Food Chemistry 377 (2022) 132001

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

A novel glucofucobiose with potential prebiotic activity prepared from the

exopolysaccharides of Clavibacter michiganensis M1
Mengshi Xiao a, Xinmiao Ren a, Jinzheng Cui d, Rong Li e, Zhemin Liu a, Lin Zhu a, c, Qing Kong a,
Xiaodan Fu b, *, Haijin Mou a, *
a
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, Shandong, People’s Republic of China
b
State Key Laboratory of Food Science and Technology, China-Canada Joint Laboratory of Food Science and Technology (Nanchang), Nanchang University, 235
Nanjing East Road, Nanchang, Jiangxi 330047, People’s Republic of China
c
Weihai Deepsea Biotechnology Co., Ltd, Weihai 264300, Shandong, People’s Republic of China
d
School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, Shandong, People’s Republic of China
e
Qingdao Women and Children Hospital, Qingdao 266003, Shandong, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Fucose and fucosylated oligosaccharides have important applications in various industries owing to their pre­
Fucosylated oligosaccharide biotic, anti-inflammatory, anticoagulant, and antiviral activities. Here, we aimed to obtain fucosylated oligo­
2′ -Fucosyllactose saccharides using the acidolysis method to depolymerize exopolysaccharides extracted from Clavibacter
Bacterial exopolysaccharides
michiganensis M1. Based on structural analysis, the prepared glucofucobiose was found to consist of D-glucose and
Glucofucobiose
L-fucose, with a molecular weight of 326 Da and a structure of D-Glcp-β-(1→4)-L-Fucp. The prebiotic activity of
Structure
Prebiotic activity glucofucobiose was compared with that of 2′ -fucosyllactose (2′ -FL), the most abundant oligosaccharide in human
milk. According to the results, glucofucobiose could significantly promote the proliferation of six probiotic
strains, and short-chain fatty acid production of five probiotic strains on glucofucobiose was substantially higher
than that on 2′ -FL at 48 h of fermentation. Overall, this study proposed a new technology for obtaining fuco­
sylated oligosaccharides. The prepared glucofucobiose was found to exhibit potential prebiotic activity and
should be further assessed.

1. Introduction capabilities of the brain have been well established (Matthies,

Fucose is an industrially useful sugar with high economic potential
and diverse physiological activities, such as antibacterial, anticoagulant,
and anticancer activities (Torres et al., 2012). In addition to being an
important component in eukaryotic cells, fucose is also commonly found
in natural plants, including brown algae and tragacanth gum, and it is
widely used in the pharmaceutical and cosmetic industries (Silchenko
et al., 2018; Abbasi et al., 2019). Further, its importance in the regula­
tion of intestinal microbiota and ability to improve perception
Schroeder, Smalla, & Krug, 2000). However, it is unrealistic to obtain
fucose via chemical synthesis to meet the needs of large-scale industrial
production. Therefore, new sources of fucose need to be explored.
Although the intestinal microbiota of adults is complex, that of
healthy full-term infants is relatively simple comprising primary colo­
nizers of the genus Bifidobacterium, which can persist until early child­
hood (Bäckhed et al., 2015). In the first six months following birth, a
decrease in bifidobacteria or an increase in other bacteria may consid­
erably change the natural composition of the microbial community,

Abbreviations: EPS, exopolysaccharides; HMOs, human milk oligosaccharides; 2′ -FL, 2′ -fucosyllactose; HCl, hydrochloric acid; TFA, trifluoroacetic acid; GF,
glucofucobiose; TLC, thin-layer chromatography; SCFAs, short-chain fatty acids; HPLC, high-performance liquid chromatography; ESI-MS, electron spray ionization
mass spectrometry; ESI-CID-MS, electron spray ionization in tandem with collision-induced dissociation tandem mass spectrometry; NMR, nuclear magnetic reso­
nance; CMM1, Clavibacter michiganensis M1; BLI, Bifidobacterium longum subsp. infantis ATCC 15697; BB, Bifidobacterium breve ATCC 15700; BL, Bifidobacterium
longum ATCC BAA-999; LC, Lactobacillus casei ZX147; LB, Lactobacillus plantarum CGMCC 1.19; CB, Clostridium butyricum YX038.
* Corresponding authors at: State Key Laboratory of Food Science and Technology, China-Canada Joint Laboratory of Food Science and Technology (Nanchang),
Nanchang University, 235 Nanjing East Road, Nanchang 330047, Jiangxi, People’s Republic of China (X. Fu). College of Food Science and Engineering, Ocean
University of China, No. 5 Yushan Road, Qingdao 266003, Shandong, People’s Republic of China (H. Mou).
E-mail addresses: 18227591863@163.com (M. Xiao), m13992623179@163.com (X. Ren), 2970565907@qq.com (J. Cui), lirong81@163.com (R. Li),
ocean2013@126.com (Z. Liu), zhulin5566@126.com (L. Zhu), kongqing@ouc.edu.cn (Q. Kong), luna_9303@163.com (X. Fu), mousun@ouc.edu.cn (H. Mou).

