polymers

Article
Two-Step Isolation, Purification, and Characterization
of Lectin from Zihua Snap Bean
(Phaseolus vulgaris) Seeds
Bin Jiang, Xiaojing Wang, Linlin Wang, Xiaomeng Lv, Dongmei Li, Chunhong Liu and
Zhibiao Feng *
Department of Applied Chemistry, Northeast Agricultural University, NO.600 Changjiang Road Xiangfang District,
Harbin 150030, China; jiangbin@neau.edu.cn (B.J.); neauwxj@163.com (X.W.); neauwll@163.com (L.W.);
18846173287@163.com (X.L.); lidongmei@neau.edu.cn (D.L.); liuchunhong@neau.edu.cn (C.L.)
* Correspondence: fengzhibiao@neau.edu.cn; Tel.: +86-4515-519-0222




Received: 9 April 2019; Accepted: 30 April 2019; Published: 2 May 2019


Abstract: A two-step method based on an aqueous two-phase system and Sephadex G-75 was used
to separate and purify lectin from the seeds of the Zihua snap bean. The preliminary properties and
bioactivity of the Zihua snap bean lectin were characterized by different instrumental methods, such as
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE), liquid chromatography-nano
electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS), and Fourier transform infrared
spectroscopy (FTIR). The hemagglutinating activity of the Zihua snap bean lectin could not be
inhibited by glucose, N-acetyl-d-glucosamine, d-galactose, N-acetyl-d-galactosamine, fructose, sucrose,
d-maltose, d-trehalose, and lactose. It was found that the hemagglutinating activity of the lectin
showed strong dependence on Mn2+ and Ca2+ . The thermal and pH stability of the Zihua snap bean
lectin was studied by FTIR and fluorescence spectroscopy. Relatively good stability was observed
when the temperature was not higher than 70 ◦ C, as well as in the pH range of 2.0 to 10.0. Digestive
stability in vitro was investigated. The untreated lectin was relatively stable to pepsin and trypsin
activity, but heat treatment could significantly reduce the digestive stability in vitro. Moreover, the
lectin showed an inhibitory effect on the tested bacteria (Staphylococcus aureus (S. aureus), Escherichia
coli (E. coli), Bacillus subtilis (B. subtilis)), and it also showed a certain inhibitory effect on the growth of
Phytophthora infestans (P. infestans) at higher concentrations.

Keywords: Zihua snap bean; lectin; stability; preliminary property; bioactivity

1. Introduction
Lectins are glycoproteins that are characterized by their capability to attach carbohydrates such as
mannose, galactose, lactose, N-acetyl glucosamine, N-acetyl galactosamie, fucose, and rhamnose with
significant specificity [1]. They bind specifically and reversibly to different types of glycoproteins or
carbohydrates [2,3]. Due to these advantages, some sensitive dielectric sensors based on Concanavalin
A lectin were used for the specific identification of glycoproteins and carbohydrates [4,5].
Exploratory studies on their health benefits have been initiated, because lectins have important
physiological roles, including insecticidal action [3,6], antibacterial activity [7], and antifungal effects [8],
antihuman immunodeficiency virus [9], antitumor activity [10–12], and analgesic activity [13]. Plants
are the main source of lectins that are found in different parts of plants such as seeds, leaves, bark, roots,
tubers, and fruits [14]. It has been known for a long time that lectins occur in legumes, where they
can be a major food source for both humans and animals [15]. However, lectin has an antinutritional
factor [16]. Its antinutritional properties are most likely caused by their impairment of the integrity

Polymers 2019, 11, 785; doi:10.3390/polym11050785 www.mdpi.com/journal/polymers

Polymers 2019, 11, 785 2 of 20

of the intestinal epithelium, and thus also the absorption and utilization of the nutrients that are
present in the legumes [17]. So, lectins containing foods are frequently consumed cooked or otherwise
processed to reduce the level of the antinutritional factors and improve the utilization efficiency of
legumes [18]. In addition, the lectins that are thought to survive gastric digestion have been a minor
allergen, such as soybean seeds [19,20]. Therefore, its relevance to food safety requires intensive
research to determine the digestibility of lectins from legumes, especially regarding the consumption
of high levels of unprocessed or undercooked beans [21]. Thus, more and more legume lectins are
being studied intensively. Lectin with a molecular mass of approximately 60 kDa and two different
subunits was isolated from the ground bean (Vigna sesquipedalis cv. ground bean) [22]. Lectin with a
molecular mass of 67 kDa, and two identical subunits, was purified from Phaseolus vulgaris (P. vulgaris)
cv. dark red kidney bean [23]. He et al. [24] extracted a lectin with the molecular weight of 49.24 kDa
from small black kidney bean (P. vulgaris) using a reversed micellar system.
The Zihua snap bean is a high quality bean that is distributed in northeast China. The protein
content in Zihua snap bean seeds exceeds 20% [25], of which 2.4–5% is lectin [15]. In our previous
studies, lectin was separated from the Zihua snap bean (P. vulgaris) seeds by an aqueous two-phase
system (ATPS), which was effectively applied for the extraction and purification of proteins and other
biomolecules [26–28].
In this study, seeds of the Zihua snap bean, which is an endemic species in northeast China, were
chosen to separate and purify lectin by a two-step method based on an aqueous two-phase system and
Sephadex G-75. On this basis, the partial properties of lectin were investigated. In addition, the in vitro
digestion characteristics and antimicrobial activity of the lectin were also discussed. The purpose of
the present work was to establish a two-step method for purifying lectin from the Zihua snap bean
in local special crops and explore new sources of lectin. Furthermore, system information about the
reduction of antinutritional factors of lectin by heat treatment and the effect of lectin preheating on
digestion were provided. These laid the foundation for the deep processing of the Zihua snap bean to
improve the economic value of the Zihua snap bean.

2. Materials and Methods

2.1. Instruments
An ultraviolet-visible spectrophotometer was from Beijing Purkinje General Instrument Co., Ltd.
(Beijing China). An A150011 vortex mixer (Nanjing Jiajun Biological Co., Ltd., Nanjing, China) and a
SC-3610 low speed centrifuge (Anhui Zhongke Zhongjia Scientific Instrument Co., Ltd., Hefei, China)
were applied to treat the sample. The Biorad Mini-PROTEAN Tetra Cells 4-Gel 165-8004 was from
Bio-Rad Co., Ltd. (Guangzhou China). The pH of solution was measured by an FE201EL20 pH meter
(Yidian Scientific Instrument Co., Ltd., Shanghai, China), and the weight of the sample was determined
by an AL-04 electronic analytical balance (Mettler Toledo Instruments Co., Ltd., Shanghai, China).

2.2. Reagents
Zihua snap bean (P. vulgaris) seeds were from Harbin Xiangfang District Gongbin Seed Company
(Harbin, China). A 2% rabbit red blood cell suspension was obtained from Beijing Baiaolaibo Technology
Co., Ltd. (Beijing, China). All the chemicals were obtained from Aladdin (Shanghai, China) and were
of analytical grade, and all the solutions were prepared using ultrapure water obtained from Northeast
Agricultural University.

2.3. Extraction and Purification of Zihua Snap Bean Lectin

Dried Zihua snap-bean seeds were ground to a fine powder passed through a 50-mesh sieve. Then,
the powder was mixed with phosphate-buffered saline (PBS, 10 mM, pH 7.0) by agitation overnight at
4 ◦ C. Afterwards, the extract was filtered and centrifuged to get a supernatant as the crude extract.
Polymers 2019, 11, 785 3 of 20

The crude extract was purified using the aqueous two-phase system (ATPS) method as previously
described [25]. ATPS was formed by mixing 0.75 g of ammonium sulfate, 0.9 g of polyethylene glycol
600 (PEG 600), and 0.4 g of NaCl, adding 1 mL of crude extract solutions. The total weight of the
system was 5 g with the pH set on 7.5. After separation by ATPS, lectin and proteins in the top phase
were collected and dialyzed against deionized water for 24 h by a dialysis bag with 7000-Da molecular
weight cutoffs to remove salt and PEG 600.
The lectin was further purified by gel chromatography. A 10 mg/mL lectin solution was prepared
by concentrating the dialyzed lectin solution and then filtering it through a 0.45-µm membrane. Then,
5 mL of the filtered liquid was applied to a Sephadex G-75 column pre-equilibrated with the 0.02-M
phosphate buffer (pH 7.2). The column was washed with eluent at a flow rate of 0.5 mL/min. The peak
fraction prepared for the determination of hemagglutinating activity was collected and pooled, and
then dialyzed and lyophilized to obtain lectin lyophilized powder for use.

