Structural Variation and Microrheological Properties of a Homogeneous Polysaccharide from Wheat Germ

Article abstract of DOI:10.1021/acs.jafc.7b04730

A novel polysaccharide (WGP) was purified from crude wheat germ polysaccharide by Sephacryl S-500HRgel filtration. The molecular weight of WGP was determined as 4.89 × 10⁶ Da and consisted of arabinose, xylose, glucose, and galactose. Methylation analysis and 1D/2D nuclear magnetic resonance was used to analyze the structural characterization of WGP. WGP was mainly a backbone composed of (1 → 4)-linked-β-d-Xylp (19.01%) and (1 → 3, 4)-linked-β-d-Xylp (26.27%) residues, which was branched of (1 → 5)-linked α-l-Araf (28.09%) and (1 → 3,6)-linked β-d-Galp (12.11%) with β-d-Glcp (14.52%) as terminal unit. The calculated values of Turbiscan stability indexes suggested that WGP (0.1-0.5 mg/mL) is a stable system. Microrheology results showed that WGP can form gel behavior when the concentration of WGP ranges from 0.1 to 3 mg/mL. Results of in vitro assays showed that WGP could cause the proliferation of RAW264.7 macrophages, upregulating the release of TNF-α and IL-8 in the lymphocytes.

Full text of DOI:10.1021/acs.jafc.7b04730

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Article
Structural variation and microrheological properties
of a homogeneous polysaccharide from wheat germ
liyuan Yun, Tao Wu, rui Liu, kun Li, and Min Zhang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04730 • Publication Date (Web): 19 Jan 2018
Downloaded from http://pubs.acs.org on January 19, 2018
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Journal of Agricultural and Food Chemistry
Structural variation and microrheological properties of a homogeneous
polysaccharide from wheat germ
Liyuan Yun#, Tao Wu#, Rui Liu, Kun Li, Min Zhang*
State Key Laboratory of Food Nutrition and Safety (Tianjin University of Science and
Technology); Key Laboratory of Food Nutrition and Safety, Ministry of Education,
Tianjin 300457, China
# These authors contributed to the work equally and should be regarded as coꢀfirst
authors.
Corresponding Author: Min Zhang
Telephone: +86ꢀ22ꢀ60912430
Eꢀmail address: zm0102@sina.com; wutaoxx@gmail.com
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Abstract
A novel polysaccharide (WGP) was purified from crude wheat germ
polysaccharide by Sephacryl Sꢀ500HRgel filtration. The molecular weight of WGP
was determined as 4.89×106 Da and consisted of arabinose, xylose, glucose and
galactose. Methylation analysis and 1D/2D nuclear magnetic resonance was used to
analysis the structural characterization of WGP. WGP was mainly a backbone
3
4
5
6
7
composing of (1→4)ꢀlinkedꢀβꢀ
residues, which was branched of (1→5)ꢀlinked αꢀLꢀAraf (28.09%), and
(1→3,6)ꢀlinked βꢀ ꢀGalp (12.11%) with βꢀ ꢀGlcp (14.52%) as terminal unit. The
D
ꢀXylp (19.01%), (1→3, 4)ꢀlinkedꢀβꢀDꢀXylp (26.27%)
8
9
D
D
10
11
12
13
14
15
16
17
calculated values of Turbiscan stability indexes suggested that WGP (0.1–0.5 mg/mL)
is a stable system. Microrheology results showed that WGP can form gel behavior
when the concentration of WGP range from 0.1 to 3 mg/mL. Results of in vitro assay
showed that WGP could cause the proliferation of RAW264.7 macrophages,
upregulate the release of TNFꢀα and ILꢀ8 in the lymphocytes.
Keywords: Wheat germ polysaccharide; structural features; microrheology; stability;
immunoregulatory activity
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Introduction
Wheat is the most significant staple diet in the world especially in Asia and has
been used as a major segment of different kinds food products.1 Wheat germ is a
highly nutritive byꢀproduct of the wheat processing. Wheat germ provides more
protein,2 minerals,3 Eꢀvitamins,4 dietary fiber, and unsaturated fatty acids5 than wheat
flour. The antioxidants separated from wheat germ have been applied to prevent
cardiovascular diseases and cancer.6 Therefore, wheat germ serves as a proper
medium to transmit nutritional ingredient to human diet. However, wheat germ is
almost removed during milling 7, 8and is widely applied to animal feed, which has few
commercial uses and causes energy waste.
