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Inulin acetates attract attention as the novel drug carrier. However, the acetylated derivаties of fructooligosaccharides (FOSs) (a low molecular fraction of inulin) were not evaluated. The study aimed to obtain FOSs acetyl esters and to evaluate their foaming properties, water-and oil-holding capacities (WHC and OHC), as well as their antimicrobial activity. One-pot acetylation of chicory FOS with two different degrees of polymerization (DP = 7-9 and 9-12) was performed. The resulting FOSs esters presented white, bitter, water-insoluble substances. The spectroscopic techniques as ultraviolet (UV), Fourier-transform infrared (FTIR), and nuclear magnetic resonance (NMR) spectroscopy were used for characterization and structural elucidation. The antimicrobial activity of acetylated FOSs (1 mg/mL) was tested against 16 microorganisms (Gram-positive and negative bacteria, yeasts, and fungi). Foams prepared with 0.2% FOSs acetates demonstrated the formation of highly stable foams (50-70%). FOSs acetates showed antifungal activity against Fusarium oxysporum and Aspergillus niger and inhibited the growth of yeasts Candida albicans 8673. The inhibition against Gram-positive (Bacillus subtilis 46/H1 and Bacillus subtilis ATCC 6633) and negative (Salmonella abony and Escherichia coli ATCC 8739) bacteria were not observed. However, FOSs acetate with DP 7-9 were active against E. coli 3398, Salmonella typhy 745, and Staphylococus aureus 745 against-which other acetates with DP = 9-12 were inactive. These results demonstrate the potential applications of FOS acetates as a foaming agent and an antifungal substance in pharmaceutical and cosmetic preparations.
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633
Physicochemical Properties and Antimicrobial
Activity of Acetylated Chicory Fructooligosaccharides
Nadezhda Tr. Petkova1*, Radka D. Arabadzhiva1, Yulian D. Tumbarski2,
Mina M. Todorova1, Ivanka P. Hambarlyiska1, Ivan G. Ivanov1,
Sevginar F. Ibryamova3, and Tsveteslava V. Ignatova-Ivanova3
1Department of Organic Chemistry and Inorganic Chemistry; 2Department of Microbiology
University of Food Technologies, 26 Maritza Blvd., Plovdiv 4002 Bulgaria
3Department of Biology, Shumen University “Konstantin Preslavski”
115 Universitetska Str., Shumen 9700 Bulgaria
Inulin acetates attract attention as the novel drug carrier. However, the acetylated derivаties of
fructooligosaccharides (FOSs) (a low molecular fraction of inulin) were not evaluated. The study
aimed to obtain FOSs acetyl esters and to evaluate their foaming properties, water- and oil-
holding capacities (WHC and OHC), as well as their antimicrobial activity. One-pot acetylation
of chicory FOS with two different degrees of polymerization (DP = 7–9 and 9–12) was performed.
The resulting FOSs esters presented white, bitter, water-insoluble substances. The spectroscopic
techniques as ultraviolet (UV), Fourier-transform infrared (FTIR), and nuclear magnetic
resonance (NMR) spectroscopy were used for characterization and structural elucidation. The
antimicrobial activity of acetylated FOSs (1 mg/mL) was tested against 16 microorganisms
(Gram-positive and negative bacteria, yeasts, and fungi). Foams prepared with 0.2% FOSs
acetates demonstrated the formation of highly stable foams (50–70%). FOSs acetates showed
antifungal activity against Fusarium oxysporum and Aspergillus niger and inhibited the growth
of yeasts Candida albicans 8673. The inhibition against Gram-positive (Bacillus subtilis 46/H1 and
Bacillus subtilis ATCC 6633) and negative (Salmonella abony and Escherichia coli ATCC 8739)
bacteria were not observed. However, FOSs acetate with DP 7–9 were active against E. coli 3398,
Salmonella typhy 745, and Staphylococus aureus 745 against – which other acetates with DP = 9–12
were inactive. These results demonstrate the potential applications of FOS acetates as a foaming
agent and an antifungal substance in pharmaceutical and cosmetic preparations.