2.4. Determination of the Hemagglutinating Activity

The determination of the hemagglutinating activity (HA) was performed in microtiter plates
according to Jiang [25]. The lectin lyophilized powder was formulated into lectin solution (0.5 mg/mL).
Then, 50 µL of lectin solution was twofold serially diluted with stroke-physiological saline, and 50 µL
of 2% rabbit red blood cell suspension were added. The results were read after 45 min at room
temperature, when the negative control was fully sedimented.
The sugar specificity of lectin was analyzed in a manner analogous to the hemagglutination
test. The sugar base to be tested was formulated into a certain concentration of aqueous solutions.
Equal volumes (25 µL) of the sugar solution and the lectin solution were mixed, and stored at room
temperature for 1 h. Then, the mixed solutions were diluted to detect hemagglutinating activity. Water
was set as a blank control to observe the inhibition of the lectin hemagglutinating activity by different
glycosyl groups. Each experiment was repeated three times.

2.5. The Properties Experiment of the Zihua Snap Bean Lectin

2.5.1. Metal Ions Dependence

The lyophilized powder of the Zihua snap bean lectin was dissolved in 10 mmol/L phosphate
buffer (pH 7.2) to prepare a lectin solution of 0.5 mg/mL, and the hemagglutinating activity of lectin
solution was determined. Lectin solution was dialyzed in a phosphate buffer solution (10 mmol/L,
pH 7.2) containing 50 mmol/L ethylenediaminetetraacetic acid (EDTA) until no hemagglutinating
activity was detected. Then, it was dialyzed against phosphate buffer solution to remove EDTA, and
the obtained sample solution was subjected to a hemagglutinating activity analysis to confirm that the
hemagglutinating activity was lost. Lectin solution without hemagglutinating activity was mixed with
different metal salt solutions (100 mmol/L), and then diluted to detect hemagglutinating activity to
study the effect of metal salt ions on the recovery of hemagglutinating activity. Each experiment was
repeated three times.

2.5.2. Thermal Stability of Zihua Snap Bean Lectin

A thermal stability experiment followed He’s method with minor modification [29]. The method
of lectin solution configuration was the same as in Section 2.5.1. The lectin solution in 10-mL centrifuge
tubes was heated to the desired temperature using a constant temperature water bath. All the tubes
were completely submerged in the water bath throughout the investigation and ensured to prevent the
evaporation of water or loss of solution. A series of thermal treatments were carried out at 50 ◦ C, 60 ◦ C,
70 ◦ C, 80 ◦ C, and 90 ◦ C for 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, and 30 min. At the end of the
thermal treatment, it was immediately cooled in cold water and stored at 4 ◦ C until further analysis.
Polymers 2019, 11, 785 4 of 20

2.5.3. pH Stability of Zihua Snap Bean Lectin

The lectin lyophilized powder was dissolved in buffers of desired pH to formulate a solution
of 0.5 mg/mL. After standing at 4 ◦ C for 12 h, the acid–base stability of the lectin was studied by a
hemagglutinating experiment and fluorescence spectroscopy, respectively. Glycine–HCl buffer solution
at pH 2.0, citrate buffer solution at pH 3.0 to 5.0, phosphate buffer at pH 6.0 to 8.0, and glycine–sodium
hydroxide buffer solution at pH 9.0 to 11.0 were used to maintain the pH.

2.5.4. In Vitro Digestion Assay of Zihua Snap Bean Lectin

Simulated gastric fluid (SGF) was prepared according to He [21], which consisted of 2.0 g/L of
NaCl and 3.2 g/L of pepsin. The pH value of the SGF solution was adjusted to 1.2 with hydrochloric
acid. Lectin solution (5 mg/mL) was mixed with the SGF solution (pH 1.2) in a water bath at 37 ◦ C to
start the digestion reaction. The pepsin reaction was terminated by adding Na2 CO3 solution with a
pH of 11.0 after 0 min, 10 min, 20 min, 30 min, 40 min, 50 min, and 60 min, respectively.
Simulated intestinal fluid (SIF) was prepared according to He [21] with minor modifications. First,
10 g of trypsin and 6.8 g of KH2 PO4 were dissolved in a moderate amount of water. The pH was
adjusted to 6.8 with 0.2 mol/L NaOH solution or 0.2 mol/L hydrochloric acid solution, and then diluted
to 1000 mL with water. Then, the test lectin solution (5 mg/mL) was mixed with the SIF solution
(pH 6.8) in a water bath to start the tryptic digestion reaction. The trypsin reaction was immediately
terminated by heating to boiling after a certain time.

2.5.5. Antibacterial Experiment of Zihua Snap Bean Lectin

Antibacterial activity was investigated by the disc diffusion method [30]. S. aureus, E. coli, and
B. subtilis were used. Bacteria were grown in shaker flasks containing potato medium and placed at
28 ◦ C for 24 h. After diluted to 107 colony-forming units/mL (CFU/mL), 100 µL of bacterial suspension
was smeared onto the surface of an inoculated medium. Four filter papers with diameters of 5 mm
were placed on the surface of the inoculated medium of the plate. Then, 20 µL of lectin solution with
concentrations of 0.5 mg/mL, 1 mg/mL, and 2.5 mg/mL was added to the filter papers, respectively. A
stroke-physiological saline solution was used as a blank control. Then, 20 µL of phenol solution with
concentrations of 0.5 mg/mL, 1 mg/mL, and 2.5 mg/mL was used as a positive control. After being
incubated at 28 ◦ C for 12 h, the plate was taken out for observation and recording. A transparent ring
around the filter paper revealed antimicrobial activity.

2.5.6. Antifungal Experiment of Zihua Snap Bean Lectin

P. infestans was carried out in a petri plate containing tomato juice agar and incubated in a dark
incubator at 22 ◦ C for seven days. After the mycelial colony had developed, two filter papers with
diameters of 5 mm were placed in front of the mycelial colony. Then, 20 µL of lectin solution (5 mg/mL)
and 20 µL of stroke-physiological saline solution were added to the two filter papers, respectively.
The plate was incubated at 22 ◦ C for seven days and taken out of the plate for observation.

2.6. Electrophoresis
SDS-PAGE was used to identify lectin and evaluate the digestion of lectin by 12% bis-acrylamide
homogeneous gel. The gel was run for 95 min using TRIS–glycine–SDS running buffer at constant
voltages of 80 V for the stacking gel and 120 V for the separating gel. After the electrophoresis was run,
the gel was washed with several volumes of distilled water for several times, and then stained for
30 min with Coomassie brilliant blue R-250 and destained with destaining solution.

2.7. Nano LC-ESI-MS/MS Analysis

NanoLC-ESI-MS/MS analysis of the digested lectin sample was carried out by a high-pressure
liquid chromatography (HPLC) system (Agilent, Palo Alto, CA, USA) with an Agilent C18 column
Polymers 2019, 11, 785 5 of 20

(75 µm × 8 cm, 3 µm). Mobile phase A consisted of 97.5% water, 2% acetonitrile, and 0.5% formic acid,
whereas mobile phase B consisted of 9.5% water, 90% acetonitrile, and 0.5% formic acid. The gradation
time from 2% mobile phase B to 90% mobile phase B was 60 min, plus 20 min for sample loading
and 20 min for column washing. The injection volume was 3 µL. The HPLC system was online
coupled with a linear ion trap mass spectrometer (LTQ, Thermo, San Diego, CA, USA) in a way
that a sample eluted from an HPLC column was directly ionized by an electrospray ionization (ESI)
process and entered into the mass spectrometer. The ionization voltage was often optimized in the
instrument-tuning process, and normally in a range of 1.5kv to 1.8kv. The capillary temperature was
set at 100 ◦ C. The mass spectrometer was set at the data-dependent mode to acquire MS/MS data via a
low-energy collision-induced dissociation (CID) process. The default collision energy was 33%, and
the default charge state was three. One full scan with one microscan with a mass range of 350 amu
to 1650 amu was acquired, followed by nine MS/MS scans of the nine most intense ions with a full
mass range and three microscans. The dynamic exclusion feature was set as following: repeat count
of one and an exclusion duration of 1 min. The exclusion width was 4 Da. The mass spectrometric
data was used to search against the most recent non-redundant protein database (NR database, NCBI
(Wellington, DE, USA)) with ProtTech’s ProtQuest (Philadelphia, PA, USA) software suite.

