**7.3 Cholesterol removal by** *Lactococcus* **in the conditions of simulated intestinal fluid**

As stated by Ziarno and Bartosz [113], cholesterol removal by lactic acid bacteria in intestinal fluid is less pronounced than in culture broth. This is further confirmed by the experiments of Ziarno [4] conducted under in vitro conditions with *Lactococcus* isolates originating from industrial starter cultures. The mentioned cultures were grown at 37°C for 6 h in a simulated intestinal fluid with addition of cholesterol. The tested *Lactococcus* cultures resulted in a reduction of cholesterol from the initial content of 0.543 g/dm3 to the level between 0.011 and 0.087 g/dm3 . In the majority of the tested cultures, the influence of biomass concentration on the degree of cholesterol removal was statistically significant; however, 10-fold concentrated biomass did not remove 10 times more cholesterol than onefold concentrated

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

the alimentary tract or food products.

in the range from 1 to 3 log CFU/cm3

surviving at the level of 6 log CFU/cm3

**human gastrointestinal tract**

, only 2–3 log CFU/cm3

cm3

resistance of the cell to stress factors [50]. Doubtlessly, this is significant for the survival of lactic acid bacteria in various environments they inhabit, such as

The strains which are not probiotics exhibit lower survival rate of their cells in model gastric fluid as compared with probiotic strains [41, 108]. Ziarno and Margol [109] examined the capacity of bacteria from several mesophilic starter cultures to survive in a simulated gastric fluid. Also in their study, industrial starter cultures containing bacteria from the genus *Lactococcus* were used, which, after propagation, were kept in a simulated gastric fluid with pH of 2.4 for 3 h at 37°C. The study demonstrated that the present streptococci were not resistant to the environment of a simulated gastric fluid [109]. On the other hand, intestinal fluid has a more complex enzymatic and chemical composition than broths used by other researchers, but its influence on lactic acid bacteria cells is typically referred to probiotic strains of thermophilic lactic acid bacteria [38–40, 108, 110, 111]. Ziarno [4] tested the viability of *Lactococcus* in model conditions of the alimentary tract in the presence of cholesterol, separately for the simulated gastric fluid and simulated intestinal fluid. *Lactococcus* isolated from industrial starter cultures were used for the experiments. No influence of addition of cholesterol on the viability of *Lactococcus* cultures in a simulated gastric fluid could be demonstrated, although reduction of live cells

tolerance also to the conditions of simulated intestinal fluid, considerably lower than the simulated gastric fluid. From the initial cell population of average 6–7 log CFU/

The lack of influence of cholesterol in simulated intestinal fluid on the survival rate of lactic acid bacteria cells was also demonstrated in earlier research [114, 115]. The study conducted by Ziarno [114, 115] utilized bacteria cultures isolated from commercial pharmaceutical preparations and commercially available dairy products or dairy starter monocultures. Cells of lactobacilli tolerated conditions of simulated

The good tolerance of bacterial cells to the conditions of simulated digestive fluids can be explained by the occurrence of these bacteria in the alimentary tract of humans and animals. Numerous factors determine lactic acid bacteria viability, including pH, temperature, oxygenation, and presence of toxic substances toward bacterial cells [101–103]. Bacteria not forming the natural intestinal microflora do not possess the natural resistance to the conditions of the intestinal fluid [106]. Viability of bacterial cells determines the level of cholesterol removal. It seems obvious that the count of live and dead bacterial cells holds significance for the removal of cholesterol under the conditions of a human alimentary tract. Thus, a hypothesis can be formed that the factors determining survival rate of bacterial cells further influence the cholesterol removal level by lactic acid bacteria and bifidobacteria cells. Such relationships may further impede interpretation of the results of experiments realized under in vitro or in vivo conditions and may prevent interpolation of results

intestinal fluid better than bifidobacteria cells and *Lactococcus lactis* cells.

obtained in vitro onto the conditions of human or animal organisms.

**7. Cholesterol uptake and release by** *Lactococcus* **in the simulated** 

**7.1 Cholesterol uptake by** *Lactococcus* **under conditions of simulated gastric fluid**

Cholesterol uptake by *Lactococcus* cells in simulated gastric fluid depends on the amount of biomass [4]. Ziarno [4] carried out in vitro experiments with the use of

was observed. *Lactococcus* cells exhibited low

, independent of the addition of cholesterol.

remained after 6 h of experiment, with few exceptions

**86**

biomass. Therefore, Ziarno [4] demonstrated that in not all of the tested *Lactococcus* cultures the degree of biomass concentration had a significant influence on the amount of cholesterol removed. This can be explained by the activity of enzymes such as BSH, which caused bile hydrolysis and coprecipitation of cholesterol with released bile acids, independent of the amount of cells in the culture.

The chemical composition of simulated intestinal fluid seems to be of significance for the obtained results [18]. This indicates additional methodological factors influencing the results obtained in laboratory experiments conducted under in vitro conditions. In order to prepare simulated intestinal fluid, cattle bile was also used containing conjugated and deconjugated bile salts; therefore bile salt hydrolase activity (produced by the majority of intestinal lactic acid bacteria strains) was not necessary for cholesterol precipitation with free bile acids to occur [30, 35, 39, 40, 45]. Active BSH enzyme results in hydrolysis of bile salts, whereas cholesterol molecules may coprecipitate with the released bile acids [16, 17, 21, 30, 35, 39, 40, 45, 49, 50]. Such phenomenon has been observed in numerous lactic acid bacteria species, but not in *Lactococcus* thus far [49, 53]. It is known that coprecipitates of cholesterol with bile acids are formed at a low pH below 5.5 [17, 18, 21, 39, 49, 52]. However, with a renewed increase of pH to over 5.5, such coprecipitates were rapidly dissolved [15, 18, 28, 39]. Bile secreted from the liver is introduced to the duodenum, where it neutralizes the acidic food pulp that leaves the stomach and then the pH in the small intestine has a value of over 6.0. Under these conditions, the coprecipitates of bile acids and cholesterol are dissolved. Thus, the hypocholesterolemic effect caused by cholesterol coprecipitation with deconjugated bile acids is probably impossible to occur under in vivo conditions.
