**5. Results and discussion**

#### **5.1. Characteristics of physico-chemical properties of used preparations**

Preparations of pure β-glucans and the preparation of dried spent brewer's yeast examined in this work have proven to be efficient in correcting blood lipid metabolism, which has been shown in biological experiment on rats, which were given atherogenic diet, containing 20% of fat and 1% of cholesterol, and at the same time preparations of beta glucans: soluble (BG-CMG) and insoluble (BG-HP) or dried, spent brewer's yeast (SBY).

These preparations differed in physico-chemical properties, i.e. particle size, solubility, the ability of water absorption and viscosity. These properties had a significant influence on the final effects obtained after their use in the experiment.

During the examination of distribution of particle size of powdered β-glucans in comparison with spent brewer's yeast preparations, it has been established that BG-HP preparation contained 60% of all particles of 59.4 µm in diameter and only about 5% of about 5 µm in diameter. The preparation of soluble β-glucan BG-CMG contained at least three clusters of particles of varied size, i.e. appropriately 0.2 µm, 3.5 µm and 90.2 µm, whereas the majority of particles (about 20% of all) had a diameter of 90.2 µm. Dried spent brewer's yeast were characterised by three groups of varied size, and their greatest share (over 40%) had the size of about 63.6 µm. Dried spent brewer's yeast particles were similar in size to particles of βglucan BG-HP preparation.

Table 2 presents the percentage cumulated participation of particles in preparation content (e.g. d5 means that 5% of particles has the size below the value shown in the table, d50 = 50% of particles has the size not exceeding the value presented in the table, etc.).

Particles of all examined preparations absorbed water during hydration. Preparation of native β-glucan BG-HP from brewer's yeast and dried spent brewer's yeast did not dissolve or dissolved only partially creating unstable water solutions with the tendency to create residue. Water solutions of β-glucan BG-CMG preparations were very viscid (65.2 mPas for 1% water solution and 574.9 mPas for 3% solution), whereas the preparations of β-glucan HP preparations and dried spent brewer's yeast, dissolved in water only partially, creating nonviscid or only slightly viscid solutions (0.4-1.4 mPas) - tab. 3.

272 Lipid Metabolism

at P<0.05.

cholesterol content.

*4.2.5. Statistical analysis* 

**5. Results and discussion** 

glucan BG-HP preparation.

**Atherogenic index - atherogenic** index was calculated as the relation of assessed lipid fraction (HDL-Chol or LDL-Chol) to total cholesterol content (Total-Chol). This index allowed for defining the changes of these fraction participation in relation to total

Data is presented as means ± standard deviation (SD). Obtained results were statistically analysed with STATGRAPHIC programme for Windows (v. 4.1.). The data was analysed using one-way analysis of variance (ANOVA). When a significant F ratio was found, Tukey's multiple-comparison tests were conducted. Differences were considered significant

Preparations of pure β-glucans and the preparation of dried spent brewer's yeast examined in this work have proven to be efficient in correcting blood lipid metabolism, which has been shown in biological experiment on rats, which were given atherogenic diet, containing 20% of fat and 1% of cholesterol, and at the same time preparations of beta glucans: soluble

These preparations differed in physico-chemical properties, i.e. particle size, solubility, the ability of water absorption and viscosity. These properties had a significant influence on the

During the examination of distribution of particle size of powdered β-glucans in comparison with spent brewer's yeast preparations, it has been established that BG-HP preparation contained 60% of all particles of 59.4 µm in diameter and only about 5% of about 5 µm in diameter. The preparation of soluble β-glucan BG-CMG contained at least three clusters of particles of varied size, i.e. appropriately 0.2 µm, 3.5 µm and 90.2 µm, whereas the majority of particles (about 20% of all) had a diameter of 90.2 µm. Dried spent brewer's yeast were characterised by three groups of varied size, and their greatest share (over 40%) had the size of about 63.6 µm. Dried spent brewer's yeast particles were similar in size to particles of β-

Table 2 presents the percentage cumulated participation of particles in preparation content (e.g. d5 means that 5% of particles has the size below the value shown in the table, d50 = 50%

Particles of all examined preparations absorbed water during hydration. Preparation of native β-glucan BG-HP from brewer's yeast and dried spent brewer's yeast did not dissolve or dissolved only partially creating unstable water solutions with the tendency to create residue. Water solutions of β-glucan BG-CMG preparations were very viscid (65.2 mPas for

of particles has the size not exceeding the value presented in the table, etc.).

**5.1. Characteristics of physico-chemical properties of used preparations** 

(BG-CMG) and insoluble (BG-HP) or dried, spent brewer's yeast (SBY).

final effects obtained after their use in the experiment.


**Table 2.** The comparison of cumulated distribution of particle size of examined β-glucans and powdered spent brewer's yeast.

β-glucan particles isolated from spent brewer's yeast, in the initial phase of hydration increased their volume from 3 (soluble BG-CMG) to about 5 times (insoluble BG-HP). These glucans were characterised by the fact that in the final stage of hydration, these particles decreased. The increasing, and then decreasing sizes of particles in the initial stage of hydration, may constitute the proof of tearing hydrogen and covalency bindings of helix of pre-hydrated β-glucans.


**Table 3.** The characteristics of viscositya/ of 1% and 3% water solutions of β-glucan preparations in comparison with 1% and 3% viscosity of water solutions of spent brewer's yeast.

a/ identical letter signs in columns equal the lack of a significant difference between compared mean values

Limited solubility in water of native β-glucan HP might be explained with the presence in the structure of long, side chains with bindings β-(1→6), which can cause high crystality and insolubility of this β-glucan [6].

Weak hydration of complex structure of helix of high molecular weight β-glucans is the reason for their mutual intermolecular interactions between β-(1→3)-D and β-(1→6)-Dglucan bindings, of strenght exceeding the interactions between bindings of β-glucan and water particle bindings or another solvent. Lowering of degree of polymerisation of beta glucans with β-(1→3)/(1→6)-D-glucan bindings to DP below 20, results in the weakening of intermolecular interactions, therefore, creating new bindings between β-glucan particles and solvent, causing its dissolution [2,4].