2.8. Fourier Transform Infrared Sectroscopy

The infrared spectrum of the Zihua snap bean lectin was obtained with a Fourier transform infrared
spectrometer (Shimadzu, Kyoto, Japan) within the wave number range of 400 to 4000 cm−1 . First, 2 mg
of the tested lectin lyophilized powder were ground together with 20.0 mg of spectroscopic grade KBr
powder and pressed into a 1-mm pellet. The experiment was repeated three times. The spectra was
processed using Peakfit v4.12 software (SeaSolve, San Jose, CA, USA).

2.9. Fluorescence Spectroscopy

The lectin lyophilized powder was dissolved in 10 mmol/L of phosphate buffer (pH 7.2) and
measured with a fluorescence spectrometer (PerkinElmer LS55, Fremont, CA, USA) at fluorescence
excitation wavelengths of 280 nm and 295 nm, respectively. The emission spectrum were recorded
between 300–400 nm. The slit widths of the excitation and emission monochromators were set to
5 nm. Fluorescence spectral baseline subtraction was performed using a sample buffer system as a
blank control.

2.10. Statistical Analysis

The data were expressed as the mean or the percent mean ± standard deviation (SD). All the
experiments were tested and analyzed in triplicate. An analysis of variance (ANOVA) was identified
to determine the significant differences (P < 0.05) between means.

3. Results and Discussion

3.1. Purification of Zihua Snap Bean Lectin

The elution curve of the lectin purified by a two-step method based on an aqueous two-phase
system and Sephadex G-75 column was shown in Figure 1. As indicated in Figure 1, two peaks
could be obtained on the elution curve. Solutions corresponding to the two peaks were collected for
the hemagglutinating test, and it was found that the solution that corresponded to peak 1 showed
hemagglutinating activity, while the solution that corresponded to peak 2 did not. The active solution
was collected and dialyzed to remove salts, and then subjected to SDS-PAGE. The electrophoresis
result is shown in Figure 2.
Polymers 2019, 11, 785 6 of 21

Polymers
Polymers2019,
2019,11,
11,785
785 66of
of21
20

Figure 1. Gel chromatography of lectin extracted by an aqueous two-phase system (ATPS) on

Sephadex G-75.
Figure 1.
Figure Gelchromatography
1. Gel chromatographyofoflectin
lectinextracted
extractedby
byan
anaqueous
aqueoustwo-phase
two-phasesystem
system(ATPS)
(ATPS) on
on
SephadexG-75.
Sephadex G-75.

Figure 2. SDS-PAGE of the lectin after Sephadex G-75 purification: A, sample; B, marker.
Figure 2. SDS-PAGE of the lectin after Sephadex G-75 purification: A, sample; B, marker.
As indicated in Figure 2, a band (lectin tape) at 35 kDa and a band (unknown tape) at 18.4 kDa
Figure 2. SDS-PAGE of the lectin after Sephadex G-75 purification: A, sample; B, marker.
As indicated
were found in theinSDS-PAGE
Figure 2, a of
bandthe (lectin tape)Sephadex
lectin after at 35 kDa G-75.
and a The
bandSDS-PAGE
(unknownsuggested
tape) at 18.4 kDa
that the
were
lectinfound in
existed the
as a SDS-PAGE
monomer of
with the
a lectin
molecularafter Sephadex
weight of G-75.
about 35 The
kDa, SDS-PAGE
which is suggested
similar to that that the
obtained
As indicated in Figure 2, a band (lectin tape) at 35 kDa and a band (unknown tape) at 18.4 kDa
lectin existed
by Jiang [25].as awasmonomer with athat
molecular weight of about 35 kDa,be whichtoisthe
similar todissociation
that obtainedof
were found in Itthe conjectured
SDS-PAGE the band
of the lectin afterofSephadex
18.4 kDa might
G-75. The due
SDS-PAGE partial
suggested that the
by Jiang
the [25].
lectin It wasCompared
subunit. conjecturedto that
the the band
band at ofkDa,
18.4 18.4 the
kDaband
mightat be
35 due to
kDa maythebepartial
due todissociation
the of
incomplete
lectin existed as a monomer with a molecular weight of about 35 kDa, which is similar to that obtained
the lectin
unfolding subunit.
of It
thewas Compared to
non-aggregated the band
lectin at 18.4 kDa, the band at 35 kDa may be due to the incomplete
by Jiang [25]. conjectured that thethat
bandoccurs
of 18.4in kDa
the presence
might beofdue SDS
todenaturing conditions [31].
the partial dissociation of
unfolding
Thelectin
band, of the
unknown non-aggregated
tape, atto
18.4lectin that occurs in the presence of SDS denaturing conditionsfor [31].
the subunit. Compared thekDabandwas notkDa,
at 18.4 sure.the
Weband
defined
at 35itkDa
as an
mayunknown
be due tosample
the incompletemass
The band, unknown
spectrometric tape, at 18.4 kDa was not sure. We defined it as an unknown sample for mass
analysis.
unfolding of the non-aggregated lectin that occurs in the presence of SDS denaturing conditions [31].
spectrometric analysis.
The band, unknown tape, at 18.4 kDa was not sure. We defined it as an unknown sample for mass
3.2. Identification of Zihua Snap Bean Lectin by Tandem Mass Spectrometry
spectrometric analysis.
3.2. Identification of Zihua Snap Bean Lectin by Tandem Mass Spectrometry
The lectin sample tape and the unknown sample tape in Section 3.1 were subjected to liquid
3.2. The lectin sample
Identification
chromatography-nano of Zihuatape
Snapand
Bean
electrospraytheLectin
unknown sample
by Tandem
ionization mass Masstape in Section
(Nano3.1
Spectrometry
spectrometry were subjected
LC-ESI-MS/MS), to liquid
respectively.
chromatography-nano electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS),
3.2.1.The lectin
Lectin
respectively. Tape sample tape and the unknown sample tape in Section 3.1 were subjected to liquid
Analysis
chromatography-nano electrospray ionization mass spectrometry (Nano LC-ESI-MS/MS),
The database UniProt was used to analyze the peptides hydrolyzed by sequencing grade modified
respectively.
trypsin (Promega). The molecular weight of the Zihua snap bean lectin was 29,742.25 Da, and its
relative abundance was 57.3%. There were also other proteins, most of which were undetermined, as
3.2.1. Lectin Tape Analysis
The database UniProt was used to analyze the peptides hydrolyzed by sequencing grade
modified trypsin (Promega). The molecular weight of the Zihua snap bean lectin was 29742.25 Da,
and its2019,
Polymers relative
11, 785 abundance was 57.3%. There were also other proteins, most of which 7were of 20
undetermined, as shown in Table 1. In addition, a few were α-amylase inhibitors with a molecular
weight of 27190.47 Da and a relative abundance of 6.1%.
shown in Table 1. In addition, a few were α-amylase inhibitors with a molecular weight of 27,190.47
Da and a relative
Table abundance
1. The test results of of
the6.1%.
lectin sample by liquid chromatography-nano electrospray ionization
mass spectrometry (Nano LC-ESI-MS/MS).
Table 1. The test results of the lectin sample by liquid chromatography-nano electrospray ionization
Protein(Nano
mass spectrometry Molecular
LC-ESI-MS/MS). Relative
Hits Number of Peptides Link
Weight/Da Abundance
Hits
1 Protein Molecular Weight/Da
29,742.25 Number
246 of Peptides Link
Q8RVX6 Relative Abundance
57.3%
12 39,105.93
29,742.25 65 246 V7BFT4
Q8RVX6 57.3%8.6%
23 35,564.65
39,105.93 56 65 V7AIB2
V7BFT4 10.8%
8.6%
34 35,564.65
27,190.47 27 56 V7AIB2
P02873 10.8%6.1%
45 27,190.47
36,314.02 26 27 P02873
V7BPP1 6.1%2.9%
56 36,314.02
97,527 26 26 V7BPP1
V7BX14 2.9%1.6%
67 97,527
97,769.06 23 26 V7BX14
V7BZK0 1.6%0.6%
7 97,769.06 23 V7BZK0 0.6%
8 71,495.53 20 V7C9P5 0.1%
8 71,495.53 20 V7C9P5 0.1%

