foods

Article
Chemical Composition of Turmeric (Curcuma longa L.) Ethanol
Extract and Its Antimicrobial Activities and Free Radical
Scavenging Capacities
Huan Wu 1,2, * , Zhihao Liu 2,3 , Yaqiong Zhang 4 , Boyan Gao 4 , Yanfang Li 2 , Xiaohua He 5 ,
Jianghao Sun 3 , Uyory Choe 2 , Pei Chen 3 , Ryan A. Blaustein 2 and Liangli Yu 2

1 Key Laboratory of Xin’an Medicine, Ministry of Education, Anhui University of Chinese Medicine,
Hefei 230038, China
2 Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA;
[email protected] (Z.L.); [email protected] (Y.L.); [email protected] (U.C.);
[email protected] (R.A.B.); [email protected] (L.Y.)
3 Methods and Application of Food Composition Laboratory, Beltsville Human Nutrition Research Center,
Agricultural Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA;
[email protected] (J.S.); [email protected] (P.C.)
4 Institute of Food and Nutraceutical Science, School of Agriculture and Biology, Shanghai Jiao Tong University,
Shanghai 200240, China; [email protected] (Y.Z.); [email protected] (B.G.)
5 Western Regional Research Center, Agricultural Research Service, United States Department of Agriculture,
Albany, CA 94710, USA; [email protected]
* Correspondence: [email protected]

Abstract: Turmeric (Curcuma longa L.) is a perennial tuberous plant from the genus Curcuma (Zin-
giberaceae) and has been widely used in foods for thousands of years. The present study examined
the ethanol extract of turmeric for its chemical composition, antimicrobial activity, and free radi-
cal scavenging properties. UHPLC-MS/MS analysis tentatively identified eight compounds in the
turmeric extract. Potential antimicrobial effects of 0.1, 1.0, and 10 mg turmeric equivalents (TE)/mL
Citation: Wu, H.; Liu, Z.; Zhang, Y.; were evaluated in vitro against a variety of Gram-negative bacteria (i.e., Escherichia coli, Klebsiella
Gao, B.; Li, Y.; He, X.; Sun, J.; Choe, U.; pneumoniae, and Pseudomonas sp.) and Gram-positive bacteria (i.e., Enterococcus faecalis, Listeria in-
Chen, P.; Blaustein, R.A.; et al. nocua, and Staphylococcus aureus). Concentrations of 0.1 and 1.0 mg TE/mL inhibited the growth of
Chemical Composition of Turmeric S. aureus and significantly suppressed that of Pseudomonas sp., E. faecalis, and L. innocua. The growth
(Curcuma longa L.) Ethanol Extract and of all strains, including E. coli, was inhibited by 10 mg TE/mL. Moreover, free radical scavenging
Its Antimicrobial Activities and Free
capacities were determined using HO• , ABTS•+ , and DPPH• (HOSC, ABTS, and RDSC, respectively)
Radical Scavenging Capacities. Foods
radicals. The turmeric ethanol extract had a TPC value of 27.12 mg GAE/g, together with HOSC,
2024, 13, 1550. https://doi.org/
RDSC, and ABTS values of 1524.59, 56.38, and 1.70 µmol TE/g, respectively. Our results suggest that
10.3390/foods13101550
turmeric extract has potential applications for use in functional foods to reduce microbial burdens
Academic Editor: Barbara and oxidative stress-related health problems.
Laddomada

Received: 21 April 2024 Keywords: turmeric; UHPLC-MS/MS; antimicrobial activity; antioxidant; curcumin; demethoxycurcumin;
Revised: 7 May 2024 bisdemethoxycurcumin
Accepted: 10 May 2024
Published: 16 May 2024

1. Introduction
Spices have been an integral part of the human diet for thousands of years. More
Copyright: © 2024 by the authors.
recently, the growing interest in the relationship between diet and health has elevated the
Licensee MDPI, Basel, Switzerland.
importance of using spices in the food arena and understanding the mechanisms behind
This article is an open access article
their beneficial properties [1]. Turmeric (Curcuma longa L.) is a perennial tuberous plant
distributed under the terms and
conditions of the Creative Commons
from the genus Curcuma (Zingiberaceae) and is commonly distributed in East, South, and
Attribution (CC BY) license (https://
Southeast Asia [2]. The rhizomes of turmeric are well-developed, clumped, branching,
creativecommons.org/licenses/by/ elliptic, or cylindrical and are widely used in many foods, such as canned beverages, baked
4.0/). products, dairy products, ice cream, yogurt, yellow cakes, milk, orange juice, biscuits,

Foods 2024, 13, 1550. https://doi.org/10.3390/foods13101550 https://www.mdpi.com/journal/foods

Foods 2024, 13, 1550 2 of 13

popcorn, cereals, sauces, and others, in the form of powder for over 4000 years [3,4]. The
use of turmeric in foods adds a distinctive flavor compared to other spices. Previous
chemical analysis of turmeric has shown that it mainly contains terpenoids, curcuminoids,
and other phenolic compounds [5]. Among these components, curcuminoids were the
major bioactive constituents and recognized by the U.S. Food and Drug Administration as
a safe tolerable class of components, at doses up to 8 g per day [6]. Additionally, the main
component of turmeric, curcumin, has an orange-yellow color and is an important food
colorant. Today, with an increasing interest in applications for food and beverages, as well
as nutraceuticals, the global curcumin market is projected to grow from USD 85.77 million
in 2023 to USD 165.10 million by 2030 [7].
In addition to enhancing flavor and color in food products, turmeric has applications
to support human health, such as through anti-inflammatory, anti-cancer, and antimicrobial
effects [8]. For example, ethnopharmacological studies have reported turmeric use as a
medical herb for knee osteoarthritis [9], radiodermatitis [10], and cancers [11]. Curcumin
has notable antibacterial properties, as it has been shown to inhibit the growth of Escherichia
coli, Klebsiella pneumoniae, and Staphylococcus aureus, as well as suppress the formation of
mixed-community biofilms and aid in the body’s ability to clear bacteria [12,13]. Curcumin
has also been reported to support immunologic response through reducing inflammation,
limiting the overexpression of cytokines, and improving the removal of reactive oxygen
species, which may lower risks for developing chronic disease [14]. Nevertheless, turmeric,
rather than pure curcumin, is typically used as a food ingredient. Characterizing the
chemical components and bioactivities of turmeric could provide a foundation for its
improved use in food system safety and health-promoting foods and may add profitability
to the turmeric industry.
This research aims to understand the antibacterial and radical scavenging activities
of turmeric and the chemical components that may contribute to these properties. The
antibacterial effects of turmeric extract were evaluated against several Gram-positive and
Gram-negative bacteria strains. The radical scavenging properties were examined against
ABTS•+ , DPPH• , and HO• radicals according to our published laboratory protocols. In
addition, the total phenolic content (TPC) was determined for the extracts as they are
potential contributors to overall bioactivity. The results from this study have important
implications for the potential utilization of turmeric as an active ingredient in foods to
enhance food safety and human health.

