**5. Conclusions**

This study contributes to updating data in the literature concerning the effect of bacteriocin-producing and beneficial strain *E. faecium* CCM8558, and its enterocin M on the quality and mineral content of rabbit meat. Higher phosphorus and iron levels were recorded during both CCM8558 strain and EntM administration in rabbits. During EntM application, improved pH48, lightness, and water content was noted. In general, physicochemical parameters and nutritional value of rabbit meat were not negatively influenced during bioactive compounds' administration. This study has impact for basic research primarily due to spreading knowledge regarding the rabbit meat's mineral composition and secondarily due to the opportunity to compare the beneficial effect of different natural additives—probiotics and enterocins in rabbits under in vivo conditions. Nevertheless, further investigations are needed to assess the efficacy of CCM8558 and EntM in rabbit husbandries.

**Author Contributions:** Conceptualization, A.L. and M.P.S.; methodology, L'.C.; validation, A.L.; investigation, M.P.S., L'.C. and A.L.; resources, A.L.; data curation, L'.C. and M.P.S.; writing—original draft preparation, M.P.S.; writing—review and editing, M.P.S.; visualization, M.P.S.; supervision, A.L.; project administration, A.L. and M.P.S.; funding acquisition, A.L. and M.P.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Scientific Grant Agency of the Ministry for Education, Science, Research and Sport of the Slovak Republic and the Slovak Academy of Sciences VEGA, grant number 2/0006/17 and 2/0005/21.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the State Veterinary and Food Administration of the Slovak Republic on 1 December 2016 (approval numbers SK CH 17016 and SK U 18016).

**Data Availability Statement:** Data are available upon reasonable request to the corresponding author.

**Acknowledgments:** We are grateful to P. Jerga for his skillful technical assistance and to J. Pecho for slaughtering. We thank A. Billingham for his English language editing.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


**2. Polydextrose studies in animals, human and** *in vitro***: Contribution of polydextrose in lipid metabolism**

Polydextrose is a fermentable non-viscous fiber, and has been shown to exhibit lipid metabolism regulating effects [5]. Typically these effects have been associated with two physiochemical properties of soluble fibers: viscosity and fermentability. Viscous soluble fibers may work by slowing down gastric emptying and prevention of bile salt reabsorption which would increase the secretion of bile acids and neutral sterols into feces and interruption of the enterohepatic circulation of bile acids [35, 36]. Soluble fiber can also decrease intestinal cholesterol absorption by affecting micelle formation and mobility [37, 38], and reduce glycemic response leading to lower insulin stimulation and hepatic cholesterol synthesis [39]. Fibers can also promote satiety [40]. Additionally, colonic fermentation products of these fibers, short chain fatty acids (SCFAs), mainly propionate, have been shown to inhibit hepatic fatty acid synthesis [41]. Polydextrose has been reported to confer lipid modulating effects in human clinical intervention studies, as well as in animal studies. However, some of the characteristics of polydextrose are different to other soluble fibers, such as low viscosity, and sustained fermentation throughout the colon [11].

#### **2.1. Polydextrose studies in animals**

234 Lipid Metabolism

25] and iron [26].

stool consistency and ease of defecation, and reduced fecal pH [7]. It is safe to use, and well tolerated, with a mean laxative threshold of 90 g/day, or 50 g as a single bolus dose [14-16].

**Figure 1.** Structure for the polydextrose. The letter R can be either hydrogen (H), sorbitol, sorbitol bridge, of more polydextrose. Polydextrose has highly branched complex three-dimensional structure with all different combinations of α- and β-linked 12, 13, 14 and 16 glycosidic linkages.

Several beneficial effects have been linked to the consumption of polydextrose. Consumption of polydextrose promotes the growth of beneficial bifidobacteria and lactobacilli while preventing the growth of harmful ones, such as clostridia [17, 18]. It has been suggested to possess anti-inflammatory actions and to improve the signs of osteoarthritis in canines [19], to increase IgA amount in the rat cecum [20], to reduce cyclooxygenase 2 expression in pigs distal colon, and to reduce lesions in rat colitis model [21]. Furthermore, it has been suggested to improve the absorption of magnesium, calcium [22-

Soluble fiber, both viscous (e.g. gums, pectin and β-glucan) and non-viscous (e.g. polydextrose, resistant maltodextrin and inulin), has been suggested to have beneficial metabolic advantages. These include increasing satiety and reduction of body weight, control of postprandial glycemic and insulin responses, and hypocholesterolemic effects on serum lipid parameters [5, 27]. The inverse relationship of higher HDL to coronary artery disease risk has been recognized and is evident across numerous populations, and the increment of its relative amount over LDL has been generally accepted as a hallmark of better cardiovascular health [28]. Soluble fiber has been associated inversely with serum total and LDL cholesterol, while HDL cholesterol concentration has been reported to either slightly decrease or remain unchanged [29, 30]. This effect has been attributed as an effect of soluble viscous fibers, as insoluble fibers do not appear to affect serum cholesterol concentrations [31, 32]. The ability of soluble fibers to reduce serum triglyceride levels is also controversial, as in some studies an inverse association has been suggested, while in many studies no effect has been observed [29, 33]. Soluble viscous fibers have a characteristic of being hypocholesterolemic, reducing serum cholesterol by about 5-10 % for a 5-10 g dose in subjects with hypercholesterolemia, whereas insoluble fibers have not shown this effect [34].

The ability of polydextrose to modulate triglycerides, total, LDL, and HDL cholesterol has been studied in animals both in normal diets without additional lipid load or in diets in which lipids have been included as part of the normal diet. There is a clear difference between the types of studies, as the two studies without lipid load have not shown any effect on the blood lipid values. In a 6-week feeding study in normal rats with 5 % (w/w) inclusion of polydextrose no change in plasma triglycerides, total cholesterol, and HDL cholesterol or liver cholesterol, triglycerides and phospholipids was observed [42]. Another, 15-day feeding trial with rats, did not show differences in serum total and free cholesterol, triacylglycerols, and phospholipids even though 3 % polydextrose was administered together with 3 % pectin or 3 % cellulose [43].