Figure 3 showed the peptides in the Zihua snap bean lectin. As indicated in Figure 3b, the peak
Figurewas
m/z 855.49 3 showed the peak
the strong peptides
of theiny the Zihuapeptide
cleavage snap bean lectin. As
GLFNNYK, indicated
while the peakinm/z
Figure 3b,was
685.40 the
peak m/z 855.49 was the strong peak of the y cleavage peptide GLFNNYK, while
the stronger peak of the y cleavage peptide FNNYK. The peak m/z 511.32 was the weaker peak of thethe peak m/z 685.40
was
b the stronger
cleavage peptidepeak of theThe
GGLLG. y cleavage
peak m/z peptide
227.98,FNNYK.
the peakThem/zpeak m/zthe
341.25, 511.32
peakwas
m/zthe weaker
658.41, andpeak
the
of them/z
peak b cleavage
1049.44peptide GGLLG.toThe
corresponded thepeak m/z 227.98,
b cleavage the peak
peptides GGL, m/zGGLL,
341.25,and peak m/z 658.41,
the GGLLGLFNNY,
respectively. m/z 1049.44
and the peak The peak m/z corresponded
798.48 and the to the
peakb cleavage
m/z 968.57 peptides
were yGGL, GGLL,
cleavage and GGLLGLFNNY,
peptides LFNNYK and
respectively. The
LGLFNNYK, peak m/z As
respectively. 798.48
shown andin theFigure m/z the
peak 3d, 968.57
peakwere
m/zy856.48
cleavage peptides
[M+H] + wasLFNNYK and
the stronger
LGLFNNYK, respectively. As shown in Figure 3d, the peak
peak of the y cleavage peptide, GLFNNYK, and the peak m/z 686.31 [M+H] +m/z 856.48 [M+H] + was the stronger peak
stronger peak of
of the
the y cleavage
y cleavage peptide,
peptide, GLFNNYK,
FNNYK. and the peak
The identified m/z 686.31
peptides [M+H]snap
of the Zihua + was stronger
bean peak
lectin are of theiny
shown
cleavage
Table 2, peptide,
which FNNYK.
indicatedThe identified
that peptidesofof the Zihua
the peptides two snap beancleavages
primary lectin are shown in Table
of lectin were 2,
which indicated that the peptides of the two primary cleavages of lectin
GGLLGLFNNYK and DKGGLLGLFNNYK, respectively. The two peptides had overlapping portions were GGLLGLFNNYK and
DKGGLLGLFNNYK,
to obtain a complete aminorespectively. The twowhich
acid sequence, peptides hadthat
proves overlapping
the proteinportions to obtain a complete
was a lectin.
amino acid sequence, which proves that the protein was a lectin.

Figure 3. Two level mass spectrometry of the Zihua snap bean lectin (a) HIGIDVNSIK, (b) GGLLGLFNNYK,
(c) GENAEVLITYDSSTK, and (d) DKGGLLGLFNNYK.
Polymers 2019, 11, 785 8 of 20

Table 2. The peptide of the Zihua snap bean lectin as determined by Nano LC-ESI-MS/MS.

Peptide Number Amino Acid Sequence m/z

6046 HIGIDVNSIK 1094.61
6339 TTTWDFVK 996.49
6350 LTNVNDNGEPTLSSLGR 1785.89
7217 DKGGLLGLFNNYK 1437.76
7269 GNVETNDVLSWSFASK 1752.83
7304 YDSNAHTVAVEFDTLYNVHWDPKPR 2973.40
7329 GGLLGLFNNYK 1194.64
7503 TTTWDFVKGENAEVLITYDSSTK 2604.26
8487 SVLPEWVIVGFTATTGITK 2018.11
8669 YDSNAHTVAVEFDTLYNVHWDPK 2720.25
8877 LSDGTTSEALNLANFALNQIL 2204.13
9249 LLVASLVYPSLK 1301.80
11054 DKGGLLGLFNNYK 1437.76

3.2.2. Unknown Sample Tape Analysis

The results of Table 3 showed that 34.4% of relative abundance was still a lectin with a molecular
weight of 29,742.25 Da, meaning that unknown sample band with 18 KDa was due to the partial
dissociation of the lectin subunit.

Table 3. The test results of unknown sample by Nano LC-ESI-MS/MS.

Hits Protein Molecular Weight/Da Number of Peptides Login ID Relative Abundance

1 29,742.25 104 gi|19773404| 34.4%
2 24,778.15 48 gi|2780981| 18.4%
3 50,723.5 29 gi|75708857| 3.1%
4 49,241.02 25 gi|130169| 2.8%
5 49,483.71 19 gi|351727923| 0.3%
6 71,980.77 17 gi|356500683| 0.4%
7 97,136.89 16 gi|351727843| 1.1%
8 47,824.05 15 gi|374297913| 0.7%

It could be obtained from Tables 2 and 4 that the identified peptides in the unknown samples
had fewer species than the identified peptides in the lectin samples. Nano LC-ESI-MS/MS mass
spectrometry showed that the molecular weight of the Zihua snap bean lectin was 29,742.25 Da, and
the two-step method based on an aqueous two-phase system and Sephadex G-75 was suitable to
separate and purify lectin.

Table 4. The peptide of the lectin in an unknown sample as determined by Nano LC-ESI-MS/MS.

Peptide Number m/z Amino Acid Sequence

6346 1094.61 HIGIDVNSIK
6451 1785.89 LTNVNDNGEPTLSSLGR
6580 996.49 TTTWDFVK
6741 1246.67 FNETNLILQR
6991 1323.69 TSFIVSDTVDLK
7279 1752.83 GNVETNDVLSWSFASK
7326 1437.76 DKGGLLGLFNNYK
7410 1301.80 LLVASLVYPSLK
7444 2973.40 YDSNAHTVAVEFDTLYNVHWDPKPR
7496 1194.64 GGLLGLFNNYK
8307 2018.11 SVLPEWVIVGFTATTGITK
8634 2204.13 LSDGTTSEALNLANFALNQIL
Polymers 2019, 11, 785 9 of 20

3.3. The Properties of Zihua Snap Bean Lectin

3.3.1. Carbohydrate Specificity of Zihua Snap Bean Lectin

Glucose, fructose, sucrose, d-maltose, d-trehalose, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine,
d-galactose, and lactose were used to study the carbohydrate specificity of the Zihua snap bean lectin.
None of the nine glycosyl groups tested could inhibit the hemagglutinating activity of the Zihua snap bean
lectin. Consistent with the result, d-fructose, d-glucose, and sucrose failed to inhibit the hemagglutinating
activity of deep red kidney bean lectin [23]. As for Hypsizigus marmoreus lectin, glucose, mannose, L-fucose,
and lactose had no inhibitory effect [32]. According to related literature, the glycosyl group specifically
bound by some of the bean lectin is a polysaccharide with a larger molecular weight than a monosaccharide
or oligosaccharide [33].

3.3.2. Metal Ions’ Dependency of Zihua Snap Bean Lectin

It was confirmed that certain metal ions played a key role in maintaining the stability of the lectin
structure and maintaining its specific biological activity [34]. Ethylenediaminetetraacetic acid (EDTA)
is a divalent metal ion-chelating agent. When the lectin fully reacted with EDTA, the hemagglutinating
activity of lectin was lost, and the reaction was generally reversible [35].
After fully reacting with EDTA, the hemagglutinating activity of the lectin was completely lost.
The effect of metal ions on the recovery of the hemagglutinating activity was studied by adding
different metal ions containing Cu2+ , Na+ , Mn2+ , Ba2+ , Mg2+ , K+ , Fe2+ , Fe3+ , and Ca2+ . The results
showed that Cu2+ , Na+ , Ba2+ , K+ , Fe2+ , and Fe3+ ions had no effect on restoring the hemagglutinating
activity of the lectin. Meanwhile, among the ions that could restore the Zihua snap bean lectin activity,
Mn2+ and Ca2+ ions could completely restore the hemagglutinating activity of the lectin, while Mg2+
ions could only partially restore the activity. Some researchers indicated that the agglutinating activity
of Inocybe umbrinella lectin was inhibited by Ca2+ , Mg2+ , and Mn2+ ions, but was unaffected by Fe3+
ions [36]. In addition, Ca2+ ions showed a significant effect on the agglutinating activity of lectin from
Laetiporus sulphureus mushroom [37]. It could be concluded that Mn2+ and Ca2+ ions were essential
metal ions of the Zihua snap bean lectin and played an important role in maintaining its biological
activity, while Mg2+ ions could play a certain substitution role. Previous studies indicated that when
Mn2+ and Ca2+ ions in proteins were chelated with EDTA, the protein conformation of lectin gradually
changed to an open conformation. When a specific metal ion recombines with a lectin, the lectin slowly
changed to a binding conformation. The change of binding conformation and open conformation
was a cis-trans isomerization process, and the biological activity of lectin depended on the resulting
conformations [26].

