Biosynthesis of Silver Nanoparticles by Polysaccharide of Leucaena leucocephala Seeds and Their Anticancer, Antifungal Properties and as Preservative of Composite Milk Sample

Abstract

The AgNPs were synthesized using water-soluble polysaccharides extracted from the Leucaena leucocephala seeds. The UV-visible spectrum of the AgNPs showed a sharp absorption peak at 448 nm. The XRD analysis showed four major peaks of the crystalline AgNPs with planes of a face-centered cubic lattice of silver. The EDS spectrum showed a strong signal peak at ~3 keV. TEM and SEM observations showed the spherical shape of AgNPs with no particle agglomeration, and the size ranged from 8 to 20 nm. AgNPs were highly stable at -14.2 mV by zeta potential measurement. AgNPs showed significant anticancer activity against the cell lines of breast cancer, liver carcinoma, and colon carcinoma with the IC50 value of 22.5, 12.3, and 8.9 μg mL⁻¹, respectively. AgNPs at 900 μg mL⁻¹ exhibited considerable antifungal activity against ten fungal pathogens. Water-soluble polysaccharide has the ability to synthesize AgNPs keeping strong antitumor, antifungal activities. The AgNPs can slow down spoilage of composite milk samples at different temperatures. In addition, the accuracy of milk-IR-analyses was not affected by different concentrations of AgNPs.

Full text

Research Article
Biosynthesis of Silver Nanoparticles by Polysaccharide of
Leucaena leucocephala Seeds and Their Anticancer, Antifungal
Properties and as Preservative of Composite Milk Sample
Mohamed A. Taher ,
1
Ebtihal Khojah ,
2
Mohamed Samir Darwish ,
3
Elsherbiny A. Elsherbiny ,
4
Asmaa A. Elawady ,
3
and Dawood H. Dawood
1
1
Agricultural Chemistry Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt
2
Department of Food Science and Nutrition, College of Science, Taif University, P.O. Box 11099, Taif 21944, Saudi Arabia
3
Dairy Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt
4
Plant Pathology Department, Faculty of Agriculture, Mansoura University, Mansoura 35516, Egypt
Correspondence should be addressed to Mohamed A. Taher; mohamedtaher@mans.edu.eg
Received 21 November 2021; Accepted 9 January 2022; Published 25 January 2022
Academic Editor: José Agustín Tapia Hernández
Copyright © 2022 Mohamed A. Taher et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
The AgNPs were synthesized using water-soluble polysaccharides extracted from the Leucaena leucocephala seeds. The UV-visible
spectrum of the AgNPs showed a sharp absorption peak at 448 nm. The XRD analysis showed four major peaks of the crystalline
AgNPs with planes of a face-centered cubic lattice of silver. The EDS spectrum showed a strong signal peak at ~3 keV. TEM and
SEM observations showed the spherical shape of AgNPs with no particle agglomeration, and the size ranged from 8 to 20nm.
AgNPs were highly stable at -14.2 mV by zeta potential measurement. AgNPs showed significant anticancer activity against the
cell lines of breast cancer, liver carcinoma, and colon carcinoma with the IC50 value of 22.5, 12.3, and 8.9 μgmL
-1
, respectively.
AgNPs at 900 μgmL
-1
exhibited considerable antifungal activity against ten fungal pathogens. Water-soluble polysaccharide has
the ability to synthesize AgNPs keeping strong antitumor, antifungal activities. The AgNPs can slow down spoilage of
composite milk samples at different temperatures. In addition, the accuracy of milk-IR-analyses was not affected by different
concentrations of AgNPs.
1. Introduction
Nanoparticles are of great research attention as they have inno-
vative optoelectronic, magnetic, and physicochemical features,
affected by their various shape and size details [1]. Researchers
are most interested in the biosynthesis of stable nanoparticles
using green chemistry routes in which no xenobiotic or toxic
compounds are used for environmental sustainability. Green
synthesis of nanoparticles depends on natural reduction com-
ponents commonly found in bacteria, fungi, yeast, actinomy-
cetes, and plant extracts [1]. In a few recent years, the
polysaccharide-mediated green synthesis of different metal
nanoparticles had a voluminous role in nanobiotechnology
[2]. Several examples are available of stable metal nanoparticles
biosynthesis by polysaccharides which chiefly comprise gold,
silver, zinc, and palladium nanoparticles.
Among zero-dimensional nanomaterials, silver nanoparti-
cles (AgNPs) are one of the worthiest candidates for performant
and unconventional applications, with enormous outcomes in
pharmaceutical sciences, cosmetics, wound dressings, and
anti-infective coatings, antimicrobial textiles, and food packages
[3, 4]. The major role of AgNPs in medicine mostly depends on
their exceptional and wide antimicrobial activity [3, 4]. On the
other hand, the utilization of nanosilver for prolonging the shelf
life of dairy products is not probable as the minimum inhibitory
concentration of silver is at least 5 mg L
-1
in milk which was
greater than the WHO-recommended maximum concentration
of AgNPs (0.1 mg L
-1
) [5]. However, to date, there are no data in
Hindawi
Journal of Nanomaterials
Volume 2022, Article ID 7490221, 16 pages
https://doi.org/10.1155/2022/7490221

the literature that indicate the suitability of AgNPs as a potential
substitute for chemical preservative agents of milk composite
samples without any effect on the accuracy of infrared (IR)
devices to tested samples.
The area of food and agriculture research takes benefitof
wastes from agroindustrial and agricultural sources to give
them an added value, thus avoiding pollution to the environ-
ment by minimizing burning or extreme accumulation [6].
In this context, Leucaena leucocephala (Lam.) is a tropical
tree that grows in Egypt, perennial thornless, with a height
of around 8 m, and belongs to the subfamily Mimosoid eae
and family Fabaceae with bulk volumes of solid wastes in
the form of leaves, ripened fruits, pods, and seeds [7]. This
tree is commonly used as organic fertilizer, timber, gum,
fuel, forage, firewood, and raw mass for paper and pharma-
ceutical industries [8]. Moreover, the tree has different bio-
medical properties, including antiviral, antidiabetic, anti-
inflammatory, anticancer, antithrombotic, anticoagulant,
and immunostimulant properties [9, 10]. The seed gums of
L. leucocephala are used for treating gastric tract diseases
and well act as a laxative, while the whole seed has been used
as a coffee substitute.
The galactomannan gum is the predominant constituent
in the seeds of L. leucocephala with insignificant levels of
tannins, organic acids, oils, and the amino acid of mimosine
[11]. The viscous galactomannan obtained from the endo-
sperm of L. leucocephala seeds is structured as linear chains
of β-(1-4)-d-mannose units substituted by single α-d-galac-
tose units at O-6. The galactose/mannose ratio of Leucaena
gum is approximately 1 : 1.3, although the galactose/
mannose ratio is variable between species, portions, or even
fractions of Leucaena [12]. Recently, the different extracts of
L. leucocephala have been used in developing metal-based
nanoparticles such as copper, silver, and cadmium [13–15].
To our best knowledge, no documents have been
directed on the use of polysaccharides of galactomannan
precipitated from the seed wastes of L. leucocephala for
developing silver nanoparticles. Therefore, the present study
is aimed at (1) precipitating the crude polysaccharide of L.
leucocephala seeds, (2) characterizing the chemical composi-
tion of the crude polysaccharide, (3) using the crude poly-
saccharide in the preparation of silver nanoparticles, (4)
characterizing the synthesized AgNPs using polysaccharides
of L. leucocephala seeds, (5) estimating the antitumor and
antifungal activities of the biosynthesized AgNPs, (6) evalu-
ating the preservative action of the AgNPs in composite milk
samples, and (7) determining the influence of AgNPs on the
accuracy of midinfrared milk component testing.
2. Materials and Methods
2.1. General. Absolute ethanol, silver nitrate (AgNO
3
), metha-
nol, petroleum ether (60-80
°
C), sulfuric acid, 3,5-dinitrosa-
licylic acid (DNS), Folin-Ciocalteu reagent, gallic acid, phenol
crystal, and sodium carbonate (anhydrous) were purchased
from Sigma (Sigma-Aldrich Chemicals, USA). The seeds of L.
leucocephala were collected from Mansoura University Cam-
pus in Mansoura city. The plant materials were authenticated
by a plant taxonomist at the Botany Department, Faculty of
Science, Mansoura University, Egypt.
The fungal species of Alternaria alternata AUMC 10301,
Aspergillus flavus AUMC 8965, A. parasiticus AUMC 8947,
Bipolaris hawaiiensis AUMC 1120, B. spicifera AUMC 459,
Botrytis cinerea AUMC 6095, Cochliobolus cynodontis AUMC
2393, Fusarium oxysporum AUMC 9704, F. sambucinum
AUMC 1266, and Penicillium digitatum AUMC 2206 were
obtained from Assiut University Mycological Centre (AUMC),
Egypt. The bacterial strains of Escherichia coli MSD102,
Enterococcus faecalis MSD23, Lactobacillus delbrueckii subsp.
bulgaricus MSD231, and Streptococcus thermophilus AAE84
were obtained from the Laboratory of Dairy Microbiology
at Dairy Department, Faculty of Agriculture, Mansoura
University, Egypt.
2.2. Extraction of Crude Polysaccharides. The crude polysac-
charide of L. leucocephala wasextractedaccordingtothe
protocol mentioned by Dawood et al. [16]. In detail, twenty
grams of dry seeds powder were extracted with petroleum ether
(40–60
°
C) for 6 h for removal of lipid materials. The pretreated
dry powder was extracted three times each for 120 minutes
with deionized water (water-solid material; 10 : 1 mL g
-1
)at
70
°
C. The insoluble materials were detached by filtration, and
the water-soluble solution was concentrated under a vacuum
at 50
°
C. The precipitation process was then originated by the
anhydrous ethanol addition to a final concentration of 75%
(v/v) to the concentrated extract; the mixture was then kept
for 24 h at 4
°
C, followed by centrifugation at 12000 × gfor
15 min, and then the resulting precipitate was dried by air-
dried to afford crude polysaccharides named LLPs.
2.3. Quantitative Analysis of Reducing Sugar and Total
Carbohydrates Concentration. The concentration of total
carbohydrates was estimated by the phenol-sulfuric acid
method previously written [17]. D-Glucose was used as a
standard for calculating the total carbohydrate concentra-
tion. The determination of reducing sugar was carried out
according to the Miller method [18]. The reducing sugar
was subtracted from the total carbohydrate to calculate the
total polysaccharide of LLPs.
2.4. Determination of Protein Content and Total Polyphenol
Content. The protein content was determined by the Kjel-
dahl nitrogen determination method [19]. The content of
total polyphenols content of the LLPs was determined
according to Taher et al. [20]. It was expressed as a milli-
gram of gallic acid equivalent per gram of LLPs (mg GAE/g).
2.5. Identification of LLP Components by Thin Layer
Chromatography (TLC). The final residue of LLPs was
dissolved in an appropriate volume of 40% methanol, and
TLC was done using precoated silica gel GF 254 plates accord-
ing to the details written by Mittal et al. [21]. Galactose (G)
and mannose (M) were used as standards.
2.6. Nuclear Magnetic Resonance Spectroscopy. Nuclear mag-
netic resonance (NMR) spectra were obtained using a
400 MHz Bruker Avance III spectrometer with a 5 mm inverse
2 Journal of Nanomaterials