https://doi.org/10.1016/j.foodchem.2021.132001
Received 18 September 2021; Received in revised form 14 November 2021; Accepted 18 November 2021
Available online 4 January 2022
0308-8146/© 2022 Elsevier Ltd. All rights reserved.
M. Xiao et al.
leading to various negative effects on infant health, including allergy
and childhood obesity (Wampach et al., 2018). Therefore, discovering a
prebiotic that can promote the proliferation of probiotics in the infant
gut to reduce the occurrence of early infant diseases is of great signifi­
cance. Fucosylated oligosaccharides are common in the human intesti­
nal tract and mostly located at the end of mucin glycoproteins on the cell
surface (Becker & Lowe, 2003). These fucosylated oligosaccharides
coated with mucins are considered to be adhesion targets for many in­
testinal bacteria (Stahl et al., 2011). Fucose and fucosylated oligosac­
charides also serve as chemoattractants and carbon sources for gut
bacteria. Human milk oligosaccharides (HMOs) are oligosaccharides
that exist only in human milk (Stahl et al., 2011). It has been reported
that probiotic bacteria abundant in early life, such as bifidobacterial
strains, are capable of recognizing and metabolizing fucosylated HMOs
(Chambers & Townsend, 2020). 2′ -Fucosyllactose (2′ -FL) is the most
abundant oligosaccharide in human milk and it has been proved to
regulate infant intestinal microbiota (Zabel et al., 2020). However,
complex preparation methods have limited the wide application and
further studies of 2′ -FL. Nevertheless, fucosylated oligosaccharides have
also been prepared via hydrolyzing fucoidan extracted from brown algae
by fucoidanase in recent years (Silchenko et al., 2018).
Microbial exopolysaccharides (EPS) containing fucose are a pro­
spective source of fucose and fucosylated oligosaccharides. The ability
to produce EPS is an attribute widely found among various microbial
species, including bacteria, microalgae, and fungi (Vanhooren & Van­
damme, 1999). Microbial synthesis of EPS is amenable to control the
process conditions and it is unaffected by environmental factors (Alves
et al., 2010). However, only few bacterial EPS are commercially avail­
able, such as xanthan, gellan gum, and hyaluronic acid (Freitas, Alves, &
Reis, 2011). Fucose-containing EPS have been extracted from several
bacterial strains (Vanhooren & Vandamme, 1999). Colanic acid is a type
of EPS composed of fucose, glucose, galactose, and glucuronic acid,
which is generally produced by members of Enterobacteriaceae,
including Escherichia and Klebsiella spp. (Rättö et al., 2006). Clavan,
which is composed of glucose, galactose, fucose, and pyruvate, can be
produced by Clavibacter michiganensis strains (Bulk, Zevenhuizen, Cor­
dewener, & Dons, 1991). Fucogel, a fucose-rich type of EPS extracted
from Klebsiella pneumoniae I-1507, is used in the cosmetic industry
(Guetta, Mazeau, Auzely, Milas, & Rinaudo, 2003). The EPS produced
by Enterobacter sp. A47, FucoPol, also have high fucose content
(Antunes, Freitas, Sevrin, Grandfils, & Reis, 2017).
To obtain fucosylated oligosaccharides, fucose-containing EPS must
be further depolymerized. Over the last several years, hydrochloric acid
(HCl) and trifluoroacetic acid (TFA) have been used to degrade EPS to
obtain oligosaccharides. The hydrolysis of EPS by HCl is usually carried
out using a high concentration of HCl at high temperature. The HCl
acidolysis process is simple and easy to perform; however, the produc­
tion of oligosaccharides with molecular weights greater than those of
tetrasaccharides is not an easy task (Jang et al., 2005). Hydrolysis by
TFA was found to be mild, and TFA could be removed via freeze-drying
or vacuum distillation after hydrolysis, thereby avoiding salt production
during the traditional neutralization steps (Fels, Jakob, Vogel, & Wefers,
2018; Maalej et al., 2014).
Fucose-containing EPS extracted from C. michiganensis is a prospec­
tive source of fucose and fucosylated oligosaccharides. In the current
study, C. michiganensis M1 (CMM1) was used to prepare fucose-
containing EPS, and fucosylated oligosaccharides were obtained by