3.3.3. Thermal Stability of the Zihua Snap Bean Lectin

3.3.3.1. Infrared Analysis

FTIR was used to study the changes of the secondary structure of the Zihua snap bean lectin
during thermal treatment. The second derivative of the amide I region of the lectin treated at different
temperatures (70 ◦ C, 80 ◦ C, 90 ◦ C, and 100 ◦ C) was found, and the Gauss peak shape was used for
fitting. Different bands that overlapped together were analyzed, and the results are shown in Figure 4.
The corresponding relationship between each sub-peak and secondary structure meant that
~1610–1642 cm−1 was a β-fold structure; ~1642–1650 cm−1 was an irregular curly structure; ~1650–1660 cm−1
was an α-helix structure; ~1660–1680 cm−1 was the β-turn structure; and ~1680–1700 cm−1 was the β-reverse
structure [38]. According to the corresponding relationship, the relative percentages of various secondary
structures of lectin were obtained. As shown in Table 5, in the unheated lectin, the β-fold was the most
abundant secondary structures, with the relative percentages of 40.43%. As the temperature increased,
the α-helix content decreased, while the irregular curl content increased, and the most significant change
 Polymers 2019, 11, 785 10 of 20

was obtained at 90 ◦ C (p < 0.05, n = 3). It indicated that the lectin started to depolymerize under thermal
treatment,
Polymers and
2019, 11, 785the random coil structure was mainly converted from the β structure. 10 of 21

Figure 4. Second-derivative Fourier transform infrared (FTIR) spectra in the amide Ι region and
Figure 4. Second-derivative Fourier transform infrared (FTIR) spectra in the amide I region and
Gaussian curve fitting of lectin at (a) room temperature, (b) 70 °C,◦ (c) 80 °C,◦ (d) 90 °C,◦ and (e) 100 °C.◦
Gaussian curve fitting of lectin at (a) room temperature, (b) 70 C, (c) 80 C, (d) 90 C, and (e) 100 C.
The corresponding relationship
Table 5. The content of thebetween
secondaryeach sub-peak
structure andatsecondary
of lectin structure meant that
different temperature.
~1610–1642 cm was a β-fold structure; ~1642–1650 cm was an irregular curly structure; ~1650–1660
−1 −1
Temperature α-Helix β-Fold β-Turn β-Reverse Random Curl
cm−1 was an α-helix structure; ~1660–1680 cm−1 was the β-turn structure; and ~1680–1700 cm−1 was
◦
25 structure
C b 40.43 ± 0.11 a d 3.85±0.08 c 13.60 ± 0.16 a of
the β-reverse [38]. ±According
12.85 0.14 to the 29.22 ± 0.12
corresponding relationship, the relative percentages
◦
70 C b d 27.68 ± 0.17 c b 13.64 ± 0.15 a
12.84 ± 0.13 42.23 ± 0.08 3.39±0.11
various secondary◦ structures of lectin were obtained.
e As shown in c Table 5, in the unheated lectin, the
80 C 12.6 ± 0.19 b 42.68 ± 0.13 27.87 ± 0.15 3.23±0.13 ab 13.69 ± 0.08 a
β-fold was 90 the◦ Cmost abundant secondary
11.65 ± 0.17 a
structures,
41.74 ± 0.15 c
with the relative
25.59 ± 0.14 b
percentages
3.13±0.14a
of 40.43%. As the
17.87 ± 0.11 b
temperature 100increased,
◦C the α-helix
11.58 ± 0.11 a content
40.76 decreased,
± 0.19 b while
23.86 the irregular
± 0.09 a curl content
3.82±0.17 c increased,
19.98 ± 0.19 c and
the most significant change was obtained at 90 °C (p < 0.05, n = 3). It indicated that the lectin
Note: The same letter followed by the same column means that the difference is not significant (p > 0.05), and started
the
to depolymerize undermarked
difference between thermal treatment,
letters andthe
indicates that the random
difference (p < 0.05).
coil structure
is significant was mainly converted from
the β structure.
3.3.3.2. Fluorescence Spectroscopy Analysis
Table 5. The content of the secondary structure of lectin at different temperature.
Fluorescence spectroscopy could reflect the changes in the tertiary structure of proteins [39].
The maximum emission
Temperature α-Helixwavelength β-Fold
of the native lectin (0 min) was maintained
β-Turn β-Reverse between Random 328–330
Curl nm
in Figure
25 °C 5. All the tryptophan
12.85 ± 0.14 b residues in lectin
40.43 ± 0.11 a might be buried
29.22 ± 0.12 d inside the
3.85±0.08hydrophobic
c 13.60 ± 0.16 of
cavity a the

protein
70 °C[29]. As shown
12.84 ±in0.13
Figure 5a,b,
b 42.23when
± 0.08the heating
d 27.68time was short3.39±0.11
± 0.17 c or the heating temperature
b 13.64 ± 0.15 a was
low,80the
°Cfluorescence12.6 intensity
± 0.19 b was42.68basically
± 0.13 eunchanged,
27.87 ± and
0.15 cthe hemagglutinating
3.23±0.13 ab activity
13.69 of alectin
± 0.08
was90 not
°C obviously affected
11.65 ± 0.17 (Figure
a 6a,b). As
41.74 ± 0.15 cshown in Figure
25.59 ± 0.14 b 5c–e, fluorescence
3.13±0.14 a intensity increased
17.87 ± 0.11 b
when100the
°C heating11.58
time±was 0.11 long
a or40.76
the ±heating
0.19 b temperature
23.86 ± 0.09was
a high, which cmight 19.98
3.82±0.17 be related
± 0.19 to
c the

thermal
Note: The same letter followed by the same column means that the difference is not significant (p > a red
polymerization of lectin multimers caused by heat treatment [40]. In addition, there was
shift in maximum
0.05), emission
and the difference wavelength
between marked(Figure 6c–e). It represented
letters indicates a change
that the difference in the microenvironment
is significant (p < 0.05).
of the tryptophan residue, which indicated that the tryptophan residue was transferred to a hydrophilic
3.3.3.2. Fluorescence Spectroscopy Analysis
Fluorescence spectroscopy could reflect the changes in the tertiary structure of proteins [39]. The
maximum emission wavelength of the native lectin (0 min) was maintained between 328–330 nm in
Figure 5. All the tryptophan residues in lectin might be buried inside the hydrophobic cavity of the
 lectin was not obviously affected (Figure 6a,b). As shown in Figure 5c–e, fluorescence intensity
increased when the heating time was long or the heating temperature was high, which might be
related to the thermal polymerization of lectin multimers caused by heat treatment [40]. In addition,
there was a red shift in maximum emission wavelength (Figure 6c–e). It represented a change in the
microenvironment
Polymers 2019, 11, 785 of the tryptophan residue, which indicated that the tryptophan residue 11 ofwas
20
transferred to a hydrophilic environment [41]. As the heating temperature reached 90 °C, the
fluorescence intensity decreased most significantly, and the hemagglutinating activity of lectin
environment [41]. As the heating temperature reached 90 ◦ C, the fluorescence intensity decreased
dropped sharply, with all activity lost within 10 min (Figure 6e). In addition, the maximum emission
most significantly, and the hemagglutinating activity of lectin dropped sharply, with all activity lost
wavelength of the tryptophan residue was accompanied by a further red shift to 340 nm (Figure 5e).
within 10 min (Figure 6e). In addition, the maximum emission wavelength of the tryptophan residue
The maximum emission wavelength of tryptophan residues in the hydrophilic environment was 350–
was accompanied by a further red shift to 340 nm (Figure 5e). The maximum emission wavelength
360 nm. It indicated that the lectin folding structure was not fully expanded, and the lectin had good
of tryptophan residues in the hydrophilic environment was 350–360 nm. It indicated that the lectin
heat resistance.
folding structure was not fully expanded, and the lectin had good heat resistance.

