**2.4 Microbiological examination**

*Veganism - a Fashion Trend or Food as a Medicine*

The nutritional regime was determined on the basis of a questionnaire on the frequency of use of selected commodities. The questionnaire focused on the amount and frequency of consumption of 144 individual foods, food groups and recipes. Groups and recipes include soups, soups, sauces, pickled vegetables, fruits and jams. Probands responded to the consumption of 4 categories: almost never, x times a month, x times a week, x times a day. The data after dispensing into individual foods as well as after the exact expression of the amounts of consumption and conversion to daily intake were processed using the Nutrition program, which is a food data bank of the Food Research Institute in Bratislava. The calculation revealed the loss of vitamins during technological food processing. The output of the evaluation of the nutritional regime was the average daily energy intake and

Belly (mm) on caliper 24,40 ± 1,13\*\*\* 30,16 ± 1,01 Visceral fat (cm2) 70,06 ± 4,94\*\*\* 100,56 ± 4,13 % fat on InBody230 23,16 ± 0,69\* 25,93 ± 0,88 % fat on Omron BF-306 27,00 ± 0,67\* 28,27 ± 0,86

**Vegetarians Non-vegetarians**

Blood was collected in the morning on an empty stomach after standard food intake in the previous days to examine the monitored parameters. Blood collection for plasma was performed in commercial syringes with EDTA (ethylene diamine tetraacetate), which in addition to its anticoagulant properties is also an inhibitor of free radical reactions. Morning urine and feces were also collected and processed and stored at −80° C on the day of collection. Average sample 24-h. urine volume for the determination of iodine probandi brought in the morning on the day of

Total cholesterol, HDL-cholesterol, triacylglycerols, glucose and iron were determined by standard laboratory methods with a Vitros 250 automated analyzer (Johnson & Johnson, USA). The LDL-cholesterol content was determined by calculation from the Friedewald formula: LDL-cholesterol = total cholesterol - triacylglycerols/2,2 - HDL-cholesterol (6). The condition of the calculation was the value of triacylglycerols <4.5 mmol/l. The atherogenic index was calculated from the ratio of total and HDL-cholesterol. Vitamin C, E, A and β-carotene, malondialdehyde were measured by HPLC [17–20]. Protein carbonyls, conjugated dienes were determined spectrophotometrically [21]. The alkaline comet assay was used to detect DNA breaks, oxidized purines and oxidized pyrimidines in isolated lymphocytes [22–24]. Determination of 25-hydroxyvitamin D and other hydroxylated metabolites of vitamin D (both its forms - ergocalciferol D2 and cholecalciferol D3) in serum was performed by the classical equilibrium RIA method. Plasma N-carboxymethyllysine and plasma fluorescent AGEs were determined by competitive ELISA [25]. The iodine value was determined in the 24-h samples. Urine by modification of the

Collection of biological material - blood, urine and stool.

**2.2 Dietary examination**

*Subcutaneous and visceral fat.*

**Table 2.**

selected nutrients.

examination.

**2.3 Biochemical examination**

Sandell-Kolthoff reaction [26].

**44**

The qualitative and quantitative representation of the microflora was evaluated by classical microbiological methods by culturing on selective diagnostic nutrient media according to Mitsuok et al. [27]. In probands for which we did not notice differences in the qualitative representation by culture methods, we determined bacterial profiles using molecular biology methods. The presence of potentially mutagenic substances was determined by Ames plate incorporation assay using S. typhimurium TA98 (shift mutations) and TA100 (point mutations) bacterial cultures. Sterol analysis by gas chromatography was performed using a GC17-QP5000, with a split/splitless injector (Shimadzu, Kyoto, Japan).

## **2.5 Statistical processing of results**

Commercial programs - Excel 2000 and Statgraphics for Windows, version 1.4 and PASW Statistics 18 were used for computer processing of the obtained data. The comparison was supplemented by determining the percentage occurrence of risk values of monitored parameters as well as the occurrence of protective values of antioxidant vitamins in each evaluated group. This partially eliminated the fact that malnutrition probands could also be included in the traditional diet (this is a current sample of our population, whose malnutrition ultimately reflects a high incidence of morbidity and mortality from the two main diseases - cardiovascular and cancer), while in vegetarians, incorrect vegetarianism is less common (the risks are given by the one-sidedness of the diets resulting from the type of alternative diet).
