Supplement to
Effect ofdietary type and consumption of different foods on the fecal microbiota in vegetarians and omnivores in Slovenia
European Journal of Nutrition
Bojana Bogovič Matijašić, Tanja Obermajer, Luka Lipoglavšek, Iztok Grabnar, Gorazd Avguštin, Irena Rogelj
Corresponding author:Bojana Bogovič Matijašić, PhD
University of Ljubljana
Biotechnical Faculty
Department of Animal Science
Institute of Dairy Science and Probiotics
Groblje 3
SI - 1230 Domžale
Slovenia
E-mail:
Supplemental Table 1 Consumption of different groups of food, alcohol, liquids, additional salt, multivitamin and dietary supplements by vegetarians and omnivores participating in the study; data were obtained by self-administered questionnaire
Group of food / Consumption / Vegetarians / Omnivores / TotalAlcohol / Yes / 0 (0.0 %) / 17 (68.04 %) / 17 (30.9 %)
No / 30 (100.0 %) / 8 (32.0 %) / 38 (769.1 %)
Liquid / > 1 l/day / 18 (60.0 %) / 15 ( 57.7 %) / 33 ( 58.9 %)
< 1 l/day / 12 (40.0 %) / 11 (42.3 %) / 23 ( 41.1 %)
Sweets including chocolate / Yes / 28 (93.3 %) / 24 (92.3 %) / 52 (92.9 %)
No / 2 (6.7 %) / 2 (7.7 %) / 4 (7.1 %)
Additional salting / Yes / 8 (26.7 %) / 3 (11.5%) / 11 (19.6 %)
No / 22 (73.3 %) / 23 (88.5 %) / 45 (80.4 %)
Coffee / Yes / 12 (40.0 %) / 20 ( 76.9 %) / 32 ( 57.1 %)
No / 18 (60.0 %) / 6 (23.1 %) / 24 (42.9 %)
Fruits / Yes / 30 (96,8 %) / 25 (86.2 %) / 55 (91.7 %)
No / 1 (3,2 %) / 4 (13.8 %) / 5 (8.3 %)
Vegetables / Yes / 31 (100.0 %) / 23 (79.3 %) / 54 (90.0 %)
No / 0 (0.0 %) / 6 (20.7 %) / 6 (10.0 %)
Vegetables - family Solanaceae / Yes / 30 (96.8 %) / 22 (75.9 %) / 52 (86.7 %)
No / 1 (3.2 %) / 7 (24.1 %) / 8 (13.3 %)
Legumes / Yes / 29 (93.5 %) / 22 (75.8 %) / 51 (85.0 %)
No / 2 (6.5 %) / 7 (24.2 %) / 9 (15.0 %)
Seeds / Yes / 25 (80.6 %) / 15 (51.7%) / 40 (66.7 %)
No / 6 (19.4 %) / 14 (48.3 %) / 20 (33.3 %)
Nuts / Yes / 26 (83.9 %) / 15 (51.7%) / 41 (68.3 %)
No / 5 (16.1 %) / 14 (48.3 %) / 19 (31.7 %)
Cereals (grains) / Yes / 28 (90.3 %) / 23 (79.3 %) / 51 (85.0 %)
No / 3 (9.7 %) / 6 (20.7 %) / 9 (15.0 %)
Rice / Yes / 28 (90.3 %) / 24 (82.8 %) / 52 (86.7 %)
No / 3 (9.7 %) / 5 (17.2 %) / 8 (13.3 %)
Corn / Yes / 24 (77.4 %) / 19 (65.5 %) / 43 (71.7 %)
No / 7 (22.6 %) / 10 (34.5 %) / 17 (28.3 %)
Red meat / Yes / 0 (0.0 %) / 24 (82.8 %) / 24 (40.0 %)
No / 31 (100.0 %) / 5 (17.2 %) / 36 (60.0 %)
White meat / Yes / 0 (0.0 %) / 26 (89.7 %) / 26 (43.3 %)
No / 31 (100.0 %) / 3 (10.3 %) / 34 (56.7 %)
Fish and seafood / Yes / 0 (0.0 %) / 21 (72.4 %) / 21 (35.0 %)
No / 31 (100.0 %) / 8 (27.6 %) / 39 (65.0 %)
Eggs / Yes / 0 (0.0 %) / 23 (79.3 %) / 23 (38.3 %)
No / 31 (100.0 %) / 6 (20.7 %) / 37 (61.7 %)
Milk / Yes / 5 (16.1 %) / 24 (82.8 %) / 29 (48.3 %)