Figure 5. The intrinsic fluorescence spectra of lectin at (a) 50 ◦ C, (b) 60 ◦ C, (c) 70 ◦ C, (d) 80 ◦ C, and (e) 90 ◦ C.
Figure 5. The intrinsic fluorescence spectra of lectin at (a) 50 °C, (b) 60 °C, (c) 70 °C, (d) 80 °C, and (e)
90 °C.
3.3.4. pH Stability of Zihua Snap Bean Lectin
The endogenous fluorescence of the Zihua snap bean lectin at different pH conditions was
measured at excitation wavelengths of 280 nm and 295 nm, respectively. The experimental results
were shown in Figure 7.
Polymers
Polymers 11, 785
2019,2019, 11, 785 12 of 2112 of 20

Polymers 2019, 11, 785 13 of 21

Hemagglutinating activity changes of lectin at (a) ◦ C, (b) 60 ◦ C, (c) 70 ◦ C, (d) 80 ◦ C, and (e) 90 ◦ C.
Figure 6.Figure 6. Hemagglutinating activity changes of lectin at 50
(a) 50 °C, (b) 60 °C, (c) 70 °C, (d) 80 °C, and
(e) 90 °C.

3.3.4. pH Stability of Zihua Snap Bean Lectin

The endogenous fluorescence of the Zihua snap bean lectin at different pH conditions was
measured at excitation wavelengths of 280 nm and 295 nm, respectively. The experimental results
were shown in Figure 7.

Figure The
Figure7. 7. emission
The fluorescence
emission spectra
fluorescence of lectin
spectra of from
lectinthe Zihua
from thesnap beansnap
Zihua at various
bean pH conditions
at various pH
atconditions
the excitation wavelengths
at the of (a) 280 nmofand
excitation wavelengths (a) (b)
280295
nmnm.and (b) 295 nm.

As shown in Figure 7a,b, as the pH increased in the range of 3.0 to 10.0, the fluorescence intensity
gradually decreased, and the maximum absorption wavelength gradually increased. However, the
change was not significant. The maximum emission wavelength of the Zihua snap bean lectin in the
range of pH 3.0 to 10.0 was 329 ± 1.5 nm; there were a blue shift at 2.0 and a red shift at pH 11.0. It
was indicated that most of the tryptophan residues in the lectin were in a non-polar environment.
Polymers 2019, 11, 785 13 of 20

As shown in Figure 7a,b, as the pH increased in the range of 3.0 to 10.0, the fluorescence intensity
gradually decreased, and the maximum absorption wavelength gradually increased. However, the
change was not significant. The maximum emission wavelength of the Zihua snap bean lectin in the
range of pH 3.0 to 10.0 was 329 ± 1.5 nm; there were a blue shift at 2.0 and a red shift at pH 11.0. It was
indicated that most of the tryptophan residues in the lectin were in a non-polar environment. When
the protein was fully expanded, the maximum emission wavelength of the exposed tryptophan residue
was between 350–360 nm. Thus, the acid-induced development of the Zihua snap bean lectin did not
cause the tryptophan residue to directly contact with water. The fluorescence intensity decreased at
pH 2.0 and 11.0 in Figure 7. However, it cannot be explained simply by the denaturation of proteins
in acidic or alkaline environments. The reduction of fluorescence intensity at low pH values may be
caused by fluorescence quenching or the neutralization of COO– groups on acidic amino acids near
the fluorophore [40]. In general, the fluorescence spectra of nectarines had a peak shift at lower or
higher pH conditions, and the difference in the position of the peak shift was not significant. It was
indicated that the lectin structure did not show significant differences within the tested pH range.
Figure 8 indicates that the Zihua snap bean lectin has hemagglutinating activity in a wide pH
range (2.0 to 10.0). Unlike temperature and denaturant, pH-induced protein unfolding was achieved by
protonation or relatively few discrete sites of protonation [42], whereas perturbing a small number of
residues did not allow the protein to fully unfold. It has been reported that lectins maintain their tertiary
structure stability mainly through non-covalent forces such as hydrogen bonding, ionic interaction,
hydrophobic
Polymers 2019, interaction,
11, 785 van der Waals force, and disulfide bond covalent linkage [43]. The change in21
14 of
the tertiary structure of the acid-induced Zihua snap bean lectin might be due to the interaction of ions
or hydrogen bonds. However, this change had no effect on the hemagglutinating activity of the lectin.

Figure 8. The hemagglutinating activity of the lectin from the Zihua snap bean at various pH conditions.
Figure 8. The hemagglutinating activity of the lectin from the Zihua snap bean at various pH
The pH stability of the Zihua snap bean lectin was studied using the denaturing agent guanidine
conditions.
hydrochloride (GdnHCl). Some models suggested that GdnHCl could migrate to the interior of
the protein
The pH to stability
form hydrogen bonds
of the Zihua snapto bean
reduce thewas
lectin hydrophobic effect
studied using theofdenaturing
the protein [44].guanidine
agent Under
physiological
hydrochloride conditions
(GdnHCl).of pH 7.2,models
Some the lectin reacted with
suggested that different
GdnHCl concentrations
could migrate ofto GdnHCl for of
the interior 24 the
h.
The results of the conformational changes are shown in Figure 9. The fluorescence
protein to form hydrogen bonds to reduce the hydrophobic effect of the protein [44]. Under intensity gradually
decreased with the
physiological progressive
conditions of pHincrease
7.2, theinlectin
GdnHCl concentration
reacted (Figure
with different 9a). The maximum
concentrations of GdnHClemission
for 24
wavelength of lectin
h. The results didconformational
of the not change obviously
changesbetween 0–3 mol/L
are shown of GdnHCl,
in Figure while the maximum
9. The fluorescence intensity
emission
graduallywavelength
decreasedofwith
lectinthe
showed a significant
progressive increase
increase between 3–6
in GdnHCl mol/L GdnHCl
concentration (Figure
(Figure 9a). 9b).
The
When the concentration
maximum of GdnHCl of
emission wavelength reached
lectin 6did
mol/L, the fluorescence
not change obviouslyintensity
between decreased
0–3 mol/L by of
two-thirds
GdnHCl,
while the maximum emission wavelength of lectin showed a significant increase between 3–6 mol/L
GdnHCl (Figure 9b). When the concentration of GdnHCl reached 6 mol/L, the fluorescence intensity
decreased by two-thirds compared to 0 mol/L, and the maximum emission of lectin was a red shift to
337 nm, which was much smaller than the maximum emission wavelength (350–360 nm) of the
tryptophan residue. It indicated that the tryptophan residue was not completely exposed to the
 protein to form hydrogen bonds to reduce the hydrophobic effect of the protein [44]. Under
physiological conditions of pH 7.2, the lectin reacted with different concentrations of GdnHCl for 24
h. The results of the conformational changes are shown in Figure 9. The fluorescence intensity
gradually decreased with the progressive increase in GdnHCl concentration (Figure 9a). The
maximum
Polymers 2019, 11,emission
785 wavelength of lectin did not change obviously between 0–3 mol/L of GdnHCl,
14 of 20
while the maximum emission wavelength of lectin showed a significant increase between 3–6 mol/L
GdnHCl (Figure 9b). When the concentration of GdnHCl reached 6 mol/L, the fluorescence intensity
compared to 0 mol/L, and the maximum emission of lectin was a red shift to 337 nm, which was much
decreased by two-thirds compared to 0 mol/L, and the maximum emission of lectin was a red shift to
smaller than the maximum emission wavelength (350–360 nm) of the tryptophan residue. It indicated
337 nm, which was much smaller than the maximum emission wavelength (350–360 nm) of the
that the tryptophan residue was not completely exposed to the solvent, and the folding structure of the
tryptophan residue. It indicated that the tryptophan residue was not completely exposed to the
lectin was not fully opened. The result showed that the acid–base stability of the Zihua snap bean
solvent, and the folding structure of the lectin was not fully opened. The result showed that the acid–
lectin was high.
base stability of the Zihua snap bean lectin was high.

Figure 9. The fluorescence spectra (a) and maximum emission wavelength (b) of the lectin at different
Figure 9. The
Polymers 2019, 11, 785 fluorescence spectra (a) and maximum emission wavelength (b) of the lectin at different
15 of 21
GdnHCl
GdnHCl concentrations.
concentrations.
3.4.In
3.4. InVitro
VitroStudies
Studiesofofthe
theDigestibility
DigestibilityofofZihua
ZihuaSnap
SnapBean
BeanLectin
Lectin

3.4.1.The
3.4.1. TheDigestibility
DigestibilityofofNative
NativeLectin
LectinIn
InVitro
Vitro
Sincethe
Since theZihua
Zihuasnap
snapbean
beanlectin
lectincan
canagglutinate
agglutinaterabbit
rabbitred
redblood
bloodcells,
cells,ititisisprobably
probablyaasensitizing
sensitizing
protein.Therefore,
protein. Therefore, the
thein
invitro
vitrodigestion
digestionsimulation
simulationexperiment
experimentof ofthe
theZihua
Zihuasnap snapbean
beanlectin
lectinwas
was
studied. SDS–PAGE has been widely used in simulated gastric fluid (SGF) analysis
studied. SDS–PAGE has been widely used in simulated gastric fluid (SGF) analysis and simulated and simulated
intestinalfluid
intestinal fluid(SIF)
(SIF)analysis
analysis[45].
[45].SDS–PAGE
SDS–PAGEanalyses
analysesof
ofnative
nativelectin
lectinfrom
fromthe theZihua
Zihuasnap
snapbean
beaninin
SGF and SIF are shown in Figure
SGF and SIF are shown in Figure 10. 10.

