**4. Cholesterol binding sites by bacterial cells**

As stated above, binding (adhesion or assimilation) by bacteria cells is one of the major mechanisms for the removal of cholesterol by bacteria from the environment. Hosono and Tono-Oka [24] have suggested that it is the chemical nature and structure of peptidoglycan present in bacterial cell wall that fulfill a major role in cholesterol binding. This hypothesis was confirmed by Usman and Hosono [43]. They further suggested that a portion of cholesterol could be embedded into the bacterial cell walls. The possibility for incorporating cholesterol into the cell membrane of lactic acid bacteria was demonstrated in the study of Noh et al. [19].

The phenomenon of cholesterol binding by the cell wall has been indicated by similar research conducted on the binding of aflatoxin B1 (AFB1) by lactic acid bacteria cells [81–86]. Many researchers point out to the phenomenon of AFB1 aflatoxin binding by live and dead lactic acid bacteria cultures, which do not possess the capacity to metabolize this compound [81, 83–85, 87, 88].

As it is known, Gram-positive bacteria are characterized by a thick cell wall. The cell wall of Gram-positive bacteria comprises of peptidoglycan (murein) and its associated teichoic and/or teichuronic and lipoteichoic acids and proteins [89]. The wall thickness ranges between 15 and 50 nm, corresponding to 20–30 individual murein layers. Murein is built of saccharide chains comprising of alternately arranged N-acetylglucosamine and N-acetylmuramic acid, joined with a β-(1 → 4)-glycoside bond [89]. Apart from the saccharide chains, murein contains short peptides. The free carbonyl group of muramic acid forms the acceptor for the first peptides amino acid. Typically, l-alanine is the first amino acid. The protein portion of murein exhibits considerably greater diversity than its saccharide part, as its composition depends on the bacteria species, environmental conditions, and even the cell age. In Gram-positive bacteria, the cell wall further contains proteins active in various physiological and biochemical processes—energy transfer, electron and proton transport, cell casing synthesis, etc. [89]. Moreover, various types of polymers are associated with murein, such as teichoic acids (teichoic and lipoteichoic acid) and teichuronic acid. Considering the manner in which these acids are attached, they are sometimes referred to as secondary (after murein) polymers of bacterial cell wall. The importance of these acids has not been fully explained, although numerous assumptions have been made explaining the presence of these compounds in the bacterial cell wall. It is possible that these acids play a certain

**81**

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

role in bacteria adhesion, biofilm formation, tolerance to environmental acidity, resistance to antibiotics, bacteriophages, or UV radiation. Lactic bacteria synthesize teichoic and lipoteichoic acids simultaneously or lipoteichoic acids only. The characteristic feature of lipoteichoic acids is the presence (at the end of the chain) of a glycolipid anchored into the cytoplasmic membrane, and the structure of this connection depends on bacteria species. The qualitative composition of cytoplasmic membrane phospholipids depends on environmental factors, such as availability of nutrients, temperature, pH, and presence of toxic materials. The fatty acids profile changes also depending on the genus and species of bacteria and their growth phase, which has been used for microorganism grouping and classification attempts [69, 74, 90]. C16 fatty acids are the most common, while C12, C14, and C18 fatty acids are less frequently found. Methylated, hydroxylated, and branched fatty acids or those containing cyclopropane ring are common. Lactobacillic acid—a fatty acid containing cyclopropane ring—was first detected in the cytoplasmic membrane of

