The adhesion capacity of probiotic microorganisms to intestinal epithelial cells is one of the key features of microorganisms required for their subsequent pro-health effects on the human body. It enables the direct contact of probiotic bacteria with the host cells, and also extends the time they stay in the digestive system. Therefore, in addition to research on the sensitivity to the influence of low pH or the presence of bile salts and digestive enzymes, it is one of the basic indicators of the effectiveness of probiotic preparations.
In addition to probiotic products, the CaCo-2 cell line isolated from human colon cancer cells and the HT-29 line from colorectal adenocarcinoma were used for research. Both cell lines were from the American Collection of Cell Cultures (ATCC). CaCo-2 and HT-29 intestinal epithelial cells were cultured in DMEM medium containing essential amino acids and gentamicin.
First, the appropriate volume of the cell suspension was collected as a probiotic product, containing 6.0 × 108 cfu / ml of bacteria. The whole was supplemented with DMEM medium and an antibiotic. CaCo-2 and HT-29 cells were cultured in an incubator at 37 ° C and a gas atmosphere of 4% CO2 and 96% air, changing the medium every 24 h.
The cultivation was carried out for 21 days for the CaCo-2 culture and 14 days for the HT-29 culture. The number of CaCo-2 and HT-29 cells was determined under a microscope. Prior to the test, CaCo-2 and HT-29 cells were washed with PBS solution and DMEM medium containing probiotic bacteria cells was introduced. Cultures were incubated for 2.5 h at 37 ° C. Triton X-100 solution was then added to each well and CaCo-2 and HT-29 cells were lysed. Lysates were transferred to Eppendorff tubes and centrifuged. The supernatant was removed and the pellet was suspended in saline. Selected dilutions were inoculated in three parallel replications. Cultures were performed in MRS medium with 2% agar addition at 35 ° C for 48-72 h.
A 21-day CaCo-2 cell culture and a 14-day HT-29 colon epithelial cell culture were used to determine the adhesive properties of the probiotic strains included in the probiotic products. They are the most widely used cell models used to determine the adhesive capacity of microorganisms. After the intestinal transit was completed, the number of bacterial cells associated with intestinal epithelial cells was determined. The adhesion capacity of probiotic bacteria was expressed as the level of adhesion, i.e. the number of microorganism cells associated with 100 cells of the CaCo-2 and HT-29 lines.
The figures below present the results of the adhesive capacity of probiotic microorganisms in the product after digestion in the small intestine. The obtained results indicate the highest affinity to the epithelial cells of the bacterial preparation with the addition of a food matrix in the form of milk substitute.
The number of bacteria associated with 100 intestinal epithelial cells was 258 for CaCo-2 and 409 for HT-29, respectively. The lowest bound number of cells, as in the case of the analysis carried out with CaCo-2 cells, was observed for the product without the use of a food matrix. In the latter variant, in contrast to others (with the use of a food matrix), both with the use of the CaCo-2 and HT-29 lines, the number of bound cells decreased after incubation in conditions simulating the digestive process in the small intestine.
Comparing the number of bacteria associated with 100 CaCo-2 and HT-29 cells, it can be concluded that the cells included in the probiotic product have a higher affinity for HT-29 cells. Comparing the samples with the largest number of bacterial cells adhering to CaCo-2 and HT-29 cells, it can be noticed that about 100 more cells adhere to HT-29 cells.
Rysunek 1. The number of probiotic bacteria associated with 100 HT-29 and CaCo-2 cells after in vitro digestion
The same conclusions can be drawn from the percentage of adhesion determined at the beginning of the analyzes, as shown in the figure below. This parameter describes the ratio of the number of bacterial cells that adhered to the number of all introduced cells. The obtained results of the degree of adhesion also indicate that the probiotic bacteria included in the product have a high ability to adhere to the model epithelial cells of the HT-29 line. This value depends on the food matrix used in the experiment. Clearly, the greatest efficiency of adhesion can be seen in the sample enriched with a food matrix in the form of substitute milk. The lowest degree of adhesion to HT-29 cells is seen in the variant in which the product was subjected to experiments without the food matrix. The degree of adhesion of bacterial cells to the CaCo-2 line is kept at a low level of about 6% in all four tested variants.
Rysunek 2. Degree of adhesion of undigested probiotic bacteria before intestinal transit (%)
The highest level of microbial adhesion with the addition of a food matrix in the form of powdered milk is associated with the presence of a number of protective substances in this product. As already mentioned, substitute milk is rich in many substances of a protective nature including polyols, disaccharides, polysaccharides, amino acids, as well as protein hydrolysates, minerals, organic acid salts and many others. In the discussed study, the most effective protection of adhesive properties was shown by substitute milk, where the number of bacteria adhering to CaCo-2 cells and HT-29 cells was the highest.
It is worth mentioning that milk is a commonly used substance to protect the survival and functional properties of bacterial cells. It is a natural environment for lactic acid bacteria, also due to the content of proteins, numerous vitamins and minerals, but above all, lactose, which is a substrate for these microorganisms. Lactose contained in milk increases its stability by creating hydrogen bonds with proteins of the cell membrane. In addition, thanks to its buffer capacity, milk reduces the acidity of the gastric environment, thus contributing to the reduction of the death rate of probiotic bacteria cells during the intestinal transit.
Source: Research on the evaluation of the quality and biofunctionality of the probiotic products of Living Food Sp. z o.o .. Scientific Monograph, 2019.