**5.3 Influence of** *Lactococcus* **live cell biomass concentration on cholesterol removal**

Usman and Hosono [43] determined that cholesterol binding was significantly dependent on the amount of bacterial cell biomass and it increased proportionate to the increase of the cell count. Furthermore, Liong and Shah [38] observed that the amount of cells has a significant impact on the differences in the amount of cholesterol bound by lactic acid bacteria, whereas the growth dynamics for individual strains determines the amount of cell biomass and differences in experimental results. Ziarno [4] verified the manner in which the concentration level of live cell biomass originating from monocultures and multi-species cultures of *Lactococcus* influences the capacity of cultures to remove cholesterol from M17 culture broth. As expected, the highest amount of cholesterol was removed in the cultures containing 10-fold concentrated *Lactococcus* biomass. At this cell biomass concentration, the

*Prebiotics and Probiotics - Potential Benefits in Nutrition and Health*

unsaturated acids (oleic and linoleic acids) than the samples, to which cholesterol was added. According to Boggs [93] cholesterol forms hydrogen bonds with the amide group N▬H of bile acids and oxygen molecules of hydroxyl groups of saccharides in fatty acids. It is likely that the same bonds connect the cholesterol with phospholipids and glycolipids of bacterial cell membrane [50]. However, according to other literature data, no strict relationship exists between lactic acid bacteria

It can be stated that the phenomenon of cholesterol binding and removal by bacterial cells is complex. It can be concluded that the contribution of the phenomenon of cholesterol molecule assimilation or adhesion by lactic acid bacteria cells depends on a wide range of factors, which are not always possible to reproduce or replicate in subsequent experiments. Perhaps this depends on the different chemical structure of the cell wall, particularly peptidoglycan, as well as lipid profile of phospholipids

**5.1 Influence of cholesterol concentration on cholesterol removal by** *Lactococcus*

to between 11.4% and 14.9%, depending on the strain. In turn, in the experiments of Kimoto et al. [25], bacterial cells from *Lactococcus lactis* subsp. *lactis* and *Lactococcus lactis* subsp. *lactis* biovar. *diacetilactis* strains removed 53.9–86.7% and 31.0–97.3% of cholesterol, respectively, from GM17-THIO broth, containing addi-

Moreover, Ziarno [4] examined the capacity of isolates from the genus *Lactococcus* originating from fermented dairy products to remove cholesterol depending on the concentration of cholesterol in culture broth (in a range from slightly above 0 g/dm3

however the dynamics of this removal was far less pronounced than in broths with lower cholesterol concentration. The earlier research indicates a different capacity of lactic acid bacteria cultures to remove cholesterol from culture media [6, 94, 95]. The differences were observed between individually tested cultures and between individual replications for the same culture. This is a confirmation of observations

lize cholesterol, its loss from post-culture liquid can be seen as the amount of cholesterol removed and bound by bacterial cells. Ziarno [4] demonstrated that the amount of cholesterol removed by bacterial cells is determined by the preliminary concentration of this substance in the culture medium. In general, the more cholesterol was introduced to the culture broth, the more of it was removed by bacterial cells. However, the above statement is true only for low cholesterol concentrations in culture broth. With higher concentration of cholesterol in the culture broth,

). Considering it is known that lactic acid bacteria do not metabo-

, its removal by bacterial cells was still observed;

of medium.

tion of 0.2% sodium taurocholate and 0.070 g cholesterol per 1 dm3

The capacity of lactic streptococci to reduce the cholesterol level under in vitro conditions was also tested by Hosono and Tono-Oka [24] and Kimoto et al. [25]. The cited researchers carried out cultures at 37°C for 24 h. In the study of Hosono and Tono-Oka [24], the percentage of cholesterol bound by *Lactococcus lactis* subsp. *lactis* 12007 and 12546 strains was 25.1 and 30.3%, respectively. Four strains of *Lactococcus lactis* subsp. *cremoris* bound from 14.2 to 20.9% of cholesterol and two strains of *Lactococcus lactis* subsp. *lactis* biovar. *diacetilactis*—29.7 and 33.9%, respectively. The capacity to remove cholesterol from culture broth was demonstrated also in the case of *Leuconostoc mesenteroides* subsp. *cremoris*, and it amounted

resistance to bile salts and their capacity to bind cholesterol [46, 48].

