**4. Research material and conditions of biological experiment**

Material consisted of:

268 Lipid Metabolism

delay the process of cell aging [23,44].

troublesome waste for the brewing industry.

obtaining beta glucans.

binding properties.

**3. The purpose and scope of work** 

Sparse research on β-glucans isolated in the laboratory from cell walls of baker's yeast *Saccharomyces cerevisiae,* shows varied biological activity depending on the used technology of their isolation. They can strengthen the immune system, show antioxidant properties, and

Due to a high content of β-glucans in spent brewer's yeast *Saccharomyces cerevisiae*, remaining after alcohol fermentation in the process of beer production, it seems that they

Fig. 8 presents the diagram showing the use of spent brewer's yeast, which constitute

can be an effective and inexpensive material for obtaining β-glucan preparations.

**Figure 8.** The possibilities of using spent brewer's yeast *Saccharomyces cerevisiae*.

Beta glucans might be obtained as a byproduct in the production of preparations enhancing flavour (yeast extracts), and the remains after fibre extraction constitute a good material for

So far no research has been conducted on pro-health β-glucans obtained from spent brewer's yeast *Saccharomyces cerevisiae*, which constitute a natural, often a troublesome byproduct of the brewing industry, waste product after alcohol fermentation in beer production. Sparse research on β-glucans obtained from this type of material is conducted on a laboratory scale and aims mostly to examine their technological properties, conditioning their usage as additional substances of thickening, gelling and/or water

This work has aimed to assess pro-health activity of β-glucans isolated from a new, uninvestigated within this scope source, i.e. spent brewer's yeast *Saccharomyces cerevisiae*.


#### **4.1. Biological experiment and its progress**

The experimental animals were growing male rats (Wistar) with an initial body weight of about 100 g. Prior to the start of the experiment, animals were given water and commercial, standard rat food LSM® ad libitum for 7 days to adapt them to the experimental conditions. Later the animals were randomly divided into 6 groups (7 rats in one group) in relation with diet composition:

	- BG-CMG10 (10 mg/kg of body mass),
	- BG-CMG100, (100 mg/kg of body mass),
	- BG-HP10 (10 mg/kg of body mass),
	- BG-HP100 (100 mg/kg of body mass),

The average body mass of rats in each group was similar. Diets (semisynthetic, isocaloric) were prepared in accordance with the recommendations of the American Institute of Nutrition [45]. Mineral mixture AIN-93G-MX and vitamin mixture AIN-93G-MV were used. The composition of experimental diets is presented in Table 1.

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

lasted 6 weeks, after which blood from myocardium was collected. Animals were put to

The size of particles was analysed with the method of laser difraction with the use of Mastersizer S analyser of Malvern Instruments Ltd., Malvern, Great Britain. Particle size distribution of examined preparations was measured using the wet process in oil emulsions

Rheometric measurements were conducted with the use of rheo-viscometer of Brookfield DV III+ using ULA spindle and DIN-82. Measurement device was coupled with RHEOCALC for Windows version 2.l. computer programme, from which dynamic viscovity

Antioxidant properties of preparations were marked with spectrophotometric method with the use of synthetic radicals ABTS•+ [46]. Antioxidant activity of examined preparations was expressed as the ability of an examined preparation to de-activation of cation radicals ABTS•+ and as TEAC (Trolox Equivalent Antioxidant Capacity), i.e. µM of Trolox for 1g of

**Total cholesterol -** methyl esters have been prepared, from which 0.5% of the solution has been made using toluene as solvent. 1µl was collected from prepared solutions, which was dosed with a µ-syringe on the Hewelett-Packard HP 6890 Series GC System Plus gas chromatographer with a flame ionization detector (FID) and capilary column with polar stationary phase of 30 m length. Stigmasterol produced by Sigma was used as

**Lipid fractions -** particular lipid fractions: HDL-cholesterol, and triacylglycerols (TG) in the plasma (mmol/L) were enzymatically measured with INTEGRA analyser. The concentrations of LDL cholesterol (mg/dL) were calculated as LDL cholesterol = total cholesterol – HDL cholesterol - (triacylglycerols/5). Cholesterol was converted from mg/dL to mmol/L by multiplying by 0.0259. VLDL-cholesterol was calculated as 1/5 TG [48]. In order to calculate the amount of cholesterol expressed in mg/dl for mmol/l, 0.0258 multiplier

and/or water solutions of concentration within the range of 0.01 – 0.05%.

