**3. Mechanism of cholesterol level reduction by lactic acid bacteria including** *Lactococcus* **in humans**

The assumption that lactic acid bacteria may cause reduction of the cholesterol level directly in fermented milk products or live organisms was made on the basis of numerous in vivo and in vitro studies demonstrating that certain lactic acid bacteria produced a reduction of the cholesterol level in the blood serum of experimental animals or human volunteers or in model culture media. This type of research has been conducted since the 1970s [5, 7, 8, 14, 31–36]. The majority of these studies concern the influence of consumption of fermented products or products containing lactic acid bacteria strains, including primarily probiotic strains. In that time, several scientific hypotheses were formed on the mechanisms through which the phenomenon of cholesterol level reduction performed by lactic acid bacteria may occur. Literature data lists here primarily cholesterol binding, enzymatic deconjugation of bile salts, production of exopolysaccharides, and synthesis of short-chain fatty acids (SCFAs) [15, 21, 23–25, 28, 29, 36–45].

Cholesterol binding by the bacterial cell wall and its incorporation into the cell wall or cytoplasmic membrane of bacterial cells are listed among the major mechanisms [9, 19, 23–25, 43]. It is known that cholesterol binding may have different paths. Certain bacteria incorporate cholesterol into the cell wall, as exhibited by

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

documented probiotic traits but also some "traditional" lactic acid bacteria used in the production of cheese, cream, or fermented milk products. The role of *Lactococcus* in dairy fermentation is mostly down to the production of lactic acid; however these bacteria utilize less than 0.5% of lactose from milk. Only *Lactococcus lactis* is applicable in the dairy industry, with its two subspecies: *Lactococcus lactis* subsp. *lactis* and *Lactococcus lactis* subsp. *cremoris*. These subspecies comprise the basic component of dairy mesophilic starter cultures, used for the production of cream, buttermilk, cottage cheeses, cheeses, and fermented milk [1–3]. In the process and functional terms, *Lactococcus* possess all the traits required for starter cultures: the capacity to ferment lactose, resistance to low pH, low temperature, and high concentrations of cooking salt. They are characterized by stability and suitable survival time during lyophiliza-

tion and freezing and in the storage process of starter cultures [1, 2, 4].

Furthermore, lactic acid bacteria have the capacity to reduce the level of cholesterol in simulated conditions, i.e., culture media. It is known that lactic acid bacteria are not capable of metabolizing cholesterol, meaning its transformation into other compounds. It has been noted that bacterial cells are capable of binding cholesterol, consisting in adhesion of substances by the cell wall or its assimilation into cell wall. It has also been suspected that lactic acid bacteria are capable of deconjugating bile salts being the component of bile, followed by coprecipitation of cholesterol with deconjugated bile acids. Furthermore, tests on gnotobiotic animals demonstrated that hydrolysis of bile enhances its secretion and thus may contribute to reduction of the cholesterol level in blood serum. Moreover, the cholesterol level in the human organism may be also influenced by exopolysaccharides (EPS) produced by numerous lactic acid bacteria species. It is believed that these bacteria, similar to fiber, can bind cholesterol and bile acid molecules present in intestines and remove them from

In many cases, results of in vitro studies are not unambiguous, or lack of their reproducibility has been determined. It turns out that also in vivo tests conducted on human volunteers or experimental animals do not produce unambiguous results or that their results are divergent. Considering that it is difficult to explain as to why this happens, such studies are frequently criticized for methodological and techni-

The extensive collection of scientific publications devoted to health-promoting properties of lactic acid bacteria includes articles presenting studies on the possibility of reducing the cholesterol level in human and animal organisms through consumption of fermented milk products including traditional and probiotic strains

As early as in 1974, Mann and Spoerry [5, 6] determined the reduced level of cholesterol in the blood serum of men from the African Maasai, which stemmed from the consumption of high amounts of fermented milk containing wild lactic acid bacteria strains. This research enabled researchers to look for the methods of reducing the cholesterol level in the human organism, although the first reports on the positive impact of fermented milk drinks on the reduction of the cholesterol level in live organisms were criticized due to their methodological and technical errors. However, these studies opened a new route for researchers in terms of the search of methods of cholesterol level reduction in the human organism, increasing the chances of the modern human populations in the combat with cardiovascular

**2. Influence of lactic acid bacteria, including** *Lactococcus***, on the** 

**76**

disorders [7–13].

the human organism.

of lactic acid bacteria.

cal errors and lack of reproducibility.

