Food Chemistry: X 20 (2023) 100901

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Food Chemistry: X
journal homepage: www.sciencedirect.com/journal/food-chemistry-x

The potential mechanism of low-power water bath ultrasound to enhance

the effectiveness of low-concentration chlorine dioxide in inhibiting
Salmonella Typhimurium
Wei Luo a, Jie Tang a, *, Beibei Wang a, Di Wu a, Jinqiu Wang a, Lei Cheng b, *, Fang Geng a, c, *
a
Institute for Egg Science and Technology, School of Food and Biological Engineering, Chengdu University, 2025 Chengluo Avenue, Chengdu, China
b
School of Food and Health, Beijing Technology & Business University (BTBU), Beijing 100048, China
c
Meat Processing Key Laboratory of Sichuan Province, School of Food and Biological Engineering, Chengdu University, No. 2025 Chengluo Avenue, Chengdu 610106,
China

A R T I C L E I N F O A B S T R A C T

Keywords: This chapter presents a systematic study of the inhibition effect of chlorine dioxide treatment alone and in
Chlorine dioxide combination with ultrasound treatment of Salmonella and the physiological metabolic processes within the
Low-power ultrasound treated cells. The low-power ultrasound (0.03 W/mL) significantly enhanced the effectiveness (110.00 %) of low
Salmonella
concentrations of chlorine dioxide (0.25 mg/L) in inhibiting Salmonella, which, in turn, would significantly
Substance metabolism
Energy metabolism
reduce the potential environmental impact. In addition, further studies found that low-power ultrasound may
enhance the structural and functional damage of chlorine dioxide on Salmonella cell membranes (significant
increase in permeability of the outer and inner cell membranes) and disrupt intracellular substance metabolism
(small molecule and nucleotide metabolism) and energy metabolism (significant reduction in ATP content and
ATPase activity) balance to improve the bacterial inhibitory effect of chlorine dioxide. The results of the study
will provide a theoretical basis and methodological guidance for the implementation of “cleaner production” in
the food industry.

1. Introduction a key component of cleaner production in the food industry.

As a highly effective chemical disinfectant, chlorine dioxide has been
With the rapid growth of the global population and rising con­ used in the disinfection of drinking water, surface cleaning of fruits and
sumption levels, the food industry has entered a phase of rapid devel­ vegetables, and disinfection of industrial waste water (Bridges et al.,
opment. However, with the rapid growth of the food industry, it is also 2020). In addition, chlorine dioxide solutions are often chosen to reduce
putting enormous pressure on resources and the environment. During bacterial hazards during slaughter and cleaning in the poultry industry
the production and processing process, a large amount of “clean water” (Anang et al., 2006). However, the excessive use of chlorine dioxide in
is required to ensure food quality and safety and to avoid “secondary food processing can have an impact on the quality of some foods and
contamination”, both for pre-treatment and for further subsequent pose a health risk to consumers. For example, studies have shown that
processing and cleaning of the environment and equipment (Shrivastava high doses of chlorine dioxide can lead to significant production of toxic
et al., 2022). Secondly, food is a complex system that is rich in various by-products such as chlorite and chlorate ions (Ayyildiz et al., 2011). In
nutrients, and wastewater from the food industry is highly susceptible to addition, excessive chlorine residues in wastewater are potentially
carrying various harmful microorganisms (e.g., E. coli, Salmonella, etc.) harmful to the production environment and the natural environment,
(Sto i et al., 2016). If left unchecked, industrial effluent will result in having a potential impact on aquatic ecosystems and resulting in the
huge amounts of pollution entering the water environment, posing a accelerated emergence of some drug-resistant strains of bacteria (Roy &
serious health risk (Pratap et al., 2023). An energy-efficient supply of Ghosh, 2017).
“clean water” and subsequent wastewater treatment steps are therefore In recent years, physical processing technologies such as ultrasound

* Corresponding authors at: Institute for Egg Science and Technology, School of Food and Biological Engineering, Chengdu University, 2025 Chengluo Avenue,
Chengdu, China.
E-mail address: gengfang@cdu.edu.cn (F. Geng).

https://doi.org/10.1016/j.fochx.2023.100901
Received 5 May 2023; Received in revised form 24 July 2023; Accepted 23 September 2023
Available online 25 September 2023
2590-1575/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
W. Luo et al. Food Chemistry: X 20 (2023) 100901