2. Materials and Methods

2.1. Materials
Turmeric (Curcuma longa L.) root was gifted from Frontier Co-op (Norway, IA). DPPH• ,
ABTS, Folin–Ciocalteu’s phenol reagent (F9252), gallic acid (G7384), fluorescein (FL), (±)-
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), ferric chloride (FeCl3 ),
and hydrogen peroxide (H2 O2 ) were purchased from Sigma-Aldrich Corporation (St. Louis,
MO, USA). Acetonitrile (LC-MS grade) and formic acid (HPLC grade) were purchased from
Merck (Darmstadt, Germany). Other chemicals used in this study were purchased from
Fisher Scientific (Hampton, NH, USA), at analytical grade. Bacteria used in the antimicro-
bial assays included E. coli K-12 and strains of Enterococcus faecalis, K. pneumoniae, Listeria
innocua, Pseudomonas sp., and S. aureus, maintained in glycerol stocks at the University of
Maryland, College Park.

2.2. Sample Preparation

Turmeric root was grounded to powder using a Micromil Grinder (Wayne, NJ, USA).
The grinding process was repeated until all the turmeric root powder passed No. 40 mesh
(0.420 mm). Next, 1 g of turmeric powder was accurately weighed and mixed with 10 mL
of ethanol for 24 h at ambient temperature. After 24 h, a centrifuge (3500 rpm) was used to
obtain turmeric extract having a concentration of 100 mg dry turmeric equivalents (TE)/mL
and stored at −20 ◦ C.
Foods 2024, 13, 1550 3 of 13

2.3. Chemical Compositions of Turmeric (Curcuma longa L.) Ethanol Extract

The chemical compositions of turmeric ethanol extract were analyzed with a Vanquish
UHPLC- Orbitrap Fusion ID-X Tribrid mass spectrometer. The sample was separated in an
Agilent Eclipse Plus-C18 column (150 mm × 2.1 mm i.d., 1.8 µm). Mobile phase consisted
of 0.1% formic acid in acetonitrile (v/v) (A) and 0.1% formic acid in water (v/v) (B). The
gradient elution procedure is as follows: 0 min (2%A), 15 min (10%A), 35 min (40%A),
55 min (95%A), and 60 min (95%A), then re-equilibration with 2%A for 10 min. Conditions
were as follows: Injection volume, 1 µL. Flow rate, 0.3 mL/min. Mass Spectrometry
conditions: Resolution, 60,000. Scan range, m/z 120 to 1200. Ion transfer tube temperature,
300 ◦ C. Ion source temperature, 275 ◦ C. The capillary voltages were 3.9 kV (positive ion
mode) and 2.5 kV (negative ion mode). Peaks were tentatively identified by their precision
mass of excimer ions, MS/MS data and fragmentation patterns, and data from the literature.

2.4. Antimicrobial Activities

Antimicrobial effects of turmeric were evaluated against three Gram-negative (E. coli,
K. pneumoniae, Pseudomonas sp.) and three Gram-positive (E. faecalis, L. innocua, S. aureus)
bacteria. Isolates from pure cultures of the respective strains were individually grown in
500 µL trypticase soy broth (TSB) incubated at 37 ◦ C for 24 h. The pre-grown cultures
were vortexed, and 10 µL was transfer-inoculated into 180 µL TSB and that which was
augmented with varying concentrations of turmeric ethanol extract dissolved in DMSO
(i.e., 0.1, 1, and 10 mg turmeric equivalents mL−1 ) in 96-well plates. Since DMSO can have
antimicrobial effects at high concentrations [15], TSB containing 0, 0.1, 1, and 10% DMSO
with no turmeric was included as assay controls. All plates were incubated at 37 ◦ C shaking
at 180 rpm for 24 h. Optical density absorbance at 600 nm (OD600 ) was measured with a
Multiscan FC plate reader (Thermo Scientific, Waltham, MA) at 0, 3, 6, 12, and 24 h. OD600
values reflecting bacterial growth were normalized against negative control blanks that
contained no bacteria. All assays were performed with three biological replicate plates, in
which technical replicates for each treatment were averaged.

2.5. Total Phenolic Content (TPC)

The TPC of turmeric ethanol extract was measured using a laboratory protocol, as
previously described [16]. Briefly, 3 mL of water was mixed with 50 µL of solvent (blank),
standard, or sample. To the mixture, 250 µL of Folin–Ciocalteu’s phenol reagent was added.
After the vortex, 750 µL of 20% (w/v) Na2 CO3 was added to initiate the reaction, and the
reaction mixture was maintained in ambient temperature under dark for 2 h. After the
incubation, the absorbance at 765 nm was measured. The results were calculated based on
the standard curve generated using gallic acid and reported in milligrams of gallic acid
equivalents (GAE) per gram of dry turmeric sample (mg GAE/g turmeric).