Keywords: acetylation, antimicrobial activity, fructooligosacchrides, physicochemical properties
*Corresponding Author: petkovanadejda@abv.bg
INTRODUCTION
Inulin is a polysaccharide of plant origin and belongs to the
fructan family. FOSs are short-chained inulins consisting
of 3–10 fructose units with terminal fructose linked to a
glucose residue by β-(1→2) glycosidic bonds. FOSs and
inulin are classified based on their DP, which is based
on the number of monosaccharide units. FOSs have a
DP < 10, while inulin has DP between 2–60 (Barclay et
al. 2010; Kumar et al. 2016). Synthesis of long-chained
inulin esters was demonstrated in many reports (Damian
et al. 1999; Wu and Lee 2000; Poulain et al. 2003;
Starbird et al. 2007; Hartzell et al. 2013; Jain et al. 2014;
Kumar et al. 2016; Walz et al. 2018). However, most of
the acetylation methods include the use of toxic solvents
as N,N-dimethylformamide and pyridine (Damian et al.
1999; Wu and Lee 2000; Poulain et al. 2003; Starbird
Philippine Journal of Science
150 (4): 633-642, August 2021
ISSN 0031 - 7683
Date Received: 04 Jan 2021
634
et al. 2007; Jain et al. 2014). Inulin acetate presented a
novel adjuvant (Kumar et al. 2016) that is not recognized
singularly by dendritic cells. However, when presented in
an appropriately sized particle, inulin acetate acts as an
efficient immune modulator (Hartzell et al. 2013; Kumar
et al. 2016, 2017). Moreover, inulin acetate presents
an encapsulating agent (Wu and Lee 2000; Poulain et
al. 2003; Starbird et al. 2007; Hartzell et al. 2013; Jain
et al. 2014; Kumar et al. 2017) for the formation of
microparticles (Damian et al. 1999; Hartzell et al. 2013;
Kumar et al. 2016). Biodegradation of these polymers
includes hydrolysis of ester bonds to inulin and acetic
acids without enzymes, as well as degradation of inulin
acetates by inulinase (Damian et al. 1999; Hartzell et al.
2013). In addition, in our earlier research, the influence of
the length of the inulin chain and the acetyl residues on the
growth of different microorganisms was demonstrated for
the first time in the scientific literature for inulin acetates
(Petkova et al. 2017b, 2020).
Until now, the antimicrobial activity of short-chain
inulin acetates was not studied in detail. To the best
of our knowledge, there are no scientific data about
WHC and OHC of FOSs acetates with a high degree of
esterification, as well as their application in the cosmetic
and pharmaceutical industry. Moreover, the information
about solubility and foaming properties of FOSs acetates
still remains unrevealed. Therefore, this research
aimed to obtain acetylated FOSs and to evaluate their
physicochemical properties and antimicrobial activity.
MATERIALS AND METHODS
Materials
Short-chain chicory inulin Frutafit® CLR with an average
DP = 7–9 and middle-chain inulin Frutafit® HD with
an average DP = 9–12 (Sensus, the Netherlands) were
used as two FOSs fraction for the modification. Acetic
anhydride (Sigma Aldrich, USA) and anhydrous sodium
acetate (Merck, Germany) were used for the esterification
reaction. Ethanol (95% v/v, Merck, Germany) was applied
during re-crystallization and purification of synthesized
FOSs esters. All other reagents and solvents were of an
analytical grade scale.
Synthesis of FOSs Acetate
FOSs (short-chain chicory inulin Frutafit® CLR with
an average DP = 7–9 and middle-chain inulin Frutafit®
HD with an average DP = 9–12, respectively) (16 g)
was vigorously stirred in a round bottom flask with 9.6
g of anhydrous sodium acetate. Then 96 mL of acetic
anhydride was added to the flask. The molar ratio FOSs:
sodium acetate: acetic anhydride was 1:10:1. The flask
was connected to the reflux with fixed anhydrous calcium
chloride on top. The reaction was performed for 1 h in
boiling under direct heating using a hot plate magnetic
stirrer (Figure 1). After finishing the reaction, the mixture
was poured into a 200-mL water-ice mixture, mixed
vigorously, and with the sample left at –18 °C for 12 h.
FOSs acetates were precipitated in an excess of cold water
as a white solid, filtered, and then purified by washing with
Figure 1. One-pot synthesis of FOSs acetates.