probe. The spectra were noted using a 1% (w/v) solution of
LLPs in deuterium oxide (D
2
O).
2.7. Synthesis of AgNPs. To synthesize AgNPs, 50 mg of LLPs
was dissolved in 100 mL of distilled water and mixed with
200 mL of 0.1 mM AgNO
3
, and the solution was stirred with
a magnetic stirrer for 30 min at 60
°
C under constant stirring.
Bioreduction occurs quickly as shown by a reddish-brown
color after 36 h representing the AgNPs formation (AgNPs
were regularly checked by visual assessment of the solution
as well as by ultraviolet-visible spectroscopy), followed by
repeated centrifugation at 12000 × gfor 20 min to collect
AgNPs. The pellets were collected and dried [22].
2.8. Characterization of Biosynthesized AgNPs. The character-
ization of biosynthesized silver nanoparticles was determined
by an ultraviolet-visible spectroscopy spectrophotometer
(UV-2550, Shimadzu, Japan) with a spectral resolution of
1 nm over a wavelength ranging from 300 to 600nm. For iden-
tification of the functional groups and chemical bonding in the
silver nanoparticles, 300 mg of fine potassium bromide (KBr)
was mixed with one milligram of each sample. The prepara-
tion of thin pellets was carried out using hydraulic pellet press,
followed by subjecting to the Fourier-transform infrared
spectrophotometer (FTIR) ranging between 500 and
4000cm
-1
at a resolution of 4 cm
−1
(Bruker Vector 22).
The crystalline structure of the biosynthesized AgNPs
was analyzed by the X-ray diffractometer (D8 Advance, Bru-
ker, Germany) operating at 40 kV using Cu-Kαradiation
with λof 1.54 Å and scanning rate 0.1
°
in the 2θrange from
5
°
to 80
°
. The elemental composition of the AgNPs was iden-
tified by energy-dispersive spectroscopy (EDS) attached with
scanning electron microscopy (JEOL JSM-6510LV) between
0 and 20 kV.
The morphology of the synthesized AgNPs was carried
out with a JEOL JSM-6510LV scanning electron microscopy
(SEM) operating at an electron accelerating voltage of 30kV.
A very small sample size was put on a carbon-coated copper
grid. The film on the grid was then dried at room temperature
before SEM analysis. The transmission electron microscopy
(TEM, Model JEM 2100LV, JOEL, Japan) was used to analyze
the size and shape of the AgNPs. A drop of biosynthesized
AgNPs was put on the carbon-coated copper grid and then
loaded onto a specimen holder before the sample observation.
The stability of Ag nanoparticles was evaluated using the Zeta-
Sizer Nano-ZS90 (Malvern Instruments Ltd., UK).
2.9. Antitumor Activity. The antitumor activity was conducted
in vitro on liver carcinoma (HEPG-2), colon carcinoma
(HCT-116), and breast cancer (MCF-7) cell lines (ATCC,
Minnesota, USA) using the sulforhodamine B stain (SRB)
assay [23].
In detail, the cells were cultured in RPMI-1640 medium
enriched with 10% fetal bovine serum and 1% streptomycin/
penicillin. Different concentrations of AgNPs and 5-
fluorouracil (5-FU), as a reference drug (6.25, 12.5, 25, 50,
and 100μgmL
-1
) were prepared in the culture medium. The
cells were added to 96-well microtiter plates at the concentra-
tion of 3×10
3cells per well in a 150 μL fresh medium and left
for 24 h to attach to the plates. Then, 100 μL of 5-FU (positive
control) and each AgNP concentration was added to every
well except the control wells. After 48h, the cells were fixed
with 50μL of ice-cold trichloroacetic acid (10% w/v), followed
by incubation at 4
°
C for 1 h. The addition of 50 μLofSRB
(0.4% w/vin 1% aqueous acetic acid) stained the TCA-fixed
cells. The plates were incubated for 30min at room tempera-
ture. The acetic acid (1% v/v) was used for washing the plates
four times to remove the unbound, followed by drying. The
bound SRB was solubilised by adding 200μLof10mMTris
Base (pH 10.5) to each well and shaking for 5-10 min. The
absorbance of microplates at 570 nm was determined by a
96-well plate reader. The percentage of cell viability inhibition
was estimated in comparison to the control.
2.10. Antifungal Activity. The silver nanoparticles were indi-
vidually tested against the previously mentioned ten fungal
strains. A mycelial disk of each of the fungi (5 mm diameter)
was placed in the center of each potato dextrose agar (PDA)
plate (90 mm diameter) containing the final concentrations
of AgNPs at 0 (control), 300, 500, 700, and 900μgmL
-1
.All
plates were incubated at 25 ± 2°Cuntil the growth in the
control reached the edge of the plates. Each treatment was per-
formed in triplicate, and the experiment was repeated twice.
The inhibition of mycelial growth was calculated as follows:
Mycelial growth Inhibition ð%Þ=½ðC−TÞ/C×100,whereC
(mm) and T(mm) are the mean growth diameter in the con-
trol and treatment, respectively [24].
2.11. Correlation between Log of Cell Numbers (CFU/mL)
and Milk Acidification (pH). The spread plate method was
carried out to determine the numbers of bacterial cells using
MRS and M17 agar for Lactobacillus delbrueckii subsp. bul-
garicus MSD231and Streptococcus thermophilus AAE84,
respectively, at 42
°
C for 24 h, while using TSA for Escheri-
chia coli MSD102 and Enterococcus faecalis MSD23 at 37
°
C
for 24 h. Acidification of milk using the growth of the previ-
ously mentioned four bacterial strains was determined by a
pH meter (Hanna Hi2210). A correlation between log values
of cell numbers (CFU) and pH was performed by the Table-
Curve 2D software [5].
2.12. The Practical Parameters for the Assessment of AgNP
Preserved Composite Milk Samples
2.12.1. Milk Samples and Preservation. Reconstituted milk was
made from powder full cream milk obtained from a local mar-
ket in Mansoura city, Egypt. The process for producing the
reconstituted milk was carried out according to Braun, Ilberg,
Blum, and Langowski [5]. The reconstituted milk was divided
into 4 equal parts. The first, second, third, and fourth parts
were individually inoculated with Escherichia coli MSD102,
Enterococcus faecalis MSD23, Lactobacillus delbrueckii subsp.
bulgaricus MSD231, and Streptococcus thermophilus AAE84,
respectively. Every part was redivided into 6 groups, where
the first and second groups were set as a negative control
(without AgNPs) and positive control (formalin 0.4%), respec-
tively. The 25, 50, 100, and 200μgmL
-1
of AgNPs were indi-
vidually added to the 3rd, 4th, 5th, and 6th groups. Every
group was redivided into two subgroups, where the first and
3Journal of Nanomaterials