**cholesterol level in humans**

such genera as *Micrococcus*, *Bacillus*, *Proteus*, or *Mycoplasma*. In the case of lactic acid bacteria, it has been thus far believed that cholesterol is solely attached by the cell through physical adhesion and it is not subject to subsequent metabolism. However, in vitro tests demonstrated that lactic acid bacteria are also capable of incorporating cholesterol into the cell wall [19, 23, 28, 30]. Many scientists have determined that the amount of cholesterol bound by lactic acid bacteria cells depends on, among others, genus, species, and culture of bacteria, growth phase, viability, and cell count [5, 10, 23–25, 38–40, 43]. Research results demonstrate that the strains commercially used in fermented food production are less efficient in binding cholesterol in comparison with the strains isolated from the alimentary tract of humans and animals [16, 46]. According to the majority of literature data, the phenomenon of cholesterol binding by lactic acid bacteria occurs primarily in anaerobic conditions and with the presence of bile salts [5, 17, 35, 47]. However, scientific reports have been published indicating a lack of or poor correlation between tolerance of bile salts and the capacity to bind cholesterol [22, 46, 48].

Another proposed mechanism for cholesterol level reduction in the human organism by lactic acid bacteria is the deconjugation of bile salts, associated with the activity of bile salt hydrolase (BSH) enzyme [16, 17, 21, 30, 39, 40, 45, 49, 50]. Bile salt hydrolase also referred to as cholylglycine hydrolase EC.3.5.1.24 catalyzes hydrolysis (also known as deconjugation) of the amide bond in bile acids conjugated with taurine or glycine, with the release of primary bile acids and amino acids, taurine or glycine [30, 45]. Hydrolysis of bile salts conjugated with taurine or glycine is one of the best known microbiological biotransformations of bile salts. BSH activity is observed for certain bacteria species isolated from the alimentary tract of humans and animals, i.e., strains from genera *Lactobacillus*, *Enterococcus*, *Peptostreptococcus*, *Bifidobacterium*, *Clostridium*, and *Bacteroides*, that is, microflora from environments rich in conjugated bile acids [14, 30, 35, 39, 40, 45, 51, 52]. A study conducted by Tanaka et al. [53] demonstrated BSH activity also in *Lactococcus lactis*, *Leuconostoc mesenteroides*, and *S. thermophilus* strains. The details on the function of BSH are unknown. It is believed that a relationship exists between BSH activity and the natural environment of bacteria. It is likely that hydrolysis of bile salts catalyzed by BSH constitutes a protection mechanism against the toxic effect of these salts, present in the natural environment of the bacteria. As demonstrated in the literature data, the influence of bile salts on the surface of bacterial cells may result in changes in the metabolism and structure of the cell wall and membrane [23, 54–56]. Such changes have been observed in *Lactobacillus* bacteria, among others. However, some researchers believe that bile acids released by BSH are even more toxic toward bacterial cells than their forms conjugated with taurine or glycine [48, 53, 57–61]. Recently, a mechanism has been proposed, according to which BSH facilitates incorporation of cholesterol or bile salts into bacterial cell membrane [62]. The positive effects stemming from the capacity of lactic acid bacteria for bile salt hydrolysis are sometimes understated in the literature. It is believed that deconjugated bile salts may return to the liver and then to the intestines, where the intestinal microflora transforms them into secondary bile salts (SBS), which are considered cytotoxic [63]. Deoxycholate and lithocholate are examples of such secondary bile salts and are formed by removing the 7α-hydroxyl group from primary bile salts, cholane and chenodeoxycholate, respectively [28, 30, 64, 65]. Removal of the 7α-hydroxyl group from primary bile salts is catalyzed by an enzyme known as 7α-dehydroxylase. It is suspected that BSH along with 7α-dehydroxylase plays a significant role in the gallstone formation [53]. However, no 7α-dehydroxylase activity could be found for *Lactobacillus* strains isolated from humans or dairy products [64, 65]. This debunks the myth that lactic acid bacteria and bifidobacteria contribute to the formation of secondary bile acids and gallstones.