The apparent improvement of β-glucan solubility in water solutions, without polymer degradation, can be achieved by activities stabilizing their scattering in water environment. [49]. It is possible to achieve stable scattering of β-glucans in water solutions, e.g. through

#### 274 Lipid Metabolism

microwave heating, with temperature range 100-121oC and increased pressure within 4-10 min. Quite advantageous effects in the modification of physico-chemical properties of βglucans are also obtained while using ultrasounds, as the method does not change the chemical structure of polymer's particle size, resulting only in the decrease in the particle size, through breaking of the most sensitive chemical bindings. This method is used to obtain the so-called micronized β-glucan preparations. The process of micronization improves the distribution and stability of slightly soluble powders in water environment. The method also leads to permanent reduction of solution viscosity. According to the definition in the Polish Pharmacopoeia, micronized powders should contain particles of average diameter below 10 µm, at admissible 20% particle content of up to 50 µm diameter.

Spent Brewer's Yeast and Beta-Glucans Isolated from Them as Diet Components

The preparation of spent brewer's yeast showed over 50-100 times higher antioxidant activity than β**-**glucan BG-HP isolated from them and β**-**glucan BG-CMG subject to chemical modification. Such high antioxidant activity of spent dried yeast preparation might result from squalene contained in the preparation, whose presence was confirmed in parallel

In numerous research conducted on animals and in clinical research on people, hypocholesterolemic influence of β-glucans of cereal origin containing (1→3)/(1→4)-β bindings has been proven. This influence has been confirmed in case of β-glucans isolated from fungi, containing (1→3)/(1→6)-β bindings, however, it has been weaker in comparison with cereal β-glucans. However, there is no literature data on the influence of β-glucans from spent brewer's yeast on the lipid metabolism of both experimental animals and people [52, 53]. During biological experiment, animals given diets supplemented with β**-**glucans HP and CMG and dried spent brewer's yeast grew at a comparable pace. Body mass of animals after the experiment ranged from 390.8 to 412.3 g, and daily growth ranged from 6.5 to 7.2 g. Slight differences between particular groups were not statistically significant. In the groups of animals receiving β**-**glucans or spent brewer's yeast, feed efficiency ratio (FER) did not

CONTROL 18.1a ± 0.4 0.38a ± 0.03

BG-CMG10 18.0a ± 04 0.38a ± 0.03 BG-CMG100 17.7a ± 0.5 0.36a ± 0.03 BG-HP10 17.7a ± 0.7 0.37a ± 0.04 BG-HP100 17.9a ± 0.4 0.38a ± 0.02 SBY – spent brewer's yeast 17.9a ± 0.9 0.38a ± 0.03  *SEM 2/ (p3/) 0.067 (0.710) 0.004 (0.874)*  **Table 4.** The comparison of intake and nutritious efficiency of diets supplemented with β-glucan

1/ mean values ± standard deviation for n = 7 or 8, 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05, 4/ Feed efficiency ratio: body mass growth (g/day) x diet intake-1 (g/day)-1, identical letter signs in columns

Table 5 and Fig. 10-11 show the results determing the influence of β-glucans and dried spent brewer's yeast on total cholesterol concentrationm HDL and LDL fraction cholesterol and

Total cholesterol concentration in the blood of rats in control group, given a model atherogenic diet (1% cholesterol, 20% fat) amounted to 3.79 mmol/l and was significantly

research at the rate of 125 mg per 100 g of powder.

differ significantly and ranged from 0.36 to 0.38 (tab. 4).

Group – experimental factor Diet intake

equal the lack of a significant difference between compared mean values

preparations and spent brewer's yeast1/ (n=63).

triacylglycerols in rat blood plasma.

**5.2. Blood lipid metabolism** 

β-glucans

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 275

[g/day] Feed efficiency ratio FER4/

CMG β-glucan preparation of modified chemical properties is the example of β-glucan examined in this work. HP native beta-glucan obtained from spent brewer's yeast underwent the process of carboxymethylation, and in turn led to obtaining BG-CMG β-glucan preparation of modified viscosity, dozen times higher than the viscosity of HP native β-glucan (tab. 3).

Carboxymethylated β-glucan CMG strongly absorbed water creating solutions of very high viscosity and stability, but also not showing properties of sedimentation. Native HP β-glucan did not dissolve in water, created nonviscous solutions, and moreover, strongly sedimented. Higher viscosity of carboxymethylated CMG β-glucan than native HP β-glucan, despite comparable particle size, might be explained by the fact that carboxymethylated CMG β-glucan might be characterised by higher particle mass resulting from additional methyl groups contained in their structure, which in turn enabled ist stronger hydration and viscosity increase.

Many biological experiments showed that the change of physicochemical parameters of βglucans as a result of chemical or enzymatic modification might lead to the change of their pro-health influence, changes in sensoric quality [50, 51], therefore, it is extremely important to conduct research in case of each newly obtained preparation.

During the evaluation of bioactive substance properties very often the evaluation of antioxidant properties is conducted, which are also checked for native β-glucan HP and βglucan BG-CMG modified through carboxymethylation. The preparations of these β-glucans were characterised by varied antioxidant activity expressed as TEAC (the number of Trolox milimols for each 1 g of preparation) - Fig. 9.

The preparation of spent brewer's yeast showed over 50-100 times higher antioxidant activity than β**-**glucan BG-HP isolated from them and β**-**glucan BG-CMG subject to chemical modification. Such high antioxidant activity of spent dried yeast preparation might result from squalene contained in the preparation, whose presence was confirmed in parallel research at the rate of 125 mg per 100 g of powder.