2.6. Free Radical Scavenging Capacities

The 2,2′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical (ABTS•+ ) values
were detected following a laboratory protocol [16]. Briefly, 160 µL of solvent/standards
(5 to 300 µmol/L Trolox solution)/clove extracts was mixed with 2 mL of ABTS•+ working
solution by vortexing for 30 s. Following a 60 s reaction, the absorbance was detected at
734 nm with a Genesys 20 visible spectrophotometer (Thermo Fisher Scientific, Norristown,
PA, USA). The relative 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging capacity
(RDSC) values were detected based on previous studies [17,18]. Briefly, 100 µL of solvent,
standards (7 to 36 µmol/L Trolox solution), or sample was mixed with 100 µL of freshly
prepared 0.2 mM DPPH solution in the wells of a 96-well plate. Detection wavelength
was 515 nm, and the samples were detected every minute for 90 min using a Tecan M200
Pro microplate reader (Tecan Group Ltd., Mannedorf, Switzerland). The hydroxyl radical
scavenging capacity (HOSC) values were detected, as previously described [19]. Briefly,
170 µL of freshly prepared fluorescein working solution (92.8 nM) was mixed with 30 µL of
either solvent, standard, or sample in the wells of a 96-well plate. Then, 40 µL of freshly
Foods 2024, 13, 1550 4 of 13

prepared H2 O2 working solution (199 mM) and 60 µL of FeCl3 (3.43 mM) were added to
start the reaction. The fluorescence intensities were recorded (excitation: 485 nm, emission:
535 nm) every five minutes for 8 h with a Tecan M200 Pro microplate reader (Tecan Group
Ltd., Mannedorf, Switzerland). In all three assays, Trolox was used as a standard, and
results were expressed as micromoles of Trolox equivalents (TE) per gram of dry turmeric
sample (µmol TE/g turmeric).

2.7. Statistical Analysis

To characterize the antimicrobial effects of turmeric, area under the curve (AUC) for
logistic bacterial growth (OD600 ) was approximated using Growthcurver [20]. ANOVA
with Tukey’s post hoc test was applied to evaluate differences in AUC, as well as in
OD600 for each time point (i.e., 3, 6, 12, 24 h), based on the augmented concentrations
of turmeric and/or DMSO in the growth media. The Student’s t-test was further used
to determine differences in AUC between the turmeric treatments and respective DMSO
controls (e.g., compare 10 mg ml−1 turmeric dissolved in DMSO in TSB to 10% DMSO
in TSB, as both contained equal volumes DMSO in the bacterial growth media). These
statistical analyses were performed in R.v.4.3.1. For radical scavenging properties and TPC,
t-test was performed using the GraphPad Prism 9 software.

3. Results and Discussion

3.1. Chemical Compositions of Turmeric Ethanol Extract
Eight compounds (Table 1) were tentatively identified in turmeric ethanol extract using
tandem mass spectrometry and literature data. All eight compounds were phenolic com-
pounds. Compound 1 and compound 5 were identified as coumaric acid [21] and calebin
A, respectively. Both coumaric acid and calebin A were reported in turmeric ethanol extract
for the first time. Compounds 2–4 were keto forms of bisdemethoxycurcumin, demethoxy-
curcumin, and curcumin, while compounds 6–8 were enol forms of bisdemethoxycurcumin,
demethoxycurcumin, and curcumin [22,23]. These results agreed with the previous reports
that curcuminoids are the major bioactive compounds found in turmeric root [24]. In
addition, our study showed that the ion intensities of the enol form of curcuminoids were
higher than the keto form of curcuminoids. Compound 6 (bisdemethoxycurcumin) had
the highest intensity, followed by compound 7 (demethoxycurcumin) and compound 8
(curcumin) in the total ion chromatogram (TIC) (Figure 1).

Table 1. Characterization of chemical compounds in turmeric (Curcuma longa L.) ethanol extract.

Negative Ionization Mode Positive Ionization Mode

Ion In-
Peak tR Mass Mass Tentative
Product Product Formula tensity Ref.
No. (min) [M−H]− Error [M+H]+ Error Identification
Ions Ions (×105 ) *
(mmu) (mmu)
1 18.49 163.0386 119.0489 −1.55 ND ND ND C9 H8 O3 Coumaric acid 12 [21]
187.0386
225.0894 Bisdemethoxycurcumin
2 31.44 307.0952 143.0490 −2.15 309.1097 −2.40 C19 H16 O4 217 [22]
147.0429 (keto form)
119.0492
255.0994
217.0483 245.0789
Demethoxycurcumin
3 32.21 337.1056 173.0588 −2.58 339.1205 239.1071 −2.22 C20 H18 O5 135 [22]
(keto form)
143.0485 231.1277
146.3248
299.1252
217.0484 285.1097
4 33.28 367.1156 173.0589 −3.08 369.1309 259.0942 −2.40 C21 H20 O6 Curcumin (keto form) 75 [22]
135.0436 245.0787
175.0737
217.0487
173.0592 261.0737
5 37.53 383.1111 −2.57 385.1257 −2.45 C21 H20 O7 Calebin A 210
165.0542 177.0531
158.0358
Foods 2024, 13, 1550 5 of 13

Table 1. Cont.

Negative Ionization Mode Positive Ionization Mode

Ion In-
Peak tR Mass Mass Tentative
Product Product Formula tensity Ref.
No. (min) [M−H]− Error [M+H] +
Error Identification
Ions Ions (×105 ) *
(mmu) (mmu)
187.0383
225.0892 Bisdemethoxycurcumin
6 39.08 307.0954 143.0488 −2.15 309.1100 −2.09 C19 H16 O4 1810 [23]
147.0428 (enol form)
119.0491
217.0488 255.0994
187.0384 245.0787 Demethoxycurcumin
7 39.79 337.1056 −2.58 339.1205 −2.19 C20 H18 O5 1230 [23]
173.0593 175.0738 (enol form)
143.0489 147.0426
299.1256
217.0486
285.1100
8 40.44 367.1157 175.0383 −3.05 369.1309 −2.41 C21 H20 O6 Curcumin (enol form) 506 [23]
245.0789
Foods 2024, 13, x FOR PEER REVIEW173.0591 175.0739
5 of 14

tR represents retention time; ND represents not detected; * generated in negative mode.

Figure
Figure 1. Turmeric ethanol
1. Turmeric ethanolextract’s
extract’stotal
totalionion chromatogram
chromatogram (TIC)
(TIC) operated
operated innegative
in the the negative ion
ion mode.
mode. Numbers
Numbers (blue) shown
(blue) shown on of
on the top thethe
top of the
peak peakthe
indicate indicate thetentatively
order of order of tentatively identified
identified compounds
compounds
according toaccording to their
their retention retention
time shown time shown
in Table 1. in Table 1.