acid hydrolysis of fucose-containing EPS. The acidolysis conditions for
CMM1 EPS were optimized to select the degradation conditions result­
ing in degradation products with single-component and low mono­
saccharide contents. The structure of the glucofucobiose (GF) obtained
by acidolysis was analyzed and the in vitro prebiotic activity of GF on six
probiotic strains was compared with that of 2′ -FL and CMM1 EPS. By
introducing a novel preparation method for fucosylated oligosaccha­
rides and comparing the prebiotic activity of 2′ -FL and GF prepared
using the bacterial fermentation method, the present study may serve as
2
Food Chemistry 377 (2022) 132001
a reference strategy for exploring potential fucosylated oligosaccharides
with functional abilities similar to HMOs.
2. Materials and methods
2.1. Bacterial strains and chemicals
The strains used in the present study, CMM1 isolated from decaying
tomato leaves, Lactobacillus casei ZX147 (LC), and Clostridium butyricum
YX038 (CB) isolated from feces of infants, were provided by the Applied
Microbiology Laboratory, Ocean University of China. Bifidobacterium
longum subsp. infantis ATCC 15697 (BLI), Bifidobacterium breve ATCC
15700 (BB), and Bifidobacterium longum ATCC BAA-999 (BL) were pur­
chased from the American Type Culture Collection (ATCC, Rockville,
MD, USA). Lactobacillus plantarum CGMCC 1.19 (LP) was purchased
from the China General Microbiological Culture Collection Center
(CGMCC, Beijing, China). Bio-gel P4 was acquired from Bio-Rad (Her­
cules, CA, USA). Standards for short-chain fatty acids (SCFAs) (lactic
acid, propionic acid, butyric acid, formic acid, acetic acid, and iso­
caproic acid) and monosaccharides (glucose, galactose, fucose,
mannose, rhamnose, glucuronic acid, and galacturonic acid) were pur­
chased from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Lactose,
mannotriose, mannotetraose, mannose pentasaccharide, and mannose
hexasaccharide were purchased from Solarbio Technology Co., Ltd.
(Beijing, China). Thin-layer chromatography (TLC) silica gel was pur­
chased from Merck (Darmstadt, Germany). All reagents were of
analytical grade.
2.2. Extraction and purification of CMM1 EPS
The CMM1 strain preserved in 30% (v/v) glycerin was inoculated
into a 250-mL shake flask containing 100 mL YPG medium (yeast extract
5 g/L, peptone 10 g/L, glucose 5 g/L; pH 7.0–7.2) and cultivated for 24 h
at 27 ◦ C. The fermentation medium used for CMM1 EPS production was
prepared according to the method described by Bulk et al. (1991), with
the following modifications: glucose 20 g/L, yeast extract 4 g/L, CaCO3
5 g/L, (NH4)2SO4 0.3 g/L, KH2PO4 1.3 g/L, and K2HPO4 1.7 g/L; pH
7.0–7.2. The CMM1 strain was inoculated into 100 mL of the fermen­
tation broth at a concentration of 1% (v/v) in a 250-mL conical flask.
The cultures were incubated at 27 ◦ C for 5 days. After the incubation
period, in order to remove the bacteria and insoluble substances, the
fermentation broth was centrifuged at 6000 ×g for 10 min at 4 ◦ C. After
centrifugation, the supernatant was evaporated to 1/4 of the original
volume at 50 ◦ C. Three volumes of 95% ethanol were then added, and
the supernatant was allowed to stand overnight. The supernatant was
centrifuged at 8000 ×g for 10 min at 4 ◦ C to collect the precipitate,
which was washed with 95% ethanol. The precipitate was redissolved in
25 mL of ultrapure water, and the protein in the precipitate was removed
using the Sevag method (Sevag, Lackman, & Smolens, 1938). The su­
pernatant was dialyzed using a 10 kDa dialysis bag at 4 ◦ C for 72 h.
Finally, CMM1 EPS was obtained after vacuum freeze-drying.
2.3. Optimization of the acidolysis conditions for CMM1 EPS
Fucosylated oligosaccharides were prepared by degrading CMM1
EPS via acid hydrolysis (acidolysis). To select the degradation conditions
resulting in degradation products with single-component and low
monosaccharide contents, the acidolysis conditions need to be opti­
mized. In the present study, the acidolysis reaction was carried out by
treating CMM1 EPS with HCl (0.2 mol/L HCl or 0.1 mol/L HCl) or TFA
(0.2 mol/L TFA or 0.1 mol/L TFA) at 80 ◦ C or 90 ◦ C, respectively. The