*4.2.3. Antioxidant properties of preparations with in vitro method* 

**Total lipid content** was marked with a modified method of Folch et al. [47].

sleep with the peritoneal injection of Thiopental.

*4.2.1. Particle size and swelling ability* 

*4.2.2. Viscosity of water solutions* 

preparation.

a standard.

was used.

*4.2.4. Lipid content* 

values were read at varied shear velocity.

**4.2. Research methods** 

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 271

The research was conducted on control group, which did not receive beta glucan additive. Diet intake and body weight growth of research animals were controlled during the research. Ethics Committee approved of the research. Energy derived from fat, protein and carbohydrates was 40.1; 15.8 and 44.1%, respectively.


**Table 1.** Composition of 100 g control and model diet enriched with β-glucans.

a/ Sample weight of prepared earlier diet were mixed with fresh cholesterol in the proportion 99:1, directly before being served to animals; b/ β-glucans were weighed in the amounts adequate for each rat's body mass, then mixed with small diet portions (5 g) and given to each animal individually. After having eaten, the animals were served diet and water *ad libitum.*

During the whole experimental period (6 weeks) rats were housed in individual cages with 24 h access to water. The premises, in which the experimental rats were housed had a 12:12h light cycle with temperature of 21-22oC and humidity of 55-65%. The proper experiment lasted 6 weeks, after which blood from myocardium was collected. Animals were put to sleep with the peritoneal injection of Thiopental.

## **4.2. Research methods**

270 Lipid Metabolism

Components

β-glucans from spent brewer's yeast, mg/kg of body mass/dayb/:

Protein, g/100 g of diet

Carbohydrates, g/100 g

Fat, g/100 g of diet

Energy value (EV),

The average body mass of rats in each group was similar. Diets (semisynthetic, isocaloric) were prepared in accordance with the recommendations of the American Institute of Nutrition [45]. Mineral mixture AIN-93G-MX and vitamin mixture AIN-93G-MV were used.

The research was conducted on control group, which did not receive beta glucan additive. Diet intake and body weight growth of research animals were controlled during the research. Ethics Committee approved of the research. Energy derived from fat, protein and

> BG-CMG100

Diet

0 10 100 10 100 0

BG-HP10 BG-HP100 SBY100

The composition of experimental diets is presented in Table 1.

Control BG-

CMG10

Wheat starch, g 49.0 49.0 49.0 49.0 49.0 48.5 Casein, g 20.0 20.0 20.0 20.0 20.0 20.0 Soya oil, g 20.0 20.0 20.0 20.0 20.0 20.0 α-cellulose, g 5.0 5.0 5.0 5.0 5.0 5.0 DL-methionine, g 0.3 0.3 0.3 0.3 0.3 0.3 Choline bitartrate, g 0.2 0.2 0.2 0.2 0.2 0.2 Mineral mix, g 3.5 3.5 3.5 3.5 3.5 3.5 Vitamin mix, g 1.0 1.0 1.0 1.0 1.0 1.0 Cholesterola/, g 1.0 1.0 1.0 1.0 1.0 1.0 Spent brewer's yeast, g 0 0 0 0 0 0,5

(% of EV) 18.0 (15.8%) 18.1 (15.9)

(% of EV) 20.3 (40.1%) 20.3 (40.2)

of diet (% of EV) 50.2 (44.1%) 49.9 (43.9)

kJ (kcal) 1904 (455,5) 1900.5 (454.7)

During the whole experimental period (6 weeks) rats were housed in individual cages with 24 h access to water. The premises, in which the experimental rats were housed had a 12:12h light cycle with temperature of 21-22oC and humidity of 55-65%. The proper experiment

a/ Sample weight of prepared earlier diet were mixed with fresh cholesterol in the proportion 99:1, directly before being served to animals; b/ β-glucans were weighed in the amounts adequate for each rat's body mass, then mixed with small diet portions (5 g) and given to each animal individually. After

**Table 1.** Composition of 100 g control and model diet enriched with β-glucans.

having eaten, the animals were served diet and water *ad libitum.*

carbohydrates was 40.1; 15.8 and 44.1%, respectively.