#### **5.2. Blood lipid metabolism**

274 Lipid Metabolism

microwave heating, with temperature range 100-121oC and increased pressure within 4-10 min. Quite advantageous effects in the modification of physico-chemical properties of βglucans are also obtained while using ultrasounds, as the method does not change the chemical structure of polymer's particle size, resulting only in the decrease in the particle size, through breaking of the most sensitive chemical bindings. This method is used to obtain the so-called micronized β-glucan preparations. The process of micronization improves the distribution and stability of slightly soluble powders in water environment. The method also leads to permanent reduction of solution viscosity. According to the definition in the Polish Pharmacopoeia, micronized powders should contain particles of average diameter below 10 µm, at admissible 20% particle content of up to 50 µm diameter. CMG β-glucan preparation of modified chemical properties is the example of β-glucan examined in this work. HP native beta-glucan obtained from spent brewer's yeast underwent the process of carboxymethylation, and in turn led to obtaining BG-CMG β-glucan preparation of modified viscosity, dozen times higher than the viscosity of HP native β-glucan (tab. 3).

Carboxymethylated β-glucan CMG strongly absorbed water creating solutions of very high viscosity and stability, but also not showing properties of sedimentation. Native HP β-glucan did not dissolve in water, created nonviscous solutions, and moreover, strongly sedimented. Higher viscosity of carboxymethylated CMG β-glucan than native HP β-glucan, despite comparable particle size, might be explained by the fact that carboxymethylated CMG β-glucan might be characterised by higher particle mass resulting from additional methyl groups contained in their structure, which in turn enabled ist stronger hydration and viscosity increase. Many biological experiments showed that the change of physicochemical parameters of βglucans as a result of chemical or enzymatic modification might lead to the change of their pro-health influence, changes in sensoric quality [50, 51], therefore, it is extremely important

During the evaluation of bioactive substance properties very often the evaluation of antioxidant properties is conducted, which are also checked for native β-glucan HP and βglucan BG-CMG modified through carboxymethylation. The preparations of these β-glucans were characterised by varied antioxidant activity expressed as TEAC (the number of Trolox

**Figure 9.** The antioxidant activity of β-glucan preparations in comparison with spent brewer's yeast.

TEAC (milimol of Trolox/1 g of preparation)

0 0.5 1 1.5 2 2.5 3

2.67

to conduct research in case of each newly obtained preparation.

milimols for each 1 g of preparation) - Fig. 9.

0.054

SBY

BG-HP

BG-CMG

0.018

In numerous research conducted on animals and in clinical research on people, hypocholesterolemic influence of β-glucans of cereal origin containing (1→3)/(1→4)-β bindings has been proven. This influence has been confirmed in case of β-glucans isolated from fungi, containing (1→3)/(1→6)-β bindings, however, it has been weaker in comparison with cereal β-glucans. However, there is no literature data on the influence of β-glucans from spent brewer's yeast on the lipid metabolism of both experimental animals and people [52, 53].

During biological experiment, animals given diets supplemented with β**-**glucans HP and CMG and dried spent brewer's yeast grew at a comparable pace. Body mass of animals after the experiment ranged from 390.8 to 412.3 g, and daily growth ranged from 6.5 to 7.2 g. Slight differences between particular groups were not statistically significant. In the groups of animals receiving β**-**glucans or spent brewer's yeast, feed efficiency ratio (FER) did not differ significantly and ranged from 0.36 to 0.38 (tab. 4).


**Table 4.** The comparison of intake and nutritious efficiency of diets supplemented with β-glucan preparations and spent brewer's yeast1/ (n=63).

1/ mean values ± standard deviation for n = 7 or 8, 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05, 4/ Feed efficiency ratio: body mass growth (g/day) x diet intake-1 (g/day)-1, identical letter signs in columns equal the lack of a significant difference between compared mean values

Table 5 and Fig. 10-11 show the results determing the influence of β-glucans and dried spent brewer's yeast on total cholesterol concentrationm HDL and LDL fraction cholesterol and triacylglycerols in rat blood plasma.

Total cholesterol concentration in the blood of rats in control group, given a model atherogenic diet (1% cholesterol, 20% fat) amounted to 3.79 mmol/l and was significantly

#### 276 Lipid Metabolism

higher than in all experimental groups. A significantly higher (p=0.043) concentration of LDL fractions (1.8 mmol/l) was also observed in the control group. Total cholesterol concentration in the blood of rats given β-glucans preparations from spent brewer's yeast in the daily amount equalling 100 mg/kg of bady mass or preparation of spent brewer's yeast ranged from 2.82 to 2.97 mmol/l.The differences between these three groups were, however, insignificant statistically. The concentration of LDL-cholesterol fraction ranged in these groups respectively from 1.09 to 1.11 mmol/l (tab. 5).

Spent Brewer's Yeast and Beta-Glucans Isolated from Them as Diet Components

22.2% (in the group with β-glucan BG-CMG100) and of 25.6% (in the group with β-glucan BG-HP100) – Fig. 10. In these groups, LDL-cholesterol fraction was respectively lower of 38.3

The type of β-glucans from spent brewer's yeast (BG-CMG and BG-HP) did not influence significantly the level of total cholesterol in blood (p = 0.638), whereas their dosage (p = 0.002) had a significant influence. In rats from control group on a model athrogenic diet (1% cholesterol, 20% fat), cholesterol of HDL fraction amounted to 41.7% of total cholesterol, and LDL fraction cholesterol – 47.5%. The use of examined preparations in diet supplementation caused changes in the configuration of these fractions leading to lowering the LDL fraction participation and increasing percentage participation of HDL fraction cholesterol. Changes of these fractions in total cholesterol (LDL-cholesterol fraction below 40% of total cholesterol), were particularly visible in groups of rats given CMG and HP β-glucan preparations from spent brewer's yeast in the amount equalling 100 mg/kg of body mass

**Figure 11.** Reduction ratio of LDL and HDL-cholesterol fractions and triacylglycerols in peripheral blood of experimental animals on atherogenic diets caused by diet supplementation with β-glucan


Table 6 presents the ratios of atherogenic factors of examined preparations. The ratio of HDL fraction to Chol-C and HDL to LDL fractions, after the experiment completion was in each case significantly higher in comparison with control group, which suggests that each of the examined preparations, regardless of the type and dosage, significantly improves the


LDL HDL

4.4b




BG-CMG10 BG-CMG100 BG-HP10 BG-HP100

SBY

In groups with beta glucan participation, the concentration of triacylglycerols ranged from 0.67 to 0.76 mmol/l plasma, i.e. was lower in the control group from 14.8 to 25.6% (tab. 7, Fig.

