Introduction
Materials and methods
Harvesting of L. digitata
Polysaccharide extraction
Crude seaweed powder production
Hot acid extraction
Purification and further processing of extract
Ethanol precipitation
Soluble and insoluble dietary fiber analyses
Depolymerization of extract
Simulated human gastrointestinal digestion
Fecal fermentations
Basal growth media
Fecal sample collection and processing
Fermentation vessel setup and sampling
Enumeration, isolation and identification of Bifidobacteria and Lactobacillus species
Sample dilution
Analysis of short-chain fatty acids
Internal standard and external standard curve generation
Sample processing
GC-FID analysis
454 Pyrosequencing
DNA extraction
Generation of 16S rDNA amplicons
Bioinformatics analysis
Statistical analysis
Results
Dietary fibre analysis
Effects on bacterial composition as assessed by 454 pyrosequencing
Crude extract (CE) (Table 1)
0 h | 24 h | ∆ | Effect |
P
| ||||
---|---|---|---|---|---|---|---|---|
Cellulose (%) | CE (%) | Cellulose (%) | CE (%) | Cellulose | CE | |||
Phylum | ||||||||
Fibrobacteres | 0 (0) | 0.009 (0.006) | 0.04 (0.01) | 0.012 (0.009) | + 0.04 (0.005) | + 0.003 (0.005) |
↓
| 0.026 |
Family | ||||||||
Porphyromonadaceae | 1.305 (0.176) | 1.232 (0.175) | 2.856 (0.703) | 5.941 (0.546) | + 1.551 (0.813) | + 4.709 (0.713) |
↑
| 0.043 |
Fibrobacteraceae | 0 (0) | 0.009 (0.006) | 0.04 (0.01) | 0.012 (0.009) | + 0.04 (0.005) | + 0.003 (0.005) |
↓
| 0.026 |
Streptococcaceae | 0.067 (0.043) | 0.06 (0.004) | 0.097 (0.056) | 0.028 (0.016) | + 0.03 (0.014) | − 0.033 (0.012) |
↓
| 0.025 |
Lachnospiraceae | 32.303 (2.438) | 32.55 (1.878) | 16.718 (0.458) | 26.182 (2.781) | − 15.585 (1.992) | − 6.368 (1.03) |
↑
| 0.015 |
Genus | ||||||||
Parabacteroides | 0.488 (0.152) | 0.511 (0.05) | 1.293 (0.35) | 5.085 (0.674) | + 0.805 (0.255) | + 4.574 (0.633) |
↑
| 0.005 |
Fibrobacter | 0 (0) | 0.009 (0.006) | 0.04 (0.01) | 0.012 (0.009) | + 0.04 (0.005) | + 0.003 (0.005) |
↓
| 0.026 |
Streptococcus | 0.06 (0.041) | 0.06 (0.004) | 0.097 (0.056) | 0.028 (0.016) | + 0.037 (0.015) | − 0.033 (0.012) |
↓
| 0.022 |
Ruminococcus | 2.235 (0.983) | 2.058 (0.524) | 2.204 (1.291) | 0.675 (0.377) | − 0.032 (0.369) | − 1.383 (0.148) |
↓
| 0.027 |
Dialister | 0.589 (0.107) | 0.504 (0.169) | 0.132 (0.084) | 0.463 (0.136) | − 0.457 (0.037) | − 0.042 (0.062) |
↑
| 0.005 |
γ B38UC | 0.019 (0.009) | 0.007 (0.007) | 0.029 (0.01) | 0.041 (0.003) | + 0.01 (0.008) | + 0.034 (0.004) |
↑
| 0.045 |
Depolymerized extract (DE) (Table 2)
0 h | 24 h | ∆ | Effect |
P
| ||||
---|---|---|---|---|---|---|---|---|
Cellulose (%) | DE (%) | Cellulose (%) | DE (%) | Cellulose | DE | |||
Phylum | ||||||||
Actinobacteria | 1.499 (0.299) | 0.873 (0.218) | 1.955 (0.284) | 0.607 (0.13) | + 0.456 (0.068) | − 0.266 (0.171) |