have become a new research hotspot in the field of water treatment 2.2. Evaluation of antibacterial effect
research. Among other things, it has been suggested that ultrasound may
be used to inhibit/kill microorganisms through the physical and chem­ The number of viable cells of Salmonella after different treatments
ical effects of cavitation (Dai et al., 2020). However, it can also be found (CK, CD, WU, CD-WU) was calculated using the standard plate counting
that the ultrasonic treatment conditions (time and power) required to method (Bi et al., 2020). Specifically, 1 mL of the treated sample was
achieve a certain level of bacterial inhibition are demanding. For diluted with a 0.85 % (w/v) NaCl solution gradient (1:10), and 0.1 mL of
example, in a previous study, it was found that treatment with 3.46 W/ the dilution was aspirated and applied (plate counting agar) in dupli­
mL for 15 min or 13.85 W/mL for 5 min resulted in only about 0.6 log cate. Scientific counts were carried out at 37 ◦ C after 24 h of resting
reduction in Salmonella (Luo et al., 2022a). The consequently high cost incubation. Finally, log(N) was calculated on the results of the colony
of equipment purchase and huge energy consumption limit the practical counts (N, CFU/mL) to determine the inhibition effect.
use of ultrasound in industrial production (Al-juboori et al., 2015).
As mentioned above, the limited effectiveness of a single inhibition
method, the harsh treatment conditions, and the constraints of energy 2.3. Observation of cell surface and microstructure
and environment have led to a new research trend of using multiple
inhibition methods together. For example, some recent studies have The samples (CK, CD, CD-WU) were processed in the same way as in
preliminarily found that ultrasound can effectively increase the anti­ the previous study (Luo et al., 2022a), and then the surface structure of
bacterial effect of some natural oils (He et al., 2021). However, there are the Salmonella cells was observed via scanning electron microscopy
still few studies on the inhibition of bacteria by ultrasound in combi­ (SEM, Thermo scientific Apreo 2C, Thermo Fisher Scientific (China)
nation with chlorine dioxide, especially those examining the deeper Ltd., China). The analysis of each treatment sample was repeated three
molecular mechanisms, which further limits the development and times, and a typical image was selected for presentation.
application of related technologies (Zhou et al., 2016). After processing the samples (CK, CD, CD-WU) in the same way as
In addition, the outbreak and spread of Salmonella, a typical food­ described in the previous study (Luo et al., 2022a), the internal struc­
borne pathogen frequently found in food, has not only caused huge tural changes exhibited by the Salmonella cells were observed via
losses to the industry’s production, but also poses a great threat to the transmission electron microscopy (TEM, Hitachi-7800, Hitachi Scienti­
lives and health of consumers, seriously affecting the orderly develop­ fic Instruments Ltd., Japan). This was repeated three times for each
ment of the social economy (Sarjit et al., 2019). Therefore, in this study, treatment sample, and a typical image was selected for presentation.
the conditions for the implementation of an efficient means of bacterial
inhibition and the mechanism of action behind the inhibition effect are
investigated, using Salmonella as the subject of the study. The results of 2.4. Cell outer membrane permeability assay
this study provide scientific guidance and an application basis for the
efficient provision of clean water and the scientific management of The effect of ultrasound combined with chlorine dioxide treatment
wastewater in the food industry, and they lay the foundation for the on the permeability of the outer membranes of Salmonella cells was
implementation of “clean production” in the food industry. investigated using N-phenyl-1-naphthylamine (NPN) (He et al., 2021).
NPN fluoresces once it enters the interior of a bacterial cell that has been
2. Materials and methods damaged by the outer membrane and has failed to function (Mellegård
et al., 2011). The suspensions of the Salmonella treated under different
2.1. Microbial preparation and treatment conditions (CK, CD, CD-WU) were mixed thoroughly with NPN
(Shanghai Maclean’s Biochemistry Co., Ltd., China) solution. The final
The same method as that used in the previous study (Luo et al., concentration of NPN was 8 µM. The fluorescence intensity was then
2022b) was used to activate Salmonella typhimurium CGMCC1.1859 monitored using a fluorescence spectrophotometer (FL970, Shanghai
(The China General Microbiological Culture Collection Center, Beijing, Tianmei Scientific Instruments Co., Ltd., China) until the fluorescence
China). Then, 0.2 mL of the activated Salmonella solution was added to intensity stabilised and the corresponding values were recorded. The
20 mL of fresh nutrient broth medium and incubated for 11 h, and initial excitation wavelength was set to 350 nm and the emission wavelength to
stable growth was obtained. A volume (3–6 mL) of stable growth Sal­ 420 nm, and the analysis of each treatment sample was repeated three
monella was aspirated, centrifuged at 1685 × g for 5 min and resus­ times.
pended in an equal volume of PBS (pH 7.2). Finally, 1 mL of the bacterial
resuspension was added to 98.75 mL of sterilised deionised water to
produce a final concentration of 1.50 × 107-2.50 × 107 CFU/mL of 2.5. Flow cytometry
Salmonella working solution. Chlorine dioxide (Beijing Kangmu Veteri­
nary Pharmacy Centre) was added to sterilised deionised water to pro­ The effect of ultrasound combined with chlorine dioxide treatment
duce a chlorine dioxide stock solution (100 mg/L). For bacteriostatic on the permeability of the inner membranes of Salmonella cells was
treatment, 0.25 mL of the chlorine dioxide stock solution was added to further validated using SYTO9 (S34854, Thermo Fisher Scientific
99.75 mL of the Salmonella working solution (the final concentration of (China) Ltd., China) and PI (C0080, Beijing Sollerbauer Technology Co.,
chlorine dioxide was 0.25 mg/L). China) stains (Bridges et al., 2020). First, 500 μL of the sample (CK, CD,
Finally, the working solution without (WU) and with (CD-WU) added CD-WU) was centrifuged in a 2 mL centrifuge tube at 4 ◦ C for 3 min at
chlorine dioxide was subjected to 150 W sonication for 5 min in a water 6790 × g. The supernatant was discarded, and the wash was repeated.
bath (SB-5200D, frequency 40 kHz, water bath volume 4.5 L, Ningbo Then, 400 μL of 0.85 % Nacl was added to resuspend the cells; 200 μL of
Xinzhi Biotechnology Co.). Meanwhile, the untreated (CK), chlorine bacterial resuspension was removed from each tube and placed into a
dioxide-treated (CD) Salmonella working solutions were placed under new 2 mL centrifuge tube, before being vortexed and mixed well for use.
magnetic stirring for 5 min at 400 r/min. The temperature of all SYTO 9 and PI were then diluted 10 times with pure water, and 2 μL of
experimental treatments was controlled at 25 ◦ C. The power density of SYTO 9 stain was added to the samples for 20 min, followed by 2 μL of PI
water bath ultrasound (D, 0.03 W/mL) was calculated as D = P/V, where stain. After 20 min at room temperature with protection from light,
P is the input power and V is the total volume of the bathing water and apoptotic changes were detected using the Green-B and Red-B channels
Salmonella working solution (Li et al., 2017). of the flow cytometer (Guava EasyCyte, Luminex, USA), and the flow
data were analysed using FlowJo. The analysis of each treatment sample
was repeated three times.