**79**

conducted in vivo.

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

Another probable mechanism of cholesterol level reduction in the blood serum is associated with the capacity of numerous lactic acid bacteria to synthesize exopolysaccharides. However, this mechanism remains among the group of hypotheses that have been poorly understood and studied [28, 29, 42, 66, 67]. It is suspected that EPS influence the absorption of cholesterol, free bile acids, or salts from the intestines through binding and removing them from the organism via the same principle as it is performed by nutritional fiber or plant polysaccharides [42, 68]. Nakajima et al. [67] demonstrated that the level of cholesterol in the blood serum was lowest in the rats fed with milk containing EPS-producing streptococci. Similarly, the HDL cholesterol fraction ratio to its total content was highest in the rats fed with diet including these streptococci. This shows that EPS produced by *Lactococcus lactis* subsp. *cremoris* SBT 0495 had a positive impact on the metabolism of cholesterol in rats. Moreover, results of in vitro tests carried out by Pigeon et al. [42] suggested that bile acid binding by EPS could influence reduction of the cholesterol level via its usage in the synthesis of new bile acids in the place of those associated with EPS and thus removed from the system. Moreover, they formed a hypothesis that the full EPS efficiency in terms of cholesterol or bile acid removal requires the activity of BSH-type enzymes in lactic acid bacteria and bifidobacteria. However, these researchers did not verify whether this relationship is also present with regard to conjugated bile acids (e.g., glycocholic acid, taurocholic acid), as then it would be possible that the phenomenon of bile salt binding by EPS does not require activity of bile salt hydrolase and it may occur in the conditions prevailing in the intestine. Perhaps the cholesterol removal by EPS-producing bacteria is even more complex than in the case of bacteria that do not produce these substances. Moreover, it is unknown whether the cholesterol bound by EPS is biologically available to human organism, as the literature lacks information as to whether this research has been

Another mechanism, associated with production of short-chain fatty acids, has been mentioned among other possible mechanisms of cholesterol level reduction in the human organism by lactic acid bacteria [28, 29, 36, 69]. In the human organism, propionic acid penetrates to the liver and inhibits the hypercholesterolemic effect of acetate, the precursor of cholesterol and a product of fermentation activity of lactic acid bacteria. Thus far no in vivo tests have been conducted to confirm this phenomenon. St-Onge et al. [36] further point out to the fact that synthesis of acetate

The aforementioned supposed mechanisms concern reduction of the level of cholesterol in the blood serum by lactic acid bacteria. It is presumed that considering lactic acid bacteria and bifidobacteria do not metabolize cholesterol, then it is possible that only binding (adhesion and/or assimilation) of cholesterol by cell wall or cytoplasmic membrane occurs in food products. Thus far, it has not been demonstrated that lactic acid bacteria are capable of metabolizing cholesterol, although the literature provides examples of studies on the introduction of genes encoding activity of such genes to the cells of lactic acid bacteria [70–73]. It is known that many other microorganisms produce enzymes that decompose cholesterol to other compounds, e.g., cholesterol reductase or cholesterol oxidase [70, 71, 74, 75]. Worth noting are intestinal microorganisms producing the enzyme of cholesterol reductase that transforms cholesterol into coprostanol (5β-cholestan-3β-ol). In the human organism, the anaerobic intestinal microflora transforms cholesterol primarily to 5β-coprostanol [76]. It should be noted that coprostanol is poorly absorbed in the gastrointestinal tract and it is easily eliminated from the organism [14]. *Eubacterium coprostanoligenes* is a bacteria species that includes cholesterol reductase. These bacteria could be used for production of probiotic foods with a naturally reduced cholesterol level, and such attempts have been made, yet thus far

by lactic acid bacteria predominates synthesis of other SCFAs.