Compound
Compound55had hadm/z m/z values
valuesof of
383.1111 andand
383.1111 385.1257
385.1257in negative
in negativeand and
positive ion
positive
mode, respectively (Table 1). These values coincided with the molecular formula of
ion mode, respectively (Table 1). These values coincided with the molecular formula of
C2121H2020
C H O7 and
O 7 and it was speculated to be calebin A by searching in SciFinder. For its product
it was speculated to be calebin A by searching in SciFinder. For its product
ions (MS22))in
ions(MS in positive
positive mode mode (Table 1), the
(Table 1), m/z values
the m/z values were
were 261.0737
261.0737 [M+H–C
[M+H–C77H O22]]++and
H99O and
177.0531[M+H–C
[M+H–C +
177.0531 HH
1111 12O124O ] , which
]+,4 which are are
the the product
product ionsions ofloss
of the the of
loss of 2-methoxyphenol
2-methoxyphenol and
and methyl 3-(4-hydroxy-3-methoxyphenyl)acrylate
methyl 3-(4-hydroxy-3-methoxyphenyl)acrylate from calebin A, respectively.from calebin A, respectively.
Compound6 6showed
Compound showed thethe highest
highest intensity,
intensity, in which
in which bisdemethoxycurcumin
bisdemethoxycurcumin had m/zhad
m/z values of 307.0954 and 309.1100 in negative ([M − H] − ) and positive ([M+H]+ ) modes,
values of 307.0954 and 309.1100 in negative ([M–H] ) and positive ([M+H] ) modes, respec-
− +
respectively.
tively. These These
valuesvaluescoincidedcoincided
with with the molecular
the molecular formula
formula of Cof C19OH4 16(Table
19H16
O4 (Table 1). For
1). For its
its product ions (MS 2 ) in negative mode, m/z values of 187.0383, 143.0488, and 119.0491
product ions (MS2) in negative mode, m/z values of 187.0383, 143.0488, and 119.0491 were
were detected. It has been reported that m/z values of 187.0383 and 119.0491 were obtained
detected. It has been reported that m/z values of 187.0383 and 119.0491 were obtained by
by cleavage of the bond between C and C3 of bisdemethoxycurcumin, whereas the m/z
cleavage of the bond between C2 and2 C3 of bisdemethoxycurcumin, whereas the m/z value
value of 143.0488 was obtained by the loss of CO2 from the m/z 187.0383 fragment [22]
of 143.0488 was obtained by the loss of CO2 from the m/z 187.0383 fragment [22] (Figure
2). For bisdemethoxycurcumin’s MS2 in positive ion mode, two fragments, m/z 225.0892
and m/z 147.0428, were detected (Table 1). It has been reported that the fragment of m/z
225.0892 is generated by the loss of a 1-hydroxy-3-ketocyclobutene moiety from its pre-
cursor ion (MS), m/z 309.1100. Contrarily, an m/z 147.0428 fragment was generated by a C3
Foods 2024, 13, 1550 6 of 13

(Figure 2). For bisdemethoxycurcumin’s MS2 in positive ion mode, two fragments, m/z
225.0892 and m/z 147.0428, were detected (Table 1). It has been reported that the fragment
of m/z 225.0892 is generated by the loss of a 1-hydroxy-3-ketocyclobutene moiety from its
precursor ion (MS), m/z 309.1100. Contrarily, an m/z 147.0428 fragment was generated by
a C and C4 bond cleavage followed by a neutral loss of a 1-aryl-3-hydroxy-1,3-butadiene
Foods 2024, 13, x FOR PEER REVIEW 3 6 of 14
moiety [24]. By comparing MS and MS2 results with the previously published literature,
compound 6 was tentatively identified as bisdemethoxycurcumin.

Figure 2.
2. Identification
Identificationof
ofbisdemethoxycurcumin
bisdemethoxycurcumin(C(C
19H16O4). (A) Full-scan MS and (B) MS2 in neg-
2
Figure 19 H16 O4 ). (A) Full-scan MS and (B) MS in
ative ionization mode.
negative ionization mode.