second subgroups were incubated at 37
°
C and 43
°
C, respec-
tively. The determination of milk acidification (pH, alcohol
precipitation, and clot on boiling), proteolytic, and lipolytic
activity was used as an evaluation tool of preservative efficacy
of AgNPs in the composite milk samples.
2.12.2. Alcohol Precipitation Test. This test is carried out by
mixing equal volumes of 68% of ethanol solution and milk
in test tubes. If the acidity of the tested composite milk is
normal (0.14 to 0.18%), there will be no clotting, coagula-
tion, or precipitation. The presence of clots points out the
acidity of the tested composite milk sample ranged from
0.21 to 0.23%.
2.12.3. Clot on Boiling (COB) Test. The COB is a very easy
test used to evaluate the quality of milk. A defined volume
of composite milk sample in the test tube is boiled. The sam-
ple cannot withstand boiling, and coagulation was created
on the wall of the test tube due to high acidity of the sample
(0.23 to 0.28%).
2.12.4. Proteolytic Activity. The proteolytic activity of milk
samples was determined according to El Dessouky Abdel-
Aziz et al. [25] with slight modification. Briefly, approxi-
mately 0.5 mL buffer (4.5 mM KH
2
PO
4
; pH 7.5) was trans-
ferred into a screw-capped test tube and was mixed with
24 mg of Azocoll, followed by incubation for 5 min at 36
°
C.
Then, 0.5 mL of milk sample was added into the tube and
mixed with a vortex mixer for 45 s followed by incubation
for 30 min at 36
°
C. The screw-capped tubes were transferred
to the icebox, for stopping the reaction. The filtration of the
mixture was carried out by using Whatman No.4 filter
papers. The absorption of released azo dye was determined
by using a UV spectrophotometer at 520 nm. One unit of
activity was defined as the amount of enzyme that will cata-
lyze the release of sufficient azo dye to produce an absor-
bance change of 0.001 in 30 min at 520 nm. Results are
expressed as the time required to increase one unit of activity
compared with the initial point.
2.12.5. Lipolytic Activity of Composite Milk Sample. The lipo-
lytic activity of the tested milk samples was determined by
estimating the time required to liberate estimable free fatty
acids (FFA) according to El Dessouky Abdel-Aziz et al.
[25], with slight modification. Briefly, a volume of composite
milk sample (5 mL) mixed with 25 mL ethanol was mixed
with 10 mL, and then 3 drops of 1% phenolphthalein indica-
tor solution were added. The titration of the mixture was
performed by using potassium hydroxide (0.05 N). The titra-
tion of the mixture was at the endpoint when the pinkish
color of the mixture appeared and persisted for 30 seconds.
The calculation of % FFA depended on the following equa-
tion using the acid values and the value of the molar mass
of oleic acid for the studied samples. Results are expressed
as the time required to increase 1% of FFA compared with
the initial point.
Free fatty acid %as oleic acid
ðÞ½
=T×N× 282 × 100
W× 1000 ,ð1Þ
where Tis the volume of titrant, Nis the normality of KOH,
the molecular weight of oleic acid is 282, and Wis the
weight of the sample.
2.13. Infrared Analyzer for Composite Milk Analysis. The
chemical composition analysis of the composite milk sample
was carried out by MilkoScan (FOSS MilkoScan FT120)
according to ISO [26].
2.14. Statistical Analysis. The data were analyzed with one-
way ANOVA (analysis of variance) to determine the signif-
icant differences between the means by Tukey’s HSD test at
P<0:05 using SAS (version 9.1, SAS Institute, NC, USA).
The probit analysis was used to calculate the values of IC50
(concentrations of the silver nanoparticles that produce
50% inhibition on the cell lines viability) by SAS (version
9.1, SAS Institute, Cary, NC, USA).
3. Results and Discussion
3.1. Chemical Analysis of Crude Polysaccharides. The poly-
saccharide yield was 10.4% on the dry matter basis of the
seeds used to prepare the extract (Table 1). However, the
polysaccharide yield from L. leucocephala polysaccharide is
20% (w/w) [21]. The chemical composition of LLPs revealed
the presence of total carbohydrate content (85.9%), total
reducing sugars (10.4%), total polysaccharide (75.5%), crude
protein (4.88%), and total ash (2.05%) (Table 1). Remark-
ably, LLPs contain a low level of total polyphenols as
35.3 mg GAE/g LLPs. The chemical analysis of LLPs in this
study agreed to some extent with that obtained by the previ-
ous report [27]. In this work, L. leucocephala inactively
extracted polysaccharide that contains total carbohydrates
and total conjugated protein by 76.2 and 4.2%, respectively,
with a trace content of total phenols by 38.32 mg GAE/g
[27]. In another example, the total polyphenols in the crude
polysaccharides were 21.6 and 16 mg GAE/g when the solid-
liquid extraction method is used by methanol and ethanol,
respectively [28, 29]. Additionally, the presence of low levels
of proteins and ash in LLPs has been reported [21].
The TLC profile of LLPs presented two monosaccha-
rides. These monosaccharides were assessed as mannose
and galactose in comparison to Rf values of standard mono-
saccharides. Rf value was noted to be related to that detected
with galactose and mannose, so it can be termed as galacto-
mannan gum. Additionally, the NMR analysis was done to
confirm the galactomannan structure of LLPs (Figure 1).
1
H NMR spectrum exposed two anomeric protons at δ=
4:70 ppm and δ=4:89 ppm, assignable to H-1 of β-d-man-
nopyranosyl and α-d-galactopyranosyl residues, respectively
(Figure 1). Moreover, the peaks between δ=4:01 and 3:69
ppm corresponded to H2to H6. These
1
H NMR chemical
shifts were in line with the values obtained in the previous
studies [21, 30, 31]. These findings have confirmed the galac-
tomannan structure of the crude polysaccharide isolated
from seeds of L.leucocephala with a linear chain of D-
mannopyranosyl residue connected through β(1→4) link-
age and α-d-galactopyranosyl residue at O-6 of mannose
unit. Overall, extraction of water-soluble galactomannans
4 Journal of Nanomaterials