material to liquor ratio was set to 1:10, and the acidolysis reaction was
performed in a water bath shaker for 8 h. Samples were taken every 2 h
to characterize the variations of carbohydrate degradation and hydro­
lysate production via high-performance liquid chromatography (HPLC)
and TLC. Before HPLC and TLC analyses, the pH of the acidolysis
M. Xiao et al.
solution was adjusted to 7.0 using 1 mol/L NaOH. The hydrolysate was
then mixed with three volumes of ethanol to remove high molecular
weight precipitates, and the supernatant was collected by centrifugation
at 5000 ×g for 10 min and freeze-dried.
The above lyophilized hydrolysate was solubilized at 5 mg/mL and
then separated on a Shodex OH pak SB 802.5 HQ column (8 mm × 300
mm, 6 μm, Shodex Technology) with a refractive index detector. The
mobile phase was 100% water, the flow rate was 0.85 mL/min, and the
temperature of the column oven was set at 50 ◦ C.
The developing agents were N-butanol, acetic acid, and water at
1:1:1. The chromogenic agent was composed of 1 mL HCl, 3 mL aniline,
4 g diphenylamine, 10 mL 85% H3PO4, and 100 mL acetone. The loading
volume was 3 μL (5 mg/mL lyophilized hydrolysate solution). Glucose,
galactose, and fucose were used as the standards.
2.4. Preparation and purification of fucosylated oligosaccharides
The CMM1 EPS was degraded under the optimized conditions (0.2
mol/L TFA, 90 ◦ C, 2 h). Thereafter, the acidolyzed EPS solution was
cooled to 25 ◦ C. The remaining TFA in the solution was removed via a
dedicated electrodialysis (Sichuan Lvwo Environmental & Energy Co.,
Ltd) with the current and voltage set at 3 A and 18 V, respectively. The
acidolysis products were centrifuged at 5000 ×g for 10 min at 4 ◦ C.
Thereafter, 95% ethanol (three times the volume of supernatant) was
added to the supernatant to precipitate the high molecular weight
substances at 4 ◦ C overnight. Centrifugation was performed at 5000 ×g
for 10 min at 4 ◦ C to collect the supernatant. Finally, crude fucosylated
oligosaccharides were obtained via vacuum lyophilization.
A Bio-gel P4 column was used for the purification of the acidolysis
products. The mobile phase was 0.2 mol/L NH4HCO3, the flow rate was
0.15 mL/min, and the loading volume was 500 µL (50 mg/mL crude
oligosaccharides powder). The eluent was collected in 40 tubes with 1.5
mL of eluent per tube. The total sugar content of each tube was then
determined according to DuBois, Gilles, Hamilton, Rebers, and Smith
(1956), with slight modifications. Briefly, 200 µL of sample, 400 µL of
6% phenol solution, and 2 mL of concentrated sulfuric acid were mixed
evenly and reacted for 30 min. Thereafter, their optical density at 490
nm (OD490) was determined, and the total sugar content was calculated
using a glucose standard curve (Y = 0.0052 X - 0.0706, R2 = 0.9999).
The elution curve was plotted with the elution time as the abscissa and
the total sugar content as the ordinate. After purification, acid hydro­
lysates were collected for structural characterization.
2.5. Structural characterization of the fucosylated oligosaccharides
2.5.1. Mass spectrometry analysis
The molecular weight of the oligosaccharide samples was identified
by electron spray ionization mass spectrometry (ESI-MS) and electron
spray ionization in tandem with collision-induced dissociation tandem
MS (ESI-CID-MS), which was carried out using an Agilent 1290 Infinity
II system and 6460 triple quadrupole MS (Agilent Technologies, Santa
Clara, CA, USA). The elution was performed using 50% acetonitrile and
50% water at a flow rate of 0.35 mL/min. The two mass spectrometers
were operated in positive ion mode with a capillary voltage of 3.5 kV
and a nebulizer pressure of 40 psi. The collision energy was set to 15–60
eV, and the collision gas was nitrogen. All spectra were analyzed using
MassHunter Qualitative Analysis B.07.00 (Agilent Technologies).
2.5.2. Monosaccharide composition analysis
Seven monosaccharide standards and one lactose internal standard
were combined into a mixed standard solution with a concentration of 1
mg/mL, respectively. Ten milligrams of fucosylated oligosaccharides