Figure 10. (a) Simulated gastric fluid (SGF) digestion profiles of the native lectin from the Zihua snap
Figure 10. (a) Simulated gastric fluid (SGF) digestion profiles of the native lectin from the Zihua snap
bean. In the SDS-PAGE analysis, lane 1 was the molecular weight marker, lane 2 was the native lectin,
bean. In the SDS-PAGE analysis, lane 1 was the molecular weight marker, lane 2 was the native lectin,
lane 3 was the pepsine, and lanes 4 to 10 were the SGF digestion pattern of the native lectin at 0 min,
lane 3 was the pepsine, and lanes 4 to 10 were the SGF digestion pattern of the native lectin at 0 min,
2 min, 5 min, 10 min, 20 min, 30 min, and 60 min. (b) Simulated intestinal fluid (SIF) digestion profiles
2 min, 5 min, 10 min, 20 min, 30 min, and 60 min. (b) Simulated intestinal fluid (SIF) digestion profiles
of the native lectin from the Zihua snap bean. In the SDS-PAGE analysis, lane 1 was the molecular
of the native lectin from the Zihua snap bean. In the SDS-PAGE analysis, lane 1 was the molecular
weight marker, lane 2 was the native lectin, lane 3 was the tryptic, and lanes 4 to 10 were the tryptic
weight marker, lane 2 was the native lectin, lane 3 was the tryptic, and lanes 4 to 10 were the tryptic
digestion pattern of the native lectin at 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 90 min.
digestion pattern of the native lectin at 0 min, 10 min, 20 min, 30 min, 40 min, 60 min, and 90 min.

As indicated in Figure 10a, as the digestion time prolonged, the native lectin from the Zihua
snap bean was gradually decreased in SGF. It was difficult to observe the lectin band at 60 min. The
results of the in vitro simulated digestibility evaluation of food proteins indicated that most food
allergens were basically stable during SGF for 60 min [46]. Therefore, the Zihua snap bean lectin had
a certain anti-enzymatic ability in the gastrointestinal tract, which laid a foundation for the
Polymers 2019, 11, 785 15 of 20

As indicated in Figure 10a, as the digestion time prolonged, the native lectin from the Zihua snap
bean was gradually decreased in SGF. It was difficult to observe the lectin band at 60 min. The results
of the in vitro simulated digestibility evaluation of food proteins indicated that most food allergens
were basically stable during SGF for 60 min [46]. Therefore, the Zihua snap bean lectin had a certain
anti-enzymatic ability in the gastrointestinal tract, which laid a foundation for the application of lectin
in the field of medicine.
As shown in Figure 10b, under the tryptic digestion conditions, there was still a clear lectin
fragment that remained until 90 min. A lectin from the red kidney bean band could still be observed
after 48 h in SIF [47]. It indicated that the native lectin had good stability. The stability of the lectin
provides an estimate of whether the protein may trigger clinical symptoms of an allergic disease.

3.4.2. The Digestibility of Thermal-Treated Lectin

In vitro digestion experiments were performed on the preheated lectin from the Zihua snap bean.
Polymers
The 2019,are
results 11,shown
785 in Figure 11. 16 of 21

Figure 11. (a) SGF digestion profiles of the preheated lectin from the Zihua snap bean. In the SDS-PAGE
Figure 11. (a) SGF digestion profiles of the preheated lectin from the Zihua snap bean. In the SDS-
analysis, lane 1 was the molecular weight marker, lane 2 was the pepsine, lane 3 was the native lectin,
PAGE analysis, lane 1 was the molecular weight marker, lane 2 was the pepsine, lane 3 was the native
and lanes 4 to 10 were the SGF digestion pattern of the native lectin at 0 min, 2 min, 5 min, 10 min,
lectin, and lanes 4 to 10 were the SGF digestion pattern of the native lectin at 0 min, 2 min, 5 min, 10
20 min, 30 min, and 60 min. (b) SIF digestion profiles of the preheated lectin from the Zihua snap bean.
min, 20 min, 30 min, and 60 min. (b) SIF digestion profiles of the preheated lectin from the Zihua snap
In the SDS-PAGE analysis, lane 1 was the molecular weight marker, lane 2 was the tryptic, lane 3 was
bean. In the SDS-PAGE analysis, lane 1 was the molecular weight marker, lane 2 was the tryptic, lane
the native lectin, and lanes 4 to 10 were the tryptic digestion pattern of the native lectin at 0 min, 10 min,
3 was the native lectin, and lanes 4 to 10 were the tryptic digestion pattern of the native lectin at 0 min,
20 min, 30 min, 40 min, 60 min, and 90 min.
10 min, 20 min, 30 min, 40 min, 60 min, and 90 min.
The preheated zihua snap bean lectin could be completely digested by pepsin in SGF in 10 min
The preheated zihua snap bean lectin could be completely digested by pepsin in SGF in 10 min
(Figure 11a). Similarly, no obvious lectin bands were observed in SIF after 10 min (Figure 11b). It meant
(Figure 11a). Similarly, no obvious lectin bands were observed in SIF after 10 min (Figure 11b). It
that the digestibility of lectin from the Zihua snap bean was changed to some extent by thermal
meant that the digestibility of lectin from the Zihua snap bean was changed to some extent by thermal
treatment. Some studies proved that the partial structure of lectin after preheating treatment was
treatment. Some studies proved that the partial structure of lectin after preheating treatment was
unfolded, which was helpful to improve the in vitro enzymatic hydrolysis of lectins [16].
unfolded, which was helpful to improve the in vitro enzymatic hydrolysis of lectins [16].
3.5. Antimicrobial of Zihua Snap Bean Lectin
3.5. Antimicrobial of Zihua Snap Bean Lectin
3.5.1. Antibacterial Activity of Zihua Snap Bean Lectin
3.5.1. Antibacterial Activity of Zihua Snap Bean Lectin
The inhibition of S. aureus, E. coli, and B. subtilis by the Zihua snap bean lectin is shown in Figure 12a–c,
The inhibition
respectively. Figure of S. aureus,
12d–f were theE. coli, and B.control
positive subtilisof
byphenol.
the ZihuaThesnap beanantibacterial
in vitro lectin is shown in Figure
experiment
12a–c, respectively. Figure 12d–f were the positive control of phenol. The in vitro
indicated that the Zihua snap bean lectin showed antibacterial activity against the tested bacteria (S. aureus,antibacterial
E.experiment
coli, and B.indicated
subtilis). that the Zihua
Lectins have thesnap bean to
ability lectin showed
recognize antibacterial activity
carbohydrates against the tested
(e.g., peptidoglycan and
bacteria (S. aureus, E. coli, and B. subtilis). Lectins have the ability to recognize carbohydrates
lipopolysaccharide) on the surface of bacterial cells [48]. The antibacterial activity of lectins was attributed (e.g.,
topeptidoglycan andwith
their interaction lipopolysaccharide)
the glycans of theonbacterial
the surface of bacterial
cell wall [49]. Incells [48]. The
addition, antibacterial
antibacterial activity
lectins can
of lectinsprotein
promote was attributed
leakage to and their
theinteraction
formation with the glycans
of pores of the
in the cell wallbacterial
[50]. Ascell wall [49].
shown In addition,
in Figure 12, S.
antibacterial lectins can promote protein leakage and the formation of pores in the cell wall [50]. As
shown in Figure 12, S. aureus, E. coli, and B. subtilis were capable of being inhibited by the lectin. At
the same concentration, the antibacterial effect of lectin was stronger than that of phenol. The
corresponding diameter of inhibition halos was shown in Table 6. As the lectin addition amount
increased, the inhibition halo grew larger. Romero et al. [30] indicated that Phthirusa pyrifolia leaf
Polymers 2019, 11, 785 16 of 20

aureus, E. coli, and B. subtilis were capable of being inhibited by the lectin. At the same concentration, the
antibacterial effect of lectin was stronger than that of phenol. The corresponding diameter of inhibition
halos was shown in Table 6. As the lectin addition amount increased, the inhibition halo grew larger.
Romero et al. [30] indicated that Phthirusa pyrifolia leaf lectin (PpyLL) had an inhibitory effect on B. subtilis,
but not on S. aureus, when the addition amount was 80 µg. Lectin from the seeds of Archidendron jiringa
Nielsen was detected to have inhibition for B. subtilis and S. aureus, whereas it did not for E. coli [51]. Some
studies have shown that the lectin had a greater inhibition on Gram-positive bacteria than Gram-negative
bacteria [30,51]. Our results agreed with this observation. The difference of the peptidoglycan content of
cell walls 2019,
Polymers of Gram-positive
11, 785 bacteria and Gram-negative bacteria was the main cause of this condition.
17 of 21