from the seeds of L. leucocephala with different M/G molar
ratios has been reported [30, 32]. In this context, the molec-
ular weight of LLPs was evaluated by high-performance gel
permeation chromatography. The peak of elution was nearly
single, equivalent to the feature of homogeneous distribu-
tion. The molecular weight of LLPs was calculated to be
3:008 × 104Da (Figure 2(a)). The molecular weight of LLPs
in this study was lesser than that of galactomannan isolated
from the same part of the plant as 6:4×10
4Da [30] as well
as galactomannans isolated from other plants [33, 34].
The Fourier-transform infrared spectroscopy (FTIR) is one
of the most useful and commonly used analytical means for
identifying the functional groups of different macromolecules.
The FTIR spectra of LLPs displayed different absorption peaks
at 3449, 2922, 1642, 1406, 1255,1226, 1150, 1074, 1024, 870, and
816 cm
-1
, respectively (Figure 2(b)). The values listed below
confirmed the galactomannan structure of LLPs, where the
broad band at 3449 cm
-1
,arequalified to hydroxyl groups (O-
H) present in these materials. Peak stretching at 2922 cm
−1
was associated with the stretching vibration of C-H. The peaks
at 1255 and 1226 cm
-1
are most possibly from the C-O group of
polyols of glucans and flavones [35] The absorption peaks at
1150, 1074, and 1024 cm
−1
presented the stretching vibration
of C-OH from the mannose structures [36]. The bands at 870
and 816 cm
-1
were allocated to the anomeric configuration of
β-D-mannopyranosyl and α-D-galactopyranosyl units, respec-
tively; similar observations have been reported [34, 37]. On
the other hand, the sharp band at 1642 cm
-1
represents the pres-
ence of the carbonyl group C=O of the –NH-C=O amide bond
stretching. The relative absorption peak is at 1406 cm
-1
for N-H
and methylene bending vibration. Therefore, the peaks at 1642
and 1406 cm
-1
might be related to the low content of protein in
crude LLPs.
3.2. Characterization of the Silver Nanoparticles. Numerous
methods have been adapted for the biosynthesis of AgNPs
using plant extracts [38]. Biosynthesis of AgNPs using
macroalgae extracted polysaccharides, as reductant for silver
ions as well as stabilizing agents for the synthesized AgNPs,
has been stated [39]. Likewise, in this work, a well-stabilized
AgNP solution was prepared using the extracted polysaccha-
rides from L. leucocephala seed wastes as reducing agents for
silver ions to produce AgNPs. Practically, AgNPs show
brownish color in an aqueous solution due to excitation of
surface plasmon vibration of AgNPs [40]. Consequently,
the reduction of Ag
+
to AgNPs through trials could be con-
firmed by a color change from pale yellow to dark brown
which was noted systematically by the UV-Vis spectroscopy
(Figure 3(a)).
3.2.1. UV-Vis Spectroscopy. The ultraviolet-visible spectros-
copy is an initial step for analyzing the formation of silver
nanoparticles in an aqueous solution. Therefore, the poly-
saccharides along with AgNO
3
were subjected to bioreduc-
tion reaction, and the change in color to yellowish brown,
indicating the formation of AgNPs. The silver surface plas-
mon resonance was detected at 448 nm (Figure 3(b)). In a
previous report, Gengan et al. [41] itemized that the maxi-
mum absorbance occurred at 448 nm due to the existence
of AgNPs. The characteristic silver surface plasmon reso-
nance bands were found around 400-450 nm [42]. The
mechanism of silver ions reduction to nanoparticles using
polysaccharides extracted from L. leucocephala seed wastes
might be due to the existence of functional hydroxyl groups
in both monosaccharide units and related phenolics respon-
sible for the development of AgNPs. These results are in har-
mony with previous researchers [43, 44].
3.2.2. FTIR Analysis. FTIR spectroscopy could be efficiently
exploited for understanding the degree and nature of the
interaction between two or more chemical species during
the formation of nanoparticles. The results showed that all
identified peaks in LLPs were shifted to some extent in
AgNP-LLPs affirming the production of polysaccharide-
capped AgNPs (Figure 2(b)). The presence of a peak at
3422 cm
-1
in AgNP-LLPs is assigned for hydroxyl stretching,
offering the capping efficacy of LLPs. The shifted peak from
1406 to 1386 cm
-1
in AgNP-LLPs indicated the probable role
of the trace proteins in the formation of AgNPs. Remark-
ably, it is well known that proteins can bind to the AgNPs
Table 1: Chemical composition of LLPs and monosaccharide profile.
Chemical composition of LLPs
Yield (%) Total sugars
(%)
Reducing sugars
(%)
Total polysaccharides
(%)
Crude protein
(%)
Moisture
(%)
Ash
(%)
Total polyphenols
(mg GAE/g)
10.4% 85.9 10.4 75.5 4.88 3.95 2.05 35.3
4.9758
6.5427
7.6223
Dawood Hosni DHL-proton-WH.10.c
Dawood Hosni DHL-proton-WH
0.44
0.28
1.00
0.79
0.34
0.32
1.64
2.43
2.49
2.99
0.94
0.32
0.26
0.41
0.22
0.30
4.7298
4.6956
4.4534
4.4389
4.4237
4.3844
4.3619
4.0982
3.9459
3.9098
3.8596
3.7893
3.7585
3.7152
3.5440
3.1503
2.1784
1.8709
1.2868
1.2697
1.0974
1.0827
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5
F1 (ppm)
4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
–50
0
4.97
58
6.542
7.622
Dawood Hosni DHL-proton-WH.10.c
27
7
23
3
Dawood Hosni DHL-proton-WH
22
22
4.72
98
4.69
56
4.45
34
4.4389
4.42
37
4.3844
6
4.36
6
436
6
36
43
43
19
19
9
9
9
9
9
9
9
19
19
1
1
4.09
82
3.9459
3.9098
3.8596
3.78
93
3.75
85
3.7152
3.54
40
3.
1
503
2.1784
1.87
09
1.28
68
1.2697
1.0974
1.0827
Figure 1:
1
H-NMR of the polysaccharides extracted from Leucaena
leucocephala seeds.
5Journal of Nanomaterials

via the free amine groups in their backbone structure [45].
The changes in FTIR spectrum in this study between 1255
and 1226 cm
-1
might reflect the probable role of the C-O
group of polyols such as hydroxyl flavones in the formation
of AgNP-LLPs [35]. Overall, the results confirm the theory
that the phenolic constituents and polypeptide structure
are critical in the development and stabilization of different
metal ion nanoparticles. These functional groups may shield
the surface of silver ions as capping mediators that stabilize
the nanoparticles, but the full mechanism assumed in the
synthesis of AgNPs is still debated. Moreover, polyols like
different polyphenols and polysaccharides that reduce silver
ions may perform an identical role in the construction of
AgNPs from L. leucocephala [46, 47].
3.2.3. X-Ray Diffraction (XRD). The crystallinity of formu-
lated AgNP-LLPs was tested by XRD. Its pattern presented
diffraction peaks at 38.27
°
, 46.27
°
, 64.63
°
, and 77.66
°
which
can be indexed to the planes (111), (200), (220), and (311),
respectively (Figure 4(a)). The identified crystalline peaks
in the present work represented planes of a face-centered
cubic lattice of silver; parallel observations have been docu-
mented in different studies [48–50]. Unknown crystalline
peaks (27.89
°
, 32.30
°
, and 54.79
°
) are also evident in many
reports in which the XRD pattern contains the relevant 2θ
range [51]. These peaks are due to the organic molecules
which occur in the extract.
3.2.4. Energy-Dispersive Spectroscopy (EDS). The EDS spec-
trum revealed a strong signal peak at ~3 keV, which con-
firms the occurrence of elemental silver in the produced
nanosilver synthesized by polysaccharides (Figure 4(b)). In
a similar study, Jagtap and Bapat [52] described the forma-
tion of irregular-shaped AgNPs at 2.98 keV using the seed
extract of Artocarpus heterophyllus. The presence of a signal
peak for carbon (C) in the EDS spectrum is possibly due to
the structure of the polysaccharides present in the seeds of
L. leucocephala, which is an added benefit in the green
chemical synthesis. Our results strongly confirmed that the
polysaccharides extracted from L. leucocephala seeds might
act both as reducing and stabilizing agents in the production
of AgNPs.
3.2.5. SEM and TEM Observations. Scanning electron
microscopy (SEM) is a qualitative tool to examine the
surface morphology of polysaccharides [53]. The surface of
polysaccharides (LLPs) is relatively homogeneously smooth
and consists mainly of randomly distributed particles
under observations by SEM (Figure 5(a)). Meanwhile, the
1e3
0.6
0.5
0.4
0.3
0.2
0.1
1e4
Molar mass (D)
W (log M)
1e5
(a)
525526
816
870
815
1024
1074
1150 1255
1350
1406
1642
2155
2922
3449
4000
120
100
80
60
40
20
Transmittance (%)
0
3500 3000 2500
Wavenumber (cm–1)
2000 1500 1000 500
870
1025
1071
1149
1308
1386
1638
2925
3446
1257
LLPs
LLPs/AgNPs
(b)
Figure 2: Molecular weight of polysaccharides extracted from the seeds of Leucaena leucocephala (a). FTIR pattern of polysaccharides
extracted from the seeds of L. leucocephala and their AgNPs (b).
(A) (B)
(a)
3
2
Absorbance
0
300 400 500 600
(nm)
Control
AgNPs
1
(b)
Figure 3: (a) The reaction mixture color was changed from yellow (a) to reddish brown (b) after the addition of AgNO
3
solution (0.1 mM)
to aqueous polysaccharides of Leucaena leucocephala seeds. (b) UV-Vis spectrum of AgNPs synthesized using polysaccharides extracted
from L. leucocephala seeds.
6 Journal of Nanomaterials