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W. Luo et al. Food Chemistry: X 20 (2023) 100901

2.6. Determination of intracellular ATPase activity and total ATP content compound/). In addition, P-values from hypergeometric tests were
used to identify significantly enriched pathways.
The ATP content in Salmonella cells after treatment under different
conditions (CK, CD, CD-WU) was measured using an enhanced ATP 2.8. Statistical analysis
assay kit (Shanghai Biyuntian Biotechnology Co., Ltd., China) (Luo et al.,
2021). Chemiluminescence values were measured using a multifunc­ Data in this study are expressed as mean ± standard deviation.
tional enzyme marker (Synergy HTX, BioTek Instruments, Inc., Additionally, statistical analysis via one-way analysis of variance was
Winooski, VT, USA), and analysis of each treatment sample was performed using GraphPad Prism 8.0 software. P < 0.05 indicates sig­
repeated three times. nificant differences.
The changes in intracellular ATPase activity of Salmonella after
treatment under different conditions (CK, CD, CD-WU) were measured
3. Results and discussion
using an ultra-trace total ATPase test kit (Nanjing Jiancheng Institute of
Biological Engineering, Nanjing, China) (Luo et al., 2022a). The OD636
3.1. Low-power ultrasound treatment enhances bacterial inhibition of
values were first measured using a multifunctional enzyme marker
chlorine dioxide at low concentrations
(Synergy HTX, BioTek Instruments, Inc., Winooski, VT, USA), and then
the ATP enzyme activity was calculated according to the formula given
In this study, the number of viable Salmonella cells did not decrease
in the kit (ATPase activity = Total ATPase activity/Sample protein
significantly (by approximately 0.06 log) after low-power ultrasound
concentration). In this case, the assay was repeated three times for each
treatment alone (WU group, 0.03 W/mL). Chlorine dioxide treatment at
treatment sample.
0.25 mg/L alone (CD group) only caused a 1.51 log decrease in viable
Salmonella cells. The combined treatments of 0.25 mg/L chlorine dioxide
2.7. Intracellular metabolic changes—widely targeted metabolomic
and low-power ultrasound (CD-WU group) caused a 3.17 log decrease in
analysis
viable Salmonella cells (Fig. 1). Therefore, low-power ultrasound
significantly enhanced the effect of low concentrations of chlorine di­
2.7.1. Sample preparation and metabolites extraction
oxide on the inhibition of Salmonella. In other studies, it has been found
Samples stored at − 80 ◦ C were thawed on ice. Then, 500 μm of
that treatment with 17 mg/L chlorine dioxide alone only reduces the
methanol/water solution (4:1, V/V, containing internal standard) was
number of Salmonella cells by about 2.00 log (initial concentration of
added to the Salmonella samples and vortexed for 3 min. The samples
approximately 1.30 × 107) (Paula Rossi et al., 2021). In another previ­
were each placed in turn on liquid nitrogen and dry ice for 5 min each,
ous study, it was also found that ultrasound at 13.85 W/mL for 25 min
then thawed on ice and vortexed for 2 min. The freeze–thaw cycle was
only resulted in a decrease in the number of Salmonella cells by
repeated a total of three times and then centrifuged at 16904 × g for 10
approximately 2.87 log (Luo et al., 2022a). The low-power ultrasound
min (4 ◦ C). A 300 μL volume of supernatant was collected and left at
combined with the low-concentration chlorine dioxide treatment con­
− 20 ◦ C for 30 min. It was then centrifuged again at 16904 × g for 3 min
ditions optimised in this study are therefore not only effective at
(4 ◦ C). Then, 200 μL of supernatant was aspirated for analysis via ultra-
reducing the potential for chlorine residues in “clean water” in the food
performance liquid chromatography–tandem mass spectrometry. The
industry, but also at reducing energy consumption. This is a further
analysis of each treatment sample was repeated four times.
indication of its potential application in the food industry. A previous
study also found that lower power levels and treatment times were the
2.7.2. Data acquisition
most important parameters for achieving energy-efficient, cost-effec­
The data acquisition instrumentation system consisted mainly of
tive, and effective bacterial inhibition in continuous sonication mode
ultra-performance liquid chromatography (ExionLC AD, https://sciex.
(Al-juboori et al., 2015). In addition, not only does it enhance the
com.cn/, UPLC) and tandem mass spectrometry (Tandem mass spec­
antibacterial effect of chlorine dioxide, but ultrasound has also been
trometry, QTRAP®, https://sciex.com/, MS/MS).
found to enhance the antibacterial effect of some natural antibacterial
For chromatographic acquisition, a Waters ACQUITY UPLC HSS T3
substances (He et al., 2021). However, there is a lack of understanding
C18 column (1.8 µm, 2.1 mm*100 mm) was selected. Mobile phase A
was ultrapure water and B was acetonitrile, both of which contained 0.1
% formic acid. The elution gradient program consisted of 0 min for
water/acetonitrile (95:5 V/V), 11.0 min for 10:90 V/V, 12.0 min for
10:90 V/V, 12.1 min for 95:5 V/V and 14.0 min for 95:5 V/V. In addi­
tion, the flow rate was 0.35 mL/min; the column temperature was 40 ◦ C;
and the injection volume was 2 μL.
The electrospray ion source temperature in the mass spectrometry
acquisition was 500 ◦ C, the mass spectrometry voltages were 5500 V and
− 4500 V, the ion source gas I was 50 psi, gas II was 50 psi, the gas
curtain gas was 25 psi and the parameters for collision-induced ionisa­
tion were high. In the triple quadrupole, the ion pair was scanned for
detection based on the declustering voltage and collision energy (after
optimisation).