However, in two other rat feeding studies in which polydextrose was accompanied with a lipid load, reduced lipid levels were reported. In one study rats were given two different dosages of corn oil, 10 % and 20 %, to represent a moderate or high fat diet, for 8 weeks, with or without 5 % polydextrose [44]. Rats in the polydextrose group showed decreased serum triglycerides as compared to a guar gum control in the high-fat diet, and increased levels of serum HDL cholesterol both in the moderate fat and high fat diet [44]. Serum total lipids and cholesterol remained at the level of the control [44]. One study has been done with gerbils: in the 4-week study the gerbils were fed with 0.15 % cholesterol with 30 % of the energy coming from fat and with inclusion of 6 % polydextrose [45]. Both liver and plasma total cholesterol as well as free and esterified cholesterol from liver decreased in the polydextrose group [45]. The effect was presumed by the authors to be related to the reduction of VLDL and LDL, since no change in HDL was observed [45]. In the same gerbil study, it was additionally investigated whether polydextrose can remove cholesterol from

n.a. not available; d days; w weeks; m months; a↓ ↑ ↓↑ reduced; increased; Trend or Trend only a tendency for reduced or increased values were observed; N.C. no change

a Indirect evidence, LDL and VLDL were not directly measured but authors concluded that as total cholesterol decreased and there was no change in HDL, then preferential target is then VLDL and/or LDL.

b 0.4 % cholesterol administered to the gerbils 2 weeks prior the feeding with polydextrose in order to expand the endogenous cholesterol pools.

c 0.4 % cholesterol administered to the gerbils 2 weeks prior the feeding with polydextrose in order to expand the endogenous cholesterol pools.  Polydextrose in Lipid Metabolism 237

endogenous pools by first artificially expanding the endogenous cholesterol pools of the gerbils with a preload of 0.4 % cholesterol for two weeks before 6 % polydextrose was fed for an additional three weeks. Polydextrose was shown to hasten the endogenous clearance of cholesterol pools, and to reduce liver and plasma total cholesterol, esterified cholesterol

The acute response of polydextrose on serum lipid values has also been studied in rats, but together with lactitol [46]. Polydextrose was administered as a 28 % solution in a dose of 3 ml/200 g of body weight in a solution also containing 26 % lactitol and a fat emulsion which was comparable to one in chocolate. The rats showed reduced serum triglyceride levels, and an increase in luminal triglyceride levels in the cecum after 150 minutes of ingestion of polydextrose, which would indicate that the combination of polydextrose and lactitol reduced either the level of fat absorption in the earlier part of small intestine or promoted the transit time of fat through the intestine [46]. No change in total, HDL, or LDL cholesterol

These studies are summarised in Table 1. There is an indication from these studies that polydextrose can lower total cholesterol and has a tendency to lower LDL cholesterol and

Clinical intervention studies with polydextrose have been conducted either with healthy adults, with individuals having hypercholesterolemia or with individuals with impaired

In a human study with normal healthy adults with no reported hypercholesterolemia a reduction in the amount of total HDL by administration of 15 g of polydextrose for two months with concomitant decrease in apolipoprotein A-I, which is the main component of the HDL cholesterol, has been observed [47]. Apolipoprotein B levels, found in all atherogenic apolipoprotein particles, and the LDL cholesterol levels itself had also a tendency to be reduced after the 2-month and 1-month intervention period, respectively [47]. In another study with healthy adults, administration of 10 g of polydextrose for 18 days was shown to decrease LDL cholesterol and total cholesterol values with no effect on HDL cholesterol or triglycerides [48]. There are also contradictory results with healthy humans, as administration of polydextrose in an amount from 4 to 12 g per day for 29 days did not affect triacylglycerol, or cholesterol [49]. However, in that study no data was shown, and in

In hypercholesterolomic individuals, the effect of polydextrose has been studied in a 4-week study with administration of 15 g and 30 g polydextrose daily [45]. The study was quite small, with only 12 subjects participating, and each individual subject serving as a control for him/herself. In this study it was noted that 5 of the 6 individuals ingesting 30 g of polydextrose were in a separate responder group, and in this group the LDL cholesterol values declined significantly, and there was a tendency for reduced total cholesterol, but no change in HDL cholesterol. However, when all 6 individuals were studied together, no

**2.2. Polydextrose lipid metabolism studies in clinical intervention studies**

addition to which the cholesterol type measured was not specified.

change compared to control was observed.

from liver and plasma HDL cholesterol [45].

were found in that study.

glucose tolerance.

tendency to increase HDL cholesterol.

**Table 1.** Polydextrose studies in relation to lipid metabolism done in animals.

endogenous pools by first artificially expanding the endogenous cholesterol pools of the gerbils with a preload of 0.4 % cholesterol for two weeks before 6 % polydextrose was fed for an additional three weeks. Polydextrose was shown to hasten the endogenous clearance of cholesterol pools, and to reduce liver and plasma total cholesterol, esterified cholesterol from liver and plasma HDL cholesterol [45].

236 Lipid Metabolism

Cholesterol

Study design

Ref Animals

[42]

Sprague-Dawley rats

5 %

6 w

 No load

 N.C.

 n.a.

 N.C.

 N.C.

Lipids meas-

ured both from plasma and liver

[43]

Sprague-Dawley rats

3 %

15 d

 No load

 N.C.

 n.a.

 n.a.

 N.C.

Together with pectin or cel-

lulosa

Dose

 Time

Lipid load

 Total

 LDL

 HDL

 TG

 Remarks

The acute response of polydextrose on serum lipid values has also been studied in rats, but together with lactitol [46]. Polydextrose was administered as a 28 % solution in a dose of 3 ml/200 g of body weight in a solution also containing 26 % lactitol and a fat emulsion which was comparable to one in chocolate. The rats showed reduced serum triglyceride levels, and an increase in luminal triglyceride levels in the cecum after 150 minutes of ingestion of polydextrose, which would indicate that the combination of polydextrose and lactitol reduced either the level of fat absorption in the earlier part of small intestine or promoted the transit time of fat through the intestine [46]. No change in total, HDL, or LDL cholesterol were found in that study.