were dissolved in 2 mL of 2 mol/L TFA, reacted at 110 ◦ C for 4 h, and
then cooled to 25 ◦ C. After removing TFA, the pH was adjusted to 7.0
using a solution of NaOH (6 mol/L). Thereafter, the hydrolysate and
monosaccharide standards were mixed with L-cysteine methyl ester and
3
Food Chemistry 377 (2022) 132001
pyridine. The mixture was heated at 60 ◦ C for 1 h, and arylthiocyanate
was added to the mixture. The reaction was allowed to proceed for 1 h
(Tanaka, Nakashima, Ueda, Tomii, & Kouno, 2007). Then, the deriva­
tization of these samples was performed as described in Fu, Li, Zhang, Li,
and Mou (2018). The monosaccharide composition was determined
using an HPLC system equipped with a ZORBAX 300 XDB-C18 column
(4.6 mm × 250 mm, 5 μm, Agilent Technologies). The mobile phase
consisted of mobile phase A (acetonitrile) and mobile phase B (0.05
mol/L KH2PO4 buffer solution) at a ratio of 17:83. The UV detection
wavelength was 245 nm, and the flow rate was 1 mL/min. The sample
injection volume was 20 µL.
2.5.3. Nuclear magnetic resonance spectroscopy analysis
Samples (15 mg) were exchanged twice in 99.9% D2O with inter­
mediate lyophilization, and finally dissolved in 500 µL D2O. One-
dimensional nuclear magnetic resonance (NMR) spectra (1H, 13C,
DEPT 90◦ and 135◦ ) and two-dimensional NMR spectra (1H–1H COSY,
1
H–1H TOCSY, 1H–13C HSQC, and 1H–13C HMBC) were recorded on an
Agilent DD2-500 spectrometer (Agilent Technologies), with a frequency
of 500 MHz and a temperature of 25 ◦ C. 1H and 13C chemical shifts are
expressed in ppm in reference to internal acetone (2.225 ppm for 1H and
31.07 ppm for 13C). All spectra were analyzed using MestReNova 6.1
(Mestrelabs Research SL, Santiago de Compostela, Spain), with Whit­
taker Smoother baseline correction.
2.6. In vitro prebiotic activity of fucosylated oligosaccharides
MRS broth medium containing 0.50 g/L cysteine hydrochloride was
used to activate three bifidobacterial strains (BLI, BB, and BL) and two
lactobacillus strains (LC and LP). RCM medium, purchased from Qing­
dao Hope Technology Co., Ltd. (Qingdao, China), was used to activate
CB. Each strain was activated three times at 37 ◦ C prior to fermentation
experiments. In vitro fermentation was performed in a simulated intes­
tinal medium, which was prepared according to the methods described
by Fu et al. (2020), with slight modifications. The amounts of yeast
extract and peptone were adjusted to 1 g/L and 2 g/L, respectively. EPS,
2′ -FL, and GF were added to anaerobic bottles with 50 mL of simulated
intestinal medium at a final concentration of 10 mg/mL. In vitro
fermentation was performed at 37 ◦ C for 48 h.
Fermentation samples were collected at 0, 12, 24, 36, and 48 h. The
samples were centrifuged at 13000 ×g for 10 min at 4 ◦ C in a refriger­
ated centrifuge (Thermo Fisher Scientific, Waltham, MA, USA). The
bacterial precipitation was eluted with phosphate-buffered saline buffer
(8 g/L NaCl, 0.2 g/L KCl, 1.44 g/L KH2PO4 and 0.24 g/L K2HPO4; pH
7.2) and the OD600 of this eluent was determined. The centrifuged su­
pernatant was collected for further pH, SCFAs, and TLC analyses. The
SCFAs content was determined as described previously (Wei et al.,
2020). The TLC analysis procedures were the same as those described in
Section 2.3.
3. Results and discussion
3.1. Optimization of the acidolysis conditions on CMM1 EPS
Different acidolysis conditions were used to degrade the CMM1 EPS,
and samples were taken every 2 h for TLC and HPLC analyses. For the
EPS samples treated with an equal amount of acid concentration, the
color of monosaccharide spots gradually deepened with an increase in
acidolysis temperature and time (Fig. S1). EPS were degraded more
completely by HCl than by TFA of the same concentration under the
same acidolysis temperature and time conditions. Thus, more mono­
saccharides were produced during the degradation of EPS by HCl than
during the degradation of EPS by TFA.
The effects of the acidolysis conditions on the molecular weight
distribution of CMM1 EPS are shown in Fig. 1 and Table S1. The CMM1
EPS was effectively hydrolyzed, generating substances with molecular
M. Xiao et al. Food Chemistry 377 (2022) 132001