morphological change after AgNP formation is not fully clear
(Figure 5(b)). The SEM images of AgNP-impregnated LLPs
were roughly spherical in shape and confirm the existence of
silver in the polysaccharides. In other words, the silver nano-
particles from LLPs are monodisperse with a spherical shape
and tribute among the polysaccharides. Similarly, the spherical
shape of silver nanoparticles in polysaccharide-rich extracts
has been reported [54, 55].It has been exposed thatthe smaller
size of AgNPs at pH7 is more effective concerning its prospec-
tive applications [50]. So, the size, shape, and surface morphol-
ogy of the produced AgNPs were characterized using
transmission electron microscopy (TEM). The TEM images
of AgNPs made by polysaccharides extracted from L. leucoce-
phala seeds show that the particles are almost spherical with a
size ranging from 8 to 20 nm (Figures 5(c) and 5(d)). These
findings also confirm the monodisperse nature of AgNPs.
Interestingly, the size range of AgNPs in this study was lower
than those obtained using other polysaccharide-rich extracts
[56, 57]. The size dissimilarity shown in Figure 5 may be due
to the attendance of other organic ingredients in LLPs which
are complicated in reducing and stabilizing AgNPs during
their producing stage [58]. Overall, submicroparticles with a
size below 1000nm are adequate nanometric carriers used to
deliver drugs or different types of biomolecules [59].
0
3000
27.89°
38.27°
32.30°
46.27°
54.79°
64.64°77.66°
311
220
200
111
2500
2000
1500
1000
500
Intensity (a.u.)
10 20 30 40
2 theta degree
50 60 70 80 90
(a)
C
Cl
Cl
(keV)
0246810121416
O
Fe
Ag
Fe Fe
(b)
Figure 4: XRD spectrum of AgNPs synthesized by reducing 0.1 mM AgNO
3
using polysaccharides extracted from Leucaena leucocephala
seeds (a). EDS pattern of the synthesized AgNPs from polysaccharides of L. leucocephala seeds (b).
(a) (b)
100 nm
(c)
20 nm
(d)
Figure 5: SEM micrograph of polysaccharides extracted from Leucaena leucocephala seeds (a). SEM micrograph of AgNPs synthesized by
adding 0.1 mM AgNO
3
to polysaccharides of Leucaena leucocephala seeds (b). TEM images of biologically synthesized Ag nanoparticles
using polysaccharides of L. leucocephala seeds (C&D).
7Journal of Nanomaterials

3.2.6. Zeta Potential Measurement. Aggregation of particles is
a key aspect affecting the suspension stability of the debris
particles in aqueous solutions. The aggregation or dispersion
of colloidal particles is assessed by the balance between the
Van der Waals force and electrical double-layer repulsive
forces when particles closely approach one another [60]. The
zeta potential is used to describe the surface charge and stabil-
ity of nanoparticles in aqueous media. It is recorded at or near
the border of the diffuse film on the surface of the particle;
therefore, the stability of any suspension is closely related to
the zeta potential of suspended particles [61]. Additionally, it
is well known that a suspension with a zeta potential value
closer to zero has no force to stop the aggregation of the
particles. Contrarily, a suspension with a higher negative or
positive zeta potential value has a higher tendency of its parti-
cles to repel with each other, and therefore, no accumulation
of particles is expected. In neutral pH, the value of zeta poten-
tial for AgNPs in this study was found to be -14.2mV with a
single peak revealing the stability of the biosynthesized silver
nanoparticles (Figure 6), and the related values have been
described by Wang et al. [60] and Elamawi et al. [62]. In other
words, the value of the zeta potential of AgNPs synthesized by
polysaccharides extracted from L. leucocephala seeds reflects
the presence of a notable electrostatic repulsion between parti-
cles which can block the aggregation and precipitation of
debris particles of AgNPs. This was confirmed by our experi-
mental test displaying the debris could not be aggregated by
high-speed centrifugation at 8000 × gfor 45min. Overall,
the stability of nanoparticles in an aqueous solution is very
essential regarding antimicrobial applications as unstable
AgNPs will not be able to disperse homogeneously, hence
decreasing the efficiency.
3.3. Antitumor Activity. In the recent decade, polysaccharide-
based metal nanoparticles are delivering remarkable applica-
tions in nanomedicine, including avoidance, diagnosis, and
Zeta potential (mV): –14.2
Zeta deviation (mV): 4.14
Conductivity (mS/cm): 0.342
Result quality: Good
Result
Mean (mV)
Zeta potential distribution
Apparent zeta potential (mV)
70000
60000
50000
40000
30000
Total counts
20000
–100 100 2000
10000
0
Peak 1: –14.2 100.0
Peak 2: 0.00
Peak 3: 0.00
0.0
0.0
4.14
0.00
0.00
Area (%) St dev (mV)
Figure 6: Zeta potential measurement of AgNPs synthesized from polysaccharides of the seeds of Leucaena leucocephala.
Table 2: Antitumor activity of silver nanoparticles synthesized by polysaccharides extracted from Leucaena leucocephala seeds against
breast cancer (MCF-7), liver carcinoma (HEPG-2), and colon carcinoma (HCT-116) cell lines at different concentrations.
Concentration (μgmL
-1
)
Cell inhibition (%)
MCF-7 HEPG-2 HCT-116
AgNPs 5-FU LLPs AgNPs 5-FU LLPs AgNPs 5-FU LLPs
6.25 29:6±0:4a27:4±0:5b0±0:0c34:1±0:9a30:6±0:7b3:3±0:2c32:4±0:8a26:2±0:8b0±0:0c
12.5 42:9±0:9b51:9±0:3a0:3±0:1c55:5±0:9b60:5±0:6a3:6±0:2c57:1±0:9b59:4±0:6a1:2±0:2c
25 58:8±1:4b70:6±0:7a3:6±0:7c71:8±0:8b78:6±0:6a15:2±0:6c65:6±0:3b73:9±1:0a3:0±0:4c
50 77:3±0:8b87:3±0:8a7:6±1:0c83:2±0:7b90:7±1:0a29:9±1:2c78:7±0:9b83:6±1:2a6:5±0:6c
100 88:9±0:5b96:6±0:7a4:3±0:5c90:1±1:0a98:4±0:6a53:6±0:7c87:3±1:1b91:9±0:9a15:7±0:7c
IC50 (μgmL
-1
) 22.5 11.9 >100 12.3 10.8 90.7 8.9 7.3 >100
Silver nanoparticles (AgNPs), 5-fluorouracil (5-FU), and polysaccharides extracted from Leucaena leucocephala seeds (LLPs). Results represent means ±
standard error (SE) for two experiments. Different letters in the same row are significantly different according to Tukey’s HSD test at P<0:05 for each cell line.
8 Journal of Nanomaterials