These studies are summarised in Table 1. There is an indication from these studies that polydextrose can lower total cholesterol and has a tendency to lower LDL cholesterol and tendency to increase HDL cholesterol.

#### **2.2. Polydextrose lipid metabolism studies in clinical intervention studies**

Clinical intervention studies with polydextrose have been conducted either with healthy adults, with individuals having hypercholesterolemia or with individuals with impaired glucose tolerance.

In a human study with normal healthy adults with no reported hypercholesterolemia a reduction in the amount of total HDL by administration of 15 g of polydextrose for two months with concomitant decrease in apolipoprotein A-I, which is the main component of the HDL cholesterol, has been observed [47]. Apolipoprotein B levels, found in all atherogenic apolipoprotein particles, and the LDL cholesterol levels itself had also a tendency to be reduced after the 2-month and 1-month intervention period, respectively [47]. In another study with healthy adults, administration of 10 g of polydextrose for 18 days was shown to decrease LDL cholesterol and total cholesterol values with no effect on HDL cholesterol or triglycerides [48]. There are also contradictory results with healthy humans, as administration of polydextrose in an amount from 4 to 12 g per day for 29 days did not affect triacylglycerol, or cholesterol [49]. However, in that study no data was shown, and in addition to which the cholesterol type measured was not specified.

In hypercholesterolomic individuals, the effect of polydextrose has been studied in a 4-week study with administration of 15 g and 30 g polydextrose daily [45]. The study was quite small, with only 12 subjects participating, and each individual subject serving as a control for him/herself. In this study it was noted that 5 of the 6 individuals ingesting 30 g of polydextrose were in a separate responder group, and in this group the LDL cholesterol values declined significantly, and there was a tendency for reduced total cholesterol, but no change in HDL cholesterol. However, when all 6 individuals were studied together, no change compared to control was observed.

n.a. not available; d days; w weeks; m months; a↓

change

**Table 1.** Polydextrose studies in relation to lipid metabolism done in animals.

[45] Gerbils

[45] Gerbils

[46]

Wistar rats

ml/200 g

body weight

28 % as 3

Acute

Fats as in

n.a.

 n.a.

 n.a.

Together with

25.7 % lactitol

chocolate

6 %

3 w

n.a.

N.C.

Liver total ,

lesterol

0.4 % choleterol preloadb

6 %

4 w

 30 % en-

 (or VLDL)a

N.C.

 N.C.

Liver total , free cholesterol

Total to HDL ratio trend in plasma

ergy from fat, 0.15 % from cho-

lesterol

[44]

Sprague-Dawley rats

5 %

8 w

N.C.

 n.a.

Compared to guar gum group

10 % and 20 % from corn oil

preferential target is then VLDL and/or LDL.

 ↑

a Indirect evidence, LDL and VLDL were not directly measured but authors concluded that as total cholesterol decreased and there was no change in HDL, then

b 0.4 % cholesterol administered to the gerbils 2 weeks prior the feeding with polydextrose in order to expand the endogenous cholesterol pools.

c 0.4 % cholesterol administered to the gerbils 2 weeks prior the feeding with polydextrose in order to expand the endogenous cholesterol pools.

only a tendency for reduced or increased values were observed; N.C. no

↓↑ reduced; increased; Trend or Trend

In diabetic patients, diets containing high amounts of fiber have improved plasma lipid control [50, 51]. The effect of polydextrose on lipid values has been of interest in two studies with individuals showing abnormal glucose metabolism or type 2 diabetes. In subjects with impaired glucose metabolism, polydextrose administered for 12 weeks at 16 g/day has been observed to lower LDL cholesterol, increase HDL cholesterol and cause no change in triglycerides [52]. In this study, LDL cholesterol decreased also in the control group probably because of simultaneous nutrition consultation by a nutritionist [52]. In a combination study with 7 g polydextrose and 3 g oligofructose administered daily for 6 weeks in adults with type 2 diabetes, a decrease in total cholesterol, triglycerides, VLDL cholesterol, and ratios of total cholesterol to HDL cholesterol, and LDL cholesterol to HDL cholesterol was observed, while HDL cholesterol increased [53].

Polydextrose in Lipid Metabolism 239

n.a. not available; d days; w weeks; m months; ↓

**Table 2.** Polydextrose clinical intervention studies in which lipid values have been measured.

[45]

With hypercholes-

terolemia

[52] [53]

With type 2 dia-

betes

13.2 g/day

 6 w

With impaired glu-

cose metabolism

16 g/day

 12 w

N.C.

  With 6.8 g oli-

gofructose/day. Total/HDL cho-

lesterol , VLDL choles-

terol

N.C.

Total to HDL ratio

day

15 or 30 g/

4 w

 Healthy

15.1 % in 46 g of chololate

Acute

n.a.

 n.a.

 n.a.

Together with 19.4 % lactitol

Lipids from chocolate

Trend c c

N.C.d

N.C.d

Study design

Ref

[47] Healthy

[69] Healthy

[48] Healthy

[49] Healthy

15 g/day

10 g/day

4, 8, or 12 g/

day

28 d

 18 d

 4 w

Trendb

N.C.

Measured lipid values were not [46]

 N.C.

 N.C.

 N.C.

N.C.

 N.C.

Trendb

N.C.

 N.C.

ApoB

Dose

15 g/day

 2 m

 Time

Lipid load

 Total

N.C.

 N.C.a

HDL2 HDL3

N.C.a

ApoA-I ApoA-II

 LDL

 HDL

 TG

 Remarks

Cholesterol

a had a tendency to be reduced 1 month after intervention

b Total cholesterol and LDL cholesterol were reduced by 6 % but not significantly

c When studied form a group that responded to the 30 daily dose of polydextrose

d Both in the responder group that ingested 30g/day polydextrose and in a group in which in addition to responders, a non-responder was included.