Fig. 1. GPC spectra of the degradation of CMM1 EPS by hydrochloric acid (HCl) and trifluoroacetic acid (TFA). (A) The molecular weight distribution of CMM1 EPS
degraded by 0.2 mol/L TFA (A), 0.1 mol/L TFA (B), 0.2 mol/L HCl (C), and 0.1 mol/L HCl (D) at 80 or 90 ◦ C for 2, 4, 6, and 8 h, respectively. Mannose hex­
asaccharide (1), mannose hexasaccharide (2), mannotetraose (3), mannotriose (4), lactose (5), glucose (6), and fucose (7) were used as standards.

weight less than 1000 Da. However, different types and concentrations
of acids and different acidolysis temperature and time led to different
degrees of CMM1 EPS degradation. CMM1 EPS was degraded more
thoroughly under higher concentration of acid and acidolysis tempera­
ture and longer acidolysis time. There was one main peak in the
spectrum when the EPS was degraded by 0.2 mol/L TFA at 90 ◦ C for 2 h,
which was consistent with the TLC analysis (Fig. S1). The retention time
of the peak was close to that of lactose, suggesting that the peak may
correspond to a disaccharide. Considering the preparation of fucosylated
oligosaccharides with single-component and low monosaccharide

4
M. Xiao et al. Food Chemistry 377 (2022) 132001

contents, TFA (0.2 mol/L) was selected to degrade CMM1 EPS at 90 ◦ C
for 2 h.
3.2. Purification of the acidolysis products
After acidolysis of CMM1 EPS, a Bio-gel P4 gel column was used to
separate and purify the resulting products. As shown in Fig. S2, three
peaks, P1, P2, and P3, were obtained, with P2 as the main peak. The
fractions corresponding to the three peaks were collected and purified
for subsequent analyses.
3.3. Structural characterization
3.3.1. Mass spectrometry analysis of the three peaks
After several separation and purification steps, the three peak frac­
tions were analyzed by positive ion ESI-MS to determine the molecular
weight. For P1, only one peak was observed at m/z 511.1, as shown in
Fig. 2A, suggesting that peak P1 may be a trisaccharide (511.1 = 180 +
180 + 164 – 36 + Na). The MS result of peak P2 is shown in Fig. 2B; two
main peaks, at m/z 327.3 and m/z 349.1, which were [M + H]+ and [M
+ Na]+, respectively, were observed, indicating that peak P2 may be a
disaccharide. As shown in Fig. 2C, for P3, two peaks (m/z 165.5 and
181.1), which were assigned to [F + H]+ and [G + H]+, were observed,
indicating that peak P3 was composed of monosaccharides with mo­
lecular weights of 180 and 164 Da. Owing to the low content of peaks P1
and P3, peak P2 was mainly collected for structural characterization and
activity studies. Further, ESI-CID-MS was performed to verify the
composition of peak P2, as shown in Fig. 2D. After ionization, the peak
at m/z 349.1 was knocked out, resulting in a charge peak at m/z 169.2,
which was expected to be [M + Na − 180]+. Altogether, the above re­
sults confirmed that peak P2 was a disaccharide with a molecular weight
of 326 Da.
3.3.2. Monosaccharide composition of peak P2
The mixed standard monosaccharides and peak P2 were analyzed by
HPLC after derivatization using lactose as the internal standard (Fig. S3).
Based on the results, peak P2 comprised D-glucose and L-fucose with an
approximate molar ratio of 1:1 according to the peak area. Based on the
results of monosaccharide composition and MS, peak P2 was presumed
to be a disaccharide composed of D-glucose and L-fucose. 2′ -FL is the
most abundant oligosaccharide in human milk, and it improves immu­
nity and promotes brain development in infants (Thongaram, Hoef­
linger, Chow, & Miller, 2017). The monosaccharide composition of 2′ -FL
was D-glucose and L-fucose, which is the same as that of the disaccharide
obtained in the present study. To identify the accurate structure of the
disaccharide, further analysis using NMR spectroscopy was performed,
which was beneficial for further assessments of its biological activities.
3.3.3. NMR analysis of peak P2
The signals of anomeric protons in the 1H NMR spectrum could be
easily identified between δ 4.3 ppm and 5.5 ppm (Hehemann, Boraston,
& Czjzek, 2014). The 1H NMR spectrum of GF is displayed in Fig. 3A. The
peaks around 1.5 ppm (1.517 ppm and 1.482 ppm) were assigned to the
chemical shifts of C6 in L-fucose, suggesting that L-fucose may be at the
reducing end of the disaccharide. There were three terminal protons
(5.407, 4.780, and 4.669 ppm) with relative integral ratios of
0.37:0.64:1.02, which were assigned to the H-1 of α-L-fucose, β-L-fucose,
and β-D-glucose, respectively. The coexistence of α- and β-anomers was

Fig. 2. Positive ion mode in the ESI-MS/CID-MS spectra of acidolysis products after purification with a Bio-gel P4 column. (A) ESI-MS spectrum of peak P1. (B) ESI-
MS spectrum of peak P2. (C) ESI-MS spectrum of peak P3. (D) ESI-CID-MS spectrum of peak P2 at m/z 349.1.

5
M. Xiao et al. Food Chemistry 377 (2022) 132001

Fig. 3. 1D NMR spectra of peak P2. (A) 1H spectrum; (B) 13

C spectrum; (C) DEPT 135◦ ; and (D) DEPT 90◦ .