treatment of different tumors. Its sophisticated targeting
scheme and multifunctional characters have notable advantages
in the enhancement of pharmacokinetics and pharmacody-
namics profiles compared to traditional therapeutics [56].
Therefore, the application of different polysaccharide-based
metal nanoparticles in cancer treatment has been developed as
a promising area in nanotechnology [60].
In the current study, the antitumor property of the
different AgNPs concentrations ranging between 6.25 and
100 μgmL
-1
was examined in vitro against liver carcinoma
(HEPG-2), colon carcinoma (HCT-116), and breast cancer
(MCF-7) cell lines and compared with 5-fluorouracil (5-
FU). The silver nanoparticles obtained from L. leucocephala
polysaccharides exhibited cytotoxic activity mostly equiva-
lent to the positive control (5-FU) against all cancer cell lines
in a concentration-related manner (Table 2). Our results
displayed that HCT-116 cell proliferation was significantly
inhibited by AgNPs with an IC50 value of 8.9 μgmL
-1
.
Besides, AgNPs exhibited considerable anticancer activity
against HEPG-2 and MCF-7 cell lines with IC50 of 12.3
and 22.5 μgmL
-1
, respectively. In this context, Tran et al.
[57] recorded the slightly higher antitumor activity of chito-
san with AgNPs against HEPG-2 and MCF-7 cell lines with
IC50 values of 6.09 and 5.71 μgmL
-1
, respectively. Likewise,
chitosan and silver nanocomposite showed a great inhibition
on lung cancer cells (A549) with a considerably low value of
IC50 by 29.35 μgmL
-1
[63].
In general, AgNPs with a smaller size range showed higher
biological activities [64]. Consequently, the discrepancy
regarding cytotoxicity IC50 values of polysaccharide-based
AgNPs is highly associated with their type, the size of exam-
ined cells, and the bioactive molecules tied to them since sec-
ondary metabolites may have antitumor efficacy. The mode
of action of AgNPs against tumor cell lines is not entirely
understood, but cell oxidative stress initiated by the propaga-
tion of ROS can be caused by the cellular uptake of AgNPs
[64]. Apoptosis is reflected as one of the biotic actions that dis-
rupt the abnormal cell and is a suitable sign for the investiga-
tion of cytotoxicity [65, 66].
The results of the current study presented that the
galactomannan-rich polysaccharides recorded a relatively
low value of IC50 against HEPG-2 cells compared with other
cell lines, indicating its higher selectivity to liver cancer. This
might be due to the presence of numerous side-chain galac-
tose units that might easily bind with galactose-specific
receptors of the cancer cell surface, modify the cancer sur-
face physiology, and probably influence the distribution of
bioactive ingredients and drugs to the cancer cells [67].
Moreover, galactomannan-rich polymers have different bio-
logical activities such as immunostimulation, anticancer,
and cancer chemopreventive [9, 68, 69].
According to the results obtained, galactomannan-rich
polysaccharides recorded the highest IC50 values, which
reflected their weak antitumor activity against all cell lines.
Therefore, the transformation of L. leucocephala
galactomannan-rich polysaccharides into silver nanoparti-
cles significantly increased their antitumor potential against
all tested cell lines without any selectivity. In the same way,
L. leucocephala galactomannan-sulfated derivatives recorded
high cytotoxicity against HEPG-2, MCF-7, and 1301 cell
lines [27].
3.4. Antifungal Activity. Metal nanoparticles display a great
ratio of surface area-to-volume and show antimicrobial
activity because of their ease of interaction with cellular
membranes [70]. Many silver-containing compounds, par-
ticularly AgNPs, have been used as antimicrobial agents to
inhibit or to kill the growth of the plant and human patho-
gens [71]. There is an information frame on the antimicro-
bial potential of AgNPs biosynthesized by different species
of plants, but insufficient attention has been focused on their
potential antifungal properties [71]. Therefore, this study
Table 3: Effect of silver nanoparticles synthesized by polysaccharides extracted from Leucaena leucocephala seeds on mycelial growth of
fungal pathogens at different concentrations.
Fungi Mycelial growth inhibition (%)
300 μgmL
-1
500 μgmL
-1
700 μgmL
-1
900 μgmL
-1
Alternaria alternata 0:0±0:0d0:0±0:0d15:6±1:2gh 25:6±8:0g
Aspergillus flavus 0:0±0:0d2:9±6:0d17:0±1:2fgh 42:2±1:2f
Aspergillus parasiticus 0:0±0:0d0:0±0:0d21:9±0:8ef 37:8±0:5f
Bipolaris hawaiiensis 28:5±0:9a40:7±1:2a52:6±0:8b85:6±0:6a
Bipolaris spicifera 0:0±0:0d0:0±0:0d11:5±0:9h28:5±0:6g
Botrytis cinerea 5:9±0:9c19:3±1:1b32:2±1:2c68:1±0:8cd
Cochliobolus cynodontis 4:4±0:6c16:3±0:9b25:9±1:2de 71:5±1:2c
Fusarium oxysporum 0:0±0:0d16:7±1:2b28:5±0:9cd 52:9±0:9e
Fusarium sambucinum 0:0±0:0d11:5±0:9c20:4±1:1efg 63:7±1:4d
Penicillium digitatum 12:2±1:1b40:7±0:8a65:9±1:4a78:5±0:9b
Results represent means ± standard error (SE) for two experiments. Different letters in the same column are significantly different according to Tukey’s HSD
test at P<0:05.
9Journal of Nanomaterials

focused on evaluating the antifungal activity of AgNPs
against different fungal strains.
The results of the current showed that the inhibitory
effect of AgNPs on the mycelial growth of all tested fungi
was positively correlated to the concentration of AgNPs used
(Table 3). At the concentration of 900 μgmL
-1
, in particular,
AgNPs caused a significant reduction in the growth of B.
hawaiiensis,P. digitatum,C. cynodontis, and F. sambucinum
by 85.6, 78.5, 71.5, and 63.7%, respectively. Furthermore, the
results pointed to the fungus A. alternata being the most tol-
erant fungi, while the fungus B. hawaiiensis being more sen-
sitive to the concentrations of AgNPs.
As documented by Bahrami-Teimoori et al. [72], the
maximum of the antifungal activity of the biosynthesized
AgNPs using the plant extract of A. retroflexus against differ-
ent plant pathogenic fungi was reached at lower concentra-
tion (400 μgmL
-1
). This could be explained by the
presence of bioactive metabolites owning to the antifungal
activity in the plant extracts.
AgNPs synthesized and stabilized by polysaccharides
have effective antimicrobial properties on both Gram-
positive and Gram-negative bacteria as well as a considerable
antifungal activity [73]. Gram-negative bacteria were more
probably to be affected than other microbes due to their
membrane structure characterized by the negatively charged
cell wall, which made it easier to attach the released Ag
+
,
which resulted in cell death [74]. The silver ions rapidly bind
with the thiol (-SH) group of the lipid bilayer or membrane-
bound proteins causing ion leakage and cell rupture as a
result of membrane destabilization [75].
In fungi, the changes related to silver nanoparticles
intensely suppress the regulatory functions of cell membrane
ergosterol, predominantly during the normal binding pro-
cess, as well as disrupt the electron transport chain (ETC,
respiratory chain) by forming insoluble constituents in the
cell wall due to the inactivation of the sulfhydryl group
[76]. However, the full mechanism complicated in the anti-
fungal property of AgNPs is still not entirely understood.
The propagation of ROS and other free radicals and modu-
lation of microbial signal transduction pathways have been
identified as the most well-known modes of antimicrobial
property of AgNPs penetrated inside the microbial cell [77].
In this context, a few studies have been conducted to
estimate the antifungal activities of green biosynthesized
AgNPs. Silver nanoparticles from microbial filtrates or plant
extracts showed strong antifungal activity against several
phytopathogenic fungi, including F. oxysporum [78], Macro-
phomina phaseolina,A. alternata,Rhizoctonia solani,Sclero-
tinia sclerotiorum,B. cinerea,Curvularia lunata [79], A.
flavus, and A. fumigatus [80]. Interestingly, the antifungal
potency of transparent nanocomposites composed of the
polysaccharide of pollutant and silver against A. niger has
been also evaluated [81].
The strains of the genus Cochliobolus and its asexual
state Bipolaris largely spread and are both saprophytic as
well as pathogenic to more than 60 different hosts [82]. They
are accountable for critical spoilage to grasses and some eco-
nomically important crops worldwide, comprising root rot,
leaf spots in wheat and southern leaf blight of maize, black
kernels of rice, spot blotches in barley and wheat, and eye-
spots and brown stripes in sugarcane [82, 83]. Besides, Bipo-
laris species are probably able to infect humans, particularly
the species of B. spicifera which causes fungal peritonitis,
fungal sinusitis, disseminated infection, keratitis, and men-
ingitis; meanwhile, phaeohyphomycosis can also occur by
B. hawaiiensis [82]. Hitherto, the present study is the first
work reporting the strong antifungal effect of green synthe-
sized AgNPs against the fungi of B. hawaiiensis and C.
cynodontis.
3.5. Correlation between the Log of Cell Numbers (CFU/mL)
and Milk Acidification (pH). The bacterial growth curves of
Escherichia coli MSD102, Enterococcus faecalis MSD23, Lac-
tobacillus delbrueckii subsp. bulgaricus MSD231, and Strep-
tococcus thermophilus AAE84 presented a typical bacterial
growth curve. This matched with the pH curve taken from
pH measurements during the time, pointing out that pH
values act as a surrogate sign for bacterial activity
(Figure 7). The correlation between log CFU/mL and pH
values was carried out. The selected function appreciated
the data with an R2=99:5, 99:3, 99:6, and 99:6%for bacterial
growth curves of E. coli,En. faecalis,L. delbrueckii subsp.
bulgaricus, and S. thermophilus, respectively, and their
course has corresponded to the growth curve of bacterial
culture. Bacteria were grown in batch culture, where most
waste is not eliminated and no nutrients are added, follow-
ing a reproducible pattern of growth indicated as the growth
curve. The initial phase is called the lag phase, where pH
values did not change, and log CFU/mL was low. The expo-
nential phase started, where the log CFU/mL of bacteria
began to increase, which was associated with a decrease in
pH values. The highest cell number was not yet until the
examined pH of 4.6. However, this was not critical, since
merely the starting of acidification was determined. More-
over, the decline phase did not play any role in this study
and was ruled out.
3.6. Practical Parameters for the Assessment of AgNPs
Preserved Composite Milk Samples. Milk acidification was
regarded as a measure of microbial growth, and the starting
of milk acidification coincides with the point at which the
pH decreases by 0.1 unit relative to the initial pH. Moreover,
the alcohol precipitation and clot on boiling (COB) tests are
used on the composite milk sample to present whether it will
coagulate on both tests. These tests are particularly signifi-
cant for monitoring the development of acidity in the milk.
The clot on boiling (COB) test is less sensitive than the alco-
hol test. It is dependent on the milk protein tendency to get
unstable because of disturbance in the salt balance of milk.
On the other hand, lipolytic (production of free fatty acids
“FFA”) and proteolytic activity by some microorganisms
during storage of composite milk samples were used as an
important indicator for the stability of milk samples. The
proteolytic and lipolytic activity was regarded as an indicator
of microbial activity and the beginning of proteolytic or lipo-
lytic activity corresponded to the point which the activity of
proteolytic or lipolytic increases by 1.0 unit or 1% (as FFA),
respectively, relative to the initial point. The milk
10 Journal of Nanomaterials