Many researchers recently focused on wheat germ agglutinin activity,9, 10 germ
oil properties11, 12, and wheat protein structure studies,13–15 whereas few studies
identified the nonstarch polysaccharides of wheat germ. Meanwhile, polysaccharides,
such as dietary fiber and starch, have been mixed in protein, suspensions, and
emulsions to enhance their stability with respect to creaming or sedimentation. Many
studies reported that the immunostimulatory activity of polysaccharide may be one of
the mechanisms underlying its antitumor, anticomplementary, and antiꢀinflammatory
functions.16 Therefore, the physicochemical properties (stability, thermal, and
rheological) and bioactivity of the wheat germ polysaccharide must be characterized.
Accordingly, the immunostimulatory activity could be a significant biological activity
of polysaccharides.16
In this report, a waterꢀsoluble wheat germ polysaccharide (WGP) was first
separated from wheat germ and characterized for its chemical structure. Physical
stability of WGP was assessed by an optical analyzer (Turbiscan), diffusing wave
spectroscopy (DWS), and thermogravimetric analysis (TGA). The immune activity of
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WGP was evaluated.
The structure, physicochemical properties, and immunostimulatory activity help
in characterizing wheat germ polysaccharides. In addition, the results are essential to
predict their farꢀreaching utilization in the industrial spheres.
Materials and methods
Materials. Wheat germ was provided by FADA Flour Group in Shandong, China.
Reagents and solvents in the study were of analytical purity (AR) grade.
Sample preparation. Wheat germ was ground by a highꢀspeed disintegrator into
a powder and sieved through a 60 mesh screen. Petroleum ether was used to remove
the fat of WGP (1:3 g/mL) in a Soxhlet apparatus by reflowing at 60 °C for 7 h.
Afterward, the powder was dried at room temperature for subsequent experiments.
Extraction of polysaccharides from wheat germ. After the amylase enzymatic
hydrolysis, precipitates were collected via centrifugation (4000 r/min, 10 min).The
precipitates were extracted with hot water (1:5, w/v) for three times at 70°C and 44
min for each time. Solutions were concentrated with rotary evaporator (REꢀ52AA,
Shanghai, China) at 60°C under vacuum and then 95% ethanol (1:4, v/v) was used to
precipitate at 4°C overnight. Precipitation was obtained by centrifugation and
reꢀdissolved in distilled water. Protein was removed according to Sevage method,
followed by exhaustive dialysis with distilled water for 72 h. After being lyophilized,
solution was concentrated under reduced pressure to produce the crude polysaccharide
(cWGP).17 Each cWGP sample was weighted with an analytical balance (CPA225D,
Beijing, China), according to the following equation to calculated the extraction yield
(Y) :18
weight of the cWGP
Y% =
× 100
66
(1)
weight of sample
.
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Isolation and purity of WGP. In brief, 2 mL of cWGP (40 mg/mL) was purified
by Sephacryl Sꢀ500 HR chromatography column (1.6 cm×45 cm). The eluents with
distilled water were collected in test tubes (2 mL/tube) and detected by using
phenolꢀsulfuric acid method.19 Two fractions were gathered and the first fraction
(WGP) was studied in this part the second fraction was studied in the following study.
WGP was further purified with Sepharose 4B column by using water as an eluent.
Highꢀperformance liquid chromatography (HPLC) was used to analysis the
homogeneity and molecular weight determination of WGP. 20 ꢁL WGP (5 mg/mL)
aqueous solution was subjected to a Waters LCꢀ20AT HPLC system (Shimadzu
Company, Japan) with OHpak SBꢀ805 HQ column (8.0 mm×300 mm) and equipped
with a refractive index detector (Shimadzu RIDꢀ10A, Kyoto, Japan). Ultrapure water
was used to elute at a flow rate of 0.8 mL/min. According to the calibration curve,
which was provided by the dextran standards (5300, 3755, 2400, 2000, 500, 110, 70,
40, and 10 kDa), the average molecular weight of WGP was computed.
Determination of chemical composition of WGP. The carbohydrate content
was analyzed by the method of phenolꢀsulfuric acid,19 and xylose was used a standard.
The content of nitrogen was determined using Kjeldahl method and multiplied by a
factor of 5.45 to determine the protein content.20 The method of carbazoleꢀsulfuric
acid was used to detect the content of uronic acid, and glucuronic acid was used as a
standard. Orcinolꢀhydrochloric acid method was applied to determine the pentosan
content, and xylose was used as standard.21
Measurement of monosaccharide components. 10 mg WGP was hydrolyzed
by 1mL trifluoroacetic acid (TFA, 2M) at 120oC for 6 h. The hydrolyzed product was
dried by rotary evaporation and then added 1 mL of methanol repeatedly three times
to completely remove TFA. 30 mg sodium borohydride (NaBH4) and 2 mL distilled
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water was added to the hydrolyzed product, then completely reaction at room
temperature for 1.5 h. After that, acetic acid was used to consume the superfluous
NaBH4. The mixture solution was concentrate to dry by rotary evaporation and added
2 mL 0.1% (v/v) hydrochloric acid methanol solution to the dried mixture production
repeatedly three times. Finally, 0.5 mL pyridine and 0.5 mL acetic anhydride was
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added to the mixture production and reacting at 105 C for 1 h. The acetylated
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derivatives were produced and filtered through a 0.22 ꢁm membrane for gas
chromatography (GC) analysis. 22 The GC equipped with an Agilent 7890A instrument
and an OVꢀ1701 capillary column (30 m×0.32 mm×0.5 um).