6
M. Xiao et al.
attributed to the random rearrangement of the anomeric protons during
the degradation process (Hehemann et al., 2014; Ulaganathan et al.,
2017).
The resonance scope of anomeric carbon C-1 was distributed in the
range δ 90–110 ppm and three C-1 signals (δ 104.719 ppm, δ 97.760
ppm, and δ 93.817 ppm), assigned to the C-1 of β-D-glucose, β-L-fucose,
and α-L-fucose, respectively, were observed in Fig. 3B. In the DEPT 135◦
spectrum, the peaks of –CH and –CH3 were upward whereas those of
–CH2 were downward (Wu, Cui, Eskin, Goff, & Nikiforuk, 2011). As
shown in Fig. 3C, 62.174 ppm was inferred to correspond to the C-6 of
β-D-glucose. According to previous studies, the chemical shifts of 17.128
ppm and 17.058 ppm were assigned to the C-6 of β-L-fucose and α-L-
fucose, respectively (Xiao et al., 2021).
As shown in Fig. 4B, the three cross-peaks of 5.407/3.952 ppm,
5.407/4.074 ppm, and 5.407/3.566 ppm were considered to represent
the associations between H-1 and other H signals of α-L-fucose. The three
cross-peaks of 4.780/3.631 ppm, 4.780/3.813 ppm, and 4.780/4.160
ppm represented the connections between H-1 and other H signals of β-L-
fucose. Three other cross-peaks, 4.669/3.655 ppm, 4.669/3.924 ppm,
and 4.669/4.091 ppm, were assigned to the connections of H-1 with
other H signals of β-D-glucose. According to the 1H–1H COSY and 1H–1H
TOCSY spectra, the H signals of β-L-fucose, β-D-glucose, and α-L-fucose
were determined. Thereafter, every C signal was identified using 1H–13C
HSQC (Fig. 4C), and all 1H and 13C signals are summarized in Table S2.
The HMBC spectrum commonly shows the correlation between C and
H separated by several chemical bonds (Cescutti, Cuzzi, Herasimenka, &
Rizzo, 2013). According to the 1H–13C HMBC spectrum (Fig. 4D), the
cross peaks between H-1 of β-D-glucose (δ 4.669 ppm) and C-4 of β-L-
fucose (δ 81.575 ppm) indicated that the linkage of the disaccharide was
β-D-Glcp-(1→4)-β-L-Fucp. In addition, the C-1 of β-D-glucose (δ 104.719
ppm) and H-4 of β-L-fucose (δ 4.160 ppm) were connected as indicated in
Fig. 4D. The H-1 of β-D-glucose was linked to the C-4 of α-L-fucose (δ
Fig. 4. 2D NMR spectra of peak P2. (A) 1H–1H COSY; (B) 1H–1H TOCSY; (C) 1H–13C HSQC; and (D) 1H–13C HMBC.
7
Food Chemistry 377 (2022) 132001
77.010 ppm), and the C-1 of β-D-glucose was linked to the H-4 of α-L-
fucose (δ 3.566 ppm). In summary, the structure of the disaccharide, GF,
was identified as D-Glcp-β-(1→4)-L-Fucp.
3.4. In vitro prebiotic activity
3.4.1. Utilization profile of GF, 2′ -FL, and EPS by six probiotic strains and
their growth
Intestinal microbiota commonly demonstrate various carbohydrate
utilization preferences and metabolic behaviors during the fermentation
of non-digestible carbohydrates (Li et al., 2020). The growth charac­
teristics of the six probiotic strains (BLI, BB, BL, LC, LP, and CB) on
different carbon sources (GF, 2′ -FL, or EPS) were evaluated by
measuring the absorbance of cultures over 48 h (Fig. 5). Similar to the
blank controls, the proliferative effect of EPS on the six strains was not
distinct. The addition of GF significantly promoted the growth of all
strains compared to the blank controls. For the bifidobacterial strains,
GF exhibited a better promoting effect than 2′ -FL on the growth of BB
and BL. Although the proliferation effect of 2′ -FL on BLI was better than
that of GF during 24–36 h, the proliferation effect of 2′ -FL on BLI
decreased rapidly from 36 h to 48 h. In the present study, GF showed the
best prebiotic effect on LC, with the highest OD600 value of 0.668 at 24 h,
followed by CB, BL, BLI, BB, and LP. In addition, 2′ -FL was capable of
promoting the proliferation of BLI, BL, and LPM, but it had no obvious
effect on the proliferation of the other three strains.
TLC was used to determine whether the observed growth patterns
matched the consumption of the three sugars (Fig. S4). The variations in
spots revealed that GF was completely utilized by BL, BB, LC, and CB
within 24 h, and utilized by BL and LP within 48 h, indicating that the
three bifidobacteria, two lactobacilli, and Clostridium butyricum could
produce β-(1→4) glucosidase to hydrolyze fucosylated disaccharide
during their metabolism processes. 2′ -FL was completely utilized by BLI
M. Xiao et al.
Fig. 5. In vitro prebiotic activity of six selected bacterial strains (Bifidobacterium longum subsp. infantis ATCC 15697 (BLI), Bifidobacterium breve ATCC 15700 (BB),
Bifidobacterium longum ATCC BAA-999 (BL), Lactobacillus casei ZX147 (LC), Lactobacillus plantarum CGMCC 1.19 (LP), and Clostridium butyricum YX038 (CB)) on
different carbohydrate was assessed by cell proliferation in simulated intestinal mediums with glucofucobiose (GF), 2′ -fucosyllactose (2′ -FL), and exopolysaccharides
(EPS) as carbon sources or with no additive (control) at 0, 12, 24, 36 and 48 h. The results are presented as the mean values ± standard deviations (n = 3).
and BL within 24 h and 48 h, respectively. The points at 48 h were
significantly lower than those at 0 h during the fermentation processes
of LC and LP, suggesting that these strains were able to utilize 2′ -FL,
although the effect was not obvious. However, 2′ -FL was not degraded
by BB and CB during the 48 h of in vitro fermentation. None of the six
selected strains had the ability to metabolize and utilize EPS within 48 h,
which was consistent with the variations in the OD600 value.
Bifidobacterial and lactobacilli strains have various genes encoding
glycoside hydrolases, thus facilitating the metabolism and utilization of
different carbohydrates. The proliferation effect of 2′ -FL on BLI was
better than that of GF in the first 24 h and then decreased rapidly from
36 h to 48 h, which was attributed to the low pH of the medium, leading