Dried spent brewer's yeast given to animals in a daily dosage of 100 mg/kg of body mass contributed to lower the concentration of TG in blood in relation to control group of 27.2%

/ identical letters indicate no significant difference).

to 39.1% in comparison with control group (Fig. 11).

preparations and dried spent brewer's yeast (\*






lipid metabolism of animals given atherogenic diet.

daily.

% reduction of LDL i HDL-Chol fraction

10).

(p = 0,008) – tab. 7, Fig. 12.

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 277


**Table 5.** Selected lipid parameters of peripheral blood in experimental animals given atherogenic diets, supplemented with β-glucan preparations and spent brewer's yeast preparation1/ (n=63).

1/ mean values ± standard deviation for n = 7 or 8; 2/ SEM – standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of significant difference between compared mean values

**Figure 10.** The degree of total cholesterol reduction in peripheral blood of experimental animals on atherogenic diets caused by diet supplementation with β-glucan preparations and spent brewer's yeast (\*/ identical letters indicate no significant difference).

It was concluded that diet supplementation with spent brewer's yeast preparation, contributed to achieving lower concentration of total cholesterol of 21.6%. In groups of animals given diets containing β-glucans in a daily dosage of 10 and 100 mg/kg of body mass, the concentration of total cholesterol in blood was lower than in control group of 22.2% (in the group with β-glucan BG-CMG100) and of 25.6% (in the group with β-glucan BG-HP100) – Fig. 10. In these groups, LDL-cholesterol fraction was respectively lower of 38.3 to 39.1% in comparison with control group (Fig. 11).

276 Lipid Metabolism

Group –

β-glucans

**% total cholesterol reduction**

experimental factor

higher than in all experimental groups. A significantly higher (p=0.043) concentration of LDL fractions (1.8 mmol/l) was also observed in the control group. Total cholesterol concentration in the blood of rats given β-glucans preparations from spent brewer's yeast in the daily amount equalling 100 mg/kg of bady mass or preparation of spent brewer's yeast ranged from 2.82 to 2.97 mmol/l.The differences between these three groups were, however, insignificant statistically. The concentration of LDL-cholesterol fraction ranged in these

Control 3.79a ± 0.13 1.58a ± 0.15 1.80a ± 0.12 0.41a ± 0.03

**Table 5.** Selected lipid parameters of peripheral blood in experimental animals given atherogenic diets,

1/ mean values ± standard deviation for n = 7 or 8; 2/ SEM – standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of significant difference between compared mean values

**Figure 10.** The degree of total cholesterol reduction in peripheral blood of experimental animals on atherogenic diets caused by diet supplementation with β-glucan preparations and spent brewer's yeast


It was concluded that diet supplementation with spent brewer's yeast preparation, contributed to achieving lower concentration of total cholesterol of 21.6%. In groups of animals given diets containing β-glucans in a daily dosage of 10 and 100 mg/kg of body mass, the concentration of total cholesterol in blood was lower than in control group of



(\*/ identical letters indicate no significant difference).




supplemented with β-glucan preparations and spent brewer's yeast preparation1/ (n=63).

BG-CMG10 3.23bc ± 0.23 1.52ab ± 0.14 1.35bc ± 0.14 0.26bc ± 0.04 BG-CMG100 2.95bc ± 0.17 1.51ab ± 0.12 1.11de ± 0.15 0.33bc ± 0.03 BG-HP10 3.20b ± 0.38 1.56ab ± 0.15 1.31bc ± 0.23 0.31bc ± 0.05 BG-HP100 2.82bc ± 0.38 1.38ab ± 0.19 1.10e ± 0.19 0.30ab ± 0.16 SBY 2.97bc ± 0.36 1.39b ± 0.13 1.29bce ± 0.26 0.30bc ± 0.04 *SEM p = 0.0065 p = 0.415 p=0.043 p=0.0295* 

Cholesterol fractions HDL [mmol/l] LDL [mmol/l] VLDL

[mmol/l]

BG-CMG10 BG-CMG100 BG-HP10 BG-HP100

SBY

groups respectively from 1.09 to 1.11 mmol/l (tab. 5).

Total cholesterol [mmol/l]

The type of β-glucans from spent brewer's yeast (BG-CMG and BG-HP) did not influence significantly the level of total cholesterol in blood (p = 0.638), whereas their dosage (p = 0.002) had a significant influence. In rats from control group on a model athrogenic diet (1% cholesterol, 20% fat), cholesterol of HDL fraction amounted to 41.7% of total cholesterol, and LDL fraction cholesterol – 47.5%. The use of examined preparations in diet supplementation caused changes in the configuration of these fractions leading to lowering the LDL fraction participation and increasing percentage participation of HDL fraction cholesterol. Changes of these fractions in total cholesterol (LDL-cholesterol fraction below 40% of total cholesterol), were particularly visible in groups of rats given CMG and HP β-glucan preparations from spent brewer's yeast in the amount equalling 100 mg/kg of body mass daily.

**Figure 11.** Reduction ratio of LDL and HDL-cholesterol fractions and triacylglycerols in peripheral blood of experimental animals on atherogenic diets caused by diet supplementation with β-glucan preparations and dried spent brewer's yeast (\* / identical letters indicate no significant difference).

Table 6 presents the ratios of atherogenic factors of examined preparations. The ratio of HDL fraction to Chol-C and HDL to LDL fractions, after the experiment completion was in each case significantly higher in comparison with control group, which suggests that each of the examined preparations, regardless of the type and dosage, significantly improves the lipid metabolism of animals given atherogenic diet.

In groups with beta glucan participation, the concentration of triacylglycerols ranged from 0.67 to 0.76 mmol/l plasma, i.e. was lower in the control group from 14.8 to 25.6% (tab. 7, Fig. 10).

Dried spent brewer's yeast given to animals in a daily dosage of 100 mg/kg of body mass contributed to lower the concentration of TG in blood in relation to control group of 27.2% (p = 0,008) – tab. 7, Fig. 12.