↓
| 0.017 |
Family | ||||||||
Lachnospiraceae | 32.303 (2.438) | 30.968 (1.478) | 16.718 (0.458) | 25.724 (3.06) | − 15.585 (1.992) | − 5.244 (2.791) |
↑
| 0.039 |
Alcaligenaceae | 0.636 (0.088) | 1.071 (0.142) | 6.444 (0.569) | 3.864 (0.54) | + 5.807 (2.793) | + 2.793 (0.682) |
↓
| 0.030 |
Genus | ||||||||
Parabacteroides | 0.488 (0.15) | 0.618 (0.206) | 1.293 (0.35) | 5.64 (1.234) | + 0.805 (0.255) | + 5.022 (1.039) |
↑
| 0.017 |
Lachnospiraceae uc. | 0 (0) | 0.007 (0.003) | 0.003 (0.003) | 0.046 (0.003) | + 0.003 (0.003) | + 0.039 (0.005) |
↑
| 0.003 |
Peptostreptococcaceae IS | 0.846 (0.048) | 1.299 (0.074) | 0.32 (0.085) | 0.244 (0.074) | − 0.526 (0.051) | − 1.056 (0.147) |
↓
| 0.027 |
Dialister | 0.589 (1.234) | 0.418 (0.107) | 0.132 (0.084) | 0.277 (0.104) | − 0.47 (0.037) | − 0.141 (0.054) |
↑
| 0.008 |
Fructooligosaccharides (Table 3)
0 h | 24 h | ∆ | Effect |
P
| ||||
---|---|---|---|---|---|---|---|---|
Cellulose (%) | FOS (%) | Cellulose (%) | FOS (%) | Cellulose | FOS | |||
Phylum | ||||||||
Proteobacteria | 3.11 (0.312) | 2.881 (0.279) | 11.082 (1.226) | 3.968 (1.628) | + 7.972 (1.368) | + 1.087 (1.904) | ↓ | 0.043 |
Family | ||||||||
Streptococcaceae | 0.067 (0.043) | 0.129 (0.045) | 0.097 (0.056) | 0.003 (0.003) | + 0.03 (0.014) | − 0.126 (0.043) | ↓ | 0.025 |
Clostridium XIII IS | 0.032 (0.013) | 0.011 (0.011) | 0.069 (0.067) | 0.227 (0.063) | + 0.037 (0.016) | + 0.216 (0.053) | ↑ | 0.032 |
Lachnospiraceae | 32.303 (2.438) | 27.771 (0.886) | 16.718 (0.485) | 21.909 (1.271) | − 15.585 (1.99) | − 5.862 (0.387) | ↑ | 0.009 |
Alcaligenaceae | 0.636 (0.088) | 0.988 (0.262) | 6.444 (0.569) | 1.959 (0.612) | + 5.807 (0.612) | + 0.971 (0.848) | ↓ | 0.010 |
Genus | ||||||||
Butyricimonas | 0.072 (0.029) | 0.187 (0.046) | 0.288 (0.081) | 0.079 (0.023) | + 0.217 (0.094) | − 0.109 (0.032) | ↓ | 0.030 |
Parabacteroides | 0.488 (0.152) | 0.735 (0.11) | 1.293 (0.35) | 0.579 (0.11) | + 0.805 (0.255) | − 0.155 (0.084) | ↓ | 0.023 |
Streptococcus | 0.06 (0.041) | 0.129 (0.045) | 0.097 (0.056) | 0.003 (0.003) | + 0.037 (0.015) | − 0.125 (0.043) | ↓ | 0.023 |
Christensenella | 0.058 (0.019) | 0.041 (0.015) | 0.094 (0.027) | 0.033 (0.005) | + 0.037 (0.009) | − 0.007 (0.01) | ↓ | 0.033 |
Clostridium XIII IS UC | 0.012 (0.012) | 0.008 (0.008) | 0.023 (0.006) | 0.212 (0.052) | + 0.013 (0.016) | + 0.204 (0.045) | ↑ | 0.016 |
Flavonifractor | 0.085 (0.017) | 0.079 (0.004) | 0.282 (0.032) | 0.082 (0.006) | + 0.197 (0.046) | + 0.003 (0.008) | ↓ | 0.047 |
Sutterella | 0.451 (0.046) | 0.832 (0.302) | 5.304 (1.0) | 1.569 (0.471) | + 4.85 (0.989) | + 0.737 (0.772) | ↓ | 0.030 |
Cronobacter | 0 (0) | 0.003 (0.003) | 0.116 (0.023) | 0.02 (0.016) | + 0.116 (0.023) | + 0.017 (0.012) | ↓ | 0.019 |