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The temperature produce was set as follows: 150°C for 1 min; 10°C /min from
150 to 200 °C; 200 °C for 10 min; 5 °C /min from 200 to 220 °C; 220 °C for 5 min;
1.5 °C /min from 220 to 240 °C; 240 °C for 20 min. Injector temperature and detector
temperature was 240 °C and 280 °C, respectively. The injection volume is 5 ꢁL and
the split ratio is 1:10. The monosaccharides were analyzed by comparing with
monosaccharide standard.23
Periodate oxidation and Smith degradation analysis. The degree of branching
and provision of the residues along the polysaccharide chain was analyzed by
Periodate oxidation and Smith degradation. Briefly, 30 mg WGP was dissolved in 30
mL of sodium periodate (NaIO4, 0.02 M) and maintained in the dark at 4 °C. The
mixture was then dialyzed for 24 h24 and reduced with 30 mg NaBH4 at 4 °C for 12 h.
50% acetic acid was used to neutralize and then the mixture production was dialysis
by water. The retentate was lyophilized and hydrolyzed by 2 M TFA at 100 °C for 3 h.
Product was hydrolyzed and acetylated as described in the monosaccharide
composition analysis section and was then subjected to GC analysis.
Methylation analysis. WGP was methylated by the reported method of
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Hakomori with appropriate modification.25 Briefly, 25 mg WGP was completely
dissolved in 2 mL of dimethyl sulfoxide and 50 mg of sodium hydroxide (NaOH)
powder. After that, 1 mL of methyl iodide was gradually added to the mixed solution
maintained in dark and sonicated for 1h. The disappearance of O-H band (3200–3700
cm−1) in the infrared spectrum means that the accomplishment of methylation. After
that, methylated products were hydrolyzed, reduced, and acetylated to produce alditol
acetates, and analyzed by gas chromatography–mass spectrometry (GC–MS).26
According to mass spectra to identify the methylated products. By peak areas and
suiting response factors in GCꢀMS to analysis the relative molar ratios of products.24
FT-IR analysis. 1 mg WGP dried powder was ground with KBr powder and then
pressed into pellets for FTꢀIR measurement (Thermo Nicolet, MA, USA). The test
frequency is 4000 to 400 cm−1.
1D and 2D NMR analysis. Dried WGP was exchanged with D2O for several
times.24 In brief, 20 mg of deuteriumꢀexchanged polysaccharide was dissolved in 0.5
mL of D2O (99.8% D, Cambridge Isotope Laboratories Inc., Andover, MA, USA) and
then added in a NMR tube. 1D and 2D NMR spectra were determined by an Avance
III 400 MHz NMR spectrometer (Bruker Corporation, Switzerland).
Conformational structure analysis. Congo red was used to analysis the
conformational structure of polysaccharides solution. 2 mL WGP (1 mg/mL) aqueous
solution was mixed with 2 mL Congo red (80 ꢁmol/mL). The mixed solution was
dissolved with NaOH (0-0.5 M). The λmax of the Congo red–polysaccharide
solutions at different NaOH concentrations was used to evaluate the transition from a
tripleꢀhelical preparation to a singleꢀstranded conformation. 27
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Morphology feature analysis. Scanning electron microscopy (SEM) and atomic
force microscopy (AFM) were used to measure the morphology features.
The SEM images of WGP (500×, 1000×, and 2000× magnifications) were
analyzed by a Hitachi SU-1510 system (Hitachi, Pleasanton, CA, 170 USA). Sputter
coater (Hummer XP, Anatech, CA, USA) was used to coat a thin layer of gold onto
the sample.
AFM has been recently used to recognize the surface atoms.28 WGP aqueous
solution (0.1 ꢁg/mL) was dropped into the surface of mica plate then dried at room
temperature for overnight.29 Subsequently, the images of WGP were determined by
AFM (JEOL, Japan) with a tapping mode. The imaging force was approximated to be
0.05–3.0 nN, and the resonant frequency was 2 kHz.30
Physicochemical property
Sample preparation. WGP was treated with distilled water in different
concentrations (0.1, 0.5, 1, 3, and 5 mg/mL) with gentle stirring at room temperature
until dispersion.