No / 26 (83.9 %) / 5 (17.2 %) / 31 (51.7 %)
Dairy products / Yes / 7 (22.6 %) / 25 (86.2 %) / 32 (53.3 %)
No / 24 (77.4 %) / 4 (13.8 %) / 28 (46.7 %)
Yoghurts (except probiotic yoghurts) / Yes / 6 (20.0 %) / 243(88.4 %) / 29 (51.8 %)
No / 24 (80.0 %) / 3 (11.6 %) / 27 (48.2 %)
Probiotic yoghurts and drinks (dairy) / Yes / 1 (3.4 %) / 14 (63.6 %) / 15 (29.4 %)
No / 28 (96.6 %) / 8 (36.4 %) / 36 (70.6 %)
Soya yoghurts / Yes / 9 (31.0 %) / 1 (4.3 %) / 10 (23.8 %)
No / 20 (69.0 %) / 22 (95.7 %) / 42 (76.2 %)
Multivitamin dietary supplements / Yes / 6 (19.4 %) / 5 (17.2 %) / 11 (18.3 %)
No / 25 (80.6 %) / 24 (82.8 %) / 49 (81.7 %)
Data are expressed as number (%) of subjects who answered to each of the questions.
Supplemental Table 2 Relativequantificationby real-time PCR of bacteria in thefeces of vegans (20), lacto-vegetarians (11) andomnivores (29); results are presented as medians (range)
Target bacteria(species, genus or group)a,b,c / Vegans
(n =20) / Lacto-vegetarians
(n = 11) / Omnivores
(n = 29) / p
Bacteroides-Prevotella group a / 5.74E+10 (2.16E+08-3.91E+11) / 9.15E+10 (5.48E+09-1.30E+12) / 2.52E+10 (2.89E+08-5.79E+11) / 0.080
Clostridium leptumgroup a / 4.45E+11 (5.79E+09-1.75E+12) / 2.37E+11 (3.44E+10-3.06E+12) / 4.31E+11 (3.21E+08-6.44E+12) / 0.977
Clostridium coccoides group a / 4.43E+11 (6.65E+10-1.08E+12) / 3.52E+11 (8.46E+10-1.59E+12) / 4.92E+11 (6.12E+10-3.92E+12) / 0.256
Enterobacteriaceae a / 1.50E+08 (4.51E+06-1.75E+10) / 1.85E+07 (3.98E+05-2.44E+09) / 6.07E+07 (8.02E+05-1.34E+9) / 0.079
Bifidobacterium a / 2.52E+09 (1.86E+06-2.22E+10) / 4.39E+09 (3.03E+08-8.08E+10) / 8.03E+09 (1.81E+06-6.19E+10) / 0.141
Clostridium clostridioforme a / 4.27E+09 (3.94E+08-1.87E+10) / 3.10E+09 (8.51E+08-6.82E+09) / 3.10E+09 (4.10E+08-2.12E+10) / 0.342
Bacteroidesthetaiotaomicron a / 1.65E+09 (1.02E+07-8.45E+10) / 1.009E+09 (6.07E+07-1.38E+10) / 6.04E+08 (6.05E+05-5.04E+09) / 0.223
Faecalibacteriumprausnitziic / 5.21E+10 (6.61E+09-1.60E+11) / 7.07E+10 (2.30E+10-1.12E+11) / 4.72E+10 (4.22E+09-4.51E+11) / 0.490
All bacteria a / 5.61E+12 (1.07E+12-1.29E+13) / 5.78E+12 (2.66E+12-1.49E+13) / 5.49E+12 (1.36E+12-4.75E+13) / 0.785
Bacteroides-Prevotella group / All bacteria (%)b / B1.174 (0.020-5.667) / C1.780 (0.112-14.797) / B.C0.506 (0.011-5.267) / 0.026*
Clostridium leptumgroup / All bacteria (%)b / 7.456 (0.541-22.625) / 3.374 (0.681-52.896) / 5.830 (0.021-37.201) / 0.732
Clostridium coccoidesgroup / All bacteria (%)b / B7.364 (3.378-13.072) / C5.446 (2.641-12.652) / B.C8.892 (3.805-16.765) / 0.025*
Enterobacteriaceae / All bacteria (%)b / 0.004 (0.000-0.141) / 0.000 (0.000-0.061) / 0.001 (0.000-0.025) / 0.055
