Supplementary Data1. Primers used for qPCRTable S1. Primers used for qPCR

Gene Primers

16S-F 5' GGTAGTCCACACAGTAAACGATGAA 3'

16S-R 5' CCCGTCAAATTCCTTTGAGTTTC 3'

BT 2620-F 5' ATACATGCAGCCTTGCGTCA3'

BT 2620-R 5' TCAATGCGCTGCCTATGGTA 3'

BT 2824-F 5' CTCCGACTGGAACGACGAAA 3'

BT 2824-R 5' GCCATTGGGGTAACTGGTGA 3'

BT 3683-F 5' CCGGAAAGAGCAGAACCGTA 3'

BT 3683-R 5' GCCACAGGAATACGAGCCTT3'

BT 2550-F 5'CAATCAGACGCTGCCCAATG3'

BT 2550-R 5' CCATTTGGCATTTTCGGGCA 3'

BT 4671-F 5' GGTGGTGCATCACTTAACGC 3'

BT 4671-R 5' AGCATCTTTCAGCACGGTCA 3'

BT 0339-F 5' CTGACGGCAAACGGGAAATC 3'

BT 0339-R 5' CGGCTCTCGAAAGAAAGGGT 3'

BT 3661-F 5' CCGGAAAGAGCAGAACCGTA 3'

BT 3661-R 5' GCCACAGGAATACGAGCCTT 3'

BT 3703-F 5' TGCGTACAGAAGCGGAAGTT 3'

BT 3703-R 5' CGGCAAACATTCTGCCAACA 3'

2. DEPT spectrum of CDA-0.05 and CDA-0.05-05I

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Fig. S1. DEPT spectrum of CDA-0.05(A), CDA-0.05-05I (B).

3. FT-IR spectrum analysisMethod: FI-IR spectrum was detected based at previous report (Cong et al., 2016). 2 mg

polysaccharide was ground with dried KBr powder and pressed into pellets to conduct by KBr

pellet form on a Perkin Elmer 599B FT-IR spectrometer (Waltham, MA, USA), and scanned from

4000 to 600 cm-1.

Result: According to the FT-IR spectrum of CDA-0.05, typical characteristic absorption

peaks of the polysaccharide were observed (Fig. S2), such as the strong stretching band around

3391 cm-1 and narrow weak peak around 2926 cm-1 could be assigned to O-H and C-H stretching

vibrations, respectively. The relatively strong absorption at 1645 cm-1 represented associated water.

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The strong bands at 1415 cm-1 and 1020 cm-1 were assigned to C-H bending and non-symmetric C-

O-C stretching vibration, respectively.

Fig. S2. IR spectrum of CDA-0.05.

4. Partial acid hydrolysis of CDA-0.05

1) Methods:

The polysaccharide CDA-0.05 was dissolved in 0.5 M TFA (10 mg/mL) and incubated at 100

oC for 1 h. Subsequently, the hydrolysate was co-evaporated with methanol for several times to

give the dry residue which was re-dissolved in water and dialyzed (molecular weight cut off =

1,000 Da) against deionized water (1 L × 5). Finally, the retentate (designated CDA-0.05-05I) was

obtained.

To elucidate the polysaccharide after degradation using acid, molecular weight and

homogeneity, monosaccharide composition analysis and NMR spectroscopy of CDA-0.05-05I

were done as “Section 2.3, 2.4, 2.6” in the main text.

2) Results:

a: CDA-0.05 (100.0 mg) was hydrolyzed with 0.5 M TFA to obtain the CDA-0.05-05I (39.6

mg, yield: 39.6%). HPLC analysis suggested that the average molecular weight of CDA-0.05-05I

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was 3.2 kDa (Fig. S3B). And for your convenience, we also provided the HPLC results of CDA-

0.05 (Fig. S3A) and CDA-0.05-05I (Fig. S3B) as shown below, and the retention time of CDA-

0.05 and CDA-0.05-05I were 25.1 min and 26.8 min, respectively, while the small peak with the

retention time of 30.3 min was the solvent peak (NaNO3). The Mw of CDA-0.05-05I was

estimated as 3.2 kDa according to the standard curve, indicating that the polysaccharide CDA-0.05

was degraded by 0.5 M TFA successfully. Monosaccharide composition analysis showed that

CDA-0.05-05I was composed of Gal and Glc in a molar ratio of 3.1: 96.9. Comparing with CDA-

0.05 (Gal: Glc = 3.6: 96.4), the relative amount of Gal and Glc in CDA-0.05-05I showed a similar

monosaccharide composition, indicating that the backbone of CDA-0.05 might be composed of

Gal and Glc. This result suggested that CDA-0.05-05I might be regarded as repeating units of core

structure of CDA-0.05. It was also reasonable to suggest that CDA-0.05-05I might represent the

core structure of CDA-0.05.