#### 278 Lipid Metabolism


Spent Brewer's Yeast and Beta-Glucans Isolated from Them as Diet Components

Lipids Total cholesterol

mg/1 g of wet

tissue mg/g of lipids

± 42.6

± 26.4

± 28.1

± 38.8

Two-factor analysis showed the lack of significant influence of the type of β-glucan (soluble and insoluble) (p=0.091). Also the dosage of these beta glucans did not influence the

Table 8 compiles the total content of lipids and cholesterol in liver of rats on atherogenic diets supplemented with β-glucans and dried spent brewer's yeast. The content of lipids was presented both in the equivalent of the so-called wet tissue and dry mass, whereas cholesterol content was presented in the equivalent of wet tissue and in reference to total

> g/100 g s.m. of the liver

Control 20.9a ± 2.8 47.3bc ± 1.3 82.5a ± 7.5 432.1ab ± 23.7

SBY 19.1a ± 2.7 43.4ab ± 4.4 65.1bc ± 5.0 369.1bc ± 30.1 *SEM2/ (p3) 0.110 (0.62) 0.513 (0.007) 14.4 (0.05) 8.16 (0.001)* 

BG-CMG10 19.5a ± 3.3 42.0ab ± 2.7 69.9b ± 6.0 352.1c

BG-CMG100 18.6a ± 3.5 39.6a ± 4.1 67.5bc ± 6.5 340.9c

BG-HP10 18.4a ± 1.9 42.5ab ± 3.9 66.0bc ± 1.1 361.4c

BG-HP100 18.7a ± 34 39.1a ± 3.7 65. 1bc ± 5.4 348.3c

**Table 8.** The content of lipids and cholesterol in the liver of rats on atherogenic diets supplemented

1/ mean values ± standard deviation for n = 5 or 6; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05, identical letter signs in columns equal the lack of a significant difference between compared mean

Total lipid content in fresh liver mass of rats did not differ significantly (p=0.110) in all groups and ranged from 18.4 to 20.9 g/100 g of wet tissue. Statistical analysis showed that the type of tested β-glucans (p=0.287) did not influence the concentration of lipids in the liver, whereas significantly better effects were observed when a higher dosage (p=0.003) was

In comparison with cholesterol level, it has been stated that neither the type (p=0,444) nor the amount of β-glucans (p=0.720) in the diet, significantly influence the cholesterol

Table 9 presents the percentage participation of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in total lipid pool in livers. The concentration of these groups of fatty acids was not influenced by the

The type of β-glucan from spent brewer's yeast did not significantly influence the ratio of blood lipid parameters metabolism in rats on atherogenic diet containing 1% of cholesterol. The concentration of HDL-cholesterol fraction and triacylglycerols in rat blood in this case

reduction ratio of TG in rat blood (p=0.786).

with β-glucans and dried spent brewer's yeast.

used β-glucans, but by their dosage (p=0.018).

depended neither on the type of β-glucan nor its dosage.

g/100 g of wet tissue

lipid content.

factor

β-glucans

values

used.

concentration in the liver.

Group – experimental

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 279

**Table 6.** The comparison of atherogenic factors characteristic of blood of experimental animals given atherogenic diets with β-glucan additive and spent brewer's yeast1/.

1/ mean values ± standard deviation for n = 7 or 8; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of significant difference between compared mean values


**Table 7.** Selected lipid parameters of peripheral blood of test animals on atherogenic diets, supplemented with β-glucans and the preparation of dried brewer's yeast 1/.

1/ mean values ± standard deviation for n = 7 or 8; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of a significant difference between compared mean values

**Figure 12.** The percentage of lowered (with regard to the control group) the concentration of triacylglycerols (TG) in blood plasma in rats on atherogenic diets supplemented with β-glucans and dried spent brewer's yeast (\*/ identical letters indicate no significant difference).

Two-factor analysis showed the lack of significant influence of the type of β-glucan (soluble and insoluble) (p=0.091). Also the dosage of these beta glucans did not influence the reduction ratio of TG in rat blood (p=0.786).

278 Lipid Metabolism

β-glucans

β-glucans

Group – experimental factor Atherogenic factors

CONTROL 0.42a ± 0.03 0.88a ± 0.12

SBY 0.47b ± 0.03 1.09b ± 0.15 *SEM2/ (p3/) 0.006 (0.0001) 0.035 (0.0001)* 

BG-CMG10 0.49bc ± 0.03 1.16± 0.15b BG-CMG100 0.52cd ± 0.04 1.39c

BG-HP10 0.49bc ± 0.02 1.23bc ± 0.12

BG-HP100 0.52d ± 0.03 1.36c

**Table 6.** The comparison of atherogenic factors characteristic of blood of experimental animals given

1/ mean values ± standard deviation for n = 7 or 8; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of significant difference between compared mean values

Group – experimental factor TG – triacylglycerols [mmol/l]

β-glucan BG-CMG10 0.72b ± 0.06 β-glucan BG-CMG100 0.67b ± 0.04 β-glucan BG-HP10 0.74b ± 0.02 β-glucan BG-HP100 0.76b ± 0.04 SBY – spent brewer's yeast 0.66b ± 0.09

**Table 7.** Selected lipid parameters of peripheral blood of test animals on atherogenic diets,

1/ mean values ± standard deviation for n = 7 or 8; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of a significant difference between compared mean values



**Figure 12.** The percentage of lowered (with regard to the control group) the concentration of triacylglycerols (TG) in blood plasma in rats on atherogenic diets supplemented with β-glucans and


dried spent brewer's yeast (\*/ identical letters indicate no significant difference).




**% TG reduction**

supplemented with β-glucans and the preparation of dried brewer's yeast 1/.

CONTROL 0.90a ± 0.07

*SEM2 (p3) 0.019 (0.008)* 

atherogenic diets with β-glucan additive and spent brewer's yeast1/.