2.7.3. Data processing

First, principal component analysis (PCA) and partial least squar­
es–discriminant analysis (PLS-DA) were carried out with the functions in
R (www.r-project.org). In addition, variable importance in projection
Fig. 1. Low-power ultrasound treatment enhances the inhibition of Salmonella
(VIP) values were extracted from the PLS-DA results. Differential me­ with low concentrations of chlorine dioxide. CK, untreated group; WU, ultra­
tabolites (DMs) that changed significantly between groups were then sound alone (0.03 W/mL for 5 min); CD, chlorine dioxide alone (0.25 mg/L for
identified by VIP ≥ 1 and FC > 1.2 or FC < 0.83 (FC, fold change). 5 min); CD-WU, 0.25 mg/L chlorine dioxide combined with 0.03 W/mL ultra­
Finally, an annotated analysis of differential metabolites was performed sound for 5 min. Significant differences (P < 0.05) between groups are indi­
using the KEGG compound database (https://www.kegg.jp/kegg/ cated by different letters (a–c).

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with respect to the molecular mechanisms behind the inhibition effect. shaped with intact surface morphology and structure. However, some of
Therefore, further investigation of the mechanisms behind the enhanced the cells in the CD group had depressed surfaces, and a large number of
bacterial inhibition of chlorine dioxide by low-power ultrasound treat­ Salmonella cells in the CD-WU group had depressed surfaces and the cell
ment will help to develop more efficient and production-appropriate structures were completely destroyed, accompanied by the exudation of
sterilisation technologies. intracellular material. The TEM results also showed a similar trend
(Fig. 2C, D). Salmonella cells in the CK group had a clear wall membrane
3.2. Low-power ultrasound treatment enhances damage to Salmonella cell structure and an intact electron-transparent zone in the cytoplasm.
membrane structure caused by chlorine dioxide However, cells in the CD group basically had an intact wall membrane
structure, but some cells had an enlarged electron-transparent zone and
The structural and functional integrity of bacterial cell membranes is abnormal aggregation of major components. In the CD-WU group, the
essential for the maintenance and protection of the normal physiological electron-transparent zone in the cytoplasm of a large number of Sal­
activities of bacterial cells, as they selectively control the entry and exit monella cells was further enlarged, the abnormal aggregation of major
of substances into and out of the cell and protect the normal conduct of components increased, and the wall membrane structure was disrupted.
intracellular physiological reactions (Barák & Muchová, 2013; Strahl & The destruction of the Salmonella cell structure and the loss of material
Errington, 2017). In this study, the mechanisms behind the enhanced under each treatment also showed results consistent with those of the
bacterial inhibition of chlorine dioxide by low-power ultrasound were inhibition assay of each treatment. In addition, the electron microscope
further investigated through visualisation of the Salmonella cell micro­ results suggest that low concentrations of chlorine dioxide may not
structure and an examination of the function of the cell membrane. directly damage the cell wall membrane structure of Salmonella, but only
have a serious effect on the composition of the intracellular material.
3.2.1. Damage to the cell membrane structure Similar results have been found in other studies (Bridges et al., 2020).
SEM and TEM were used to observe changes in the microscopic The disruption of the Salmonella cell membrane structure and the
morphology and internal structural components of Salmonella cells after composition of intracellular material was further enhanced by treatment
treatment with chlorine dioxide alone (CD group) and combined with with low-power ultrasound. In previous studies, it has been found that a
low-power ultrasound (CD-WU group). The SEM results (Fig. 2A, B) single low-power ultrasound treatment has a potentially detrimental
showed that most of the cells in the untreated group (CK group) were rod effect on the structural integrity of cell membranes and the control of

Fig. 2. Low-power ultrasound treatment enhances damage to Salmonella cell membrane structure with chlorine dioxide treatment. (A, B) Unzoomed and zoomed
SEM images of CK, CD, and CD-WU-treated Salmonella. (C, D) Unzoomed and zoomed TEM images of CK, CD, and CD-WU-treated Salmonella. CK, untreated group;
CD, chlorine dioxide alone (0.25 mg/L for 5 min); CD-WU, 0.25 mg/L chlorine dioxide combined with 0.03 W/mL ultrasound for 5 min.