Fig. S3. HPLC results of CDA-0.05 (A) and CDA-0.05-05I (B).

b: In addition, NMR analysis of CDA-0.05-05I was performed and the results showed the

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similar signals as that of CDA-0.05. For your convenience, the 1D-NMR spectra of CDA-0.05 and

CDA-0.05-05I was provided as shown in Fig 2. Compared the 13C NMR and 1H NMR spectra of

CDA-0.05 (Fig. S4A-a, Fig. S4B-a) and CDA-0.05-05I (Fig. S4A-b, Fig S4B-b), they had similar

signals. After partial acid hydrolysis, the signals of galactose still could be seen from NMR

spectrum of CDA-0.05-05I. The major 13C and 1H NMR chemical shifts assigned for CDA-0.05-

05I were listed in Table S2. The 13C NMR spectrum (Fig. S4A-b) of CDA-0.05-05I includes the

anomeric carbon region and the sugar ring carbon region. There are four peaks in the anomeric

region at δ 105.61, δ 101.11, δ 100.83 and δ 99.80, which were assigned to C-1 of 1, 4-linked β- D-

Galp, 1, 4-linked α-D-Glcp, 1, 4, 6-linked α-D-Glcp and T-linked α-D-Glcp, respectively. In DEPT

NMR spectrum, the inverted signal at δ 61.97 could be assigned to C-6 of 1, 4-linked β-D-Galp.

The inverted signals at δ 61.76, δ 68.47 and δ 61.68 could be assigned to C-6 of 1, 4-linked α-D-

Glcp, 1, 4, 6-linked α-D-Glcp and T-linked α-D-Glcp, respectively. Based on the chemical shifts of

anomeric carbon, and combined the HSQC (Fig. S5A) and 1H-1H COSY spectra (Fig. S5C) of

CDA-0.05-05I, δ 4.70 in the 1H NMR spectrum (Fig. S4B-b) was assigned to H-1 of 1, 4-linked β-

D-Galp, which correlated to C-1 of 1, 4-linked β-D-Galp (δ 105.61). δ 5.44 and δ 5.39 were

assigned to H-1 of 1, 4-linked α-D-Glcp and 1, 4, 6-linked α-D-Glcp, correlated to C-1 of 1, 4-

linked α-D-Glcp (δ 101.11) and C-1 of 1, 4, 6-linked α-D-Glcp (δ 100.83) in HSQC spectrum

respectively. And δ 5.03 was assigned to H-1 of T-linked α-D-Glcp, which correlated to C-1 of T-

linked α-D-Glcp (δ 99.80). δ 3.89 was assigned to H-6 of T-linked α-D-Glcp, which correlated to

C-6 of T-linked α-D-Glcp (δ 61.68). δ 3.91 and δ 3.92 were assigned to H-6 of 1, 4-linked α-D-

Glcp, which correlated to C-6 of 1, 4-linked α-D-Glcp (δ 61.76). δ 3.95 and δ 3.83 were assigned

to the H-6 of 1, 4, 6-linked α-D-Glcp and 1, 4-linked β-D-Galp, which correlated to C-6 of 1, 4, 6-

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linked α-D-Glcp (δ 68.47) and 1, 4-linked β-D-Galp (δ 61.97), respectively. The chemical shifts

were listed in Table S2.

Table S2.1H and 13C NMR spectral assignments for CDA-0.05-05I (ppm)

Residues 1 2 3 4 5 6

1, 4-β-Gal C 105.61 72.90 75.73 79.10 70.71 61.97

H 4.70 3.75 3.81 4.23 3.88 3.83

1, 4-α-Glc C 101.11 72.43 72.77 78.10 74.58 61.76

H 5.44 3.71 3.91 3.71 4.03 3.91/3.92

1, 4, 6-α-Glc C 100.83 72.77 72.99 78.94 74.22 68.47

H 5.39 3.67 3.90 3.73 4.01 3.95

T-α-Glc C 99.80 71.61 72.57 73.99 70.58 61.68

H 5.03 3.65 3.89 3.75 3.48 3.89

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Fig. S4. 1H and 13C NMR spectra of the polysaccharide CDA-0.05 and CDA-0.05-05I. (A) 13C NMR spectra of

CDA-0.05 (a), CDA-0.05-05 (b) (B) 1H NMR spectra of CDA-0.05 (a), CDA-0.05-05I (b).