 ↑

only tendency for reduced or increased values were observed; N.C. no change

↓↑ reduced; increased; Trend or Trend

The acute effect of polydextrose has been studied in humans too, together with lactitol, in the triglyceride levels after consumption of 46 g of chocolate supplemented with 15.1 % of the weight with polydextrose. The intervention group had reduced serum triglycerides in comparison to control chocolate during the 150 minutes the triglycerides were measured from the blood [46]. During intense exercise, polydextrose has been shown to reduce the amount of free fatty acids in plasma [54]. In addition, an abstract has been published about ingestion of 12.5 g polydextrose during a hamburger meal observing a reduction in total postprandial hypertriglyceridemia by 25 % [55].

#### *2.2.1. Effect of polydextrose on different HDL subfractions*

HDL is highly heterogeneous, and subfractions of it can be identified on the basis of density, size, charge, and protein composition [56]. Different fractions of HDL can be identified by ultracentrifugation, gradient gel electrophoresis, and nuclear magnetic resonance (NMR) spectroscopy, and these different fractions could play diverse roles in protective function [56, 57]. It is thought that certain fractions of HDL cholesterol could be better predictors of cardiovascular diseases and its risk. However, controversy about the role of the different forms still exists, as in [58] and in [59] increased HDL2 and apoA-I levels were associated as protective against coronary heart disease. In other studies, in turn, such as in [60], and [61], the HDL3 has had a stronger inverse association with coronary heart disease. The early studies, however, more strongly indicate that reduced levels of HDL2 over HDL3 is associated more strongly to CVD risk [62].

The different subfractions of HDL and the impact of ingestion of polydextrose on their distribution has been investigated in one study [47], and in this study HDL3 was increased during the 2-month intervention period and one month after finishing the study. At the same time, a reduced level of HDL2, apoA-I and LCAT activity was observed. In other studies with soluble fibers, such as with guar gum, no changes in HDL2 and HDL3 subfractions or their ratio were observed [63, 64]. No difference between serum HDL2 and HDL3 cholesterol subfractions could be observed between high-fiber, consisting partially of soluble fiber from psyllium, and low-fiber diets [65] or with beta-glucan as an oat fiber extract [66]. In addition, lack of standardization among the analytical methods that are used to measure size distribution of different HDL2 and HDL3 subfractions may cause approximation of HDL subclass levels [67, 68].

238 Lipid Metabolism

In diabetic patients, diets containing high amounts of fiber have improved plasma lipid control [50, 51]. The effect of polydextrose on lipid values has been of interest in two studies with individuals showing abnormal glucose metabolism or type 2 diabetes. In subjects with impaired glucose metabolism, polydextrose administered for 12 weeks at 16 g/day has been observed to lower LDL cholesterol, increase HDL cholesterol and cause no change in triglycerides [52]. In this study, LDL cholesterol decreased also in the control group probably because of simultaneous nutrition consultation by a nutritionist [52]. In a combination study with 7 g polydextrose and 3 g oligofructose administered daily for 6 weeks in adults with type 2 diabetes, a decrease in total cholesterol, triglycerides, VLDL cholesterol, and ratios of total cholesterol to HDL cholesterol, and LDL cholesterol to HDL

The acute effect of polydextrose has been studied in humans too, together with lactitol, in the triglyceride levels after consumption of 46 g of chocolate supplemented with 15.1 % of the weight with polydextrose. The intervention group had reduced serum triglycerides in comparison to control chocolate during the 150 minutes the triglycerides were measured from the blood [46]. During intense exercise, polydextrose has been shown to reduce the amount of free fatty acids in plasma [54]. In addition, an abstract has been published about ingestion of 12.5 g polydextrose during a hamburger meal observing a reduction in total

HDL is highly heterogeneous, and subfractions of it can be identified on the basis of density, size, charge, and protein composition [56]. Different fractions of HDL can be identified by ultracentrifugation, gradient gel electrophoresis, and nuclear magnetic resonance (NMR) spectroscopy, and these different fractions could play diverse roles in protective function [56, 57]. It is thought that certain fractions of HDL cholesterol could be better predictors of cardiovascular diseases and its risk. However, controversy about the role of the different forms still exists, as in [58] and in [59] increased HDL2 and apoA-I levels were associated as protective against coronary heart disease. In other studies, in turn, such as in [60], and [61], the HDL3 has had a stronger inverse association with coronary heart disease. The early studies, however, more strongly indicate that reduced levels of HDL2 over HDL3 is

The different subfractions of HDL and the impact of ingestion of polydextrose on their distribution has been investigated in one study [47], and in this study HDL3 was increased during the 2-month intervention period and one month after finishing the study. At the same time, a reduced level of HDL2, apoA-I and LCAT activity was observed. In other studies with soluble fibers, such as with guar gum, no changes in HDL2 and HDL3 subfractions or their ratio were observed [63, 64]. No difference between serum HDL2 and HDL3 cholesterol subfractions could be observed between high-fiber, consisting partially of soluble fiber from psyllium, and low-fiber diets [65] or with beta-glucan as an oat fiber extract [66]. In addition, lack of standardization among the analytical methods that are used to measure size distribution of different HDL2 and HDL3 subfractions may cause

cholesterol was observed, while HDL cholesterol increased [53].

postprandial hypertriglyceridemia by 25 % [55].

associated more strongly to CVD risk [62].

approximation of HDL subclass levels [67, 68].

*2.2.1. Effect of polydextrose on different HDL subfractions*

n.a. not available; d days; w weeks; m months; ↓ ↑ ↓↑ reduced; increased; Trend or Trend only tendency for reduced or increased values were observed; N.C. no change

a had a tendency to be reduced 1 month after intervention

b Total cholesterol and LDL cholesterol were reduced by 6 % but not significantly

c When studied form a group that responded to the 30 daily dose of polydextrose

d Both in the responder group that ingested 30g/day polydextrose and in a group in which in addition to responders, a non-responder was included.