to bacterial death. According to the results, although BLI can proliferate
better in short-term culture in the medium containing 2′ -FL, it can sur­
vive more stably in the medium containing GF. A recent study evaluated
the ability of 16 bifidobacterial strains and 19 lactobacilli strains to
ferment 2′ -FL, and the findings showed that 2′ -FL was only metabolized
by four bifidobacterial strains and had no significant effect on the pro­
liferation of other bifidobacterial and lactobacilli strains (Salli et al.,
2021). Similarly, the present study also demonstrated that 2′ -FL is highly
selective in promoting the proliferation of different probiotic strains
while GF can be utilized by all the examined strains, suggesting that its
effect is less selective and more conducive to the proliferation of these
strains.
3.4.2. Changes in the pH value and SCFAs content during fermentation
As shown in Fig. S5, GF was consumed by the six probiotic strains at
48 h, which decreased the pH of the medium. Only BLI, BL, and LP were
capable of decreasing the pH of the simulated intestinal medium con­
taining 2′ -FL. The pH values of the medium with EPS as a carbon source
or without an additional carbon source were not changed significantly.
The decrease in pH during fermentation is usually caused by the accu­
mulation of some metabolites during the growth and metabolism of
bacteria, such as SCFAs, which have beneficial effects on human health
(Wei et al., 2020; Fu, Liu, Zhu, Mou, & Kong, 2019). SCFAs are a class of
organic fatty acids with one to six carbon atoms. The most important
SCFAs for human metabolism include acetate, propionate, and butyrate
8
Food Chemistry 377 (2022) 132001
(Teixeira et al., 2013). In addition to their health benefits, SCFAs are also
considered to be associated with increased satiety caused by carbohy­
drate consumption (Sleeth, Thompson, Ford, Zac-Varghese, & Frost,
2010). The adhesion of bacteria to the human intestinal tract is closely
related to the pH of the intestinal environment, which leads to variations
in the structure and physiological function of bacteria (Wu et al., 2018).
The content of SCFAs was measured as shown in Fig. 6. Not sur­
prisingly, there was no significant production of SCFAs by the six bac­
terial strains in the simulated intestinal medium with EPS or without an
additional carbon source (data not shown). 2′ -FL could promote the
proliferation of bifidobacterial strains, and it has been shown to promote
the production of SCFAs, including formate, acetate, and lactate (Zabel
et al., 2020). In the present study, the main SCFAs generated during the
fermentation of 2′ -FL by BLI, BB, BL, LC, and LP were lactate and acetate,
with a small amount of formate. The total SCFAs production by BB, BL,
LC, LP, and CB during the fermentation of GF was 2.2-, 1.8-, 2.9-, 1.6-,
and 2.9-fold than that produced during the fermentation of 2′ -FL,
respectively. And GF stimulated CB to produce 3.7-fold butyrate than 2′ -
FL. Moreover, a low level of butyrate was produced during the
fermentation of GF by BB and LP, which was not observed during the
fermentation of 2′ -FL. Recent studies have reported that butyrate plays
an important role in maintaining the stability of the intestinal environ­
ment and providing an energy source for colon cells (Hamer, Jonkers,
Venema, Vanhoutvin, & Brummer, 2008). GF, as a sugar capable of
promoting butyrate production by probiotics, has prospects as butyro­
genic prebiotics.
4. Conclusion
Fucosylated oligosaccharides in human milk, including 2′ -FL, 3-fuco­
syllactose, and difucosyllactose, play an important role in promoting
intestinal health and reducing allergic reactions. Bacterial EPS are raw
materials for the exploration of diverse functional oligosaccharides and
polysaccharides. To date, the preparation of fucosylated oligosaccha­
rides using microbial fermentation methods has only been performed in
a few studies. In the present study, CMM1 EPS were prepared by the
bacterial fermentation method. Further, based on the diversity of the
M. Xiao et al.
Fig. 6. Concentration of butyrate, acetate, formate, and lactate in simulated intestinal medium containing GF, 2′ -FL and EPS as carbon sources during the
fermentation of six selected bacterial strains at 0, 12, 24 and 48 h. Results are presented as the mean values ± SD (n = 3).
degraded components and monosaccharide content, the effects of
different acidolysis conditions on CMM1 EPS were evaluated. Finally,
TFA (0.2 mol/L) was applied to degrade CMM1 EPS at 90 ◦ C for 2 h to
prepare fucosylated oligosaccharides. Under these conditions, GF, with
the structure D-Glcp-β-(1→4)-L-Fucp, was successfully obtained. In
addition, the present study evaluated the prebiotic effects of GF and 2′ -
FL on six probiotic strains. The results suggested that GF was capable of
promoting the proliferation of three bifidobacterial, two lactobacilli,
and one Clostridium butyricum strains, and of stimulating these probiotic
strains to produce more SCFAs than 2′ -FL. Overall, GF, with a new
structure, was successfully obtained by degrading bacterial EPS, and the
present study may serve as a reference strategy for exploring potential
fucosylated oligosaccharides. Although well-designed studies with in
vitro and in vivo prebiotic activities are needed, our data point out a new
direction for the subsequent application of GF and fucosylated
oligosaccharides.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgment
This work was supported by the National Natural Science Foundation
of China (31872893) and the China Postdoctoral Science Foundation
(2021M701547).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.132001.
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