Literature data indicate that the cell wall or cytoplasmic membrane can form the cholesterol binding site. In the case of the cell wall, the bond may have physical (adhesion) or chemical character (analogous to incorporation of teichoic, lipoteichoic, and teichuronic acid incorporation). In the case of a chemical bond, we deal with cholesterol assimilation, that is, its incorporation into the cell wall. Cholesterol molecules are oriented in the cell membrane in the same manner as phospholipid molecules. The polar portion of cholesterol molecule adheres to the polar portion of phospholipid. Perhaps, in cytoplasmic membranes of bacterial cells, cholesterol molecules are located in the same manner as in membranes of eukaryotic organisms. The bacterial cytoplasmic membrane contains compounds with a structure similar to steroids, which further indicates the possibility for cholesterol incorporation into the cytoplasmic membrane of bacterial cells. However, in order to be incorporated into the cytoplasmic membrane, cholesterol molecules must be transported through the cell wall. As shown in the studies of Kurdi et al. [91], Pigeon et al. [42], and Kurdi et al. [61] on bile acids, transport of such large molecules through the cell wall is possible even if it threatens the survival of the bacteria. Cholesterol binding by the cell membrane is not neutral to the bacterial cell itself. The presence of such substances as cholesterol in the environment influences the ratio of saturated acids to unsaturated acids in the cytoplasmic wall, as well as the structure and properties of this membrane. Goldberg and Eschar [92] noted that addition of Tween 80 to culture medium increases the concentration of certain fatty acids with the concomitant influence on the ratio of saturated acids to unsaturated acids. The same happens when the cholesterol molecules are being bound. Dambekodi and Gilliland [23] proved that incorporation of cholesterol into the cell membrane of bifidobacteria was manifested by changes in its composition and resulted in an increase of the resistance of cells growing in the presence of cholesterol to ultrasonic lysis. In turn, Taranto et al. [50] demonstrated that bacterial cells growing in the presence of cholesterol or bile salts are more resistant to lysis than those growing in their absence, contrary to the cells growing in the absence of cholesterol. The cited authors observed that addition of cholesterol to culture broth resulted in an increase of saturated fatty acid content in lactic bacilli biomass from 44.3% to 56.5% of total acids and unsaturated acids from 1.26% to 43.5% of the total amount of fatty acids. Furthermore, Kimoto et al. [25] reached a conclusion that the change in the distribution of fatty acids by *Lactococcus lactis* cells growing in the presence of cholesterol is an effect of its removal from the culture medium and incorporation into the cell membrane. Liong and Shah [38] examined the influence of cholesterol on the profile of fatty acids of lactic bacilli and determined that the strains growing in the medium without addition of cholesterol demonstrated a stronger percentage content of

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

lactic bacilli [50, 69].

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

role in bacteria adhesion, biofilm formation, tolerance to environmental acidity, resistance to antibiotics, bacteriophages, or UV radiation. Lactic bacteria synthesize teichoic and lipoteichoic acids simultaneously or lipoteichoic acids only. The characteristic feature of lipoteichoic acids is the presence (at the end of the chain) of a glycolipid anchored into the cytoplasmic membrane, and the structure of this connection depends on bacteria species. The qualitative composition of cytoplasmic membrane phospholipids depends on environmental factors, such as availability of nutrients, temperature, pH, and presence of toxic materials. The fatty acids profile changes also depending on the genus and species of bacteria and their growth phase, which has been used for microorganism grouping and classification attempts [69, 74, 90]. C16 fatty acids are the most common, while C12, C14, and C18 fatty acids are less frequently found. Methylated, hydroxylated, and branched fatty acids or those containing cyclopropane ring are common. Lactobacillic acid—a fatty acid containing cyclopropane ring—was first detected in the cytoplasmic membrane of lactic bacilli [50, 69].