HDL/Chol-C HDL/LDL

± 0.29

± 0.15

BG-CMG10 BG-CMG100

BG-HP10 BG-HP100

SBY

Table 8 compiles the total content of lipids and cholesterol in liver of rats on atherogenic diets supplemented with β-glucans and dried spent brewer's yeast. The content of lipids was presented both in the equivalent of the so-called wet tissue and dry mass, whereas cholesterol content was presented in the equivalent of wet tissue and in reference to total lipid content.


**Table 8.** The content of lipids and cholesterol in the liver of rats on atherogenic diets supplemented with β-glucans and dried spent brewer's yeast.

1/ mean values ± standard deviation for n = 5 or 6; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05, identical letter signs in columns equal the lack of a significant difference between compared mean values

Total lipid content in fresh liver mass of rats did not differ significantly (p=0.110) in all groups and ranged from 18.4 to 20.9 g/100 g of wet tissue. Statistical analysis showed that the type of tested β-glucans (p=0.287) did not influence the concentration of lipids in the liver, whereas significantly better effects were observed when a higher dosage (p=0.003) was used.

In comparison with cholesterol level, it has been stated that neither the type (p=0,444) nor the amount of β-glucans (p=0.720) in the diet, significantly influence the cholesterol concentration in the liver.

Table 9 presents the percentage participation of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA) in total lipid pool in livers. The concentration of these groups of fatty acids was not influenced by the used β-glucans, but by their dosage (p=0.018).

The type of β-glucan from spent brewer's yeast did not significantly influence the ratio of blood lipid parameters metabolism in rats on atherogenic diet containing 1% of cholesterol. The concentration of HDL-cholesterol fraction and triacylglycerols in rat blood in this case depended neither on the type of β-glucan nor its dosage.

Literature data shows that hypolipemic activity of cereal β-glucans depends on the particle size and therefore, particle mass [54]. Using beta glucans of higher viscosity and higher particle mass in diet supplementation gives a better hypocholesterolemic effect [55]. Particle mass is a significant factor influencing hypocholesterolemic effect of β-glucans, but also it is essential to pay attention to the method of its designation [56]. Due to the variety of methods used by different researchers, it is difficult to compare experiment results presented by various authors. Hypocholesterolemic effect in people, resulting from cereal βglucan intake also depends on the dosage, diet supplementation period and even the age of tested people. β-glucan intake in the dosage of 3 g daily for 4 weeks, reduced the cholesterol concentration in the blood of children and teenagers with mild hypercholesterolemia of about 6-7% [57]. After 40 days of consuming diet containing from 1 to 5% of β-glucan from barley, total cholesterol concentration was reduced of 39%, LDL cholesterol fraction of 61% and triacylglycerols of 21%.

Spent Brewer's Yeast and Beta-Glucans Isolated from Them as Diet Components

the process of carboxymethylation, β-glucan underwent additional depolymerisation of particles through the use of ultrasounds. It led to a significant lowering of its particle mass,

In the research on humans using cereal β-glucans in the diet, an atherogenic factor was expressed as the ratio of HDL cholesterol to LDL cholesterol, in almost every case was increased in comparison with control group. A similar effect was obtained in the research described in this work. Diet supplementation with β-glucan isolated from spent brewer's yeast and preparation of dried yeast influenced the value of HDL/Chol-total ratio and HDL/LDL ratio. In each case of diet supplementation, these ratios were significantly more

Dried spent brewer's yeast given to animals in the diet in the amount of 0.5% diet, were as efficient as β-glucan preparations prepared from them and helped to lower the concentration in blood of: total cholesterol of 21.6%, LDL fraction – of 28.2% and

Available literature data concerning the influence of diet supplementation with yeast preparations as the source of dietary fibre concerns in most cases other types or species of yeast. In the research on obese men with hypercholesterolemia have shown that intake of 15 g of fibre from spent brewer's yeast (containing β-glucan) advantageously lowered the concentration of total cholesterol in blood, increasing the concentration of HDL fraction cholesterol. Simultaneously, the changes in the concentration of triacylogliceroles in blood were not observed. Authors, however, did not give the exact consitution of yeast fibre, which made it difficult to compare these results with the results obtained in this work [60].

Lowering of the concentration of total cholesterol in blood of experimental animals, as a result of diet supplementation with yeast, might be the result of not only β-glucan contained in them, but also the presence of squalens [61]. The ability of correcting blood lipid metabolism as a result of diet supplementation with yeast might result from the prebiotic properties of both the whole dried yeast cells and beta glucans contained in them, thanks to

As the research results show, the degree of liver fatness was significantly influenced by the dosage of beta glucans from spent brewer's yeast, whereas the solubility did not matter statistically. Both examined β-glucan preparations were given in daily dosage of 100 mg/kg

The type of β-glucans from spent brewer's yeast and their amount used in the diet did not, however, influence the cholesterol concentration in the livers. In comparison with the control group, the participation of cholesterol in liver lipids was in each case significantly lower. Two-factor analysis of variance showed that the higher dosage of β-glucans in the diet, i.e. 100 mg/kg of body mass daily, contributed to higher concentration of polyunsaturated fatty acids (PUFA) in livers and simultaneously lower concentration of saturated fatty acids (SFA). The dosage of β-glucans did not significanlty influence the concentration of monounsaturated fatty acids (MUFA). The concentration of all these

which the composition of natural bacterial flora can be additionally corrected [62].

of body mass, efficiently protected the liver against excessive fat layering.

and therefore, the direction of its pro-health influence was changed.

advantageous than in control group, which used only atherogenic diet [59].

triacylogliceroles of 27.2% in relation to control group.

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 281


**Table 9.** The percentage participation of fatty acids in total lipid pool of livers in experimental rats given β-glucan preparations and spent brewer's yeast.

1/ mean values ± standard deviation for n = 5 or 6; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of a significant difference between compared mean values

Significant results were obtained also as a result of 5% supplementation of rats' diet with βglucan from oyster mushroom (*Ostreatus Pleurotus*). Such a level in the low- and highcholesterol diet lowered the concentration of cholesterol, including LDL and VLDL cholesterol fractions, in both cases of about 30% in blood plasma and of about 50% in the liver, which was simultaneously connected with lowering of HMG - CoA reductase activity [58].