Turbiscan analysis. Turbiscan ASG (Formulaction, Union, France) was used to
determine the polysaccharide solution stability.31 The instrument is equipped with a
detection head, which scans the sample cell from top to bottom. The percentage of
backscattering32 and transmission (T) was measured by using the height of the tube to
calculate the rate of polysaccharide solution destabilization.33,34 According to scanning
the sample at preset intervals, the light flux backscattering and macroscopic
fingerprint of sample was acquired. Thus, the stability of polysaccharide solution was
determined by Turbiscan stability index (TSI).35
In this part, TSI was measured by a special computer program Turbiscan Easy
Soft. The formula as follow:
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scani (h) − scani−1(h)
∑
h
165
(2)
TSI =
∑
H
i
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scani(h), the average backscattering for i time; scani−1(h), the average backscattering
for i−1 time; H, the height of sample. Clearly, TSI considers all individual scans in an
assay. The value of sample is calculated from the averaging. Low TSI indicates a
steady system.36
Microrheological behavior. 20 mL WGP aqueous solution with different
concentrations (0.1, 0.5, 1, 3, and 5 mg/mL) was added to sample cells and maintained
at 27 °C (±0.1 °C). The results were measured by Rheolaser Master (Formulaction,
l'Union, France).
Thermogravimetric analysis. The TA Instrument (model TGA Q50) was used
for the thermogravimetric analysis. 3–5 mg WGP was performed on a dried aluminum
crucible under nitrogen. The preset temperature was 30°C to 800°C and the heating
rate was10 °C/min. Manometric pressure was remained at 101 kPa.29
Differential scanning calorimetry (DSC) was equipped with a TAꢀ60WS
Collection Monitor DSC system (Shimadzu). In brief, 2 mg of WGP was placed in
aluminum pans. The test temperature was 30 °C to 200 °C, and the heating rate was
10 °C/min. Denaturation peak temperature (Tp) was analyzed by DSC thermograms.
Assay of immunomodulatory activity of WGP
The determined of cell proliferation ability. According to MTT assay16, the
influence of polysaccharides on proliferation of RAW264.7 cells was analyzed. In
brief, 100 ꢁL/well RAW264.7 cell suspension (1×106 cells/mL) was incubated in
96-well plate (37 °C, 5% CO2) for 12 h. The adherent RAW264.7 cells were incubated
with a medium containing various concentrations of WGP (10, 20, 40, 60, and 80.0
ꢁg/mL) and complete medium alone (blank control) or lipopolysaccharide (LPS) (5.0
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ꢁg/mL, positive control) for 48 h. After adding 150 ꢁL of dimethyl sulfoxide solution,
the absorbance was determined by microplate reader at 540 nm.38
Real-time reverse transcription polymerase chain reaction (PCR). The
immunocompetence of WGP in activating RAW264.7 cells was analyzed by
monitoring its effects on the release of tumor necrosis factor (TNF)ꢀα and interleukin
(IL)ꢀ8. Total RNA from WGPꢀtreated, untreated and LPSꢀtreated RAW264.7 cells
were extracted with Trizol reagent (GibcoꢀBRL) following the manufacturer’s
protocol.38 RAW264.7 cells (1×106/mL) adhered to the six-well plate with WGP (40
ꢁg/mL) for 48 h. The qPCR was prepared by a 25 ꢁL reaction volume, which
contained 12.5 ꢁL SYBR Mix, 1 ꢁL cDNA templates, 1 ꢁL of each forward and
reverse primer, and 9.5 ꢁL PCR grade sterile water. The results were shown as the
ratio of optimal density to GAPDH, which was used as an internal reference. The
primers were designed as follows: GAPDH (forward: TGGCCTTCCGTGTTCCTAC,
reverse:GAGTTGCTGTTGAAGTCGCA);TNFα(forward:AGGAGGAGTCTGCGA
AGAAGA,reverse:GGCAGTGGACCATCTAACTCG);ILꢀ8(forward:TCGAGACCA
TTTACTGCAACA，reverse: CATTGCCGGTGGAAATTCCTT).
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Statistical analysis. The results are represented by mean±standard deviation
and estimated using oneꢀway ANOVA, followed by Student's tꢀtest. Each result was
performed in triplicate. p<0.05 and p<0.01 were statistically significant.
Results and discussion
Composition analysis of WGP. Crude polysaccharides were isolated from the
defatted wheat germ with a yield of 1.16%. cWGP was purified by Sephacryl Sꢀ500
HR, and two parts were collected (Figure 1A). The firstꢀpart cWGP was further
purified by a Sepharose 4B gelꢀpermeation chromatography column (Figure 1B).