**Table 2.** Polydextrose clinical intervention studies in which lipid values have been measured.

Table 2 summarizes the different human clinical intervention studies done with polydextrose in relation to HDL, LDL, total cholesterol and triglycerides. From these studies it can be concluded that polydextrose in the diet can lower serum total and LDL cholesterol and triglycerides. There are two studies in which definite increases in HDL have been observed.

Polydextrose in Lipid Metabolism 241

adults at 8 g/day for three weeks was not found to increase fecal excretion of total bile acids and secondary bile acids, but rather decreased values were observed during the intervention period compared to the run-in period before [77]. A similar observation was made in normal rats, in which administration of 5 % polydextrose for 6 weeks did not increase the fecal output of bile acids [42]. The low ability of polydextrose to bind bile acids is not, however, surprising. In order for a fiber to bind bile acids, it is required to be viscous in nature, and polydextrose is lower in viscosity for instance in comparison to pectin and guar gum which have high water binding capacity with higher viscosity and thus increased capability to bind bile acids [42, 78]. Polydextrose has a high capacity to bind water, and it can for instance relieve constipation presumably due to this characteristic, but there is no gel formation by

Even though no clear effect on bile acid binding can be detected, the fact that cholesterol and triglyceride absorption can still be modulated, are indications that polydextrose can retard the transportation of lipids from the intestinal lumen to the lymph. When polydextrose was infused as 5 % and 10 % to duodenum together with cholesterol and triglyceride on mesenteric lymph-fistulated rats, the amount of cholesterol and triglycerides in the lymph decreased dose-dependently, and concomitantly the amount of the unabsorbed lipids increased in the lumen of the intestine [81]. It was concluded that since most of the luminal triglyceride and cholesterol was detected in the proximal part of the small intestine, the absorption of the lipids was inhibited. There was a tendency to have increased amount of cholesterol and a significant increase of triglycerides remaining in the colon which could indicate that some of the lipids were not absorbed [81]. However, polydextrose did not seem to increase secretion of cholesterol into feces in rats even though some tendency was

In an acute response study of polydextrose in combination with lactitol in rats with lipid load similar to the composition of chocolate an increase in luminal triglyceride in the cecum was observed with concomitant decrease in serum triglycerides [46]. This would indicate that the combination of polydextrose and lactitol reduced either the level of fat absorption in the earlier part of small intestine or promoted the transit time of fat through

Cholesterol that escapes absorption is partially degraded to coprostanol and coprostanone by colon microbes [83]. A decrease of the degradation products coprostanol, coprostanon and cholestanol has been observed [77] which would indicate that the amount of cholesterol

**3.2. Role of polydextrose on intestinal microbiota and its impact on cholesterol**

When soluble fiber enters the large intestine, it is fermented by the residual microbes forming short-chain fatty acids (SCFAs), butyrate, acetate, and propionate, end-products of

entering the colon is less in humans receiving polydextrose.

polydextrose in water and little viscosity [43, 79, 80].

observed in another study [82].

the intestine [46].

**metabolism**

#### **3. Mechanisms for polydextrose action on lipid values**


4. Methods used to study glycolipids at the water-air interface

Given the significance of glycolipids in cellular processes and their association with chronic diseases, it is important to seek a clear understanding of the biophysical and biological properties of these glycolipids. In this section, we attempt to address some of the techniques which might be helpful and a survey of studies done using these methods will be discussed. Each method has pros and cons associated with it and therefore two or more complimentary methods are often employed together for the complete assurance of the result observed.

#### 4.1. Surface pressure isotherms

The plot of surface pressure, Π, versus molecular area, A, at constant temperature as the monolayer film is compressed, by closing the barrier at constant rate, after initial deposition and solvent evaporation is known as an isotherm. Surface pressure-area isotherms provide information about molecular packing, molecular stability, phase transitions and compressibility of phases. Isotherms recorded at various temperatures helps to obtain phase diagrams [21]. The data from Π-A isotherms can also be used for various thermodynamic calculations.

Water molecules on the surface are under tension due to imbalance of the force compared to the bulk where each molecule is attracted by equal force from all direction. The surface tension decreases when a monolayer is deposited at the air-water interface. The difference in surface tension before and after monolayer deposition is known as surface pressure, Π, which is given by the relation

$$
\Pi = \mathcal{V}\_0 - \mathcal{V} \tag{1}
$$

phases and phase transitions upon compression [58]. At high molecular areas there can be a gas-like phase in which molecules are widely separated and the surface pressure is very low. Upon compression, a liquid-like phase can be entered that is known as the liquid-expanded (LE) phase in which molecules are closer together and ordered like a two-dimensional liquid. A liquid-condensed (LC) phase in which molecules are well-ordered as in a two-dimensional liquid crystal, and may be oriented vertically or tilted, is entered upon further compression. Other phases are possible and the sequence of phases seen on compression depends on temperature and other conditions. Coexistence regimes between phases, such as LE + LC, are often encountered. Kinks or flat regions in an isotherm signal these phase transitions for single component monolayers. At very high surface pressures, two-dimensional solid-like phases can be observed for some compounds. Compression of the monolayer beyond its stability limit

Figure 4. Schematic showing a pressure area, Π-A isotherm with the major phases and transitions shown for a single

Monolayers of Carbohydrate-Containing Lipids at the Water-Air Interface

http://dx.doi.org/10.5772/intechopen.76440

221

Π-A isotherms mainly provide information about later stages of monolayer compression when the molecules are closer together. Surface-potential measurement allows one to probe a Langmuir monolayer at higher surface area before it has been significantly compressed. Surface potential vs. area, ΔV-A isotherms, have a region in the higher range of molecular area which marks the initial rise of the surface potential and is attributed to aggregation of microdomains present in the monolayer [59]. In addition to these advantages, surface potential measurements are used to gain information about molecular orientation, to calculate dipole moment, surface

results in collapse into three-dimensional structures.