Bifidobacterium/ All bacteria (%)b / 0.068 (0.000-0.358) / 0.076 (0.007-0.907) / 0.090 (0.000-0.700) / 0.232
Clostridium clostridioforme / All bacteria (%)b / A0.071 (0.023-0.195) / A0.038 (0.016-0.077) / 0.055 (0.016-0.197) / 0.020*
Clostridium clostridioforme /Clostridium coccoidesgroup (%)b / A.B1.132 (0.448-1.980) / A0.627 (0.162-1.226) / B0.604(0.203-1.760) / 0.005**
Bacteroidesthetaiotaomicron / All bacteria (%)b / 0.023 (0.001-1.397) / 0.027 (0.001-0.093) / 0.008 (0.000-0.055) / 0.121
Bacteroidesthetaiotaomicron / Bacteroides-Prevotella group (%)b / 2,281 (0.500-72.258) / 0.931 (0.033-11.355) / 2.161(0.210-25.056) / 0.199
Faecalibacteriumprausnitzii/ All bacteria (%)d / B0.939 (0.106-1.704) / 0.821 (0.338-1.667) / B0.682(0.059-1.373) / 0.029*
Capital letters in superscript indicate significant differences in pair-wise comparisons (Mann-Whitney U test,Holm's adjusted p < 0.05).
a Results are presented as a number of 16S rDNA copies of individual target group of bacteria in g of feces
b Results are presented as a ratio (%) of rDNA copies of individual target group of bacteria with regard to the total bacteria
c Results are presented as a number of cells of Faecalibacteriumprausnitzii in g of feces
d Results are presented as a ratio (%) of cells of Faecalibacteriumprausnitziiwith regard to the number of 16S rDNA copies of total bacteria
p, Kruskal-Wallis test (vegans vs. lacto-vegetarians vs. omnivores); asterisks indicate significant differences among three groups (* p<0.05, ** p<0.01)
Supplemental Table 3 Association of microbiota composition in a group of omnivores with alcohol consumption; results are presented as medians (range)
Target bacteria(species, genus or group)a,b,c / Alcohol consumption / p
No
(n=8) / Yes
(n=17)
Bacteroides-Prevotella group a / 3.27E+10 (1.36E+09-3.73E+11) / 1.71E+10 (2.89E+08-5.79E+11) / 0.842
Clostridium leptumgroup a / 7.76E+11 (2.95E+10-1.77E+12) / 3.05E+11 (3.21E+08-6.44E+12) / 0.628
Clostridium coccoides group a / 9.24E+11 (2.21E+11-1.42E+12) / 4.68E+11 (6.12E+10-3.92E+12) / 0.215
Enterobacteriaceae a / 9.33E+07 (2.98E+06-1.34E+09) / 6.07E+07 (8.02E+05-1.31E+09) / 0.440
Bifidobacterium a / 5.72E+09 (1.81E+06-2.91E+10) / 1.20E+10 (1.56E+08-6.19E+10) / 0.238
Clostridium clostridioforme a / 5.69E+09 (1.70E+09-1.06E+10) / 2.64E+09 (4.85E+08-2.12E+10) / 0.238
Bacteroidesthetaiotaomicron a / 6.88E+08 (2.97E+07-4.06E+09) / 6.04E+08 (6.05E+05-5.04E+09) / 0.669
Faecalibacteriumprausnitziic / 7.21E+10 (1.34E+10-1.74E+11) / 3.71E+10 (4.23E+09-4.51E+11) / 0.075
All bacteria a / 9.36E+12 (4.24E+12-1.69E+13) / 5.31E+12 (1.37E+12-4.75E+13) / 0.262
Bacteroides-Prevotella group / All bacteria (%)b / 0.490 (0.010-3.838) / 0.432 (0.014-5.267) / 0.932