This work has not confirmed significant differencies between hypocholesterolemic effect of carboxymethylated β-glucan (soluble, of higher particle mass) and native β-glucan (insoluble).

The influence of β-glucans on lipid metabolism depends to a large extent on the size of their particles. The carboxymethylated β-glucan isolated in the laboratory from baker's yeast *Saccharomyces cerevisiae,* had a very low hipocholesterolemic activity despite its good solubility and high viiscosity. It has been explained by authors by the fact that apart from the process of carboxymethylation, β-glucan underwent additional depolymerisation of particles through the use of ultrasounds. It led to a significant lowering of its particle mass, and therefore, the direction of its pro-health influence was changed.

280 Lipid Metabolism

and triacylglycerols of 21%.

BG-CMG100 15.4c

given β-glucan preparations and spent brewer's yeast.

experimental factor

Group –

β-glucans

values

(insoluble).

Literature data shows that hypolipemic activity of cereal β-glucans depends on the particle size and therefore, particle mass [54]. Using beta glucans of higher viscosity and higher particle mass in diet supplementation gives a better hypocholesterolemic effect [55]. Particle mass is a significant factor influencing hypocholesterolemic effect of β-glucans, but also it is essential to pay attention to the method of its designation [56]. Due to the variety of methods used by different researchers, it is difficult to compare experiment results presented by various authors. Hypocholesterolemic effect in people, resulting from cereal βglucan intake also depends on the dosage, diet supplementation period and even the age of tested people. β-glucan intake in the dosage of 3 g daily for 4 weeks, reduced the cholesterol concentration in the blood of children and teenagers with mild hypercholesterolemia of about 6-7% [57]. After 40 days of consuming diet containing from 1 to 5% of β-glucan from barley, total cholesterol concentration was reduced of 39%, LDL cholesterol fraction of 61%

Saturated SFA Monounsaturated

Control 20.8a ± 2.9 26.7a ± 2.2 48.6ab ± 3.7

BG-CMG10 19.1abc ± 1.9 26.7a ± 0.6 44.8bc ± 2.4

BG-HP100 15.9bc ± 0.6 29.6ab ± 2.5 45.1bc ± 0.1 SBY 15.9bc ± 0.4 27.0a ± 1.2 50.5a ± 0.7 *SEM*2/ (*p*3/) *0.578 (0.023) 0.856 (0.0001) 0.841 (0.0001)* 

BG-HP10 19.2ab ± 4.2 28.7ab ± 4.0 44.6c

**Table 9.** The percentage participation of fatty acids in total lipid pool of livers in experimental rats

1/ mean values ± standard deviation for n = 5 or 6; 2/ *SEM –* standard error mean; 3/ ANOVA, p < 0.05; identical letter signs in columns equal the lack of a significant difference between compared mean

Significant results were obtained also as a result of 5% supplementation of rats' diet with βglucan from oyster mushroom (*Ostreatus Pleurotus*). Such a level in the low- and highcholesterol diet lowered the concentration of cholesterol, including LDL and VLDL cholesterol fractions, in both cases of about 30% in blood plasma and of about 50% in the liver, which was

This work has not confirmed significant differencies between hypocholesterolemic effect of carboxymethylated β-glucan (soluble, of higher particle mass) and native β-glucan

The influence of β-glucans on lipid metabolism depends to a large extent on the size of their particles. The carboxymethylated β-glucan isolated in the laboratory from baker's yeast *Saccharomyces cerevisiae,* had a very low hipocholesterolemic activity despite its good solubility and high viiscosity. It has been explained by authors by the fact that apart from

simultaneously connected with lowering of HMG - CoA reductase activity [58].

% fatty acids in total pool of lipids

MUFA

± 1.0 31.7b ± 1.4 51.4a ± 0.7

Polyunsaturated PUFA

± 4.1

In the research on humans using cereal β-glucans in the diet, an atherogenic factor was expressed as the ratio of HDL cholesterol to LDL cholesterol, in almost every case was increased in comparison with control group. A similar effect was obtained in the research described in this work. Diet supplementation with β-glucan isolated from spent brewer's yeast and preparation of dried yeast influenced the value of HDL/Chol-total ratio and HDL/LDL ratio. In each case of diet supplementation, these ratios were significantly more advantageous than in control group, which used only atherogenic diet [59].

Dried spent brewer's yeast given to animals in the diet in the amount of 0.5% diet, were as efficient as β-glucan preparations prepared from them and helped to lower the concentration in blood of: total cholesterol of 21.6%, LDL fraction – of 28.2% and triacylogliceroles of 27.2% in relation to control group.

Available literature data concerning the influence of diet supplementation with yeast preparations as the source of dietary fibre concerns in most cases other types or species of yeast. In the research on obese men with hypercholesterolemia have shown that intake of 15 g of fibre from spent brewer's yeast (containing β-glucan) advantageously lowered the concentration of total cholesterol in blood, increasing the concentration of HDL fraction cholesterol. Simultaneously, the changes in the concentration of triacylogliceroles in blood were not observed. Authors, however, did not give the exact consitution of yeast fibre, which made it difficult to compare these results with the results obtained in this work [60].

Lowering of the concentration of total cholesterol in blood of experimental animals, as a result of diet supplementation with yeast, might be the result of not only β-glucan contained in them, but also the presence of squalens [61]. The ability of correcting blood lipid metabolism as a result of diet supplementation with yeast might result from the prebiotic properties of both the whole dried yeast cells and beta glucans contained in them, thanks to which the composition of natural bacterial flora can be additionally corrected [62].

As the research results show, the degree of liver fatness was significantly influenced by the dosage of beta glucans from spent brewer's yeast, whereas the solubility did not matter statistically. Both examined β-glucan preparations were given in daily dosage of 100 mg/kg of body mass, efficiently protected the liver against excessive fat layering.