4.2. Surface potential measurement

component lipid monolayer.

charge density and interfacial thickness.

where γ0 and γ are surface tension in the absence and presence of monolayer, respectively. Surface pressure is measured by the Wilhelmy plate method in which a plate made of platinum or thin filter paper is contacted with the water and changes in downward force are measured. A schematic Π-A isotherm is shown in Figure 4. Monolayers can exhibit a range of

Figure 4. Schematic showing a pressure area, Π-A isotherm with the major phases and transitions shown for a single component lipid monolayer.

phases and phase transitions upon compression [58]. At high molecular areas there can be a gas-like phase in which molecules are widely separated and the surface pressure is very low. Upon compression, a liquid-like phase can be entered that is known as the liquid-expanded (LE) phase in which molecules are closer together and ordered like a two-dimensional liquid. A liquid-condensed (LC) phase in which molecules are well-ordered as in a two-dimensional liquid crystal, and may be oriented vertically or tilted, is entered upon further compression. Other phases are possible and the sequence of phases seen on compression depends on temperature and other conditions. Coexistence regimes between phases, such as LE + LC, are often encountered. Kinks or flat regions in an isotherm signal these phase transitions for single component monolayers. At very high surface pressures, two-dimensional solid-like phases can be observed for some compounds. Compression of the monolayer beyond its stability limit results in collapse into three-dimensional structures.

#### 4.2. Surface potential measurement

3.4. Lipopolysaccharides

220 Cell Culture

4.1. Surface pressure isotherms

by the relation

Lipopolysaccharides (LPS), also referred to as endotoxins, are a major component of the outer membrane of gram negative bacteria and are essential for maintaining the structural integrity of the membrane. LPS have three components: lipid A, core oligosaccharide and O-antigen polysaccharide. Lipid A is the active component and under normal conditions consists of β(1–6) linked glucosamine disaccharides. The diglucosamine backbone is phosphorylated and decorated with multiple fatty acids anchoring them in the outer leaflet of the bacterial cell membrane. Lipid A, when released from the cell, is recognized by pattern recognition receptor TLR4 triggering cytokine synthesis. At low level of endotoxins, the innate immune system

Given the significance of glycolipids in cellular processes and their association with chronic diseases, it is important to seek a clear understanding of the biophysical and biological properties of these glycolipids. In this section, we attempt to address some of the techniques which might be helpful and a survey of studies done using these methods will be discussed. Each method has pros and cons associated with it and therefore two or more complimentary methods are often employed together for the complete assurance of the result observed.

The plot of surface pressure, Π, versus molecular area, A, at constant temperature as the monolayer film is compressed, by closing the barrier at constant rate, after initial deposition and solvent evaporation is known as an isotherm. Surface pressure-area isotherms provide information about molecular packing, molecular stability, phase transitions and compressibility of phases. Isotherms recorded at various temperatures helps to obtain phase diagrams [21]. The data from Π-A isotherms can also be used for various thermodynamic calculations.

Water molecules on the surface are under tension due to imbalance of the force compared to the bulk where each molecule is attracted by equal force from all direction. The surface tension decreases when a monolayer is deposited at the air-water interface. The difference in surface tension before and after monolayer deposition is known as surface pressure, Π, which is given

where γ0 and γ are surface tension in the absence and presence of monolayer, respectively. Surface pressure is measured by the Wilhelmy plate method in which a plate made of platinum or thin filter paper is contacted with the water and changes in downward force are measured. A schematic Π-A isotherm is shown in Figure 4. Monolayers can exhibit a range of

Π ¼ γ0 � γ (1)

eliminates it, but at high concentrations it can prove fatal [57].

4. Methods used to study glycolipids at the water-air interface

Π-A isotherms mainly provide information about later stages of monolayer compression when the molecules are closer together. Surface-potential measurement allows one to probe a Langmuir monolayer at higher surface area before it has been significantly compressed. Surface potential vs. area, ΔV-A isotherms, have a region in the higher range of molecular area which marks the initial rise of the surface potential and is attributed to aggregation of microdomains present in the monolayer [59]. In addition to these advantages, surface potential measurements are used to gain information about molecular orientation, to calculate dipole moment, surface charge density and interfacial thickness.

Two common methods applied to measure surface potential are the vibrating electrode and the ionizing electrode. The vibrating electrode method also known as Kelvin probe method uses a plate-like electrode placed at a certain distance above air-water interface. The electrode is connected to the reference electrode placed in the subphase. The electrode above the interface is periodically vibrated during measurements. The ionizing electrode method employs the same measurement setup as the vibrating plate method. In this method, the electrodes are coated with α-emitters such as 241Am or 210Po to increase the conductivity of the air gap [60].

is doped with an amphiphilic fluorescent probe that has different solubility in two surface phases resulting in contrast when excited using light from an arc lamp or a laser. Despite the usefulness of fluorescence microscopy, it has a few drawbacks. First the fluorophores added, although in trace amount, can alter the original monolayer if their concentration is too high. Secondly, observation of more highly ordered phases is difficult because they reject the fluorescent molecular probe [59]. Also, problems may occur due to dissolution of the probe into

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The problems associated with fluorescence microscopy due to the addition of fluorescent dye can be avoided with Brewster angle microscopy (BAM) as it does not require any probe molecule. When a light beam is directed onto a water surface a portion of it is reflected, and the reflected light intensity depends upon the angle of incidence. For plane polarized light, there exists a certain angle of incidence at which no reflection occurs known as the Brewster angle. For water at room temperature the Brewster angle is 53�. For monolayer study at the airwater interface, light is directed onto the surface at the Brewster angle and the reflected light is observed using a CCD camera. The monolayer on the water surface has a different refractive index and this leads to violation of the Brewster angle condition and increased reflectivity so

X-rays provide a very sensitive tool for the study of monolayers by virtue of their small wavelength of few Å. Unlike visible light, reflection of X-rays from a denser medium coming from rarer (gas) medium at a certain critical angle are totally reflected without any diffraction into the denser medium (solid or liquid) [60]. Grazing incident X-ray diffraction (GIXD) has been an important technique used for investigating in situ structural arrangement of amphiphiles for more than two decades. In GIXD the incident angle, α is slightly below the critical angle. The small incident angle is desirable for larger penetration of X-rays. Two commonly used X-ray techniques are X-ray reflectivity (XRR) and

XRR provides data that can be used to estimate the thickness of the monolayer, d calculated

Besides thickness of monolayer, XRR also provides information about the electron density distribution perpendicular to the interface and the roughness of the monolayer. XRR data can be used to model thickness of different segments of the monolayer. XD provides information about the two-dimensional arrangement of the molecules on the surface and the tilt angles of

nλ ¼ 2dsinθ (2)

the subphase.