Philippine Journal of Science
Vol. 150 No. 4, August 2021
Petkova et al.: Antimicrobial Activity of
Fructooligosaccharides Acetates
635
cold water. The resulting FOSs acetates were dissolved in
95% (v/v) boiling ethanol and re-precipitated with distilled
water. In the end, the obtained FOSs acetates were dried
in a vacuum oven.
Characterization of FOSs Acetates
Melting point (mp). The mp values of initial FOSs and
obtained esters were determined with a Kofler micro hot-
stage microscope (Reichert, Austria).
Hydrophilic-lipophilic balance (HLB). HLB was
calculated by the method of Griffin (1949).
Thin-layer chromatography (TLC). Silica gel Kieselgel
60 F254 plates (Merck, Germany) were used for TLC.
FOSs acetates and a standard sucrose octaacetate (Sigma
Aldrich, USA) were dissolved in 95% ethanol while
water solutions of FOSs were used, as all were, with a
concentration of 10 mg/mL. The dissolved samples were
dropped on the plate using disposable glass capillaries.
The TLC plate was developed in a mobile phase consisting
of ethyl acetate/methanol/water 17:2:1 (v/v/v). The
visualization of spots was performed by spraying with
10% sulfuric acid dissolved in methanol and the plates
were heated at 120 °C for 5 min.
UV spectroscopy. FOSs acetates in concentration 0.002
g/mL were prepared in the solvents with the different
polarity: chloroform, methanol, ethanol, acetonitrile,
dimethylformamide (DMFA), and dimethyl sulfoxide
(DMSO). The UV spectra were collected on a UV-30
SCAN spectrophotometer in the wavelength range from
190–400 nm.
FTIR spectroscopy. The FTIR spectra of the FOSs and
their acetyl esters (2 mg) were pressed into KBr tablets
and then samples were recorded on a Nicolet FTIR Avatar
(Thermo Scientific, USA) spectrometer with 132 scans
with a resolution of 4 cm−1 in the range of 4000–400 cm−1.
The absorption bands were reported in the wavenumbers
(cm−1). The degree of acetylation of esters was calculated
as relative intensity of FTIR ester bands at 1745 cm−1
divided to that at 1020 cm−1 (Starbird et al. 2007).
NMR spectroscopy. 1H and 13C NMR spectra were
recorded using a Bruker AVIII 500 MHz spectrometer
at a frequency of 500 and 126 MHz, respectively. FOSs
acetates (25 mg) were dissolved in 0.6 mL of CDCl3. The
chemical shifts (δ) were expressed in ppm. Additionally,
the degree of acetylation of the modified FOSs acetate was
calculated by 1H NMR spectroscopy (Jain et al. 2014).
Physicochemical Properties
Swelling properties. The swelling properties of FOSs and
FOSs acetates were evaluated according to the method of
Robertson et al. (2000). β-D-glucose pentaacetate was
used as a reference. Sample (100-mg dry weight) was
hydrated with 10 mL of distilled water in a calibrated
cylinder at 25 °C. After an 18-h equilibration, the bed
volume was recorded.
WHC and OHC. The WHC and OHC of FOSs, FOSs
acetates, and β-D-glucose pentaacetate (used as a
reference) were evaluated in duplicate (Holloway and
Greig 1984). Each sample (0.10 g) was added into a
pre-weighed 50-mL centrifuge tube. After that, 10 mL of
deionized water or sunflower oil was added. The samples
were closed, left 12 h at 25 °C, and then centrifuged
at 3500 rpm for 15 min. The excess water and oil was
discarded. The tubes were weighed and dried at 105 °C
to the constant weight.
Foam stability (FS) and foam ability (FA). FS and FA of
FOSs acetates were also evaluated (Zhang et al. 2014).
The aqueous dispersion of FOSs acetates (20 mL) in four
different concentrations (0.05, 0.10, 0.15, and 0.20%) was
placed in 50-mL stoppered graduated cylinders and the
initial height of each solution was checked. The solution
was vigorously shaken for 1 min, and the foam height and
the total height were measured in cm immediately. The
foam heights at 1, 5, 10, 20, 30, and 60 min were recorded
at 25 °C. All the experiments were conducted in triplicate.
