from wheat, spelt and rye wholemeal flour. Boreczek J, Litwinek D, Żylińska-Urban J, Izak D, Buksa K, Gawor J, Gromadka R, Bardowski JK, Kowalczyk M^#. 2020. doi: 10.1002/mbo3.1009.
• Contribution of plasmid-encoded peptidase S8 (PrtP) to adhesion and transit in the gut of Lactococcus lactis IBB477 strain. Radziwill-Bienkowska JM, Robert V, Drabot K, Chain F, Cherbuy C, Langella P, Thomas M, Bardowski JK, Mercier-Bonin M, Kowalczyk M^#. Applied Microbiology and Biotechnology. 2017. doi: 10.1007/s00253-017-8334-1.
• Biodiversity in traditional Polish cheese Oscypek determined by culture-dependent and -independent approaches. Alegría Á, Szczesny P, Mayo B, Bardowski JK, Kowalczyk M^#. Applied and Environmental Microbiology. 2012. doi: 10.1128/AEM.06081-11.

Współpraca

• Peter Neubauer, Chair of Bioprocess Engineering, Institute of Biotechnology, Technische Universität Berlin, Germany.
• Muriel Mercier-Bonin, Toxalim (Research Centre in Food Toxicology) INRA, Toulouse, France
• Muriel Cocaign-Bousquet and Pascal Loubiere, Toulouse Biotechnology Institute, INRA, Toulouse, France
• Muriel Thomas and Phillip Langella, INRA, MICALIS, Jouy-en-Josas, France.
• Baltasar Mayo, Departamento de Microbiología y Bioquímica, Instituto de Productos Lácteos de Asturias (IPLA) CSIC, Spain.
• Halina Gambuś, Dorota Litwinek and Krzysztof Buksa, Department of Carbohydrate Technology, Faculty of Food Technology, University of Agriculture in Krakow, Poland.
• Małgorzata Ziarno, Department of Biotechnology, Microbiology and Food Evaluation, Division of Milk Biotechnology, Faculty of Food Sciences, Warsaw University of Life Sciences – SGGW, Poland.
• BIO4EVER Sp. z o.o., Warsaw, Poland.