**5. Influence of selected factors on cholesterol removal** 

of the cytoplasmic membrane in bacterial cells.

**by** *Lactococcus* **cells**

**82**

to close to 2 g/dm3

amounting to over 1–1.5 g/dm3

studied *Lactococcus* cultures removed on average between 0.113 and 0.129 g/dm3 of cholesterol from its initial content of 0.611 g in 1 dm3 of M17 broth. In turn, bacterial biomasses with a 10-fold lower concentration (1×) produced approximately 1.3–1.6 fold reduction of the amount of cholesterol removed. From a culture broth containing a 10-fold diluted bacterial cell biomass culture, from 0.054 g/dm3 to 0.066 g/dm3 of cholesterol was removed after culture maintained for 20h, thus 1.3–1.5 times less than in the case of 1× concentrated biomasses [4]. It is worthy of emphasis that in multi-species commercial mesophilic starter cultures, used in the dairy industry, e.g., cheese and cream production, similar capacities to remove cholesterol were observed as in lactic acid bacteria monocultures. However, it should be expected that with 10-fold decrease of cell biomass concentration, the amount of cholesterol removed from culture broth will be decreased proportionately (by 10-fold). However, minor differences were observed in the amount of cholesterol removed by biomasses with selected live cell concentration levels. This can be explained with two phenomena. Firstly, the applied cultures were live and biologically active. During the experiments, bacteria propagated, significantly altering the amount of biomass capable of binding cholesterol. Microbial analyses demonstrated that the strongest increase of *Lactococcus* population was observed in the culture with the lowest initial biomass concentration (10-fold diluted). Bacteria propagation was poorest in the cultures with the highest initial concentration of biomass (10×). After completion of experiments, in the cultures containing 10-fold diluted biomass of the tested mesophilic cultures, the live cell bacteria count was determined at 7–8 log CFU/cm3 . In cultures with 10-fold concentrated biomass, an average of 6–7 log CFU/cm3 was determined [4]. The second explanation for the minor differences in the amount of cholesterol removed by *Lactococcus* biomass with the used live cell concentration levels is the concomitant adhesion and assimilation of cholesterol molecules. Most likely, with poor growth of bacterial cells, the phenomenon of cholesterol removal through its adhesion by the cell wall is predominant. And as it could be expected, this type of cholesterol binding is not durable and cholesterol is easily released. In turn, the high biological activity of bacterial cells may favor permanent embedding of cholesterol into the wall or cytoplasmic membrane of bacteria cells, which likely occurred in the experiments of this stage of research, in cultures with the lowest initial biomass concentration (diluted 10-fold), in which the greatest increase in population was observed.

The obtained study results may find implications for the explanation of hypocholesterolemic influence of products containing lactic acid bacteria. A considerable amount of literature data is available on the subject, but these are often contradictory [5–7, 10–13]. Based on the results of this study, a hypothesis can be formed that in this case the count of live bacteria in the product is important. In order for bacterial cells to assimilate cholesterol molecules, their high biological activity is required, as demonstrated by Hosono and Tono-Oka [24] for *Lactococcus lactis* subsp. *lactis* biovar. *diacetilactis* R-43 strain; the course of this phenomenon is most intensive in the logarithmic growth phase. The physical binding of cholesterol by the cell wall does not require cell activity, only a suitably long contact time between the cells and cholesterol molecules. The same team of researchers noted that not only live but also dead cells of the tested strain were capable of binding cholesterol.