#### *4.2.1. Particle size and swelling ability*

The size of particles was analysed with the method of laser difraction with the use of Mastersizer S analyser of Malvern Instruments Ltd., Malvern, Great Britain. Particle size distribution of examined preparations was measured using the wet process in oil emulsions and/or water solutions of concentration within the range of 0.01 – 0.05%.

#### *4.2.2. Viscosity of water solutions*

Rheometric measurements were conducted with the use of rheo-viscometer of Brookfield DV III+ using ULA spindle and DIN-82. Measurement device was coupled with RHEOCALC for Windows version 2.l. computer programme, from which dynamic viscovity values were read at varied shear velocity.

#### *4.2.3. Antioxidant properties of preparations with in vitro method*

Antioxidant properties of preparations were marked with spectrophotometric method with the use of synthetic radicals ABTS•+ [46]. Antioxidant activity of examined preparations was expressed as the ability of an examined preparation to de-activation of cation radicals ABTS•+ and as TEAC (Trolox Equivalent Antioxidant Capacity), i.e. µM of Trolox for 1g of preparation.

#### *4.2.4. Lipid content*

**Total lipid content** was marked with a modified method of Folch et al. [47].

**Total cholesterol -** methyl esters have been prepared, from which 0.5% of the solution has been made using toluene as solvent. 1µl was collected from prepared solutions, which was dosed with a µ-syringe on the Hewelett-Packard HP 6890 Series GC System Plus gas chromatographer with a flame ionization detector (FID) and capilary column with polar stationary phase of 30 m length. Stigmasterol produced by Sigma was used as a standard.

**Lipid fractions -** particular lipid fractions: HDL-cholesterol, and triacylglycerols (TG) in the plasma (mmol/L) were enzymatically measured with INTEGRA analyser. The concentrations of LDL cholesterol (mg/dL) were calculated as LDL cholesterol = total cholesterol – HDL cholesterol - (triacylglycerols/5). Cholesterol was converted from mg/dL to mmol/L by multiplying by 0.0259. VLDL-cholesterol was calculated as 1/5 TG [48]. In order to calculate the amount of cholesterol expressed in mg/dl for mmol/l, 0.0258 multiplier was used.

#### 272 Lipid Metabolism

**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 cholesterol content.

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

1% 3% 1% 3%

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

Preparations The size of particles in cumulated values (d) [µm] Xśr. [µm] d5 d10 d25 d50 d75 d90 d95

HP β-glucan 5.2 16.8 32.7 50.14 70.74 93.4 107.3 52.98 CM β-glucan 0.2 0.48 2.24 17.0 67.7 133.6 174.6 44.62 SBY 6.61 16.26 34.8 64.5 169.5 325 396.9 119.4

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

> Viscosity for 10 RPM [mPas]

β-glucan BG-CMG 65.2a ± 7.5 574.9a ± 36.3 96.0 ± 2.1 94.4 ± 4.7

SBY 0.4d ± 0.04 1.5d ± 0.04 84.6 ± 4.7 90.9 ± 1.6 **Table 3.** The characteristics of viscositya/ of 1% and 3% water solutions of β-glucan preparations in

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

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

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

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

± 0.2 1.9c

comparison with 1% and 3% viscosity of water solutions of spent brewer's yeast.

**Table 2.** The comparison of cumulated distribution of particle size of examined β-glucans and

nonviscid or only slightly viscid solutions (0.4-1.4 mPas) - tab. 3.

powdered spent brewer's yeast.

pre-hydrated β-glucans.

β-glucan BG-HP 1.4c

insolubility of this β-glucan [6].

solvent, causing its dissolution [2,4].

Preparation

values

Modifying Blood Lipid Metabolism Disturbed by an Atherogenic Diet 273

The level of adjustment reliance [%]

± 0.1 90.0 ± 1.3 93.6 ± 0.9

#### *4.2.5. Statistical analysis*

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 at P<0.05.