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

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

Another probable mechanism of cholesterol level reduction in the blood serum is associated with the capacity of numerous lactic acid bacteria to synthesize exopolysaccharides. However, this mechanism remains among the group of hypotheses that have been poorly understood and studied [28, 29, 42, 66, 67]. It is suspected that EPS influence the absorption of cholesterol, free bile acids, or salts from the intestines through binding and removing them from the organism via the same principle as it is performed by nutritional fiber or plant polysaccharides [42, 68]. Nakajima et al. [67] demonstrated that the level of cholesterol in the blood serum was lowest in the rats fed with milk containing EPS-producing streptococci. Similarly, the HDL cholesterol fraction ratio to its total content was highest in the rats fed with diet including these streptococci. This shows that EPS produced by *Lactococcus lactis* subsp. *cremoris* SBT 0495 had a positive impact on the metabolism of cholesterol in rats. Moreover, results of in vitro tests carried out by Pigeon et al. [42] suggested that bile acid binding by EPS could influence reduction of the cholesterol level via its usage in the synthesis of new bile acids in the place of those associated with EPS and thus removed from the system. Moreover, they formed a hypothesis that the full EPS efficiency in terms of cholesterol or bile acid removal requires the activity of BSH-type enzymes in lactic acid bacteria and bifidobacteria. However, these researchers did not verify whether this relationship is also present with regard to conjugated bile acids (e.g., glycocholic acid, taurocholic acid), as then it would be possible that the phenomenon of bile salt binding by EPS does not require activity of bile salt hydrolase and it may occur in the conditions prevailing in the intestine. Perhaps the cholesterol removal by EPS-producing bacteria is even more complex than in the case of bacteria that do not produce these substances. Moreover, it is unknown whether the cholesterol bound by EPS is biologically available to human organism, as the literature lacks information as to whether this research has been conducted in vivo.

Another mechanism, associated with production of short-chain fatty acids, has been mentioned among other possible mechanisms of cholesterol level reduction in the human organism by lactic acid bacteria [28, 29, 36, 69]. In the human organism, propionic acid penetrates to the liver and inhibits the hypercholesterolemic effect of acetate, the precursor of cholesterol and a product of fermentation activity of lactic acid bacteria. Thus far no in vivo tests have been conducted to confirm this phenomenon. St-Onge et al. [36] further point out to the fact that synthesis of acetate by lactic acid bacteria predominates synthesis of other SCFAs.

The aforementioned supposed mechanisms concern reduction of the level of cholesterol in the blood serum by lactic acid bacteria. It is presumed that considering lactic acid bacteria and bifidobacteria do not metabolize cholesterol, then it is possible that only binding (adhesion and/or assimilation) of cholesterol by cell wall or cytoplasmic membrane occurs in food products. Thus far, it has not been demonstrated that lactic acid bacteria are capable of metabolizing cholesterol, although the literature provides examples of studies on the introduction of genes encoding activity of such genes to the cells of lactic acid bacteria [70–73]. It is known that many other microorganisms produce enzymes that decompose cholesterol to other compounds, e.g., cholesterol reductase or cholesterol oxidase [70, 71, 74, 75]. Worth noting are intestinal microorganisms producing the enzyme of cholesterol reductase that transforms cholesterol into coprostanol (5β-cholestan-3β-ol). In the human organism, the anaerobic intestinal microflora transforms cholesterol primarily to 5β-coprostanol [76]. It should be noted that coprostanol is poorly absorbed in the gastrointestinal tract and it is easily eliminated from the organism [14]. *Eubacterium coprostanoligenes* is a bacteria species that includes cholesterol reductase. These bacteria could be used for production of probiotic foods with a naturally reduced cholesterol level, and such attempts have been made, yet thus far

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

salts and the capacity to bind cholesterol [22, 46, 48].

ute to the formation of secondary bile acids and gallstones.

such genera as *Micrococcus*, *Bacillus*, *Proteus*, or *Mycoplasma*. In the case of lactic acid bacteria, it has been thus far believed that cholesterol is solely attached by the cell through physical adhesion and it is not subject to subsequent metabolism. However, in vitro tests demonstrated that lactic acid bacteria are also capable of incorporating cholesterol into the cell wall [19, 23, 28, 30]. Many scientists have determined that the amount of cholesterol bound by lactic acid bacteria cells depends on, among others, genus, species, and culture of bacteria, growth phase, viability, and cell count [5, 10, 23–25, 38–40, 43]. Research results demonstrate that the strains commercially used in fermented food production are less efficient in binding cholesterol in comparison with the strains isolated from the alimentary tract of humans and animals [16, 46]. According to the majority of literature data, the phenomenon of cholesterol binding by lactic acid bacteria occurs primarily in anaerobic conditions and with the presence of bile salts [5, 17, 35, 47]. However, scientific reports have been published indicating a lack of or poor correlation between tolerance of bile