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material entry and exit in bacteria such as E. coli and Salmonella (He dioxide into the cell, increasing the risk of exposure of intracellular
et al., 2021; Luo et al., 2022b). material and energy metabolic processes to chlorine dioxide (Ofori et al.,
2018). This may also be an important reason for the consistency shown
3.2.2. Disruption of cell membrane function between the altered permeability of the outer membrane and the syn­
The uptake of NPN by Salmonella cells after chlorine dioxide (CD ergistic inhibition effect of ultrasound in combination with chlorine
group) treatment alone and in combination with low-power ultrasound dioxide, but further studies are needed to demonstrate this.
(CD-WU group) treatment is shown in Fig. 3A. The results show that Flow cytometry was employed in this study to further assess the
chlorine dioxide treatment increases the permeability of the outer damage status of the Salmonella cell membrane. The combination of
membrane of Salmonella cells to some extent (31.23 % enhancement SYTO 9, which stains all cells in the system because of its conventional
compared to CK), which is also consistent with the results of the TEM membrane permeability, and PI, which can only access cells with
images obtained in the previous section. The increase in cell membrane damaged inner and outer membranes, can often be used to characterise
permeability may be a causal factor in bacterial cell death when there is changes in cell survival (Rosenberg et al., 2019). In this study, it was
no apparent morphological damage to the cell. Chlorine dioxide has found that the CD group treatment did not cause a significant increase
been shown in previous studies to increase the permeability of the outer (about 0.9 %) in PI binding compared to the CK group (Fig. S1C).
membrane of E. coli cells and Pseudomonas aeruginosa (Ofori et al., 2017; Furthermore, by comparison with the changes in the permeability of the
Ofori et al., 2018). Moreover, this study further revealed that low-power outer membrane of Salmonella cells described above, it can be tenta­
ultrasound significantly enhances the effect of chlorine dioxide on outer tively concluded that low concentrations of chlorine dioxide only in­
cell membrane permeability (66.25 % enhancement compared to CK) of crease the permeability of the outer membrane of Salmonella cells, but
Salmonella. In our previous study, it was found that ultrasound alone do not significantly affect the permeability of the inner membrane. It
under the same conditions was also effective at increasing the outer was also found that the change in permeability of the extracellular
membrane permeability of Salmonella cells (11.96 %) (Luo et al., 2022b; membrane caused by chlorine dioxide treatment was not sufficient to
Zhang et al., 2017). Thus, combined treatment can have a more severe allow PI to enter the cell (Bridges et al., 2020). A significant increase in
damaging effect on Salmonella cell membrane function than ultrasound PI binding (about 11.10 %) was found in Salmonella cells after CD-WU
or chlorine dioxide treatment alone. In addition, a further increase in the treatment (Fig. S1D). This indicates that, with the aid of ultrasound,
permeability of the outer membrane of Salmonella, assisted by ultra­ chlorine dioxide increases the damage to the inner cell membrane, again
sound, would mean a much higher probability of entry of chlorine indicating an increased potential for chlorine dioxide to enter the cell

Fig. 3. Low-power ultrasound treatment enhances damage to Salmonella cell with chlorine dioxide treatment. (A) Outer membrane permeability of CK, CD and CD-
WU-treated Salmonella cells. (B) Electrical conductivity in extracellular supernatant after CK, CD and CD-WU treatment. (C) ATP content of the CK, CD and CD-WU
treated Salmonella cells; (D) ATPase activity of the CK, CD and CD-WU-treated Salmonella cells. CK, untreated group; CD, chlorine dioxide alone (0.25 mg/L for 5
min); CD-WU, 0.25 mg/L chlorine dioxide combined with 0.03 W/mL ultrasound for 5 min. Significant differences (P < 0.05) between groups are indicated by
different letters (a–c).