Antimicrobial Activity
Test microorganisms. Four Gram-positive bacteria
(Bacillus subtilis ATCC 6633, B. subtilis 46/H1, Listeria
monocytogenes NBIMCC 8632, Staphylococus aureus
745), four Gram-negative bacteria (Escherichia coli
ATCC 8739, Escherichia coli 3398, Salmonella typhy
745, Salmonella abony), four yeasts (Candida albicans
– clinical isolate, Candida albicans 8673, Candida
tropicalis, Saccharomyces cerevisiae) and four fungal
species (Aspergillus niger, Fusarium oxysporum,
Beauveria bassiana, Penicillium sp.) were selected for
antimicrobial screening. The test microorganisms were
from the collection of the Department of Microbiology
(University of Food Technologies, Plovdiv, Bulgaria).
Only the strains E. coli 3398, S. aureus 745, and L.
monocytogenes ATCC 8632 were obtained from the
collection of the Department of General and Applied
Microbiology, Sofia University, Bulgaria.
For the cultivation of Gram-positive and negative bacteria
and yeasts, Luria-Bertani (LBG) glucose agar medium
was prepared by dissolving 50 g of LBG-solid mixture
(10 g tryptone, 5 g yeast extract, 10 g NaCl, 10 g glucose,
and 15 g agar) in 1 L of deionized water and with the pH
corrected to 7.5. For fungi, malt extract agar medium or
MEA (20 g malt extract, 20 g dextrose, 6 g peptone, and
15 g agar per 1 L of deionized water) was used with a final
pH of 5.5. All agar media were autoclaved at 121 °C for
Philippine Journal of Science
Vol. 150 No. 4, August 2021
Petkova et al.: Antimicrobial Activity of
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636
20 min before use.
The standard agar-well diffusion method in LBG/MEA-
agar was used to evaluate the antimicrobial activity of
FOSs acetates. Most of the bacteria (except B. subtilis
ATCC 6633 and B. subtilis 46/H1) and yeasts (except
S. cerevisiae) were cultured in LBG-agar medium at 37
°C for 24 h. The tested fungi were grown on MEA at 30
°C for 1 wk or until sporulation. A bacterial counting
chamber Thoma (Poly-Optik, Germany) was used for
the determination of the number of viable cells and
fungal spores. Their final concentrations were adjusted to
1.0×108 CFU/mL for bacterial/yeasts cells and 1.0×105
CFU/mL for fungal spores, and were then inoculated in
a preliminarily melted and tempered (45–48 °C) LBG-
agar media. Then six wells (d = 6 mm) per plate were
cut. FOSs acetates (1 mg/mL) were dissolved in aqueous
methanol (80%). As the controls, methanol (80%) and
antibiotics Nystatin (40 μg/mL), Ampicillin (10 μg/mL),
Chlornitromicin (250 μg/mL), and Biseptol (400 μg/mL)
were used. All tested samples (60 μL) were pipetted in
triplicates into the agar wells. The inoculated Petri dishes
were incubated at the following conditions: 30 °C for
Bacillus and all fungi, and 37 °C for other bacteria and
all yeasts. The antimicrobial activity was determined by
the diameter of the inhibition zones around the wells at
the 24th and 48th h of the incubation. The microorganisms
with inhibition zones of 18 mm or more were evaluated
as sensitive; others were assessed as moderately sensitive
(inhibition zones from 12–18 mm); resistant (inhibition
zones up to 12 mm), or completely missing (Tumbarski
et al. 2018).
RESULTS AND DISCUSSION
Characterization of FOSs Acetates
The results of the characterization of FOS acetates with
the different DP were summarized in Table 1. All FOS
acetates presented white powder substances with a bitter
taste and insoluble in water except in some organic
solvents such as methanol, ethanol, acetone, chloroform,
DMSO, DMFA, and acetonitrile. The highest yields were
obtained for FOSs (DP = 9–12) acetates (80% yield). It
is a crystalline white solid (mp = 79–78 °C), while FOSs
(DP = 7–9) acetates demonstrated a lower mp (69–75
°C), which could be explained by the short length of
the inulin chain and lower degree of esterification (2.2).