acidification (decline of pH, COB, and alcohol precipitation)
and proteolytic and lipolytic activity by E. coli,En. faecalis,L.
delbrueckii subsp. bulgaricus, and S. thermophilus could be
slowed down by AgNP addition with different concentra-
tions (Table 4). The concentration of AgNPs is directly pro-
portional to the time required for alcohol precipitation,
COB, a decline of pH value (0.1 unit relative to the initial
pH), and lastly the increase of proteolytic and lipolytic
activity (1.0 unit for the proteolytic activity or 1% FFA for
lipolytic activity relative to the initial values) of tested com-
posite milk samples.
No change occurred in all tested parameters until the
end of this experiment (72 h), when using AgNPs at a con-
centration of 200 μg/mL and positive control with all tested
bacterial strains. However, lower concentrations (25, 50,
and 100 μg/mL) also exhibited a significant (P<0:05)effect
compared to the negative control (without chemical preser-
vative) for all tested microorganisms (Table 4). The effect of
temperature on different tested parameters depends on the
bacterial strain, where the time required for all parameters
at 43
°
C is less than incubated at 37
°
C, this is especially the
case of composite milk samples which contain S. thermophi-
lus or L. delbrueckii subsp. bulgaricus. On the contrary, the
time needed for tested parameters at 43
°
C was more than
the incubated composite milk samples containing E. coli or
En. faecalis at 37
°
C (Table 4).
A concentration of 25 μgmL
-1
AgNPs is suitable for
composite milk samples that were required short-term pre-
servative, such as for obtaining the composite milk samples
from the dairy farm to the laboratory for analysis; this con-
centration of AgNPs provided a small increase in shelf life
but is not appropriate for composite milk samples held
long-term. However , the addition of AgNPs (200 μgmL
-1
)
can extend the stability of the composite milk sample by
~3 days when compared with the other concentrations.
Using 200 μgmL
-1
of AgNPs as a long-term preservative
(longer than 3 days) will need further studies. The present
study is consistent with Braun et al. [5], who found silver
nanoparticles (100 μgmL
-1
) to have antibacterial activity at
different temperatures (23, 33, and 43
°
C). Previous measure-
ments presented that extremely more silver nitrate has to be
supplemented to milk for free Ag
+
to become available than
in water, where milk components react with Ag
+
; therefore,
the practical use of nanosilver for the manufacture of dairy
E. coli
rank 21 eqn 8008 ErfcPeak (a,b,c,d)
r2=0.99704573 DF adj r2=0.99535757 FitStdErr=0.061303195 Fstat=899.98062
a=4.6756349 b=5.3543121
c=2.0298197 d=4.4832826
8.5
8
7.5
7
6.5
Log CFU/ml
Log CFU/ml
6
5.5
4.5 5 5.5 6
pH
6.5 7
5
8.5
8
7.5
7
6.5
6
5.5
5
(a)
4.5 5 5.5 6
pH
6.5 7
8.5
8
7.5
7
6.5
Log CFU/ml
6
5.5
5
Log CFU/ml
8.5
8
7.5
7
6.5
6
5.5
5
Enterococcus faecalis
rank 3 eqn 8008 ErfcPeak (a,b,c,d)
r2=0.99617464 DF adj r2=0.99396872 FitStdErr=0.076742807 Fstat=694.43572
a=8.2246658 b=18.039081
c=1.465937 d=10.708054
(b)
4.5 5 5.5 6
pH
6.5 7
Log CFU/ml
Log CFU/ml
8.5
8
9
7.5
7
6.5
6
5.5
5
8.5
8
9
7.5
7
6.5
6
5.5
5
Lactobacillus delbruekii subsp.bulgarius
rank 19 eqn 8008 ErfcPeak (a,b,c,d)
r2=0.99637339 DF adj r2=0.99430105 FitStdErr=0.0753644 Fstat=732.63954
a=2.2760052 b=10.172707
c=0.32976668 d=8.4184226
(c)
4.5 5 5.5 6
pH
6.5 7
8.5
8
7.5
7
6.5
Log CFU/ml
6
5.5
5
4.5
4
Log CFU/ml
8.5
8
7.5
7
6.5
6
5.5
5
Streptococcus thermophilus
rank 1 eqn 8008 ErfcPeak (a,b,c,d)
r2=0.99779401 DF adj r2=0.99653345 FitStdErr=0.067021924 Fstat=1206.1656
a=1.9361487 b=8.2132391
c=1.8718426 d=5.8183991
(d)
Figure 7: Correlation between the log of the average of cell count (CFU mL
-1
) and milk acidification (pH) of (a) E. coli in milk at 37
°
C, (b)
En. faecalis in whole milk at 37
°
C, (c) Lactobacillus delbrueckii subsp. bulgaricus in whole milk at 43
°
C, and (d) Streptococcus thermophilus in
whole milk at 43
°
C. Starting of milk acidification was delineated as the point at which the pH value lowered by 0.1 units from the beginning
value.
11Journal of Nanomaterials