4.5. Brewster angle microscopy

the monolayer domains will appear bright [61].

4.6. X-ray reflectivity and scattering

diffraction (XD).

using Bragg's law:

the molecules.

### 4.3. Transfer as Langmuir-Blodgett film for study by atomic force microscopy

The deposited monolayer can be transferred to a solid support. For example, CaF2 plates are used for transmission infrared spectroscopy while germanium silicon and ZnSe plates are used for internal reflection infrared spectroscopy [21]. The substrate most commonly used for atomic force microscopy (AFM) studies is freshly cleaved mica, because of its atomically flat surface. AFM is helpful for the visualization of coexisting phases with great resolution, which can be down to a few nanometers, and not readily visualized by other methods. Care must be taken while analyzing the micrograph that the structures visualized are a true representation of what was on the deposited monolayer and are not artifacts created during the transfer process.

The monolayer can be transferred to a solid support by Langmuir-Blodgett deposition which is carried out as a vertical transfer or by Langmuir-Schaefer transfer which is carried out as a horizontal transfer. The Langmuir-Blodgett transfer method can be performed in a number of ways depending upon the nature of the substrate (hydrophobic or hydrophilic) and the number of layers desired. For a hydrophilic substrate, the transfer is performed by immersing the substrate into the subphase prior to spreading the monolayer. The monolayer is then compressed to a desired surface pressure and the substrate is lifted out of the subphase at a suitable speed while maintaining a constant surface pressure. A series of immersion and emersion cycle can be performed to generate multilayers. In the horizontal transfer method, the substrate is held horizontally above the compressed monolayer, lowered until it makes contact with the water surface and is then lifted gently thus transferring the monolayer onto the substrate.

#### 4.4. Fluorescence microscopy

Fluorescence microscopy is another important method that is often used for studying interfacial behavior of monolayers. Even though the images from AFM help visualize the structure of monolayers at a near molecular level resolution they only provide snapshots of the continuous process and require transfer to a substrate. Fluorescence microscopy has an edge over AFM or any other static visualization method, as it provides a real time picture of the events as they occur.

Fluorescence microscopy provided the first evidence of coexisting domains in the plateau region of Π-A isotherms of Langmuir monolayers. In fluorescence microscopy, the monolayer is doped with an amphiphilic fluorescent probe that has different solubility in two surface phases resulting in contrast when excited using light from an arc lamp or a laser. Despite the usefulness of fluorescence microscopy, it has a few drawbacks. First the fluorophores added, although in trace amount, can alter the original monolayer if their concentration is too high. Secondly, observation of more highly ordered phases is difficult because they reject the fluorescent molecular probe [59]. Also, problems may occur due to dissolution of the probe into the subphase.

#### 4.5. Brewster angle microscopy

Two common methods applied to measure surface potential are the vibrating electrode and the ionizing electrode. The vibrating electrode method also known as Kelvin probe method uses a plate-like electrode placed at a certain distance above air-water interface. The electrode is connected to the reference electrode placed in the subphase. The electrode above the interface is periodically vibrated during measurements. The ionizing electrode method employs the same measurement setup as the vibrating plate method. In this method, the electrodes are coated with α-emitters such as 241Am or 210Po to increase the conductivity

The deposited monolayer can be transferred to a solid support. For example, CaF2 plates are used for transmission infrared spectroscopy while germanium silicon and ZnSe plates are used for internal reflection infrared spectroscopy [21]. The substrate most commonly used for atomic force microscopy (AFM) studies is freshly cleaved mica, because of its atomically flat surface. AFM is helpful for the visualization of coexisting phases with great resolution, which can be down to a few nanometers, and not readily visualized by other methods. Care must be taken while analyzing the micrograph that the structures visualized are a true representation of what was on the deposited monolayer and are not artifacts created during the transfer

The monolayer can be transferred to a solid support by Langmuir-Blodgett deposition which is carried out as a vertical transfer or by Langmuir-Schaefer transfer which is carried out as a horizontal transfer. The Langmuir-Blodgett transfer method can be performed in a number of ways depending upon the nature of the substrate (hydrophobic or hydrophilic) and the number of layers desired. For a hydrophilic substrate, the transfer is performed by immersing the substrate into the subphase prior to spreading the monolayer. The monolayer is then compressed to a desired surface pressure and the substrate is lifted out of the subphase at a suitable speed while maintaining a constant surface pressure. A series of immersion and emersion cycle can be performed to generate multilayers. In the horizontal transfer method, the substrate is held horizontally above the compressed monolayer, lowered until it makes contact with the water

Fluorescence microscopy is another important method that is often used for studying interfacial behavior of monolayers. Even though the images from AFM help visualize the structure of monolayers at a near molecular level resolution they only provide snapshots of the continuous process and require transfer to a substrate. Fluorescence microscopy has an edge over AFM or any other static visualization method, as it provides a real time picture of the events as

Fluorescence microscopy provided the first evidence of coexisting domains in the plateau region of Π-A isotherms of Langmuir monolayers. In fluorescence microscopy, the monolayer

surface and is then lifted gently thus transferring the monolayer onto the substrate.