The obtained mp values for FOSs esters were near to
the reported in literature data that ranged from 87–92
°C typical for another inulin acetate (Wu and Lee 2000;
Poulain et al. 2003; Petkova et al. 2020). Moreover, the
initial FOSs have a high mp (Table 1). The substitution of
free hydroxyl groups with acetyl moieties reduced more
than 2.2 times the mp of FOS esters in the comparison of
initial FOSs (Table 1). Hydrophilic-lipophilic balance of
FOSs acetates was between 6.7–8.7, which classified them
as amphiphilic molecules that are water-dispersible with
potential use as wetting and spreading agents.
FOSs acetates dissolved in 95% ethanol were analyzed
by TLC (Figure 2). The spots of corresponding different
FOSs acetates (Figure 2, spots 3 and 4) were characterized
with Rf values 0.91 and 0.92 in ethyl acetate/methanol/
water 17:2:1 (v/v/v) used as mobile phase. The resulting
Rf values were comparable with Rf obtained for sucrose
octaacetates. The Rf values of initial FOSs were lower than
those esters with Rf = 0.2. This observation qualitatively
approved the successful acetylation of FOSs.
UV Spectroscopy
The UV spectra of FOSs acetates (0.002 g/mL) were
measured in solvents with different proton accepting
and proton donating properties: methanol, ethanol,
acetonitrile, chloroform, DMFA, and DMSO. The data
characterizing the absorption properties of the investigated
substances were presented in Table 2.
The absorption properties of FOSs acetates were
analogous to the absorption properties of inulin acetates in
the same solvents. Any deviations were not observed in the
absorption bands of FOSs acetates and previously reported
inulin acetate in the range of 200–266 nm. Deviations in
the range of 3–11 nm are observed in the shoulders in the
spectra of both compounds (Petkova et al. 2020).
Table 1. Characterization of fructooligosacchrides and fructooligosacchrides acetates.
Sample Yield, % Melting point (mp), ºC Degree of acetyl-
ation (DA)
HLB
Fructooligosacchrides (DP = 7–9) a154–157 –b
Fructooligosacchrides (DP = 9–12) a165–167 –b
Fructooligosacchrides (DP= 7–9) acetate 52 69–75 2.2 8.7
Fructooligosacchrides (DP = 9–12) acetate 80 75–78 3.0 6.7
aAbsent
bNot applied
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FTIR Spectroscopy
The structure of FOSs acetylated esters was confirmed
by infrared spectroscopy with the appearance of new
functional groups (Figure 3).
The broadband at 3330 cm–1 was due to the stretching
vibrations of OH groups (Figure 3A). A decrease in
the intensity of OH groups was observed after the
acetylation process (Figure 3B). The acetylation led
to the incorporation of acetyl residue as free OH was
esterified. In addition, a new strong band at 1745 cm–1
was assigned with a stretching vibration of C=O groups
from ester, 1370 cm–1 bending of C-H, and 1220 cm–1
attributed to stretching vibration of C-O from acetyl
residue. These three typical bands were due to ester bonds
of acetyl residues to the carbohydrate chains. In addition,
the presence of bands in the fingerprint region at 924 and
817 cm–1 in both spectra proved that the resulting ester
contained β-D-fructose residues linked 1→2 glycoside
bonds. The reported FTIR bands in FOSs acetate spectra
were in accordance with FTIR for inulin acetates (Poulain
et al. 2003; Starbird et al. 2007; Kumar et al. 2016;
Petkova et al. 2020).
NMR Spectroscopy
The structure of FOSs acetates was additionally confirmed
by the 1H NMR (Figure 4) and 13C NMR spectra.
In acetylated FOSs, the shifts for inulin backbone appeared
the at δ = 5.08−5.55 ppm (t/m, 3H, methine) and δ =
3.50−4.45 ppm (d/m, 4H, methylene). However, after
Figure 2. TLC chromatogram of FOSs esters with mobile phase
ethyl acetate/methanol/water 17/2/1 (v/v/v): 1) FOSs;
2) sucrose octaacetate; 3) FOSs (DP = 7–9) acetate; 4)
FOSs (DP = 9–12) acetate.
Table 2. The absorption properties of FOSs acetates in the solvents with different solvents.