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interior. The damage to the inner and outer cell membranes also in­ 3.4. Low-power ultrasound treatment enhances the effect of chlorine
creases the likelihood of further material loss or lysis of the cells, and the dioxide treatment on the metabolic processes of substances in Salmonella
electron microscopic results described above also strongly support this cells
hypothesis.
Therefore, this study initially found and confirmed that low con­ In summary, the study of the structural function of Salmonella cell
centrations of chlorine dioxide caused more sub-lethal damage to the membranes and the changes in intracellular substances revealed some
outer cell membrane by increasing the permeability, while auxiliary preliminary clues to the mechanisms by which low-power ultrasound
treatment with ultrasound not only increased the damaging effect on the treatment enhances the bacterial inhibitory effect of chlorine dioxide.
outer cell membrane, but also increased the damage to the inner cell We will continue to investigate the effects of low-concentration chlorine
membrane and increased the exchange of substances between the inside dioxide combined with low-power sonication on the intracellular
and outside of the cell (with harmful substances moving into the cell and metabolic processes of Salmonella by applying a widely targeted
functional substances moving out of the cell). This may also be an metabolomic approach to unravel the molecular mechanisms underly­
important reason why low-power ultrasound significantly enhances the ing the phenotypic changes.
lethality of low concentrations of chlorine dioxide. The trend of con­
ductivity in the extracellular supernatant further confirms this specu­ 3.4.1. Differential metabolites (DMs)
lation (Fig. 3B). In this study, a total of 1417 metabolites were identified in the Sal­
monella cells. PLS-DA analysis of the overall metabolites revealed that
the metabolite profiles of the CK group and the two treated groups (the
3.3. Low-power ultrasound treatment enhances the dysregulation of CD group and the CD-WU group) showed a clear separation (Fig. 4A).
energy supply in Salmonella cells with chlorine dioxide treatment Pairwise comparisons between the two groups were performed, and a
total of 575 differentially abundant metabolites were identified. The
Energy metabolism plays a vital role in the physiological activity, largest number of DMs (417 DMs) was identified when comparing the
molecular function, growth, and survival of living organisms. However, CD-WU and CK groups, and 69.30 % of the DMs showed a decrease in
when bacterial cells are adversely affected by external influences, the abundance; meanwhile, 411 DMs were identified between the CD and
intracellular energy balance is affected, and more energy is required to CK groups (Fig. 4B). Thus, ultrasound-assisted processing may play a
sustain some of the physiological metabolic reactions within the bac­ more critical role for the disturbance of intracellular metabolites of
terial cell (Luo et al., 2022a). In this study, a 78.63 % reduction in ATP Salmonella. In addition, 253 DMs were found in this study to respond to
content was found in Salmonella cells after treatment with chlorine di­ both CD and CD-WU treatment, and these metabolites may be potential
oxide (CD group) alone compared to the CK group (Fig. 3C). A similar markers of Salmonella response to chlorine dioxide treatment (Fig. 4C).
finding was reported in a previous study by Bridges et al., who found
that chlorine dioxide treatment could lead to a potential decrease in ATP 3.4.2. KEGG enrichment analysis
content in E. coli cells (Bridges et al., 2020). On the one hand, it is In addition, to deepen our understanding of the biological signifi­
possible that the internal physiological regulation of Salmonella requires cance of the results, KEGG enrichment analysis was performed to infer
a lot of energy in order to resist the stressful effects of chlorine dioxide. the interconnections of metabolites in biological pathways. The 411
On the other hand, it has been shown that chlorine dioxide reacts rapidly DMs of the CD group Salmonella were annotated to 102 KEGG pathways
with NADH, which in turn affects the production of ATP (Bakhmutova- and compared to the CK group. These pathways were functionally
Albert et al., 2008). In our previous study, it was also found that ultra­ related to the biosynthesis and metabolism of various amino acids, lipid
sound alone under the same conditions caused a 14.15 % decrease in biosynthesis, nucleotide metabolism, and energy metabolism, and were
intracellular ATP in Salmonella (Luo et al., 2022b). In contrast, in the significantly enriched in the ’toluene degradation’ and ’purine meta­
present study, the combined treatment (CD-WU group) was found to bolism’ pathways (P < 0.05) (Fig. S2A). When comparing the CD-WU
result in a further reduction (91.66 %) in ATP content in Salmonella and CK groups, a total of 110 KEGG pathways were enriched in all
(Fig. 3C). Thus, the dysregulation of the supply of direct energy- 417 DMs, and these pathways were also closely functionally related to
supplying substances to Salmonella cells following chlorine dioxide various types of amino acid biosynthesis and metabolism, nucleotide
treatment was further enhanced by low-power ultrasound treatment, metabolism and lipid biosynthesis (Fig. S2B).
potentially contributing to the greater degree of lethality.
In addition to the abnormal supply of ATP, a significant decrease in 3.4.3. Abnormal metabolism of small molecules
intracellular ATPase activity in Salmonella cells following chlorine di­ The normal metabolism of intracellular substances in bacteria is an
oxide treatment was also found in this study (14.32 %) (Fig. 3D). Other important basis for the maintenance of normal cellular physiological
studies have found that chlorine dioxide treatment induces a decrease in activity, where substances such as amino acids and small peptides play
ATPase activity in Nosema bombycis spore cells (Wang et al., 2010). In an important role in the intracellular material cycle and in maintaining
addition, in a previous study, it was found that high-frequency ultra­ osmotic pressure homeostasis (Díaz-Pérez et al., 2016; O’Byrne & Booth,
sound alone caused a decrease in the intracellular ATPase activity of 2002).
Salmonella (26.06 %) (Luo et al., 2022b). In addition, in the present Firstly, in this study, it was found that the total abundance of amino
study, it was further found that CD-WU treatment causes an even more acids and their derivatives in Salmonella cells increased slightly (0.97 %)
significant decrease (38.06 %) in the intracellular ATPase activity of after chlorine dioxide treatment (CD group), while the total abundance
Salmonella (Fig. 3D). This may be due to the fact that the catalytic ac­ of amino acids and their derivatives in the cells decreased somewhat
tivity of ATPase depends on the natural conformation of its active site (4.24 %) under combined treatment (CD-WU group) (Fig. 5A). Some
and the conformation of the surrounding proteins, but some physical/ differences were also observed from the previously determined changes
chemical effects may cause a change in its natural structure or dena­ in total amino acid abundance in bacterial cells after low-power ultra­
turation, which may in turn lead to a change in ATPase activity (Liu sound alone under the same conditions (Luo et al., 2022b). To some
et al., 2021). extent, this suggests that the low-power ultrasound treatment may have
Thus, the blockage of ATP accumulation and catabolism in Salmo­ exacerbated the loss of amino acids in the bacterial cells after chlorine
nella cells after chlorine dioxide treatment may be an important reason dioxide treatment. On the one hand, it is possible that the lost amino
for the massive cell death of the bacteria, with the low-power ultrasound acids and their derivatives were consumed by the subsequent reactions.
treatment greatly enhancing this process, which in turn leads to the On the other hand, it is possible that the cell structure was severely
further advancement of the cell death process in Salmonella cells. disrupted, resulting in a loss of intracellular content of small molecules

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Fig. 4. Broadly targeted metabolomics analysis. (A) Principal component analysis (PCA). (B) Differential metabolites (DMs) obtained by comparison between two
groups. (C) Venn diagram of DM distribution. CK, untreated group; CD, chlorine dioxide alone (0.25 mg/L for 5 min); CD-WU, 0.25 mg/L chlorine dioxide combined
with 0.03 W/mL ultrasound for 5 min. Significant differences (P < 0.05) between groups are indicated by different letters (a–b).