After dialysis and lyophilization of the first fraction, WGP was obtained (6.47% yield
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from the crude extracts). In Figure 1D, a single peak was observed. The low
polydispersity index (Mw/Mn=1.06) revealed that the polysaccharide is a relative
homogeneous fraction. The average molecular weight of WGP was 4.89×106 Da as
calculated by the dextran standard curves. The neutral polysaccharide (WGP) has a
high molecular weight. Meanwhile, high molecular weights of waterꢀsoluble and
waterꢀinsoluble arabinoxylans from wheat,39 barley, sorghum, and rye have been
extracted.
The contents of total sugar, protein, uronic acid and pentosan of WGP are shown
in Table 1. Figure 1C shows that WGP has weak absorption in wavelength 250–300
nm, possibly because of its protein or nucleic acid content. Results of UV analysis
agreed with the protein contents of WGP.
Monosaccharide composition analysis. WGP is mainly comprised of arabinose
and xylose with some glucose and galactose. Table 1 shows the ratio of arabinose,
xylose, galactose, and glucose. The galactose might come from arabinogalactans or
galctoarabinoxylans, which were found among the members of Graminae family.40
The minimal glucose content of WGP is probably attributed to βꢀglucan and
unhydrolyzed starch. The ratio of arabinose to xylose (A/X) is 0.69. The ratio is
different among other wheat studies40 possibly due to the differences in wheat
varieties and polysaccharide extracted methods.
Periodate oxidization and Smith degradation analysis. Upon oxidation with
periodate, 30 mg of WGP consumed 72.70 ꢁmol periodate and produced 21.71 ꢁmol
formic acid. The consumption of periodate was more than twice that of the production
of formic acid, showing that one or more of (1→6), (1→2) or (1→4)ꢀlinked
glycosidic bonds might exist. After Smith degradation, glycerol, xylose, and galactose
were found (Figure 2A). Glycerol associated with the presence of (1→2) or
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(1→6)ꢀlinked glycosidic bonds, and it also may originate from the side chains of
arabinose.41 The existence of monosaccharides mean (1→3)-linked glycosidic bonds
exist in WGP. Several precise glycosidic bonds were confirmed in the methylation
analysis.42
Methylation analysis of WGP. Glucosidic bond types of natural
polysaccharides were determined by the method of methylation. Results of
methylation analysis are shown in Table 2. According to the CCRC Spectral Database
for PMAAs, the peaks were confirmed as 2,4ꢀMe2ꢀGalp, 2,3,4,6ꢀMe4ꢀGlcp,
2,3ꢀMe2ꢀAraf, 2,3,ꢀMe2ꢀXylp, and 2ꢀMeꢀXylp with
a
molar ratio of
12.11%:14.53%:28.09%:19.01%:26.27%. The inference also agreed with the result of
Periodate oxidation and Smith degradation. Result also showed xylose residues in the
main chain with 1–4 linkages. Xylan backbone was mainly substituted at the O-3
position by arabinofuranosyl residues due to the existence of high amounts of
2-Me-Xylp in WGP. The unsubstituted, monosubstituted, and doubly substituted
xylose residues had also been studied in rice, wheat, and sorghum.43 The results
showed that two forms (unsubstituted and monosubstituted) of xylose residues are
exist in WGP.
NMR. The 1D and 2D NMR spectra were used to analysis the structure of WGP.
In the proton spectrum of monoꢀ, oligoꢀ, and polysaccharides, all chemical shifts
come out in the range of 1–6 ppm. The region of 5–6 ppm is associated with
αꢀanomeric protons, and the region of 4–5 ppm is assigned to β-anomeric protons.
The chemical shifts variation from δ 3.4 ppm to 4.0 ppm were considered as the
1
signals correlated with the protons exist on C2–C6.44 Thus, according to the H
spectrum of WGP (Figure 3A), the resonances at 5.24 ppm was definitely due to
αꢀL
ꢀarabinose residues.45 In addition, the signal in the anomeric region of 5.00 and
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4.52 ppm was related to the protons of βꢀ
D
-Xylp residues. Resonances at 4.4 ppm and
4.5 ppm were correlated with the anomeric protons of β-D
-Galp residues46 and
β-D
-Glcp residues, respectively.