Solvents ΔFa(ε, n2)
(Reichardt 2003)
λmaxb (A)
(nm)
FOSs acetates
(DPd =
7–9)
εc
(L ∙ mol–1
∙ cm–1)
λmax (A)
(nm)
FOSs acetates
(DP =
9–12)
ε
(L ∙ mol–1 ∙ cm–1)
1. Methanol 0.3110 208 0.6734 200.5
218 shoulder
264 broadband
0.2896
0.1115
0.0756
2. Ethanol 0.1548 208
256.5 broad band
1.4275
0.1730
201
212 shoulder
0.5480
0.3455
3. Chloroform 0.1458 240.5
267 shoulder
0.2507
0.1343
238.5
270.5 broad band
0.1062
0.0543
4. Acetonitrile 0.3054 194
215 shoulder
0.8612
0.3605
191.5
207
271 broadband
1.2093
1.0417
0.0999
5. DMFA 0.2745 265.5
364 broad band
0.1589
0.0154
266
382.5
0.2182
0.0366
6. DMSO 0.2634 261 0.1042 263.5
372 broad band
0.0548
0.0033
aSolvent polarity parameter; bwavelength; cmolar extinction coefficient and molar absorptivity; ddegree of polymerization
Philippine Journal of Science
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638
Figure 3. FTIR spectra of A) FOSs (DP = 9–12) and B) FOSs acetate.
Figure 4. 1H NMR spectra of FOSs (DP = 9–12) acetate.
Philippine Journal of Science
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639
acetylation, some new shifts in 1H NMR spectrum of FOSs
acetate were observed at 2 ppm that were characteristic
of the methyl side chain of the acetyl groups (Figure 4).
13C NMR (126 MHz, CDCl3) of FOS acetates (DP =
9−12) contained shifts as follow: δ 170.15 (t, J = 50.1 Hz)
(CH2CO–), 103.73 (s) (C2f), 77.53 (d, J = 41.6 Hz), 77.33
(s), 77.07 (s), 76.82 (s), 75.79 (s), 75.53–74.62 (m), 63.84
(s), 62.66 (s), 21.64–21.29 (m), 20.73 (d, J = 12.9 Hz),
20.65 (t, J = 16.5 Hz), and 20.43 (s). Three chemical shifts
characteristic for acetyl carbonyl appeared at 170 ppm.
All the methyl carbons from the acetyl residue appeared
at δ 20.5 (3×COCH3). Carbon atoms of the inulin moiety
were found in the range from 62.66−103.73 ppm.
In the anomeric region of FOSs acetates, shifts typical at
103.73 ppm were found for C2 from fructose residue in
the inulin chain that was involved in the linkage β-(2→1)-
D-fructofyranosyl-fructose. Similar chemical shifts were
reported for inulin acetates synthesized in DMF by stirring
for 24 h (Wu and Lee 2000; Starbird et al. 2007; Kumar et
al. 2016) and for inulin acetates obtained by conventional
and green synthesis only with a catalyst without the
solvent media (Petkova et al. 2017b, 2020).
Functional Properties
Swelling properties. FOS (DP = 7−9) acetates demonstrated
better swelling properties (2.99 cm) in comparison to
initial FOSs (1.37 cm) and FOSs with longer chains
(Figure 5A). The number of acetylated hydroxyl groups
influenced on swelling properties of carbohydrate
acetylated ester. The swelling properties decreased in the
following order β-D–glucose pentaacetate > FOS (DP =
7−9) acetate > FOS (DP = 9−12) acetates.
WHC and OHC. These both properties bring about
functional, flavor, and sensory properties as well as
improve the uses of modified carbohydrates in cosmetic or
food products. However, any data about WHC and OHC
of carbohydrate acetylated esters were not available. To
the best of our knowledge, the functional properties of
FOSs acetates were not investigated at all. Their WHC and
OHC were demonstrated and compared with β-D-glucose
pentaacetate and the FOSs (Figure 5A).