such as amino acids. It has been found that bacterial cells actively peptides in Salmonella was decreased after both treatments (Fig. 5B). In
engage in amino acid metabolism for stress regulation when exposed to particular, the total abundance of small peptide metabolites in Salmo­
external stresses (Mosier et al., 2014; Tripathy et al., 2014). Specifically, nella cells appeared to be significantly reduced (11.25 %) after the CD-
a total of 24 amino acids and their derivative-like metabolites were WU treatment compared to the CK group. In a previous study, it was
significantly altered (with 13 being upregulated and 11 downregulated) also found that ultrasound alone under the same conditions may
in the CD group compared with the CK group (Fig. 5A). In contrast, a potentially decrease the total abundance of small peptides in bacterial
total of 31 amino acids and their derivative-like metabolites demon­ cells (Luo et al., 2022b). Furthermore, this result is also consistent with
strated significant changes in abundance in the CD-WU group compared the trend of damage to cell structure and function and loss of intracel­
to the CK group (with 14 being upregulated and 17 downregulated). Of lular material found above. Specifically, a total of 95 small peptide
these, 13 were significantly changed in both the CD/CK and CD-WU/CK metabolites exhibited a significant change in abundance (with 61 being
groups (with 7 being upregulated and 6 downregulated), and they upregulated and 34 downregulated) after CD treatment compared to CK
showed similar trends, including, for example, L-Homocitrulline, (Fig. 5B). A total of 85 small peptide metabolites exhibted significant
γ-Aminobutyric Acid, Methylcysteine and S-methyl-L-thiocitrulline. The changes in abundance after CD-WU treatment compared to CK (with 32
differences in the abundance of these metabolites may be due to the being upregulated and 53 downregulated). It was found that the abun­
effects of a range of stress modulations by cells in response to external dance of some small peptides increased following CD treatment, which
stimuli. In particular, a significant increase in the abundance of L-Serine may be due to a series of intracellular stress modulations leading to an
and a significant decrease in the abundance of L-Arginine, L-Homocys­ increase in the synthesis of the relevant small peptides, which also
tine, L-Methionine, L-Tryptophan and Selenomethionine were also found corresponds to the decrease in amino acid abundance described above.
in the CD-WU/CK comparison. This is extremely detrimental to the For example, a significant decrease in the abundance of 3-(pyrazol-1-yl)-
physiological regulation of stress carried out by Salmonella cells. For L-alanine and a significant increase in the abundance of the related
example, it has been found that some intestinal bacteria can use argi­ Prolylalanine were found in CD/CK. The CD-WU treatment resulted in a
nine, asparagine and aspartic acid as substrates to enhance their meta­ significant decrease in the abundances of a large number of small pep­
bolism in order to adapt to changes in external conditions (Alpert et al., tide metabolites, probably due to structural and functional damage to
2008). Yang and his colleagues (Yang et al., 2022) also found a reduc­ the cell membrane. In addition, intense stress regulation also requires
tion in the abundance of important amino acids such as arginine in the consumption of a large amount of material and energy. In bacterial
bacterial cells when exploring the resistance of Yersinia pestis to the cells, small peptides are usually used as a source of amino acids and
adverse stress of lipoic acid in the small intestine. nitrogen (Newstead, 2014). In addition, although the specific physio­
Similarly, it was found that the total intracellular abundance of small logical functions of some small peptides in bacterial cells are not yet

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W. Luo et al. Food Chemistry: X 20 (2023) 100901

Fig. 5. Low-power ultrasound treatment enhances the effect of chlorine dioxide on the metabolic processes of small molecules in Salmonella cells. (A) Amino acids
and derivatives; (B) small peptides. CK, untreated group; CD, chlorine dioxide alone (0.25 mg/L for 5 min); CD-WU, 0.25 mg/L chlorine dioxide combined with 0.03
W/mL ultrasound for 5 min.

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W. Luo et al. Food Chemistry: X 20 (2023) 100901

understood, there is much evidence that small peptides play an impor­ stress regulation is initiated by the bacteria themselves. The combina­
tant role in regulating cellular metabolism, modulating the activities of tion CD-WU treatment leads to a significant loss of material from the
other proteins and controlling the cell cycle, among other key biological bacterial cells, resulting in an irreversible dysregulation of the relevant
functions (Vajjala et al., 2022). material metabolism, which in turn leads to massive cell death.
Thus, after chlorine dioxide treatment alone, the metabolism of small
molecules within the bacterial cells is disturbed, and the corresponding

Fig. 6. Low-power ultrasound treatment enhances the effect of chlorine dioxide on the nucleotide metabolism in Salmonella cells. (A) Purine metabolism; (B) py­
rimidine metabolism. CK, untreated group; CD, chlorine dioxide alone (0.25 mg/L for 5 min); CD-WU, 0.25 mg/L chlorine dioxide combined with 0.03 W/mL
ultrasound for 5 min.