Two other
Two other curcuminoids
curcuminoids (compounds
(compounds 77 and and 8) 8) that
that showed
showed the thesecond
secondand andthird
third
highest peak
highest peak intensity
intensity inin TIC,
TIC, corresponding
corresponding to to demethoxycurcumin
demethoxycurcuminand andcurcumin,
curcumin,had had
m/z values
m/z values of 337.1057 and
of 337.1057 and 367.1157
367.1157 in in negative
negative ionion mode,
mode, respectively
respectively(Table
(Table1). 1).These
These
m/z values
m/z values are
are 30
30 and
and 6060 higher
higher than
than bisdemethoxycurcumin
bisdemethoxycurcumindue duetotoone
onemethoxyl
methoxylgroup group
foundin
found indemethoxycurcumin
demethoxycurcumin and and two
two methoxyl
methoxyl groups
groups found
foundin incurcumin.
curcumin.InInthe thesame
same
way,both
way, both demethoxycurcumin
demethoxycurcumin and andcurcumin
curcuminshowed showedproduct
product ions having
ions having m/zm/zvalues of
values
and 217 compared to bisdemethoxycurcumin’s m/z
of 173 and 217 compared to bisdemethoxycurcumin’s m/z 143 and 187 fragments duetoto
173 143 and 187 fragments due
methoxyl group
methoxyl group differences
differences(Table
(Table1).1).Using
Usingthe same
the same principle
principleas described
as describedin identifying
in identify-
compound
ing compound 6, all other
6, all othercompounds
compounds found
found ininturmeric
turmericethanol
ethanolextract
extractwere
weretentatively
tentatively
identified. Intriguingly,
identified. Intriguingly, three
three curcuminoids,
curcuminoids, such such asas bisdemethoxycurcumin,
bisdemethoxycurcumin, demethoxy-
demethox-
ycurcumin,and
curcumin, andcurcumin,
curcumin,were weredetected
detected twice
twice atat different
different retention
retention times.
times.ForForinstance,
instance,
bisdemethoxycurcuminwas
bisdemethoxycurcumin wasdetected
detectedatata aretention
retentiontime timeofof31.44
31.44 and
and 39.08
39.08 min,
min, demeth-
demethoxy-
oxycurcumin
curcumin at 32.21
at 32.21 andand39.7939.79
min,min,
and, and, lastly,
lastly, curcumin
curcumin at 33.28
at 33.28 andand 40.44
40.44 minmin (Table1).
(Table
1). These phenomena had previously explained that these curcuminoids
These phenomena had previously explained that these curcuminoids exist as rapidly inter- exist as rapidly
interconverting
converting keto-enol
keto-enol tautomers
tautomers [23]. [23].
Previously, Yang et al. extracted turmeric powder using 80% ethanol and found
Table compounds,
seven 1. Characterization of chemical
including galliccompounds in turmeric (Curcuma
acid, protocatechuic longa L.) ethanol
acid, epicatechin, rutin, extract.
curcumin,
myricetin, and cinnamic acid
Negative Ionization Mode Positive Ionization Mode [25]. Only one curcuminoid (curcumin) was detected in
this study. In the present study, three curcuminoids, including bisdemethoxycurcumin, Ion
Peak tR Mass Mass Tentative
Product
demethoxycurcumin, and Product
curcumin, were detected.FormulaThese differences mayIntensity be partially Ref.
due
No. (min) [M-H]− Error [M+H]+ Error Identification
Ions Ions
to the different extraction solvents, different analytical methods, or the material (×10 )variations.
5 *
(mmu) (mmu)
1 18.49 163.0386 119.0489 −1.55
3.2. Antimicrobial ND
Activities ND ND C9H8O3 Coumaric acid 12 [21]
187.0386The antimicrobial effects of turmeric ethanol extract in a range of practical concen-
225.0894 Bisdemethoxycurc
2 31.44 307.0952 143.0490
trations−2.15
(0.1, 1,309.1097 mL−1 ) were
and 10 mg147.0429 −2.40 C19H16O
evaluated 4
against a variety 217
of Gram-negative [22]
and
umin (keto form)
119.0492
Gram-positive bacteria. The growth of all bacteria was inhibited by turmeric at the high-
est concentration of 10 mg 255.0994
mL−1 (dissolved in DMSO), while the lower concentrations
217.0483
appeared to suppress bacterial 245.0789growth in a strain-specific manner (Figure 3).
Demethoxycurcu
3 32.21 337.1056 173.0588 −2.58 339.1205 239.1071 −2.22 C20H18O5 135 [22]
min (keto form)
143.0485 231.1277
146.3248
299.1252
217.0484 285.1097
Curcumin (keto
4 33.28 367.1156 173.0589 −3.08 369.1309 259.0942 −2.40 C21H20O6 75 [22]
form)
135.0436 245.0787
Foods 2024, 13, x FOR PEER REVIEW 8 of 14
Foods 2024, 13, 1550 7 of 13

Figure Antimicrobialactivity
Figure3.3.Antimicrobial activityof
ofturmeric
turmericethanol
ethanolextract
extractdissolved
dissolvedin inDMSO.
DMSO.(A)(A)Bacterial
Bacterialgrowth
growth
(optical density at 600-nm wavelength; OD-D00) in trypticase soy broth (TSB) supplemented with
(optical density at 600-nm wavelength; OD-D00) in trypticase soy broth (TSB) supplemented with the
the dissolved extract. Error bars represent standard error. (B) Boxplots for area under the curve.
dissolved extract. Error bars represent standard error. (B) Boxplots for area under the curve. Colors
Colors correspond
correspond to treatment.
to treatment. The
The ***, **, ***,
and**, and * indicate
* indicate p < 0.001,
p < 0.001, p <and
p < 0.05 0.05pand p <respectively.
< 0.1, 0.1, respectively.