The acetylation of FOSs with different DP brought about
the increase of their OHC. The OHC of FOSs acetates
was higher than their WHC and reached 13.4−15.0 g
oil/g sample. They were close to the OHC of β-D-glucose
pentaacetate (11 g/g sample). The higher values of OHC
in FOSs acetates could be explained with more acetylated
residues in a comparison with acetate of D-glucose,
where the number of acetyl groups was 5. In comparison,
unmodified FOS showed WHC (1.2−2 g water/g sample)
and OHC (1.5–4.5 g oil/g) samples, respectively. In the
current research, the presence of acetyl residues in the
Figure 5. Physicochemical properties of FOSs with the different
chain length and their acetates: A) swelling and water-
and oil-holding capacities; B) FS of 0.20% FOSs (DP =
7–9) acetates; C) FS of FOSs (DP = 9–12) acetates at the
different concentrations (0.05, 0.10, 0.15, and 0.20%) for
60 min at 25 °C.
inulin chain increased WHC and OHC in the range of
1.5–3 times (Figure 5A). The complete substitution
of hydrophilic OH groups in the inulin backbone with
acetyl residues explained these properties. The OHC
of FOSs acetates demonstrated their potential use in
pharmaceutical and food formulations. In addition, the
swelling properties of acetylated FOSs increased in the
comparison with the initial FOSs with different DP.
Foaming properties. The foaming properties of modified
oligofructoses with the long-chained fatty acid ester (lauric
and palmitic) were reported by van Kempen et al. (2014).
In our previous research, the long-chained inulin acetates
and octa-O-acetylsucrose were evaluated (Petkova et al.
2020). However, until now, any data for the potential of
FOSs acetates as a foaming agent were not available. The
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Petkova et al.: Antimicrobial Activity of
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640
FA values for FOS (DP = 7–9) acetate in concentration 0.2%
was 85% while for FOS (DP = 9–12), the value was 12%.
FS of resulting FOS acetates was evaluated by measuring
the foam heights from 1–60 min (Figures 5B and C).
For the foams obtained with 0.2, 0.15, and 0.1% FOSs
acetates, the FS decreased by more than 10% for the first
5 min, while the decrease of foam of 0.05% inulin acetate
was with 20% for the first 5 min. The most stable foams
were observed in the high concentration of FOSs acetate.
The most stable foams were with FS of 52% for 60 min.
In comparison, FS of octa-O-acetylsucrose (0.2 g/L) did
not exceed 52–55% for 30 min (Petkova et al. 2017a). The
results demonstrated the foam formation of FOS acetates
with high stability (40–66%) in a high concentration (0.2%).
Antimicrobial Activity
Furthermore, due to the interest in modified carbohydrates
for the application as a nanomaterial, gels with potential
antimicrobial activity for pharmaceutical purposes
Table 3. Antimicrobial activity of short-chained and middle chained FOSs acetates in a concentration of 1 mg/ml expressed as diameter of
zones of inhibition in mm (dwell = 6 mm).
Test microorganism Inhibition zones, mm
FOSs acetate Controls
DPa = 7–9 DP = 9–12 Nystatin,
40 μg/mL
Ampicillin,
10 μg/mL
Chlornitromycin, 250
μg/ml
Biseptol,
400 μg/ml
Gram-positive
Bacillus subtilis 46/H1 b– n/af20 n/a n/a
Bacillus subtilis ATCC 6633 – – n/a n/a n/a 12
Listeria monocytogenes ATCC 8632 14**d12*cn/a n/a n/a 11
Staphylococcus aureus 745 14** – n/a n/a n/a 13
Gram-negative
E.coli ATCC 8739 – – n/a 14 n/a n/a
E.coli 3398 15**d– n/a n/a n/a 16
Salmonella typhy 745 16 – n/a n/a n/a 14
Salmonella abony – – n/a n/a n/a
Yeasts
Candida tropicalis – – n/a n/a n/a
Candida albicans – – n/a n/a n/a
Candida albicans 8673 16** 19** n/a n/a 19 n/a
Saccharomyces cerevisiae – 9 n/a n/a n/a
Fungi
Aspergillus niger 19 20***e12*cn/a n/a n/a
Fusarium oxysporum 8 8 n/a n/a n/a
Penicillium sp. – – 8 n/a n/a n/a
Beauveria bassiana – – n/a n/a n/a
aDegree of polymerization; bno inhibition ; *cresistant with inhibition zones up to 12 mm or completely missing; **dmoderately sensitive with inhibition zones from
12–18 mm; ***esensitive with inhibition zones of 18 mm or more; fnot applicable
constantly increased (Abdelghany et al. 2019). To the
best of our knowledge, the antimicrobial potential of
acetylated esters of FOSs was not studied in detail. In our
previous investigation, conventional esterified inulin with
different DP was tested and demonstrated slight antifungal
activity (Petkova et al. 2017b, 2020). Our research group
reported that long-chained inulin acetates obtained after
microwave-assisted acetylation showed antimicrobial
activity against L. monocytogenes 863, E. coli 3398, C.
albicans 8673, F. oxysporum, and A. niger (Petkova et
al. 2017b, 2020).