Clostridium leptumgroup / All bacteria (%)b / 6.589 (0.327-18.230) / 4.551 (0.021-37.201) / 0.932
Clostridium coccoidesgroup / All bacteria (%)b / 9.854 (4.818-12.992) / 8.246 (3.805-16.765) / 0.440
Enterobacteriaceae / All bacteria (%)b / 0.002 (0.000-0.011) / 0.001 (0.000-0.025) / 0.711
Bifidobacterium/ All bacteria (%)b / 0.062 (0.000-0.243) / 0.159 (0.010-0.700) / 0.097
Clostridium clostridioforme / All bacteria (%)b / 0.058 (0.033-0.111) / 0.052 (0.016-0.197) / 0.511
Clostridium clostridioforme / Clostridium coccoidesgroup (%)b / 0.739 (0.425-0.854) / 0.583 (0.283-1.760) / 0.887
Bacteroidesthetaiotaomicron / All bacteria (%)b / 0.008 (0.000-0.048) / 0.009 (0.000-0.042) / 0.711
Bacteroidesthetaiotaomicron / Bacteroides-Prevotella group (%)b / 2.171 (0.960-8.290) / 1.350 (0.210-25.055) / 0.315
Faecalibacteriumprausnitzii/ All bacteria (%)d / 0.752 (0.316-1.373) / 0.682 (0.058-1.053) / 0.288
a Results are presented as a number of 16S rDNA copies of individual target group of bacteria in g of feces
b Results are presented as a ratio (%) of rDNA copies of individual target group of bacteria with regard to the total bacteria
c Results are presented as a number of cells of Faecalibacteriumprausnitzii in g of feces
d Results are presented as a ratio (%) of cells of Faecalibacteriumprausnitziiwith regard to the number of 16S rDNA copies of total bacteria
p,Mann-Whitney U test
Supplemental Table 4 Identification of selected DGGE bands
Band label / Band position (% of gel length) / Phylum / Band identificationb(genus or family)
1 / 2.1% / Firmicutes / *Erysipelotrichaceae
2 / 10.2% / Bacteroidetes / Bacteroides
3 / 19.1% / Firmicutes / *Ruminococcaceae
4 / 33.4% / Firmicutes / *Lachnospiraceae
5 / 34.3% / Firmicutes / *Erysipelotrichaceae
6 / 37.6% / Firmicutes / Lachnospiraceae
7 / 37.6% / Firmicutes / Lachnospiraceae
8 / 43.9% / Firmicutes / Feacalibacterium
9 / 49.5% / Firmicutes / Streptococcus
10 / 51.8% / Firmicutes / Subdoligranulum
11 / 61.8% / Proteobacteria / *Enterobacteriaceae
12 / 68.1% / Proteobacteria / *Enterobacteriaceae
13 / 71.7% / Firmicutes / *Clostridia
14 / 71.7% / Actinobacteria / *Actinobacteria
15 / 73.6% / Actinobacteria / Bifidobacterium
16 / 73.6% / Actinobacteria / *Coriobacteriaceae
17 / 77.6% / Firmicutes / Dialister
18 / 77.6% / Firmicutes / Dialister
19 / 80.1% / Firmicutes / *Clostridia
20 / 90.1% / Actinobacteria / Bifidobacterium
21 / 90.1% / Actinobacteria / Bifidobacterium
22 / 92.3% / Actinobacteria / Bifidobacterium
23 / 92.3% / Actinobacteria / Bifidobacterium
24 / 97.5% / Actinobacteria / Collinsella
25 / 97.5% / Actinobacteria / Collinsella
26 / 97.5% / Actinobacteria / Collinsella