In COSY spectrum, an anomeric proton is a reasonable starting point of
assigning because it is linked to a carbon holding two oxygen atoms, presumably the
1
most downfield H signal. As demonstrated in the COSY spectrum of WGP (Figure
3C), the H1–2, H2–3, H3–4, H4–5, and H5-6 correlations of WGP were observed.47
As shown in 13C NMR spectrum (Figure 3B), the signals ranging from δ 90
to110 ppm are related to anomeric carbons, whereas the chemical shift in δ 60–85
ppm is linked to nonꢀanomeric carbons. The existence of C-1 signals (δ100–108 ppm)
showed that four sugar residues were all in pyran ring.48 Some research had report that
Cꢀ1 signals around 107–109 ppm is furan ring.48 The signals at approximately 108.4–
109.2 ppm are attributed to anomeric carbon atoms of αꢀlinked arabinofuranos.49
Peaks are associated with the anomeric carbon of arabinose residues due to the
diversityꢀlinked arabinosyl residues, showing slight variations in the mode of
linkage.40
1
Five pairs of H and 13C signals of anomeric CH were caused by five different
forms of glycosidic bonds. According to chemical shifts and the relevant HMQC
correlations, these bonds were assigned at δ 4.45/102.71, 4.53/103.35, 5.24/109.16,
4.65/104.18, and 5.00/107.69 ppm (Figure 3D). The result showed that five types of
glycosidic bonds existed in WGP. From the HMQC correlations, residue A was
identified as a galactose residue. The else signals of residue A were determined as
δ73.40/3.69 (CH–2), 74.29/4.09 (CH–3), 73.72/3.57 (CH–4), 76.56/4.01 (CH–5), and
62.93/3.62 (CH–6). Meanwhile, the chemistry shifts of other residues was identified,
the results were shown in Table 3.
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According to the heteronuclear multiple bond correlation (HMBC) spectrum
(Figure 3E) of WGP, five residues were associated with each other were identified.
The correlations of each sugar residues in HMBC spectrum are identified as follows:
E Hꢀ1 (δ 5.08) and E Cꢀ4 (δ 83.40), C Hꢀ1 (δ 5.24) and E Cꢀ3 (δ 84.13), A Hꢀ3 (δ 4.05)
and C Cꢀ5 (δ 61.20), and B Cꢀ1 (δ 103.35) and A Hꢀ6 (δ 3.57). In HMBC spectrum,
the peaks at δ 5.08/83.40 and δ 5.24/84.13 were observed. Residues E and E were
correlated as Eꢀ(4→1)ꢀE due to the signal at δ5.08 connected with Hꢀ1 in residue E, δ
83.40 to Cꢀ4 in residue E. Residues C and E were associated as Eꢀ(3→1)ꢀC due to the
signal at δ5.24 corresponded to Hꢀ1 in residue C, and the signal at δ84.13
corresponded to Cꢀ3 in residue E. In addition, the crossꢀpeaks at δ 4.05/61.20 was
identified to the interꢀresidue A Hꢀ3, C Cꢀ5, which showed the residues A and C were
associated as Aꢀ(3→5)ꢀC. The chemistry shift at δ3.57/103.35 were identified to the
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interꢀresidue A Hꢀ6, B Cꢀ1, which showed residues A and B were combined as Bꢀ(6→
1)ꢀA. Thus, in Figure 4, the structure of WGP was defined as a backbone made up of
(1→4)ꢀlinked βꢀDꢀxylose. The substitute at Oꢀ3 of βꢀDꢀxylose composed of
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(1→5)ꢀlinked αꢀ
terminal unit.
L
ꢀarabinose and (1→3,6)ꢀlinked βꢀ ꢀgalactose, the βꢀ
D
D
ꢀglucose as
FT-IR spectra. According to the FTꢀIR spectrum (Figure 2B) the different
absorption bands were identified.50 The broad peak appeared at 3405.11 cm−1 was
produced by O–H stretching vibrations of hydroxyl groups of polysaccharide fractions.
The weak peak at 2925.7 cm−1 was caused by C–H stretching vibration. The peak at
1639.92 cm−1 was mainly connected with absorbed water due to the hemicelluloses is
easily combined with water. Nevertheless, the macromolecules in solid state were
easily hydrated due to the disordered structures.51 Peaks at 1400–1200 cm–1 was on
behalf of the C–H deformation vibration. This signal showed variations in intensity
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with a decrease and displacement at 1039.54 in the WGP that is typical of the βꢀ(1ꢀ3)
xylans.52 In the report of Yang et al.,53 βꢀ
Dꢀglucopyranose and βꢀDꢀgalactose had
absorptive peaks at 905–876 and 876–886 cm−1, respectively. The peak at 897.72 cm−1
ascribed to the Cꢀ1 group frequency or ring frequency, this was also a typical of
βꢀglycosidic linkages.54 Band at 817.39 cm−1 was the characteristic of αꢀglycosidic
linkages. The relative strength of the peak at 817.39 cm−1 was considerably lower than
that of the 897.72 cm−1 peak, indicating that the linkages of WGP was mainly
βꢀglycosidic with the amount of αꢀglycosidic bonds.