The type of β-glucans from spent brewer's yeast and their amount used in the diet did not, however, influence the cholesterol concentration in the livers. In comparison with the control group, the participation of cholesterol in liver lipids was in each case significantly lower. Two-factor analysis of variance showed that the higher dosage of β-glucans in the diet, i.e. 100 mg/kg of body mass daily, contributed to higher concentration of polyunsaturated fatty acids (PUFA) in livers and simultaneously lower concentration of saturated fatty acids (SFA). The dosage of β-glucans did not significanlty influence the concentration of monounsaturated fatty acids (MUFA). The concentration of all these

#### 282 Lipid Metabolism

groups of fatty acids was not influenced by the type of used β-glucans. Dried spent brewer's yeast, like β-glucans, also contributed to obtaining lower concentration of cholesterol in calculation for the wet tissue of the liver.

Spent Brewer's Yeast and Beta-Glucans Isolated from Them as Diet Components

significant lowering of triacyloglicerole concentration was observed. In people, however, oligofructose administration for 4 weeks did not lead to lowering triacylogliceroles and cholesterol [68]. Especially strong influence of prebiotics on lowering VLDL fraction is

No significant differences have been observed in hypocholesterolemic effect of soluble βglucan – (CMG) and insoluble native β-glucan (HP). The results showed that after hydration, carboxymethylated β-glucan CMG was characterised by higher viscosity and mean particle size amounting to about 90 µm, whereas particles of insoluble HP β-glucan, established nonviscous solutions of particle size amounting to about 50 µm and about 320 µm. Examined β-glucans showed an effective hypocholesterolemic effect. It has been proven that they influenced lipid metabolism advantageously, especially in case of LDL fraction cholesterol and triacylglycerols (TG). An advantageous HDL/Chol-total factor and HDL/LDL factor has also been confirmed. Dried spent brewer's yeast were given to animals in a daily dosage of 100 mg/kg of body mass were as efficient as β-glucans isolated from them and they lowered the concentration in blood of: total cholesterol of 21.6%, LDL fraction – of 28.2% and triacylglycerols of 27.2% in relation to control group. The research also proves that advantageous influence of yeast on lipid metabolism and their level in blood might be linked with prebiotic properties of yeast on lipid metabolism, as in the research simultaneously conducted by the Author, the advantageous composition of intestine microflora was observed (a higher number of lactid acid bacteria of *Bifidobacterium* type was

High nucleic acid content in yeast (supplying from 12% to 25% of total nitrogen content) [70] limits their use as a traditional ingredient in human nutrition. It has been stated that the excess of nucleic acid in the diet of people and most monogastric animals is toxic and results in excessive accumulation of uric acid in organism, leading to arthritis. Therefore, it is recommended to consume their little portions as diet supplement supplying mainly

Numerous research on fish proved that diet supplementation with yeast to a particular level (in the amount providing no more than 50% of proteins in the diet), does not show disadvantageous health effects, such as abnormal growth, improper nitrogen balance or liver diseases [71]. However, only lower diet intake was observed when spent brewer's

It seems that diet supplementation with dried spent brewer's yeast *S.cerevisiae* in the amount of 0.5% of the diet contributed significantly to correcting possible disorders in lipid metabolism of rats on an atherogenic diet – it enhances lipid changes in organism,

During the research on hypocholesterolemic activity of 81 different yeast strains, showed hypocholesterolemic activity of spent brewer's yeast of male Wistar rats with their

suggested [69].

obtained*).* 

vitamins from B group.

enhancing their parameters.

yeast constituted more than 25% of the diet [70]

**6. Conclusions** 

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 283

Beta glucans, regardless of origin, serve as dietary fibre in the body of mammals, therefore, their hipocholesterolemic effect might be associated with the mechanism recognised for dietary fibre. The influence of soluble fractions of dietary fibre on the cholesterol concentration in the body is known, by binding bile acids in the intestine and consequently increases the amount of bile acids excreted in the feces. It results in decreasing the pool of bile salt able to take part in the synthesis of cholesterol in liver and disregulation of micellas in intestines, which hampers lipid absorption. Cholesterol is used in the synthesis of bile acids instead of lipoproteid synthesis, therefore, speeding its circulation, and its concentration in plasma lowers [63,64].

Hipocholesterolemic effect of tested β-glucans might be also compared with the activity of known prebiotic (inulin) and oat fibre. Inulin while undergoing fermentation in large intestine influences the proportions of produced SCFA [65], decreasing the amount of produced octan, and increasing the level of propionic and butyric acid. It is especially advantageous, as octan acts as a simulator and propionian as inhibitor of cholesterol synthesis [66]. Research *in vitro* showed that propionic acid hampers cholesterol and fatty acid synthesis in the liver. It seems that the combination of increased excretion of bile acid with faeces and slight lowering of cholesterol synthesis in liver aims to lower total cholesterol concentration and LDL fraction in blood [67].

Supplementation of rat diets with β-glucan preparations from spent brewer's yeast examined in this work and the preparation of dried brewer's yeast contributes to advantegous lowering of cholesterol concentration in blood, at simultaneous achieving a more advantegous in relations to control group content of bowel microflora, connected particularly with increased numer of *Bifidobacterium* bacteria of lactid acid, which was shown in parallelly conducted research*.* 

It is quite difficult to explain the estimated mechanism of lowering cholesterol concentration under the influence of prebiotics. However, increased excretion of cholesterol with faeces through hampering the creation of easily digested fatty micellas has been suggested. In rats, increased excretion of cholesterol in faeces has been confirmed, and similar research presents this mechanism also in people. It is possible that some bacteria of lactid acid can assimilate cholesterol directly. There is proof that fructooligosaccharides (FOS) lower the synthesis of triacylglycerols in liver, however, so far the mechanism has not been identified.

Similar significance to probiotics is also attributed to prebiotics. Prebiotics arouse even greater interest due to practical means – they are characterised by greater durability than probiotics, their activity is not conditioned by micorbe viability after intake and they might be added to many food products as one of the ingredients. There is little research available concerning the research on people, therefore, most conclusions have been drawn based on the research on animals. In rats, for example, after a 5-week inulin administration a significant lowering of triacyloglicerole concentration was observed. In people, however, oligofructose administration for 4 weeks did not lead to lowering triacylogliceroles and cholesterol [68]. Especially strong influence of prebiotics on lowering VLDL fraction is suggested [69].