The Gram-positive
Pseudonomas sp. was bacteria
the onlyhad different growth
Gram-negative strainpatterns
in which and appeared
growth more sensi-
was abrogated at
−1 (Figure 3A). At the lowest
tive to turmeric (Figure 3A). At 0.1 mg mL , OD600 E. faecalis was
0.1 and 1 mg mL −1 concentration, OD 600lower at 6 h (p < 0.001),
for Pseudonomas sp. was
that
lower L. 3innocua
of at was lower
h compared to thatatof12the
h (p = 0.039),
strain whenand grown S. aureus
thatinofTSB without was lower at
turmeric (p 6< h0.024),
(p =
0.032), 12 h (p < a0.001),
demonstrating subtleand yet 24 h (p < 0.001).
significant effectInon media containing
lag-stage time for turmeric
its growth.at 1 mg mL−1,
Similarly,
−1 , OD
OD
at 1600mg of E.
mL faecalis was 600lower at 6 h (p < 0.001)
for Pseudomonas sp. was and significantly
12 h (p = 0.007), andat
lower that3 hof(pS.=aureus
0.013),was 6h
lower at 12 h (p < 0.001) and 24 h (p < 0.001). Accordingly, the AUC values for growth
(p = 0.043), and 24 h (p = 0.025) compared to the respective control, yielding a significantly
curves
lower for AUC S. aureus in media
(p = 0.005) with3B).
(Figure all concentrations
Thus, while E.ofcoli turmeric
and K. tested
pneumoniaewere significantly
appeared to
−1 (p >0.05 for all time points and for
suppressed relative to the control (p < 0.001 for each concentration) (Figure 3B). Thus, 0.1
tolerate turmeric at equivalents of 0.1 and 1 mg mL
AUC),
to 1 mg the mLgrowth
−1 turmericof Pseudomonas
appeared to was haveslowed
at leastby the level
some lowest ofconcentration
antimicrobialand significantly
activity against
suppressed at the medium level tested.
all of the Gram-positive bacteria tested, either in the form of complete inhibition (i.e., S.
aureus) The orGram-positive
limiting kinetics bacteria had different
for bacterial growth growth
(i.e., E.patterns
faecalis,and appeared more sensitive
L. innocua).
to turmeric (Figure 3A). At 0.1 mg mL −1 , OD E. faecalis was lower at (i.e.,
6 h (p0.1 < 0.001), that
The antimicrobial effects of turmeric at the lower concentrations
600 and 1 mg
of L. innocua was lower at 12 h (p = 0.039), and that
mL ) were validated with controls in which media augmented with DMSO alone
−1 of S. aureus was lower at 6 h (p = 0.032),
at 0.1%
12 h1% (p <(i.e.,
0.001), −1 , OD
and finaland 24 h (p < 0.001).
concentration In media
equivalents to containing
the amountturmeric
used to at 1 mg mL
dissolve turmeric) had
600 of
E. faecalis was lower at 6 h (p < 0.001) and 12 h (p = 0.007), and that
no effects on bacterial OD600 at any time point (p > 0.05 for all) or on AUC (p > 0.05 for all) of S. aureus was lower at
12 h (p < 0.001) and 24 h (p < 0.001). Accordingly, the AUC values for growth curves for
(Figure S1). We note that 10% DMSO had suppressive, though not entirely inhibitory, ef-
S. aureus in media with all concentrations of turmeric tested were significantly suppressed
fects on bacterial growth (Figure S1). Thus, comparing growth curves with turmeric at 10
relative to the control (p < 0.001 for each concentration) (Figure 3B). Thus, 0.1 to 1 mg mL−1
mg mL−1 was necessary to distinguish the specific antimicrobial properties of the food in-
turmeric appeared to have at least some level of antimicrobial activity against all of the
gredient. While there were trends for a lower average AUC for all bacteria grown in tur-
Gram-positive bacteria tested, either in the form of complete inhibition (i.e., S. aureus) or
meric at 10 mg mL−1 compared to 10% DMSO, the difference in AUC was only significant
limiting kinetics for bacterial growth (i.e., E. faecalis, L. innocua).
for E. coli (p = 0.017) and S. aureus (p < 0.001) (Figure 4), perhaps reflecting variation − in
The antimicrobial effects of turmeric at the lower concentrations (i.e., 0.1 and 1 mg mL 1 )
spectrophotometer absorbance values and/or growth at the highest concentration tested.
were validated with controls in which media augmented with DMSO alone at 0.1% and 1%
Nevertheless, the antimicrobial effects on AUC for E. coli, along with concentration- and
(i.e., final concentration equivalents to the amount used to dissolve turmeric) had no effects
time-dependent effects on growth of Pseudomonas sp., E. faecalis, L. innocua, and S. aureus,
on bacterial OD600 at any time point (p > 0.05 for all) or on AUC (p > 0.05 for all) (Figure S1).
were validated. We note that determining antimicrobial potential against K. pneumoniae
We note that 10% DMSO had suppressive, though not entirely inhibitory, effects on bacterial
was
growth limited by the
(Figure S1).scope
Thus,of the assay (i.e.,
comparing the only
growth curvesstrain
with tested in which
turmeric at 10growth
mg mL abroga-
−1 was
tion could not be distinguished from that induced by DMSO). Overall,
necessary to distinguish the specific antimicrobial properties of the food ingredient. While this study demon-
strates
there were that trends
turmericforhas antimicrobial
a lower average AUC activity
for allagainst
bacteriaa wide
grown spectrum
in turmeric of commonly
at 10 mg mL oc-
−1
curring opportunistic pathogens.
compared to 10% DMSO, the difference in AUC was only significant for E. coli (p = 0.017) and
Foods 2024, 13, 1550 8 of 13

S. aureus (p < 0.001) (Figure 4), perhaps reflecting variation in spectrophotometer absorbance
values and/or growth at the highest concentration tested. Nevertheless, the antimicrobial
effects on AUC for E. coli, along with concentration- and time-dependent effects on growth of
Pseudomonas sp., E. faecalis, L. innocua, and S. aureus, were validated. We note that determining
antimicrobial potential against K. pneumoniae was limited by the scope of the assay (i.e., the
Foods 2024, 13, x FOR PEER REVIEWonly strain tested in which growth abrogation could not be distinguished from that induced 9 of 14
by DMSO). Overall, this study demonstrates that turmeric has antimicrobial activity against a
wide spectrum of commonly occurring opportunistic pathogens.

Figure 4.
Figure 4. Antimicrobial effects of
Antimicrobial effects of turmeric
turmeric ethanol
ethanol extract
extract dissolved
dissolved in
in DMSO
DMSO (color
(color boxes)
boxes) relative
relative
to DMSO controls at the same concentration (gray boxes). Colors correspond to treatment. The The
to DMSO controls at the same concentration (gray boxes). Colors correspond to treatment. **
** and
and * indicate p < 0.05 and p < 0.1, respectively.
* indicate p < 0.05 and p < 0.1, respectively.