Conformational structure of WGP. Congo red method was applied at different
concentrations of NaOH (0.05–0.5 M) solution to identify the triple helix
conformation of WGP. As shown in Figure 2C, an obvious shift of maximum
absorption wavelength from 500 nm to 520 nm was induced by the presence of the
polysaccharides in Congo red solution, indicating that polysaccharide–Congo red
compound had formed. With the increase of concentrations of NaOH, the maximum
absorption wavelength decreased slightly. With the increase of concentrations of
NaOH, the tripleꢀhelix conformation will translate into single coil conformation, and
the maximum absorption wavelength will decrease dramatically. Thus, the result
indicated that WGP did not exhibit a triple helical conformation.55
Morphological characteristics analysis. Figure 5A shows the surface
morphology of WGP determined by SEM. The surface topography of WGP showed a
reticular layer with spherical particles or oval granules. The surface morphology may
provide an opportunity to connect linkages between polymers.2
Figure 5B shows the surface morphology of WGP determined by AFM. Many
different spherical sizes existed in the images. The result indicated that the molecular
aggregation has occurred, and polysaccharide molecules have different sizes of
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spherical structure. The height of 1.73 nm showed that WGP was not a single sugar
chain molecule but a group of molecules. The hydrogen bonds in polysaccharides can
provide the interꢀ and intramolecular interactions with each other or with water
molecule, which induces a significant effects on molecular aggregation.56 These
effects entangle the polysaccharide molecules and form the collective structure.
Physicochemical property. The viscosity of polysaccharide solution was
correlated with its conformation and molecular weight.57 Therefore in this work, the
physicochemical properties of WGP had been studied.
Physical stability analyzed by Turbiscan. According to the backscattering
technology, Turbiscan was used to evaluate the effects of WGP on stability. In Figure
6 and Table 4, the stability index of WGP increased from 0.4 to 23.14. When the
concentrations increased from 3 mg/mL to 5 mg/mL, TSI exhibited a remarkable
increase. This phenomenon occurred because with the increasing of concentration, the
molecular structure may form settling or molecular crystal and cause the aggregation
of polysaccharide solution.34
Microrheological analysis. Microrheology is commonly used to analyze and
trace the motion of droplets and the interactions. MSD of droplets in different
concentrations was analyzed as a function of time. 41
Figure S1 in the supplementary materials shows MSD curves of WGP at
different aging times. The MSD curves move from long to short displacement
signifying an increase in elasticity. The MSD curves move from short to long
decorrelation time signifying an increase in the viscosity. The MSD curve showed that,
as the aging time prolongs, the elasticity of WGP increased and then decreased. At the
beginning, the elasticity increased may be due to polysaccharide and water combined
gradually and then achieved a steady state.6 The major advantage of this approach is
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that the diffusive motions of many droplets can be recorded simultaneously while
retaining the information for each of the individual droplets trajectories.58 The
intensity correlation function can be immediately converted into a timeꢀdependent
MSD,34 which is a immediate and nonꢀinvasive probe of medium properties.33
Elasticity index (EI) value indicates the elasticity strength of polysaccharide
solution.34 EI of WGP is shown in Table 4. EI of WGP increased first and then
decreased with increasing concentration (0.5–5 mg/mL). This phenomenon occurred
because when the concentration increases, the probability of precipitation also
increases.
Macroscopic viscosity index (MVI) value stands for the macroscopic viscosity
without shear34 and is associated with the inversed MSD slope in linear scale and
connected with the macroscopic viscosity.59 In Table 4, when the concentration is 0.1
mg/mL, MVI reaches to 5.78 Pa s, possibly because the molecular activity is relatively
easy in a low concentration, which can easily afford several spaces for the motion of
the chain segments60 and the wide Brownian motion.
When the concentration ranges from 0.5 mg/mL to 3 mg/mL, MVI of WGP
increases as the concentration increases. However, when the concentration reached to
5 mg/mL, the macroscopic viscosity decreased significantly. These results suggested
that as the concentration increases, the molecular chain gradually expands in the
system. A high degree of polymerization was highly viscous in aqueous solution61, but
when the concentration is too high (5 mg/mL), the movement of the molecules is
limited, resulting in separation and aggregation.