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HMBC (Fig. S5B) spectrum was employed to further confirm the structure and the

substitution sites as well as configuration of the assignments of CDA-0.05-05I. Briefly, cross peak

a at δ 78.10/5.44 showed the correlation between C-4 of 1, 4-linked α-D-Glcp and H-1 of

neighboring 1, 4-linked α-D-Glcp, and cross peak b at δ 101.11/3.71 suggested the correction

between C-1 of 1, 4-linked α-D-Glcp and H-4 of neighboring 1, 4-linked α-D-Glcp, respectively.

That indicated the presence of consecutive 1, 4-linked α-D-Glcp. Furthermore, obvious resonance

c signal at δ 101.11/3.73 suggested that C-1 of 1, 4-linked α-D-Glcp were correlated to the H-4 of

neighboring 1, 4, 6-linked α-D-Glcp, while cross peak d at δ 78.94/5.44 showed the correlation

between C-4 of 1, 4, 6-linked α-D-Glcp and H-1 of neighboring 1, 4-linked α-D-Glcp,

respectively. In addition, cross peak e at δ 105.61/3.71 suggested that C-1 of 1, 4-linked β- D-Galp

was correlated to the adjacent H-4 of neighboring 1, 4-linked α-D-Glcp, cross peak f at δ

78.10/4.70 showed the correlation between C-4 of 1, 4-linked α-D-Glcp and H-1 of neighboring 1,

4-linked β-D-Galp. Moreover, cross peaks g at δ 68.47/5.03 showed the correlation between C-6

of 1, 4, 6-linked α-D-Glcp and H-1 of neighboring T-linked α-D-Glcp, while cross peak h at δ

99.80/3.95 showed the correlation between C-1 of T-linked α-D-Glcp and H-6 of neighboring 1, 4,

6-linked α-D-Glcp, respectively. The correlation suggested that T-linked α-D-Glcp was linked to

the C-6 position of 1, 4, 6-linked α-D-Glcp. Given the above results, the main chain of CDA-0.05-

05I composed of 1, 4-linked α-D-Glcp, 1, 4, 6-linked α-D-Glcp and 1, 4-linked β-D-Galp, with

branches contained T-linked α-D-Glcp attached at C-6 of 1, 4, 6-linked α-D-Glcp residues. Since

CDA-0.05-05I showed the same repeating unit as that of CDA-0.05, suggesting that the main

chain of CDA-0.05 was composed of 1, 4-linked α-D-Glcp, 1, 4, 6-linked α-D-Glcp and 1, 4-

linked β-D-Galp. Therefore, we think that figure 3 should be showed as a proposed structure of

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this polysaccharide.

Fig. S5. 2D NMR spectra of the polysaccharide CDA-0.05-05I. (A) HSQC spectrum; (B) HMBC spectrum; (C)

1H-1H COSY spectrum.

5. α-glucosidase, β-galactosidase, α-glucanase activity test.

5.1) The enzyme activity test for β-galactosidase

a) Method and reagent:

Enzyme assay for β-galactosidase was described by Frank H. Stephenson with minor

modifications (Stephenson, 2016). BT, BO and BF stains were cultured anaerobically in minimal

medium supplemented with CDA-0.05 (2.5 mg/mL) or glucose respectively for 24 h. 1 mL

bacterial cells were collected by centrifugation at 3,000 rpm for 10 min, followed by the

supernatant and pellet collection. Then the pellet was washed by PBS. The pellet was diluted in Z

buffer (V) until the OD600 was around 1.2. After 1 h, the dilute cells were permeabilized by 100 µL

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chloroform and 50 µL 0.1% SDS (sodium dodecyl sulfate, sodium laurel sulfate). The reaction

was started by adding 0.2 mL O-nitrophenyl-β-D-galactoside (ONPG, 4 mg/mL). When the

sufficient yellow color has developed (as yellow as LB broth), the reaction was stopped by 0.5 mL

Na2CO3 (1 M). Record the time of reaction (T) and the reaction was centrifuged at 3,000 rpm for 5

min. 0.2 mL aqueous layer was transferred to 96 well plate and read at 420 nm and at 550 nm.

Calculate Miller Units as: Miller Units = 1000 x [(OD420 - 1.75 x OD550)] / (T x V x OD600) (T =

time of the reaction in minutes; V = volume of bacteria cell in mL).

b) Result:

The mean Miller Units of BT, BF, BO cultured with CDA-0.05 was 70.33, 308.10, 49.37,

respectively. And the mean Miller Units of BT, BF, BO cultured with glucose was 56.31, 205.19,

526.24. And there was almost no yellow color in supernatant of culturing. These results indicated

that BT, BF and BO could all express β-galactosidase to digest carbon sources. The expression

level of β-galactosidase was high in BF and BT strains, which was higher induced by CDA-0.05

than that by glucose.