Table 4: Milk acidification (pH, alcohol precipitation, and heat coagulation) and proteolytic and lipolytic activity changes by E. coli,En. faecalis,L. delbrueckii subsp. Bulgaricus, and S.
thermophilus at 37 ± 1°Cor 43 ± 1°Cin the presence of 25, 50, 100, or 200 μgmL
-1
AgNPs compared with negative control (lacking AgNPs) or positive control (formalin 0.4%).
Treatments
Beginning acidification time
(min)
Alcohol precipitation time
(min) Heat coagulation time (min) Beginning proteolytic activity
time (min)
Beginning lipolytic activity
(FFA production time) (min)
37
°
C43
°
C37
°
C43
°
C37
°
C43
°
C37
°
C43
°
C37
°
C43
°
C
Escherichia coli
Negative control 95 ± 1:0d106:3±0:6d212:67 ± 3:1d231:67 ± 1:2d248:67 ± 3:1d274 ± 1:5d142:7±3:1d167:3±1:5d161:3±3:1d184:3±3:1
Positive control — — — — — — ————
25 μgmL
-1
402 ± 2:0c421:7±0:6c511:33 ± 3:1c528 ± 2:0c534 ± 3:0c554:33 ± 3:0c620:7±2:5c643 ± 2:0c637:7±3:5c672:3±1:5
50 μgmL
-1
999:7±1:5b1076:7±2:1b1126:7±2:5b1148:7±1:5b1156:7±2:5b1175:7±2:5b1235 ± 3:6b1272:3±4:0b1279:3±2:5b1295:3±4:0
100 μgmL
-1
1806:33 ± 0:6a1826:67 ± 1:5a1845 ± 2:0a1866 ± 2:0a1871:33 ± 3:1a1891:67 ± 2:5a1906:7±2:5a1931:7±3:1a1961 ± 3:6a1983 ± 1:0
200 μgmL
-1
— — — — — — ————
Enterococcus faecalis
Negative control 108 ± 1:0d116:7±1:5d234:7±2:1d244:3±3:1d263:7±3:5d293:3±2:1d156:3±2:5d174 ± 3:0d171:6±2:5d195:3±2:5d
Positive control — — — — — — ————
25 μgmL
-1
388 ± 1:0c403:3±0:6c485:7±2:5c509:3±2:5c514:3±2:5c536:7±2:5c592 ± 3:0c613:7±4:0c616:6±3:5c642:3±4:5c
50 μgmL
-1
912:3±0:6b934:7±1:5b987:7±3:1b1009:3±3:5b1031:3±3:1b1049 ± 2:6b1079:3±2:5b1119:6±3:0b1157 ± 3:0b1194:3±3:5b
100 μgmL
-1
1672:67 ± 1:5a1687 ± 2:7a1721 ± 3a1737:7±2:5a1754:6±2:5a1779:7±2:5a1794:3±3:5a1807 ± 3:0a1820:7±2:5a1837 ± 3:0a
200 μgmL
-1
— — — — — — ————
Lactobacillus delbrueckii subsp. Bulgaricus
Negative control 112:5±0:5d95:5±0:5d234:7±4:0d219 ± 1:5d287:7±3:5d257 ± 4:0d171 ± 3:0d152:7±4:0d179 ± 3:0d154:7±0:5d
Positive control — — — — — — ————
25 μgmL
-1
438:3±2:5c409:3±1:5c528:6±2:5c494:7±3:5c547:3±4:5c506:3±4:0c611:7±3:5c586:3±4:0c648:6±4:0c614:7±3:5c
50 μgmL
-1
1201:33 ± 1:5b1176 ± 1:0b1283:6±3:5b1241:3±4:2b1318 ± 3:5b1305:6±3:5b1345 ± 3:6b1328:3±3:5b1370:6±3:5b1344 ± 4:0b
100 μgmL
-1
2003:67 ± 2:5a1994:7±2:1a2157:6±2:5a2124:3±3:5a2191:7±5:5a2154:6±3:0a2234:7±3:5a2215 ± 3:0a2271 ± 3:6a2253 ± 4:0a
200 μgmL
-1
— — — — — — ————
Streptococcus thermophilus
Negative control 117:2±0:3d96:3±0:6d225 ± 1:5d200:3±3:5d278 ± 3:0d245:3±4:0d178:6±3:0d157 ± 2:6d187:7±3:0d162 ± 2:0d
Positive control — — — — — — ————
25 μgmL
-1
532:7±2:1c504:7±0:6c623:6±3:0c591:6±3:2c643 ± 3:6c6:9:7±2:5c713 ± 3:0c694:6±2:5c745:3±3:0c736 ± 3:5c
50 μgmL
-1
1303 ± 2:0b1285 ± 1:0b1384:3±3:5b1354:6±3:5b1412 ± 4:5b1389:7±3:0b1435 ± 3:0b1424:6±3:5b1454 ± 3:0b1433:7±4:0b
100 μgmL
-1
2085:67 ± 2:5a2038 ± 2:0a2236:7±3:0a2223:7±4:5a2294 ± 3:0a2276:3±3:5a2341 ± 2:4a2317:3±4:7a2371:7±3:5a2337:3±4:0a
200 μgmL
-1
— — — — — — ————
Note (n=3;average ± standard diffusion): values in rows with different superscripts (a–d) have significant differences according to Tukey’s HSD test at P<0:05.–indicates that there is no change in tested
parameters until 72 h.
12 Journal of Nanomaterials

products is improbable, due to the required high concentra-
tions of silver [5]. Heretofore, our study is the first work pre-
senting the ability to use green synthesized AgNPs as a
chemical preservative for composite milk samples.
3.7. The Influence of AgNPs with Different Concentrations on
the Accuracy of Midinfrared Milk Component Testing. The
essential part of our experiment was focused on studying
the influence of AgNPs with different concentrations as
chemical composite milk sample preservatives on the accu-
racy of milk-IR-analyses. The average values of the chemical
composition parameters (fat, protein, lactose, solid not fat
“SNF,”and total solid “TS”) of unpreserved milk and pre-
served milk stored at 4
°
C for 24h are presented in Figure 8.
According to the ANOVA, there were no statistically
significant differences (P<0:05) in the average values of
chemical parameters among the concentrations of AgNPs
with analytes. A high concentration of AgNPs was used as a
chemical preservative for milk samples compared with other
preservatives, it did not affect the accuracy of milk-IR-
analyses. However, the smallest influence of potassium dichro-
mate on the milk-IR-analyses was achieved with 0.01% of
potassium dichromate according to Kaylegian et al. [84]. Zajác
et al. [85] also found that using extremely high concentrations
(0.1 to 1%) of potassium dichromate, azidiol, bronopol, and
microtabs leads to a statistically significant increase in the
deviation between laboratory results. Up to the present, the
current study is the first work to prove that the use of AgNPs
with different concentrations as a chemical preservative for
composite milk samples does not affect the accuracy of milk-
IR-analyses compared with other chemical preservatives.
4. Conclusion
Silver nanoparticles (AgNPs) were synthesized using polysac-
charides extracted from Leucaena leucocephala seeds as a bior-
eduction and capping agent. The biosynthesized AgNPs were
confirmed by the UV-Vis spectroscopy at 448nm. The XRD
pattern showed four major peaks of the crystalline AgNPs.
The presence of silver was confirmed by the ED spectrum.
The SEM and TEM images revealed that the morphology of
AgNPs was a spherical shape with no particle agglomeration,
and the size ranged between 8 and 20nm. The negative zeta
potential by 14.2mV indicated the enhanced stability of the
silver nanoparticles. The biosynthesized AgNPs showed
strong antitumor activity against the cell lines of colon carci-
noma, liver carcinoma, and breast cancer in a concentration-
dependent manner as well as remarkable antifungal effects
on the growth of the plant and human fungal pathogens.
The high concentration of AgNPs (200μgmL
-1
)providesa
longer shelf life of composite milk samples compared with
other concentrations of AgNPs. Besides, all concentrations of
AgNPs had no influence on an accurate result of a composite
milk sample analysis. This study presents an opportunity to
obtain bioactive silver nanoparticles processing strong antitu-
mor and antifungal activities from a cheap waste, i.e., seed
crude polysaccharides. Additionally, this is the first work dem-
onstrating the strong antifungal effect of green synthesized
AgNPs against the fungi B. hawaiiensis and C. cynodontis
which were critical pests for economically important crops.
Also, our study is the first work offering the ability to use green
synthesized AgNPs as the chemical composite of milk sample
preservation.
Data Availability
All data generated or analyzed during this study are available
from the corresponding author on reasonable request.
Conflicts of Interest
The authors declare no conflict of interest.
Authors’Contributions
Mohamed A. Taher is responsible for conceptualization,
investigation, formal analysis, data curation, visualization,
and writing—review and editing. Ebtihal Khojah is responsi-
ble for data curation, visualization, writing—review and edi-
ting—and funding acquisition. Mohamed Samir Darwish is
responsible for conceptualization, investigation, formal analy-
sis, data curation, visualization, and writing—review and edit-
ing. Elsherbiny A. Elsherbiny is responsible for investigation,
data curation, and writing—review and editing. Asmaa A. Ela-
wady is responsible for conceptualization, investigation,
formal analysis, data curation, and visualization. Dawood H.
Dawood is responsible for investigation, formal analysis, data
curation, and validation. Mohamed A. Taher and Ebtihal
Khojah contributed equally to this work.
Acknowledgments
We acknowledge the financial support from the Taif Uni-
versity Researchers (supporting project number TURSP-
2020/307), 438 Taif University, Taif, Saudi Arabia.
15.00
13.00
11.00
9.00
7.00
(%)
5.00
3.00
1.00
Fat
aaaaa aaaaa
aaaaa
aaaaa
aaaaa
Protein Lactose NSF T.S.
–1.00
Control
25 mg/L
50 mg/L
100 mg/L
200 mg/L
Figure 8: Average fat, protein, lactose, SNF, and TS readings for
AgNPs (25, 50, 100, and 200 μgmL
-1
) for the preserved and
unpreserved portion of the same milk.
13Journal of Nanomaterials

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