The solid–liquid balance (SLB) connecting with MSD slope at short
decorrelation time: a key value of deformation from liquid domination to solid
domination is SLB=0.5. The 0.5<SLB<1 stand for liquid behavior dominates, whereas
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0<SLB<0.5 indicates a gel behavior and solid behavior dominates.59 In Table 4, as the
concentration of WGP ranging from 0.1 mg/mL to 3 mg/mL, SLBs are smaller than
0.5. The result indicated that solid dominate the dispersions and form gel behavior.
When concentration is greater than 3 mg/mL, SLB of WGP is greater than 0.5 and less
than 1. This finding may be due to high concentration of polysaccharides (more than 3
mg/mL), resulting in flocculation and precipitation giving rise to an instability system.
Thermal analysis. Weight loss and degradation temperature are shown in Table
5. The thermal degradation curves and their first derivatives are shown in Figure 7A.
Initial weight loss in the temperature range from 30 °C to 150 °C for WGP is ascribed
to the loss of free and bound water. Temperature range of 200 °C–400 °C was due to
the decomposition of polysaccharides.2
The DSC thermograms of WGP are presented in Figure 7B. The DSC curve of
WGP pointed an obvious endothermic peak at 82.57 °C, which might be due to the
dehydration or the loss of peripheral polysaccharide chains and dehydroxylation
reactions.62
Immunomodulatory activity in vitro of WGP
Effects of WGP on the proliferation of macrophages. As shown in Figure 8A,
WGP significantly increased the viability of RAW264.7 cells from 10 ꢁg/mL to 80.0
ꢁg/mL. Compared with blank control, WGP can observably stimulated the viability of
RAW264.7 cells (p<0.01). When the concentration of WGP is 40 ꢁg/mL, the
proliferation index reaches to 92.136%±0.899%.
Effects of WGP on production of cytokines. As shown in Figure 8B, after the
treatment of WGP, the levels of TNFꢀα and ILꢀ8 cytokines increased. This result
showed that WGP significantly increased the secretion of TNFꢀα and ILꢀ8 in
RAW264.7 cells when stimulated for 48 h. The results further confirmed that WGP
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could promote the function of macrophages.
Accoridng to methylation, GC–MS, FTꢀIR, and 1D/2D NMR, the linkages of
WGP were 1,4ꢀlinked βꢀ ꢀXylp (19.01%), 1,3,4ꢀlinked βꢀ ꢀXylp (26.27%),
1,5ꢀlinked αꢀLꢀAraf (28.09%), 1,3,6ꢀlinked βꢀ ꢀGalp (12.11%), and Tꢀlinked Glcp
D
D
D
(14.52%). Physicochemical properties analysis results showed that the gel behavior
dominates the dispersions of WGP (0.1–3 mg/mL) and probably interacted with the
molecular weight, the link location of glycoside residues, and the link order of
glycoside. In vitro, WGP has a potent immunostimulating activity possibly due to
its monosaccharide composition, sugar residues, and physicochemical properties.63
Future studies should focused on the possible mechanisms of immunomodulatory
action of WGP in in vivo assay. The study of structure, physicochemical properties,
and immunostimulating activity of wheat germ polysaccharides will enrich and
expand the application of wheat germ polysaccharide.
Associated content
Supporting Information
Figure S1 The Mean Square Displacement vs time curves for WGP
(polysaccharide from wheat germ) (the first curves are in blue, then green, and lastly
in red; A, B, C, D, E was the concentration of WGP at 0.1, 0.5, 1, 3, 5 mg/mL
respectively ).
Abbreviation
WGP, wheat germ polysaccharide; HPLC, high performance liquid chromatography;
FTꢀIR, fourierꢀtransform infrared spectroscopy; NMR, nuclear magnetic resonance.
Acknowledgements
This work was supported by the Special Fund for Agroscientific Research in the
Public Interest [201303071]; Innovation of modern Agricultural Project [F17RO2];
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Fundamental Research Funds for the Tianjin Universities [2017KDZD02]; National
Natural Science Foundation of China [31501475]; Tianjin Science and Technology
Commission [15JCQNJC46300]; and the Ph.D. Training Foundation of the Tianjin
University of Science and Technology [2016002].
Conflict of interest statements
The authors state no competitive financial interest
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Table 1. Compositional, molecular and structural features of WGP isolated from
wheat germa
WGP
460
Polysaccharide
Mn/104 Da
Mw/104 Da
Pd
489
1.06
Protein (%)
Neutral sugar (%)b
5.57±0.98
81.45±0.24
Total pentosan (%)b
Uronic (%)
60.27±1.10
n.d.
Lꢀarabinose
Dꢀxylose
29.3
42.7
14.7
13.3
0.69
0.075
Dꢀgalactose
Dꢀglucose
Arabinose/xylose
Yield (%)
b
n.d., not determined; a Data are presented as the mean (n=3); Expressed as %
of sample dry matter.
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