5.2) The enzyme activity test for α-glucosidase

a) Method and reagent:

α-glucosidase was measured according to the Micro α-glucosidase（ α - GC）Assay Kit

bought from Beijing Solarbio Science & Technology company, China. Followed the manual, BT,

BO and BF stains were cultured anaerobically in minimal medium supplemented with CDA-0.05

(2.5 mg/mL) or glucose respectively for 24 h. 1 mL bacterial cells were collected by centrifugation

at 3,000 rpm for 10 min, collect the supernatant and pellet. For the pellet, add 1 mL extraction

buffer and bacteria was broken by the ultrasonic wave, followed by the supernatant collection to

perform the enzyme test. The reaction was started by adding 0.1 mL nitrophenylcarbimide into 30

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µL super-natant, incubate for 30 min at 37 oC, then incubated in a boiling water bath, followed by

cooling. Centrifugate the mixture at 8,000 g, for 5 min, then 70 µL supernatant was transferred

into 96- well plate with 130 µL color agent and read at 400 nm after 2 min. Calculate GC activity

as: U/104 cells = 0.0187 x y (y = concentration of production, calculate based on the standard

curve using PNP).

b) Result:

The mean α-glucosidase activity of BT, BF, BO cultured with CDA-0.05 was 0.23, 4.51,

11.42, respectively. And the mean α-glucosidase activity of BT, BF, BO cultured with glucose was

0.72, 7.71, 7.68. However, there was almost no yellow color in supernatant of culturing, indicating

the enzyme was mainly expressed in Bacteroides rather than in the culture filtrates. These results

suggested that all BT, BF and BO could express α-glucosidase to digest carbon sources. The

expression level of α-glucosidase was a little low in BT strain, however there was no significant

difference about this enzyme expression induced by CDA-0.05 or glucose.

5.3) The enzyme activity test for α-glucanase.

a) Method and reagent:

The α-glucanase could degrade starch. So we used traditional potassium iodide (KI) reacting

with starch to the test the enzyme activity. Briefly, the bacteria was collected to test the activity of

α-glucanase. The pellet was also broken by the ultrasonic wave, and centrifuged to collect the

supernatant. Before testing, the ultrasonic bacteria supernatant, α-amylase (positive control), and

diluted water (negative control) were incubated at 70 oC for 15 min to inactivate β- amylase, then

incubate starch solution (1%) with the ultrasonic bacteria supernatant at 40 oC for 10 min followed

by cooling. Finally, add 10 µL KI to test starch content. The absorbance was detected at 660 nm.

b) Result:

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As we know, the blue color like negative control (Fig. S6) showed the positive reaction of

starch existence after the enzyme treatment, so the color was related to enzyme activity reversely.

The results showed that absorption at OD660 of the lysis of BT, BF, BO cultured with CDA-0.05

was 0.104, 0.096, 0.302, respectively. And the OD660 absorbance of BT, BF, BO cultured with

glucose was 0.386, 0.307, 0.393. The OD660 absorbance of negative control (starch with buffer)

was 0.358, while the positive control of α-amylase was 0.069. The result indicated that α-amylase

could be induced in BT and BF strain with CDA-0.05 rather than glucose. However, just a little

enzyme activity was detected in BO with CDA-0.05. The result suggested that α-glucanase was

upregulated in BT and BF strain by CDA-0.05.

Fig. S6. α-glucanase activity test

Taken together, the BT strain could be induced by this polysaccharide to highly express α-

glucanase and β-galactosidase. And three enzymes, α-1,4-glucanase, α-1,6-glucosidase and β-1,4

galactosidase could be induced by CDA-0.05 in the BF strains. While α-glucanase and α-

glucosidase were high expressed in BO strain by CDA-0.05. And we checked the three

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upregulated genes, BT2824 (Q8A3Y0), BT3683 (Q8A1H8), and BT2550 (Q8A4P7) coded

protein in Universal Protein database, and they are reported to encode glucanase.

References:

Cong, Q., Chen, H., Liao, W., Xiao, F., Wang, P., Qin, Y., et al. (2016). Structural characterization

and effect on anti-angiogenic activity of a fucoidan from Sargassum fusiforme. Carbohydrate

Polymers, 136, 899-907.

Stephenson, F. H. (2016). Chapter 11 - Protein. In F. H. Stephenson (Ed.), Calculations for

Molecular Biology and Biotechnology (Third Edition) (pp. 375-429). Boston: Academic

Press.

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