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

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 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 addition of 0.2% sodium taurocholate and 0.070 g cholesterol per 1 dm3 of medium. 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 to close to 2 g/dm3 ). Considering it is known that lactic acid bacteria do not metabolize 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, amounting to over 1–1.5 g/dm3 , its removal by bacterial cells was still observed; 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

**83**

*Cholesterol Uptake and Survival of* Lactococcus lactis *Strains in Fluids Simulating the Human…*

of other researchers [5, 6, 10, 16, 17, 21–26, 38, 41, 46, 48, 96]. A significant effect on the diversity of the results obtained not only within the strains but also repetitions seems to be also held by the fact that the mechanism of cholesterol binding by bacterial cells can occur via adhesion of cholesterol molecules through the cell wall or by embedding it into the cell wall or membrane [9, 19, 23–25, 43]. It appears to be obvious that cholesterol adhesion does not produce strong binding, and this substance is very easily washed back to the culture broth. In turn, embedding cholesterol into the cell wall or cytoplasmic membrane is more durable. This may explain the observed considerable dispersion of results and the lack of experiment

**5.2 Influence of culture temperature on cholesterol removal by** *Lactococcus*

Usman and Hosono [43] demonstrated that *Lactococcus lactis* subsp. *lactis* biovar. *diacetilactis* bacteria are capable of binding and removing cholesterol already after culture is started, independent of its temperature in the range from 10 to 70°C. After

inhibited. The bacteria bound the highest amount of cholesterol when the pH value was about 7.0. The applied culture temperature range indicates that dead bacterial cells are also capable of binding cholesterol, which comprised the subject of further tests of this study. In turn, Noh et al. [19] demonstrated that lactic bacilli bind cholesterol in a culture with constant pH of 6.0, as well as during growth without pH value control. Ziarno [4] examined the capacity of *Lactococcus* cells isolated from industrial dairy starters to remove cholesterol in M17 culture broth with application of several temperature variants of culture (4, 25, and 30°C). The temperature of 4°C aimed at stimulating refrigeration conditions and ensuring inhibition of bioactivity of bacterial cells [97, 98]. The temperature of 30°C was utilized as the optimum conditions for the development of mesophilic bacteria. In turn, the temperature of 25°C was used to simulate room temperature conditions. It was proven that bacterial cells from all tested lactic acid bacteria cultures reduced the level of cholesterol in culture medium in the applied experimental conditions. As it could be expected, the degree at which cholesterol is removed depended on the applied temperature of lactic acid bacteria incubation. The initial

cholesterol concentration in culture broths was on average 0.606 g/dm3

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

carried out at 25°C, the discussed cultures bound from 0.065 to 0.085 g/dm3

the obtained values of removed cholesterol ranged from 0.068 to 0.104 g/dm3

low amounts of cholesterol (from 0.005 to 0.021 g/dm3

**cholesterol removal**

tures were kept at the temperature of 4°C, mesophilic cultures of *Lactococcus* removed

the temperature of 30°C, which is optimum for the development of mesophilic cultures,

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

, Ca2+, or K+

, the cholesterol binding was

. When the cul-

. In turn, at

[4].

) [4]. When the cultures were

*DOI: http://dx.doi.org/10.5772/intechopen.88462*

addition of salts of such metals as Mg2+, Na+

reproducibility.

*Cholesterol Uptake and Survival of* Lactococcus lactis *Strains in Fluids Simulating the Human… DOI: http://dx.doi.org/10.5772/intechopen.88462*

of other researchers [5, 6, 10, 16, 17, 21–26, 38, 41, 46, 48, 96]. A significant effect on the diversity of the results obtained not only within the strains but also repetitions seems to be also held by the fact that the mechanism of cholesterol binding by bacterial cells can occur via adhesion of cholesterol molecules through the cell wall or by embedding it into the cell wall or membrane [9, 19, 23–25, 43]. It appears to be obvious that cholesterol adhesion does not produce strong binding, and this substance is very easily washed back to the culture broth. In turn, embedding cholesterol into the cell wall or cytoplasmic membrane is more durable. This may explain the observed considerable dispersion of results and the lack of experiment reproducibility.

### **5.2 Influence of culture temperature on cholesterol removal by** *Lactococcus*

Usman and Hosono [43] demonstrated that *Lactococcus lactis* subsp. *lactis* biovar. *diacetilactis* bacteria are capable of binding and removing cholesterol already after culture is started, independent of its temperature in the range from 10 to 70°C. After addition of salts of such metals as Mg2+, Na+ , Ca2+, or K+ , the cholesterol binding was inhibited. The bacteria bound the highest amount of cholesterol when the pH value was about 7.0. The applied culture temperature range indicates that dead bacterial cells are also capable of binding cholesterol, which comprised the subject of further tests of this study. In turn, Noh et al. [19] demonstrated that lactic bacilli bind cholesterol in a culture with constant pH of 6.0, as well as during growth without pH value control. Ziarno [4] examined the capacity of *Lactococcus* cells isolated from industrial dairy starters to remove cholesterol in M17 culture broth with application of several temperature variants of culture (4, 25, and 30°C). The temperature of 4°C aimed at stimulating refrigeration conditions and ensuring inhibition of bioactivity of bacterial cells [97, 98]. The temperature of 30°C was utilized as the optimum conditions for the development of mesophilic bacteria. In turn, the temperature of 25°C was used to simulate room temperature conditions. It was proven that bacterial cells from all tested lactic acid bacteria cultures reduced the level of cholesterol in culture medium in the applied experimental conditions. As it could be expected, the degree at which cholesterol is removed depended on the applied temperature of lactic acid bacteria incubation. The initial cholesterol concentration in culture broths was on average 0.606 g/dm3 . When the cultures were kept at the temperature of 4°C, mesophilic cultures of *Lactococcus* removed low amounts of cholesterol (from 0.005 to 0.021 g/dm3 ) [4]. When the cultures were carried out at 25°C, the discussed cultures bound from 0.065 to 0.085 g/dm3 . In turn, at the temperature of 30°C, which is optimum for the development of mesophilic cultures, the obtained values of removed cholesterol ranged from 0.068 to 0.104 g/dm3 [4].