Figure 12. Inhibition of (a) S. aureus, (b) E. coli, and (c) B. subtilis with different concentrations (1,
Figure 12. Inhibition
stroke-physiological of (a)solution;
saline S. aureus,2,(b)
10 E.
µg;coli, and
3, 20 µg;(c)4,B.50subtilis
µg) ofwith different
lectin. concentrations
Inhibition (1,
of (d) S. aureus,
stroke-physiological saline solution; 2, 10 µ g; 3, 20 µ g; 4, 50 µ g) of lectin. Inhibition of (d)
(e) E. coli, and (f) B. subtilis with different concentrations (1, stroke-physiological saline solution; 2, S. aureus, (e)
10E. coli, and (f) B. subtilis with different concentrations (1, stroke-physiological saline solution; 2, 10 µ g;
µg; 3, 20 µg; 4, 50 µg) of phenol.
3, 20 µ g; 4, 50 µ g) of phenol.
Table 6. The bacteriostatic circle diameter of bacteria with different concentrations of lectin.
Table 6. The bacteriostatic circle diameter of bacteria with different concentrations of lectin.
Bacterial Different Addition Amount (µg) Inhibition Halo (mm)
Bacterial Different Addition Amount (µg) Inhibition Halo (mm)
10 10.5 ± 0.5
S. aureus (G )+ 20 10 10.5 ± 0.5
12.1 ± 0.8
S. aureus (G )+
50 20 12.1 ± 0.8
14.5 ± 1.1
10 50 8.2 ± 0.814.5 ± 1.1
E. coli(G− ) 20 10 10.1 ± 0.88.2 ± 0.8
E. coli (G-) 50 20 11.6 ± 0.510.1 ± 0.8
10 50 8.8 ± 1.011.6 ± 0.5
B. subtilis (G+ ) 20 10 10.70 ± 0.5
8.8 ± 1.0
B. subtilis (G+) 50 20 12.7 ± 0.7
10.70 ± 0.5
50 12.7 ± 0.7

3.5.2. Antifungal Activity of Zihua Snap Bean Lectin

As indicated in Figure 13, the Zihua snap bean lectin showed an inhibitory effect on P. infestans
when the addition amount was 100 μg (20 μL of 5 mg/mL lectin solution). Compared with the results
of antibacterial experiments, the Zihua snap bean lectin can inhibit the growth of P. infestans at a high
Polymers 2019, 11, 785 17 of 20

3.5.2. Antifungal Activity of Zihua Snap Bean Lectin

As indicated in Figure 13, the Zihua snap bean lectin showed an inhibitory effect on P. infestans
when the addition amount was 100 µg (20 µL of 5 mg/mL lectin solution). Compared with the results
of antibacterial experiments, the Zihua snap bean lectin can inhibit the growth of P. infestans at a high
concentration. According to de Santana [35], only a few lectins had significant antifungal activity. In
addition, lectins from kidney beans have inhibitory effects on four harmful fungi (Helminthosporium
maydis, Sclerotinia sclerotiorum, Gibberalla sanbinetti, and Rhizoctonia solani) in agriculture [52]. Lectins
Polymers 2019, 11, 785 18 of 21
interacting with chitin, α-mannan, and β-glucan in the cell walls can lead to growth inhibition, cell
wall
wall disruption, reduced absorption
disruption, reduced of nutrients,
absorption of nutrients, and
and interference
interference with
with spore
spore germination,
germination, which
which is is
the main
the main reason
reason forfor the
the antifungal
antifungal activity
activity of
of lectins
lectins [53].
[53].

Figure
Figure 13.
13. Inhibition
Inhibition of
of the
the P.
P. infestans
infestans with
with lectin
lectin (B)
(B) and
and stroke-physiological saline solution
stroke-physiological saline solution (A).
(A).

4. Conclusions
4. Conclusions
The properties
The properties of ofthe
thelectin
lectinisolated
isolatedandand purified
purified bybythethe two-step
two-step method
method were were studied
studied in thisin
this study. Through the experimental study on the affinity characteristics of
study. Through the experimental study on the affinity characteristics of lectin, it found that glucose, lectin, it found that
glucose, N-acetyl-d-glucosamine,
N-acetyl- D-glucosamine, D-galactose, N-acetyl-d-galactosamine,
N-acetyl-
d-galactose, D-galactosamine, fructose, fructose,
sucrose, sucrose,
D-maltose,
d-maltose, D-
and lactose could not inhibit the hemagglutinating activity of the lectin and Mn 2+ 2+
, Ca 2+ ,
trehalose, and lactose could not inhibit the hemagglutinating activity of the lectin and Mn , Ca , and
d-trehalose, 2+

and2+Mg 2+ ions can restore the hemagglutinating activity of the lectin. The results of the thermal
Mg ions can restore the hemagglutinating activity of the lectin. The results of the thermal stability
indicate that the that
stability indicate the hemagglutinating
hemagglutinating activity of activity of the
the lectin is lectin
changedis changed
with thewith the of
change change
protein of
protein conformation. Studies on the pH stability of the lectin show
conformation. Studies on the pH stability of the lectin show that the lectin maintains its that the lectin maintains its
hemagglutinating activity
hemagglutinating activityininthe
therange
rangeof of
pHpH2.0 2.0
to 10.0. TheThe
to 10.0. SDS-PAGE
SDS-PAGE of theofinthe
vitroin digestion
vitro digestionfound
that the native lectin is almost completely digested by pepsin at 60 min in SGF,
found that the native lectin is almost completely digested by pepsin at 60 min in SGF, while a distinct while a distinct lectin
band is observed in SIF for 90 min. However, the lectin pretreated is completely
lectin band is observed in SIF for 90 min. However, the lectin pretreated is completely digested by digested by pepsin
or trypsin
pepsin or in about in
trypsin 10 about
min. Antibacterial assays demonstrate
10 min. Antibacterial that the lectin
assays demonstrate thatexhibits antibacterial
the lectin exhibits
activity against S. aureus, E. coli, and B. subtilis. In addition, the lectin shows inhibition
antibacterial activity against S. aureus, E. coli, and B. subtilis. In addition, the lectin shows inhibition on the growth
of phytophthora
on the growth of infestans
phytophthora at a high concentration.
infestans at a high concentration.
Author Contributions: Data curation, X.W., L.W. and X.L.; Funding acquisition, B.J.; Investigation, D.L.;
Author Contributions: Data curation, X.W., L.W. and X.L.; Funding acquisition, B.J.; Investigation, D.L.;
Methodology, B.J., C.L. and Z.F.; Project administration, B.J. and Z.F.; Writing—original draft, X.W.; Writing—review
Methodology,
& B.J., Z.F.
editing, B.J. and C.L. and Z.F.; Project administration, B.J. and Z.F.; Writing—original draft, X.W.; Writing—
review & editing, B.J. and Z.F.
Funding: This research was funded by the National Natural Science Foundation of China (No. 31201366), the
Natural Science
Funding: Foundation
This research wasoffunded
Heilongjiang
by the Province
National (C2018019), the Research
Natural Science Science
Foundation Foundation
of China in Technology
(No. 31201366), the
Innovation of Harbin (2016RAQXJ052), and the Northeast Agricultural University Students’ Innovation and
Natural Science Foundation of Heilongjiang
Entrepreneurship Training Program (2019). Province (C2018019), the Research Science Foundation in
Technology Innovation of Harbin (2016RAQXJ052), and the Northeast Agricultural University Students’
Innovation and Entrepreneurship Training Program (2019).

Conflicts of Interest: The authors declare no conflict of interest.

References
Polymers 2019, 11, 785 18 of 20

Conflicts of Interest: The authors declare no conflict of interest.

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