*The identification on the family level was only relevant
b The same identification of bands at different positions in the DGGE gel indicates that they originated from different taxa belonging to the same genus/family
Supplemental Table 5 Association of microbiota composition with gender; results are presented as medians (range)
Target bacteria(species, genus or group)a,b,c / Gender / p
M
(n=31) / F
(n=29)
Bacteroides-Prevotella group a / 5.73E+10 (2.89E+08-1.30E+12) / 3.13E+10 (2.16E+08-3.73E+11) / 0.176
Clostridium leptumgroup a / 4.61E+11 (3.21E+08-6.44E+12) / 2.86E+11 (1.36E+09-3.06E+12) / 0.246
Clostridium coccoides group a / 6.11E+11 (6.11E+10-3.92E+12) / 4.34E+11 (6.65E+10-1.42E+12) / 0.102
Enterobacteriaceae a / 2.97E+07 (3.98E+05-6.34E+09) / 1.30E+08 (1.60E+06-1.75E+10) / 0.016*
Bifidobacterium a / 4.77E+09 (1.86E+06-8.08E+10) / 4.58E+09 (1.81E+06-5.06E+10) / 0.842
Clostridium clostridioforme a / 4.31E+09 (3.94E+08-2.12E+10) / 2.83E+09 (4.10E+08-1.87E+10) / 0.119
Bacteroidesthetaiotaomicron a / 1.36E+09 (6.05E+05-8.45E+10) / 7.64E+08 (4.71E+06-6.64E+09) / 0.297
Faecalibacteriumprausnitziic / 7.17E+10 (5.13E+07-4.51E+11) / 2.60E+10 (4.23E+09-1.33E+11) / 0.007**
All bacteria a / 8.50E+12 (1.07E+12-1.30E+13) / 5.17E+12 (1.07E+12-1.30E+13) / 0.031*
Bacteroides-Prevotella group / All bacteria (%)b / 0.875 (0.019-14.797) / 0.699 (0.011-5.718) / 0.663
Clostridium leptumgroup / All bacteria (%)b / 5.830 (0.021-22.625) / 5.461 (0.087-52.896) / 0.641
Clostridium coccoidesgroup / All bacteria (%)b / 7.339 (4.007-14.678) / 8.524 (2.641-16.765) / 0.520
Enterobacteriaceae / All bacteria (%)b / 0.001 (0.000-0.141) / 0.004 (0.000-0.141) / 0.002**
Bifidobacterium/ All bacteria (%)b / 0.074 (0.000-0.907) / 0.089 (0.000-0.700) / 0.304
Clostridium clostridioforme / All bacteria (%)b / 0.058 (0.016-0.195) / 0.058 (0.016-0.197) / 0.947
Clostridium clostridioforme / Clostridium coccoidesgroup (%)b / 0.747 (0.162-1.980) / 0.732 (0.203-1.766) / 0.877
Bacteroidesthetaiotaomicron / All bacteria (%)b / 0.015 (0.000-1.397) / 0.017 (0.000-0.222) / 0.888
Bacteroidesthetaiotaomicron / Bacteroides-Prevotella group (%)b / 1.462 (0.185-72.258) / 2.182 (0.033-25.055) / 0.482
Faecalibacteriumprausnitzii/ All bacteria (%)d / 0.821 (0.333-1.563) / 0.720 (0.058-1.704) / 0.162
p,Mann-Whitney U test
Supplemental Fig. 1 RDA biplot(redundancy analysis)showing the association of consumption of particular foods and DGGE. The numerical band designation indicates the length of the migration (%) in the polyacrylamide gradient gel;Veg. diet Vegetarian diet, fluid Liquids, salt Additional salting, solanaceVegetables - family Solanaceae, vitamins Multivitamin dietary supplements, meals Number of meals