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3.4.4. Abnormal nucleotide metabolism disruption of cellular metabolic processes, thus inducing more Salmo­
Nucleotides and their derivatives have a variety of biological func­ nella cells to become unculturable.
tions and play an important role in the normal physiological activities of
cells. Nucleotides are the precursors of nucleic acids (DNA and RNA), 4. Conclusion
while nucleotide derivatives are often involved in biosynthesis as active
intermediates (Goncheva et al., 2022). In the present study, significant The results of this study show that low-power ultrasound treatment
changes in the levels of 33 metabolites associated with nucleotides and can effectively enhance the inhibitory effect of chlorine dioxide on
their metabolites were found within Salmonella cells following chlorine Salmonella. Our studies have also found that low-power ultrasound
dioxide treatment, while combined treatment (CD-WU group) caused significantly enhances chlorine dioxide damage to the structure and
significant changes in the levels of 38 metabolites. These significantly function of the bacterial cell membrane, as well as impacting key pro­
different nucleotide-related metabolites were further targeted to specific cesses of energy supply. This, on the one hand, causes a serious imbal­
metabolic pathways to provide insight into the inhibition effects caused ance in the homeostasis of material and energy metabolism within the
by the two treatments and their differential mechanisms. bacterial cell and, on the other hand, may exacerbate the entry of
First, purine metabolism, a key component of normal cellular phys­ chlorine dioxide into the bacterial cell, thereby deepening the effects on
iology, is closely linked to a number of intracellular metabolic processes normal intracellular metabolic processes. The results of the metab­
(Daignan-Fornier & Pinson, 2019). A significant decrease in guanine and olomics study further confirmed and demonstrated the fact that low-
adenine abundance, and a potential decrease in guanosine, adenosine power ultrasound treatment exacerbates the disruption of important
and inosine, and subsequently GDP and GMP abundance, were found in physiological processes such as small molecule metabolism and nucle­
the intracellular purine metabolism of Salmonella under both conditions otide metabolism in Salmonella cells caused by chlorine dioxide. The co-
(Fig. 6A). This may be partly due to the fact that the genetic metabolic treatment of low-power ultrasound with chlorine dioxide therefore also
processes under both treatment conditions are enhanced and require the reduces the requirement for ultrasound and chlorine dioxide to be
repair of DNA damage, which in turn consumes large amounts of purine- treated separately, thereby reducing energy consumption and environ­
like metabolites. Some studies have found that chlorine dioxide treat­ mental impact. The results of the study not only provide a theoretical
ment has the potential to damage the DNA of bacterial cells, etc. (Ersoy basis for an in-depth understanding of the mechanisms associated with
et al., 2019). In addition, Bacillus subtilis has been found to undergo the role of low-power ultrasound treatment in the co-treatment process,
stress repair by upregulating purine and pyrimidine biosynthetic path­ but also provide data furthering our understanding of the mechanisms of
ways in response to DNA damage (Wozniak & Simmons, 2021). On the chlorine dioxide bacterial inhibition. It also points the way to further
other hand, it is possible that the metabolic balance of substances within “cleaner production” in the food industry.
the cell was impaired, resulting in the enhancement of related catabolic
pathways such as riboflavin metabolism and glycine, serine and threo­ Funding
nine metabolism. However, further exploration is needed. It was also
found that Salmonella cells may attempt to replenish the loss of GDP and This work was supported by the National Natural Science Foundation
GMP by increasing the abundance of ADP-ribose, ITP, IDP, Hypoxan­ of China (32072236), and the Cultivation Project of Double First-Class
thine and dAMP (Fig. 6A). In addition, the combined CD-WU treatment Disciplines of Food Science and Engineering Beijing Technology and
further attenuated the cyclic conversion process between AMP, ADP, Business University (No. BTBUKF202206).
ATP and Cyclic AMP compared to the chlorine dioxide treatment alone.
As found above, the ATP content in CD-WU-treated cells was substan­ CRediT authorship contribution statement
tially reduced. Therefore, it is possible that the intracellular ATP was
heavily depleted, in turn leading to a disruption of the cycle and also Wei Luo: Investigation, Data curation, Formal analysis, Writing –
affecting the production of RNA and DNA material, leading to the original draft. Jie Tang: Methodology, Resources. Beibei Wang:
disruption of the regulatory processes of genetic metabolism within the Methodology, Resources. Di Wu: Methodology, Resources. Jinqiu
cell, in turn increasing the lethality of the chlorine dioxide treatment. Wang: Methodology, Resources. Lei Cheng: Methodology, Resources.
Similar findings were observed for pyrimidine metabolism (Fig. 6B). Fang Geng: Conceptualization, Writing – original draft, Writing – re­
A potential decrease in the abundance of Cytosine, Uracil, Cytidine, view & editing, Supervision, Funding acquisition.
Uridine, CMP, UMP and dTTP and a potential increase in the abundance
of UDP, CDP, Thymine, dUMP and dTMP in Salmonella cells were found Declaration of Competing Interest
under chlorine dioxide treatment alone. This also reflects in part the fact
that Salmonella cells actively regulate the production of nucleic acid The authors declare that they have no known competing financial
material such as RNA and DNA within the cell. CD-WU treatment has a interests or personal relationships that could have appeared to influence
similar process, but it also inhibits the regulation of CDP and dTMP to a the work reported in this paper.
certain extent. In addition, the disruption of the balance of intracellular
substances prevents alanine, aspartate and glutamate metabolism, Data availability
β-alanine metabolism, and valine, leucine and isoleucine degradation,
thus affecting the process of intracellular pyrimidine metabolism. In a Data will be made available on request.
study by Yang et al. (Yang et al., 2022) on the antimicrobial mechanism
of lipoic acid against Yersinia pestis in the small intestine, significant Appendix A. Supplementary data
downregulation of pyrimidine metabolism, such as 3′-UMP, was found,
leading to deletion and degradation of the bases required for DNA Supplementary data to this article can be found online at https://doi.
synthesis, thus affecting DNA integrity. org/10.1016/j.fochx.2023.100901.
Therefore, it is hypothesised that chlorine dioxide treatment not only
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