4.3. Transfer as Langmuir-Blodgett film for study by atomic force microscopy

of the air gap [60].

222 Cell Culture

process.

4.4. Fluorescence microscopy

they occur.

The problems associated with fluorescence microscopy due to the addition of fluorescent dye can be avoided with Brewster angle microscopy (BAM) as it does not require any probe molecule. When a light beam is directed onto a water surface a portion of it is reflected, and the reflected light intensity depends upon the angle of incidence. For plane polarized light, there exists a certain angle of incidence at which no reflection occurs known as the Brewster angle. For water at room temperature the Brewster angle is 53�. For monolayer study at the airwater interface, light is directed onto the surface at the Brewster angle and the reflected light is observed using a CCD camera. The monolayer on the water surface has a different refractive index and this leads to violation of the Brewster angle condition and increased reflectivity so the monolayer domains will appear bright [61].

### 4.6. X-ray reflectivity and scattering

X-rays provide a very sensitive tool for the study of monolayers by virtue of their small wavelength of few Å. Unlike visible light, reflection of X-rays from a denser medium coming from rarer (gas) medium at a certain critical angle are totally reflected without any diffraction into the denser medium (solid or liquid) [60]. Grazing incident X-ray diffraction (GIXD) has been an important technique used for investigating in situ structural arrangement of amphiphiles for more than two decades. In GIXD the incident angle, α is slightly below the critical angle. The small incident angle is desirable for larger penetration of X-rays. Two commonly used X-ray techniques are X-ray reflectivity (XRR) and diffraction (XD).

XRR provides data that can be used to estimate the thickness of the monolayer, d calculated using Bragg's law:

$$n\lambda = 2d \sin \theta \tag{2}$$

Besides thickness of monolayer, XRR also provides information about the electron density distribution perpendicular to the interface and the roughness of the monolayer. XRR data can be used to model thickness of different segments of the monolayer. XD provides information about the two-dimensional arrangement of the molecules on the surface and the tilt angles of the molecules.

#### 4.7. Infrared spectroscopy

Infrared Spectroscopy is another important technique widely used to characterize the conformation and orientation of monolayers transferred onto the solid support or in situ at the airwater interface. It is desirable to carry out the experiments in situ to avoid problems due to artifacts during transfer. Infrared reflection absorption spectroscopy (IRRAS) and polarization modulated (PM)-IRRAS are two versions of Fourier transform infrared spectroscopy (FTIR) frequently employed for the study of Langmuir monolayers [62].

term inside parenthesis is the excess surface area. Negative values of ΔGexc indicate favorable interactions resulting in a more stable monolayer and positive ΔGexc indicates unfavorable interactions and possible phase separation. Furthermore, one could calculate a free energy of

where R and T are gas constant and temperature respectively. Phase separation may also occur upon compression in mixed monolayers, resulting in coexisting phases of different composi-

Gangliosides have been the subject of many studies as monolayers and we survey some of the reported results. In an early study using surface pressure isotherms of gangliosides GM1, GM2, GM3, GD1a, GD3 and GT1, it was found that increasing the number of sialic acid residues caused an increase in the prevalence of the LE phase and that the surface pressure isotherms shifted to higher molecular areas and the monolayers became more compressible [64]. This trend was attributed to increasing electrostatic repulsions with introduction of additional negatively charged sialic acids in the structures. In a related study using surface pressure and surface potential measurements, it was found that the strength of interaction of Ca2+ ions in the subphase with gangliosides depended on the number and arrangement of the sialic acid units [65]. A study using surface pressure isotherms of mixed monolayers of GM1, GD1a, and GT1 with phospholipids showed positive deviations from ideality at 30 mN m�1 for DPPE, a phosphatidylinositol and a phosphatidylserine, but negative deviations from ideality for mixtures with DPPC [66]. In contrast, the interaction of gangliosides with neutral glycosphingolipids was found to be favorable. Mixed monolayers of ceramide or of glucosyl ceramide with gangliosides were found to show favorable interactions, while those of lactosyl ceramide and gangliosides showed immiscibility [67]. The activity of the enzyme phospholipase A2 injected into the subphase against mixed monolayers of dilauroylphosphatidylcholine and gangliosides GM1, GD1a, and GT1b was found to be strongly inhibited [68]. Gangliosides were also found to inhibit the activity of phospholipase C against model membrane systems including monolayers [69]. The sensitivity of monolayer parameters to trace impurity of peptide materials in isolated gangliosides has been

tion. Occurrence of critical points and azeotropes in two-dimensions is also possible.

6. Survey of studies of monolayers containing glycolipids

emphasized along with provision of methods for rigorous purification [70].

Gangliosides are known for their ability to form microdomains in biological membranes. The partitioning of ganglioside, GM1 in phase separated DOPC/DPPC LB films transferred to

ΔGmix ¼ ΔGid þ ΔGexc (4)

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ΔGid ¼ RT xð Þ 1ln x1 þ x2ln x2 (5)

mixing, ΔGmix using the following equations:

6.1. Gangliosides

ΔGid is the ideal value that can be calculated using

In IRRAS, the sample is irradiated with an IR beam and the intensity of the reflected beam is measured as a function of wavelength. The measurements can be carried out with p- and spolarized light at various angles of incidence above or below the Brewster angle. If the samples are on metal substrates, a "surface selection" rule is followed which states that only vibrational dipole moments oriented perpendicular to the substrate are observed. IRRAS has a disadvantage while studying Langmuir films, the strong absorption of water vapor conceals the spectral regions with desired molecular information. PM-IRRAS, which is insensitive to IR absorption of water vapor, was introduced to avoid such problems. In PM-IRRAS, the incident beam is alternated between s- and p-polarization at a frequency of tens of kHz [63] and differential reflectivity is calculated. Besides these two IR spectroscopic techniques, there are others such as surface-enhanced infrared-absorption spectroscopy (SEIRA) and attenuated total reflection ATR-FTIR spectroscopy.