In the current study, the antimicrobial properties of FOSs
acetates in the concentration of 1 mg/mL were tested
against 16 microorganisms (Gram-positive and negative
bacteria, yeasts, and fungi). The results were summarized
in Table 3. It was found that all FOSs acetates inhibited the
growth of fungi F. oxysporum and A. niger and yeasts C.
albicans 8673. FOS acetates with DP = 7–9 were active
against E. coli 3398, S. typhy 745, and S. aureus 745 –
Philippine Journal of Science
Vol. 150 No. 4, August 2021
Petkova et al.: Antimicrobial Activity of
Fructooligosaccharides Acetates
641
against which FOS acetate with DP = 9–12 was inactive.
Both FOSs acetylated esters were inactive against Gram-
positive (B. subtilis 46/H1 and B. subtilis ATCC 6633) and
negative (S.abony and E. coli ATCC 8739) bacteria. FOSs
(DP = 7–9) acetates showed moderate antibacterial activity
against L. monocytogenes ATCC 8632 and S. aureus 745,
comparable with the activity of Biseptol at 400 μg/mL. In
addition, FOSs (DP = 9–12) acetates were active against
Listeria monocytogenes but did not show any antibacterial
activity against other investigated Gram-positive and
negative bacteria (Table 3). A similar observation of
higher activity against L. monocytogenes was reported
for cinnamaldehyde-loaded chitosan nanoparticles (Soto-
Chilaca et al. 2019).
CONCLUSION
FOSs acetates with different lengths of carbohydrate chain
were synthesized and characterized. It was found that
FOSs acetates exhibited good foamability, FS, swelling
properties, WHC, and OHC. In addition, FOSs acetates
in concentration of 1 mg/mL showed antifungal activity
against Fusarium oxysporum and Aspergillus niger and
yeast Candida albicans 8673. The inhibition against
two Gram-positive (Bacillus subtilis 46/H1 and Bacillus
subtilis ATCC 6633) and negative (Salmonella abony and
E. coli ATCC 8739) was demonstrated. The synthesized
FOSs acetates with their antifungal activity could find
wide and successful application in the field of agriculture
for plant protection and biocontrol, as well as in cosmetics
for antibacterial foam agents.
ACKNOWLEDGMENTS
The authors thank for the support and assistance of Nevena
Petkova and Nikola Burdjiev from University “St. Klimet
Ohridski,” Sofia for the collection of NMR spectra.
STATEMENT ON CONFLICT OF
INTEREST
The authors in this manuscript listed above certify that
they have no affiliations and relationships with any
organization or any financial interest or non-financial
interest in the subject matter or materials discussed in
this manuscript.
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... FS and FA of lactose acetates were also evaluated as all experiments were performed in triplicate [29]. The aqueous dispersion of lactose acetates (20 mL) in 0.01% concentrations was placed in 50 mL stoppered graduated cylinders, and the initial height of each solution was checked. ...
... Similar shifts were reported for lactose acetates synthesized by classical method and microwave methods [19,25]. Reported by shifts were near to Peng [19]: 13 Lactose acetates demonstrated better swelling properties (2.82 mL water/g sample) in comparison to FOSs acetates (1.37 mL water/g sample) and inulin acetates with longer chains [10,29]. The number of acetylated hydroxyl groups influenced on swelling properties of carbohydrate acetylated ester. ...
... Until now, many studies about the evaluation of WHC and OHC of lactose octaacetates were not provided, and there is the absence of information about these functional properties. . Petkova et al. [29] investigated the influence of acetylation of FOSs with different DP and found that brought about the increase of their OHC. The OHC of lactose acetate was higher than their WHC and reached 3.4 g oil/g sample ( Figure 5). ...
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