Another proposed mechanism for cholesterol level reduction in the human organism by lactic acid bacteria is the deconjugation of bile salts, associated with the activity of bile salt hydrolase (BSH) enzyme [16, 17, 21, 30, 39, 40, 45, 49, 50]. Bile salt hydrolase also referred to as cholylglycine hydrolase EC.3.5.1.24 catalyzes hydrolysis (also known as deconjugation) of the amide bond in bile acids conjugated with taurine or glycine, with the release of primary bile acids and amino acids, taurine or glycine [30, 45]. Hydrolysis of bile salts conjugated with taurine or glycine is one of the best known microbiological biotransformations of bile salts. BSH activity is observed for certain bacteria species isolated from the alimentary tract of humans and animals, i.e., strains from genera *Lactobacillus*, *Enterococcus*, *Peptostreptococcus*, *Bifidobacterium*, *Clostridium*, and *Bacteroides*, that is, microflora from environments rich in conjugated bile acids [14, 30, 35, 39, 40, 45, 51, 52]. A study conducted by Tanaka et al. [53] demonstrated BSH activity also in *Lactococcus lactis*, *Leuconostoc mesenteroides*, and *S. thermophilus* strains. The details on the function of BSH are unknown. It is believed that a relationship exists between BSH activity and the natural environment of bacteria. It is likely that hydrolysis of bile salts catalyzed by BSH constitutes a protection mechanism against the toxic effect of these salts, present in the natural environment of the bacteria. As demonstrated in the literature data, the influence of bile salts on the surface of bacterial cells may result in changes in the metabolism and structure of the cell wall and membrane [23, 54–56]. Such changes have been observed in *Lactobacillus* bacteria, among others. However, some researchers believe that bile acids released by BSH are even more toxic toward bacterial cells than their forms conjugated with taurine or glycine [48, 53, 57–61]. Recently, a mechanism has been proposed, according to which BSH facilitates incorporation of cholesterol or bile salts into bacterial cell membrane [62]. The positive effects stemming from the capacity of lactic acid bacteria for bile salt hydrolysis are sometimes understated in the literature. It is believed that deconjugated bile salts may return to the liver and then to the intestines, where the intestinal microflora transforms them into secondary bile salts (SBS), which are considered cytotoxic [63]. Deoxycholate and lithocholate are examples of such secondary bile salts and are formed by removing the 7α-hydroxyl group from primary bile salts, cholane and chenodeoxycholate, respectively [28, 30, 64, 65]. Removal of the 7α-hydroxyl group from primary bile salts is catalyzed by an enzyme known as 7α-dehydroxylase. It is suspected that BSH along with 7α-dehydroxylase plays a significant role in the gallstone formation [53]. However, no 7α-dehydroxylase activity could be found for *Lactobacillus* strains isolated from humans or dairy products [64, 65]. This debunks the myth that lactic acid bacteria and bifidobacteria contrib-

**78**

with poor effects [77]. In vivo tests on animals demonstrated that administration of *Eubacterium coprostanoligenes* has a positive influence on reduction of cholesterol in the blood serum [29, 78–80]. This is an indication that providing lactic acid bacteria cells with the capacity for cholesterol transformation into coprostanol may enable reduction of the cholesterol level already at the stage of fermented product formation.

By examining the hypocholesterolemic influence of lactic acid bacteria on the level of cholesterol in the blood serum of volunteering humans or experimental animals, it should be borne in mind that introduction of additional microflora to the intestines may significantly alter the quantitative and qualitative composition of the entire intestine ecosystem and its function. As shown in the results of the study of Hosono et al. [14], despite the fact that lactic acid bacteria cells do not possess the capacity to transform cholesterol into coprostanol, they are capable of influencing the amount at which it is excreted from the organism. This forms the effect of the influence of lactic acid bacteria on the remaining microorganisms present in the intestinal microflora.