The antimicrobial
The antimicrobial properties
properties of of turmeric
turmeric have
have important
important food food safety
safety implications.
implications.
The E.
The E. coli
coli K-20
K-20 and
and L.L. innocua
innocua strains
strains used
used inin this
this study
study are
are surrogates
surrogates for for predicting
predicting the the
behavior of enteropathogenic E. coli and Listeria monocytogenes [26,27]. Although generic
behavior of enteropathogenic E. coli and Listeria monocytogenes [26,27]. Although generic
E. coli
E. coli bacteria
bacteria are
are aa commonly
commonly occurring
occurring commensal,
commensal, pathogenic
pathogenic strains
strains linked
linked to to the
the
consumption of contaminated or improperly prepared foods, such
consumption of contaminated or improperly prepared foods, such as E. coli O157:H7, can as E. coli O157:H7,
can
causecause illnesses,
illnesses, such such as stomachache,
as stomachache, bloody bloody diarrhea,
diarrhea, and vomiting.
and vomiting. In addition,In addition,
L. mon-
L. monocytogenes and even L. innocua can cause symptoms, such as fever,
ocytogenes and even L. innocua can cause symptoms, such as fever, diarrhea, vomiting, diarrhea, vomiting,
nausea,
nausea, andand even
even death
death [28]. Listeria species
[28]. Listeria species and E. coli
and E. coli are
are often
often linked
linked to to the
the consumption
consumption
of contaminated leafy vegetable and fresh-cut produce, as
of contaminated leafy vegetable and fresh-cut produce, as well as undercooked well as undercooked meats,
meats,
among other food products [29]. Our results demonstrate that 10 mg mL −1 turmeric
among other food products [29]. Our results demonstrate that 10 mg mL turmeric was −1
was able to significantly inhibit the growth of E. coli, and lower concentrations were
able to significantly inhibit the growth of E. coli, and lower concentrations were even sup-
even suppressive to L. innocua. When considering using turmeric in food systems such as
pressive to L. innocua. When considering using turmeric in food systems such as turmeric
turmeric salad dressing, one teaspoon of turmeric powder is often used [30]. The equivalent
salad dressing, one teaspoon of turmeric powder is often used [30]. The equivalent to 5.7
to 5.7 g of turmeric powder used to prepare the salad dressing, as dissolved in ½ cup of oil
g of turmeric powder used to prepare the salad dressing, as dissolved in ½ cup of oil and
and ¼ cup of vinegar, yields a final concentration of approximately 32 mg−1 mL−1 , which is
¼ cup of vinegar, yields a final concentration of approximately 32 mg mL , which is three-
three-times higher than the highest concentration (10 mg mL−1 ) used in the current study.
times higher than the highest concentration (10 mg mL−1) used in the current study. Thus,
Thus, in addition to the well-documented beneficial properties of turmeric on consumer
in addition to the well-documented beneficial properties of turmeric on consumer health
health [8], basic use as a food ingredient may have antimicrobial applications to mitigate
[8], basic use as a food ingredient may have antimicrobial applications to mitigate the risks
the risks for foodborne pathogen contamination.
for foodborne pathogen contamination.
The other strains evaluated in this study were largely members or phylogenetic
The other strains evaluated in this study were largely members or phylogenetic rela-
relatives of ESKAPE pathogens—Enterococcus faecium, S. aureus, K. pneumoniae, Acinetobacter
tives of ESKAPE
baumanii, pathogens—Enterococcus
Pseudomonas faecium, S.species—which,
aeruginosa, and Enterobacter aureus, K. pneumoniae,
as a group,Acinetobacter
are the
baumanii,cause
leading Pseudomonas aeruginosa,
of nosocomial and Enterobacter
infections worldwide species—which,
[31]. K. pneumoniae as aisgroup,
found are the
in soil,
leading cause of nosocomial infections worldwide [31]. K. pneumoniae is
skin, and foods and has been linked to a variety of diseases, including pneumonia, urinary found in soil, skin,
and foods
tract and has
infection, been linked
diarrhea, to a variety
meningitis, and of diseases,
sepsis. including
Although pneumonia,isurinary
K. pneumoniae tract
recognized
infection, diarrhea, meningitis, and sepsis. Although K. pneumoniae is
as non-food-associated bacteria, a recent infection report indicated that the infection is recognized as non-
food-associated
possibly due to thebacteria, a recent infection
food working report indicated
as a transmission that the
vector [32]. infection
While is possibly
our results were
due to the food working as a transmission vector [32]. While our results were inconclusive
as to whether turmeric can inhibit K. pneumoniae, which appeared to tolerate low concen-
trations of the extract, trends for the highest concentration tested, although insignificant,
suggest the potential may exist. Moreover, Pseudomonas is another important Gram-nega-
tive opportunistic pathogen group, in which members such as P. aeruginosa can cause
pneumonia and sepsis in immunocompromised patients [33]. While P. aeruginosa has been
Foods 2024, 13, 1550 9 of 13

inconclusive as to whether turmeric can inhibit K. pneumoniae, which appeared to tolerate

low concentrations of the extract, trends for the highest concentration tested, although
insignificant, suggest the potential may exist. Moreover, Pseudomonas is another important
Gram-negative opportunistic pathogen group, in which members such as P. aeruginosa can
cause pneumonia and sepsis in immunocompromised patients [33]. While P. aeruginosa
has been rarely associated with foodborne diseases, it is still a common spoilage agent
in foods with high water activity and nutrient contents, such as milk, meat, fruits, and
vegetables [34]. Given the observed sensitivity of the Pseudomonas strain in this study
to turmeric, even at relatively low concentrations, applications for the extract as a flavor
supplement and preservative agent would be an interesting future direction.
The other bacteria evaluated in this study, E. faecalis and S. aureus, may have impli-
cations for foods with high salt concentrations. E. faecalis is widely found in fermented
foods such as cured meats and cheeses. Contamination by E. faecalis often occurs during
food processing [35]. For example, fermented meat products such as salami and Land-
jager are processed without heat in many cases and have been reported to carry 102 to
105 colony-forming units/g (CFU/g) of E. faecalis [36]. Even when heat is applied, E. faecalis
can survive if the population level is intrinsically high due to its stress tolerance abilities
against temperature, pH, and salinity [35]. Similarly, S. aureus is a human skin commensal
that is widespread in the built environment such as on surfaces in processing facilities [37].
Some strains of S. aureus can produce toxins that cause human illness. Because the illness
caused by S. aureus is typically due to the toxins produced, antibiotics do not work as treat-
ment [37]. A previous study reported that turmeric’s major bioactive component, curcumin
ranging from 125 to 250 µg/mL, was able to inhibit the growth of several different strains
of S. aureus [38]. Likewise, the current study found that all evaluated concentrations (0.1,
1.0, and 10 mg mL−1 equivalents) had significant inhibitory effects against S. aureus. Thus,
the extract may have applications for biocontrol across a wide array of food types, ranging
from fresh produce (E. coli and L. innocua) to high water activity (Pseudomonas) and even
salt contents (E. faecalis, S. aureus).
In summary, the turmeric extract exerted antimicrobial activities against a variety
of model Gram-negative and Gram-positive bacteria. Although there are limitations for
applications of turmeric as functional food ingredients, such as consumer acceptance and
solubility (e.g., DMSO was used here), applications for turmeric among other herbs and
spices with effective antibacterial activity warrants investigation to improve food safety
and further support preservation.