Literature data indicate that the cell wall or cytoplasmic membrane can form the cholesterol binding site. In the case of the cell wall, the bond may have physical (adhesion) or chemical character (analogous to incorporation of teichoic, lipoteichoic, and teichuronic acid incorporation). In the case of a chemical bond, we deal with cholesterol assimilation, that is, its incorporation into the cell wall. Cholesterol molecules are oriented in the cell membrane in the same manner as phospholipid molecules. The polar portion of cholesterol molecule adheres to the polar portion of phospholipid. Perhaps, in cytoplasmic membranes of bacterial cells, cholesterol molecules are located in the same manner as in membranes of eukaryotic organisms. The bacterial cytoplasmic membrane contains compounds with a structure similar to steroids, which further indicates the possibility for cholesterol incorporation into the cytoplasmic membrane of bacterial cells. However, in order to be incorporated into the cytoplasmic membrane, cholesterol molecules must be transported through the cell wall. As shown in the studies of Kurdi et al. [91], Pigeon et al. [42], and Kurdi et al. [61] on bile acids, transport of such large molecules through the cell wall is possible even if it threatens the survival of the bacteria. Cholesterol binding by the cell membrane is not neutral to the bacterial cell itself. The presence of such substances as cholesterol in the environment influences the ratio of saturated acids to unsaturated acids in the cytoplasmic wall, as well as the structure and properties of this membrane. Goldberg and Eschar [92] noted that addition of Tween 80 to culture medium increases the concentration of certain fatty acids with the concomitant influence on the ratio of saturated acids to unsaturated acids. The same happens when the cholesterol molecules are being bound. Dambekodi and Gilliland [23] proved that incorporation of cholesterol into the cell membrane of bifidobacteria was manifested by changes in its composition and resulted in an increase of the resistance of cells growing in the presence of cholesterol to ultrasonic lysis. In turn, Taranto et al. [50] demonstrated that bacterial cells growing in the presence of cholesterol or bile salts are more resistant to lysis than those growing in their absence, contrary to the cells growing in the absence of cholesterol. The cited authors observed that addition of cholesterol to culture broth resulted in an increase of saturated fatty acid content in lactic bacilli biomass from 44.3% to 56.5% of total acids and unsaturated acids from 1.26% to 43.5% of the total amount of fatty acids. Furthermore, Kimoto et al. [25] reached a conclusion that the change in the distribution of fatty acids by *Lactococcus lactis* cells growing in the presence of cholesterol is an effect of its removal from the culture medium and incorporation into the cell membrane. Liong and Shah [38] examined the influence of cholesterol on the profile of fatty acids of lactic bacilli and determined that the strains growing in the medium without addition of cholesterol demonstrated a stronger percentage content of

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

**4. Cholesterol binding sites by bacterial cells**

the capacity to metabolize this compound [81, 83–85, 87, 88].

formation.

intestinal microflora.

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

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

As stated above, binding (adhesion or assimilation) by bacteria cells is one of the major mechanisms for the removal of cholesterol by bacteria from the environment. Hosono and Tono-Oka [24] have suggested that it is the chemical nature and structure of peptidoglycan present in bacterial cell wall that fulfill a major role in cholesterol binding. This hypothesis was confirmed by Usman and Hosono [43]. They further suggested that a portion of cholesterol could be embedded into the bacterial cell walls. The possibility for incorporating cholesterol into the cell membrane of lactic acid bacteria was demonstrated in the study of Noh et al. [19]. The phenomenon of cholesterol binding by the cell wall has been indicated by similar research conducted on the binding of aflatoxin B1 (AFB1) by lactic acid bacteria cells [81–86]. Many researchers point out to the phenomenon of AFB1 aflatoxin binding by live and dead lactic acid bacteria cultures, which do not possess

As it is known, Gram-positive bacteria are characterized by a thick cell wall. The cell wall of Gram-positive bacteria comprises of peptidoglycan (murein) and its associated teichoic and/or teichuronic and lipoteichoic acids and proteins [89]. The wall thickness ranges between 15 and 50 nm, corresponding to 20–30 individual murein layers. Murein is built of saccharide chains comprising of alternately arranged N-acetylglucosamine and N-acetylmuramic acid, joined with a β-(1 → 4)-glycoside bond [89]. Apart from the saccharide chains, murein contains short peptides. The free carbonyl group of muramic acid forms the acceptor for the first peptides amino acid. Typically, l-alanine is the first amino acid. The protein portion of murein exhibits considerably greater diversity than its saccharide part, as its composition depends on the bacteria species, environmental conditions, and even the cell age. In Gram-positive bacteria, the cell wall further contains proteins active in various physiological and biochemical processes—energy transfer, electron and proton transport, cell casing synthesis, etc. [89]. Moreover, various types of polymers are associated with murein, such as teichoic acids (teichoic and lipoteichoic acid) and teichuronic acid. Considering the manner in which these acids are attached, they are sometimes referred to as secondary (after murein) polymers of bacterial cell wall. The importance of these acids has not been fully explained, although numerous assumptions have been made explaining the presence of these compounds in the bacterial cell wall. It is possible that these acids play a certain

**80**

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