Effects on culture-dependent Lactobacillus and Bifidobacteria populations
Effects on short-chain fatty acid production
Crude extract (CE) (Table 4)
Cellulose | FOS | CE | DE | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|
Mean | SEM | Mean | SEM | P value | Mean | SEM | P value | Mean | SEM | P value | |
Acetic acid | |||||||||||
5 | 11.89 | 1.195 | 39.41 | 4.810 | 0.005 | 16.80 | 2.253 | 0.126 | 19.55 | 3.386 | 0.100 |
10 | 16.10 | 1.068 | 45.95 | 4.080 | 0.002 | 30.35 | 1.183 | 0.001 | 42.10 | 5.413 | 0.036 |
24 | 17.95 | 0.101 | 51.36 | 4.492 | 0.002 | 39.71 | 2.122 | 0.009 | 48.65 | 2.538 | 0.007 |
36 | 19.48 | 0.581 | 53.70 | 4.762 | 0.002 | 45.49 | 1.929 | 0.000 | 57.62 | 8.973 | 0.013 |
48 | 20.12 | 0.596 | 55.88 | 4.512 | 0.001 | 54.11 | 11.674 | 0.044 | 54.32 | 3.772 | 0.001 |
Propanoic acid | |||||||||||
5 | 2.75 | 0.196 | 11.06 | 2.632 | 0.035 | 4.53 | 0.177 | 0.003 | 6.06 | 0.509 | 0.004 |
10 | 3.63 | 0.228 | 15.13 | 3.087 | 0.021 | 10.68 | 1.382 | 0.007 | 17.89 | 2.303 | 0.004 |
24 | 4.49 | 0.317 | 17.25 | 2.970 | 0.013 | 15.49 | 1.852 | 0.004 | 20.69 | 1.984 | 0.001 |
36 | 5.11 | 0.149 | 18.26 | 3.107 | 0.013 | 17.80 | 1.326 | 0.010 | 24.39 | 3.999 | 0.009 |
48 | 5.42 | 0.287 | 19.33 | 3.525 | 0.017 | 21.59 | 5.527 | 0.043 | 23.02 | 2.104 | 0.001 |
Butyric acid | |||||||||||
5 | 3.37 | 0.109 | 13.09 | 0.565 | 0.000 | 5.43 | 0.829 | 0.070 | 5.09 | 0.755 | 0.089 |
10 | 4.89 | 0.236 | 22.25 | 2.144 | 0.001 | 11.04 | 1.868 | 0.031 | 9.80 | 1.628 | 0.041 |
24 | 6.65 | 0.148 | 26.44 | 2.867 | 0.002 | 16.01 | 2.090 | 0.011 | 12.42 | 1.467 | 0.017 |
36 | 7.78 | 0.229 | 28.67 | 2.982 | 0.002 | 19.33 | 2.760 | 0.014 | 15.10 | 1.721 | 0.049 |
48 | 8.50 | 0.195 | 30.69 | 3.036 | 0.002 | 22.92 | 0.702 | 0.000 | 15.18 | 1.851 | 0.023 |
Total SCFA | |||||||||||
5 | 18.98 | 1.515 | 63.92 | 2.837 | 0.000 | 27.84 | 3.692 | 0.091 | 32.62 | 5.960 | 0.091 |
10 | 26.62 | 1.500 | 84.88 | 1.544 | 0.000 | 56.14 | 3.701 | 0.002 | 72.99 | 10.163 | 0.011 |
24 | 32.76 | 1.118 | 98.69 | 0.873 | 0.000 | 75.76 | 1.271 | 0.000 | 88.18 | 8.139 | 0.009 |
36 | 36.84 | 1.638 | 105.56 | 1.450 | 0.000 | 89.46 | 3.577 | 0.000 | 104.72 | 16.786 | 0.016 |
48 | 39.13 | 1.366 | 112.04 | 2.861 | 0.000 | 107.79 | 17.710 | 0.018 | 99.98 | 6.970 | 0.001 |
BCFA | |||||||||||
5 | 0.99 | 0.347 | 0.44 | 0.239 | 0.260 | 0.96 | 0.505 | 0.967 | 1.53 | 1.022 | 0.645 |
10 | 1.93 | 0.362 | 1.43 | 0.659 | 0.539 | 3.80 | 2.699 | 0.531 | 2.52 | 1.381 | 0.703 |
24 | 3.31 | 0.754 | 2.98 | 0.840 | 0.781 | 3.64 | 0.745 | 0.774 | 5.05 | 2.785 | 0.602 |
36 | 4.05 | 0.737 | 4.07 | 0.857 | 0.988 | 5.24 | 0.987 | 0.389 | 6.29 | 2.395 | 0.424 |
48 | 4.65 | 0.596 | 5.14 | 0.765 | 0.639 | 7.37 | 0.951 | 0.072 | 6.32 | 1.464 | 0.350 |