3.3. TPC and Free Radical Scavenging Capacities

The phenolic compounds are the most abundant secondary metabolites widely found
in the plant kingdom. Because their role is to protect plants from environmental stresses,
such as light, extreme temperatures, and pathogen infection, phenolic compounds can be
applied as numerous food preservatives. In the present study, the TPC of turmeric ethanol
extract was 27.12 mg GAE/g turmeric (Table 2). This value is greater than 6.57 mg GAE/g
turmeric reported in the 95% v/v ethanol extract of turmeric [39]. In another study, Akter
and others evaluated six different species and varieties of turmeric using pure methanol as a
solvent and found that TPC values were in the range of 37.9–157.4 mg GAE/g turmeric [40].
These differences in results may be related to different extraction solvents and the potential
effects of turmeric genotype and growing conditions.
The evaluation of free radical scavenging capacities is important for both food safety
and quality. In addition, radical scavenging components may benefit human health, as
redox homeostasis is a basic requirement for performing various normal cellular func-
tions. Free radicals may increase oxidative stress, which may induce the risk of many
aging-associated human diseases through the activation of related signaling pathways,
immune disorders, DNA mutations, etc. [41]. In foods, free radicals may act as initiators of
lipid peroxidation and, consequently, reduce their shelf stability. In the current study, the
turmeric extract had ABTS, RDSC, and HOSC values of 1.70, 56.38, and 1524.59 µmol TE/g
Foods 2024, 13, 1550 10 of 13

turmeric, respectively (Table 2). HOSC value was examined for turmeric ethanol extract
for the first time. The ABTS and RDSC values are greater than that of 3.25 and 7.23 µmol
TE/g turmeric, respectively, reported previously for the turmeric water extract [42]. In
addition, the HOSC of turmeric ethanol extract was much lower than that of 2181.08 µmol
TE/g for clove ethanol extract [43] but much greater than that of 364.64 µmol TE/g for
the honeysuckle ethanol extract [44]. Hitherto, the free radical scavenging capacity of
turmeric toward the hydroxyl radical has been attributed to its major bioactive compo-
nents, curcuminoids [45,46]. For instance, Agnihotri and Mishra assessed the free radical
scavenging mechanism of curcumin against the hydroxyl radical. In their study, they found
that not only curcumin itself works as an antioxidant for hydroxyl radicals but, also, its
degradation products from curcumin including ferulic acid and vanillin can work together
as antioxidants [46]. Consequently, bioactive compounds found in turmeric may extend
the shelf life of foods since many foods using turmeric are cooked with either oil or fat. In
addition, the use of turmeric in preparing foods can also aid in maintaining good health
conditions by reducing oxidative stress. Oxidative stress is caused when the body’s redox
homeostasis is imbalanced. This can happen through different factors, including smoking,
alcohol drinking, and exposure to environmental contaminants, as well as sunlight [47].
Basically, these factors generate excessive reactive oxygen species. Therefore, to prevent
oxidative stress, more antioxidants are needed. However, there is a limit to the antioxidants
that the body can produce. Considering this, extra sources of antioxidants are necessary,
and foods such as turmeric can serve as an extra source.
Table 2. Total phenolic content (TPC) and free radical scavenging capacities of turmeric
ethanol extract.

TPC Free Radical Scavenging Capacities

(mg GAE/g (µmol TE/g Turmeric)
Turmeric*) ABTS RDSC HOSC
27.12 ± 0.52 1.70 ± 0.58 56.38 ± 1.18 1524.59 ± 29.89
Turmeric* stands for turmeric ethanol extract. TPC stands for total phenolic content. ABTS, RDSC, and HOSC stand
for ABTS•+ radical scavenging capacity, relative DPPH scavenging capacity, and hydroxyl radical scavenging
capacity, respectively. The final concentrations used for ABTS, RDSC, and HOSC assays were 7.4, 50, and
10 mg dry turmeric equivalents/mL, respectively. TE stands for Trolox equivalents. The results are reported in
mean ± standard deviation (n = 3).

4. Conclusions
Eight phenolic compounds (including coumaric acid, bisdemethoxycurcumin (keto
form), demethoxycurcumin (keto form), curcumin (keto form), calebin A, bisdemethoxy-
curcumin (enol form), demethoxycurcumin (enol form) and curcumin (enol form)) were
identified in the ethanol extract of turmeric through UHPLC-MS/MS analysis in this study.
Coumeic acid and calebin A were reported in the turmeric ethanol extract for the first
time. The ethanol extract of turmeric demonstrated concentration-dependent inhibitory
effects against Gram-negative bacteria, including E. coli and Pseudomonas sp., as well as
Gram-positive bacteria, such as E. faecalis, L. innocua, and S. aureus. The antibacterial ac-
tivity of turmeric ethanol extract against E. faecalis and L. innocua are reported for the first
time in the present study. The ethanol extract of turmeric also contained higher TPC and
showed scavenging activities against HO• , ABTS•+ , and DPPH• . HO• scavenging capacity
was reported for the turmeric extract for the first time. The results suggest that turmeric
and its extracts may have applications for use as antibacterial agents in foods to prevent
food spoilage and reduce risks for food safety. At the same time, the use of turmeric in
home-cooked dishes may provide potential health benefits by quenching excessive free
radicals. Exploring how metabolites of turmeric compounds can provide potential health
benefits would be an interesting future direction.

Supplementary Materials: The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/foods13101550/s1, Figure S1: Effects of DMSO on bacterial growth.
Foods 2024, 13, 1550 11 of 13

Author Contributions: Conceptualization, X.H. and L.Y.; methodology, L.Y.; formal analysis, Y.Z.,
B.G. and P.C.; data curation, H.W., Z.L., Y.L., U.C. and R.A.B.; investigation, Z.L., Y.L. and U.C.;
validation, J.S.; writing—original draft preparation, H.W. and U.C.; writing—review and editing,
R.A.B. and L.Y.; visualization, H.W., U.C. and R.A.B.; supervision, L.Y. All authors have read and
agreed to the published version of the manuscript.
Funding: This research was partially supported by cooperative agreement with USDA-ARS (NACA
No. 58-8040-1-012, and 58-8042-3-058).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article and Supplementary Materials, further inquiries can be directed to the corresponding authors.
Conflicts of Interest: The authors declare no conflicts of interest.

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