**6. Survival of** *Lactococcus* **cells in the human gastrointestinal tract**

Literature contains studies confirming the capacity of lactic bacilli to survive under in vivo conditions in the human alimentary tract [28, 30, 35, 52, 99, 100]. The factors with a significant impact on lactic acid bacteria survivability in the alimentary tract include low gastric pH value, intestine peristalsis, presence of bile acids in the pancreatic fluid and various digestive enzymes present in the individual sections of the alimentary tract, presence of nutrients, as well as bacteria passage time through the alimentary tract and their initial count [35, 50, 101–103]. The mentioned factors result in a decrease of lactic acid bacteria survival rate, but at the same time they may constitute a criterion for the selection of probiotic strains [28, 104].

In order to determine the survival rate of lactic acid bacteria, scientists first determine their resistance to low pH present in certain sections of the gastrointestinal tract. Gastric fluid comprises of the secretion of foveolar cells secreting mucus, chief cells secreting digestive enzymes (pepsin), and parietal cells secreting hydrochloric acid. The pH of gastric fluid is between 1.5 and 3.0. Secretion of gastric fluid is inhibited when the pH drops below 2.0. The temperature inside the stomach is over 37°C, and the alimentary content, depending on the individual physiological and emotional circumstances, remains in the stomach for average 1–3 h [4]. Results of in vitro tests concern survival rate of different lactic acid bacteria strains under conditions imitating low pH of the gastric fluid [18, 35, 41, 105–109]. Strains traditionally used to manufacture dairy products have also been commonly found to survive the conditions of gastric fluid [101]. Also the study of Lankaputhra and Shah [107] indicated that numerous lactic bacilli strains survived perfectly the conditions simulating the pH of gastric fluid.

Another subject of the study is the capacity of lactic acid bacteria to survive during transport through subsequent sections of the alimentary canal. Here, a particular significance is exhibited by the section of the small intestine [102, 110, 111]. Literature data show that bile salts comprise a serious obstacle for lactic acid bacteria, as they contain toxic bile acids [19, 106, 107, 112]. Ziarno and Bartosz [113] provided evidence for the influence of cell biomass on the survival of lactic bacilli in model intestinal fluid. Cholesterol influences the composition and functioning of the bacterial cell wall and membrane, thus producing change in the relationship with the surrounding environment, such as resistance to bile acids, pH, or temperature [46, 48, 50]. Cholesterol uptake by bacterial cells is not neutral to them and results in a change of, among others, the profile of fatty acids of the cell membrane [23, 25, 38, 50]. Cell responds to stress conditions of the environment with a change of the composition of the cell membrane, and it may result in an increase of the

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

cholesterol from its initial content of 0.611 g in 1 dm3

studied *Lactococcus* cultures removed on average between 0.113 and 0.129 g/dm3

ing a 10-fold diluted bacterial cell biomass culture, from 0.054 g/dm3

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 contain-

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

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

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

The sparse literature data on cholesterol removal by inactivated cells prove that lactic acid bacteria monocultures are capable of removing cholesterol from culture media even after their thermal death [21, 24, 25, 43, 95]. The amount of cholesterol

dead cells of the tested strain were capable of binding cholesterol.

**5.4 Influence of** *Lactococcus* **dead cell biomass concentration** 

of

.

was

of M17 broth. In turn, bacterial

to 0.066 g/dm3

**84**

population was observed.

**on cholesterol removal**

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 alimentary tract or food products.

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 in the range from 1 to 3 log CFU/cm3 was observed. *Lactococcus* cells exhibited low 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/ cm3 , only 2–3 log CFU/cm3 remained after 6 h of experiment, with few exceptions surviving at the level of 6 log CFU/cm3 , independent of the addition of cholesterol. 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 intestinal fluid better than bifidobacteria cells and *Lactococcus lactis* cells.

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