LWT - Food Science and Technology 174 (2023) 114387

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LWT
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Dihydromyricetin solid dispersion: Preparation, characterization, and

preservative effects on sturgeon fillets stored at 4 ◦ C
Hao Xu a, b, Tiantian Zhao b, Fengsong Liu a, Yan Zhang a, Yijia Xie a, Xinglong Xiao a, **,
Yousheng Zhang b, *
a
School of Food Science and Engineering, South China University of Technology, Guangzhou City, Guangdong Province 510640, PR China
b
Sericultural & Agri-Food Research Institute, Guangdong Academy of Agricultural Sciences, Key Laboratory of Functional Foods, Ministry of Agriculture and Rural
Affairs, Guangdong Key Laboratory of Agricultural Product Processing, Guangzhou, 510610, PR China

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

Keywords: Dihydromyricetin (DMY) is a well-known natural flavonoid antioxidant and bacteriostat. However, its practical
Dihydromyricetin application, e.g., as a preservative, is limited by poor bioavailability due to its low solubility. To overcome this
Polyvinylpyrrolidone K30 limitation, a solid dispersion formulation of DMY (DMYSD) was prepared from polyvinylpyrrolidone (PVP) K30,
Solid dispersion
and characterized using spectroscopic, thermoanalytical, antioxidant and bacteriostatic methods. The results
Sturgeon fillets
showed that the enhanced solubility of DMYSD (64.51 mg/mL), which was more than 90 times that of DMY,
Shelf life
could be attributed to reduced crystallinity of the DMY solid dispersion. Immersion of sturgeon fillets in DMYSD
solution (5.12 mg/mL) demonstrated that the shelf life of the treated fish stored at 4 ◦ C could be extended by
more than 3 d compared with pure DMY. Bacterial enumeration and physicochemical analyses of the treated
stored fish indicated that the increased shelf life was due to reduced protein and lipid oxidation, and suppression
of bacterial growth, by the solid dispersion.

1. Introduction
Sturgeon is an ancient species of considerable biological, commercial
and cultural importance. In recent years, sturgeon farming has devel­
oped rapidly in several nations, particularly in China, which accounts
for more than 86% of the worldwide output (Wang et al., 2019).
Compared with other cold water fatty fish, such as salmon, sturgeon
flesh has higher concentrations of essential amino acids, and poly­
unsaturated fatty acids such as docosahexaenoic acid and eicosa­
pentaenoic acid (Feng et al., 2020; Zhang et al., 2021). This, together
with a high water and nutrient content, elevated endogenous enzyme
activity, and a near neutral pH, increases its susceptibility to spoilage
during processing, transportation, and storage (Gui et al., 2017).
Dihydromyricetin or ampelopsin (DMY; C15H12O8) is an important
bioactive plant flavonoid isolated from the traditional Chinese medici­
nal plant Ampelopsis grossedentata, and is also found in other fruits and
vegetables. In some studies, plant flavonoids are the conventional edible
components of common foods, and the use of plant flavonoids is
considered to be non-toxic (Elliott & Chithan, 2017, pp. 619–652).
Moreover, researchers conducted the acute toxicity test of DMY and
found that the toxicity of DMY was very slight, and the maximum
tolerated oral gavage rats was 5.0 g/kg body weight (Liu et al., 2019).
DMY is well known for a broad range of biological functions such as
antioxidant, anti-inflammatory, anti-bacteria, anti-hypertension, and
anti-cancer (Liu et al., 2019). The reduced oxidation and spoilage
observed in treated fish oil and cooked beef indicate strong development
potential for the use of DMY in the shelf life extension of meat products
(Baek et al., 2015; Ye et al., 2015). However, DMY is only slightly sol­
uble in water at room temperature (0.2 mg/mL at 25 ◦ C), which severely
restricts its bioavailability. In recent research, a self-nanoemulsion drug
delivery system increased the stability and biological activity of DMY
(Chen, Cai, et al., 2020), while nanocapsule preparations boosted the
apparent solubility of the flavonoid and its inhibition of Pseudomonas
aeruginosa biofilms (Dalcin et al., 2017).
The solid dispersion technique is a widely accepted and successful
strategy to enhance the solubility and release of low solubility phar­
maceutical products, as evidenced by the increased number of FDA-
approved pharmaceuticals in recent years (Li et al., 2014). Many

** Corresponding author.
* Corresponding author.
E-mail addresses: fexxl@scut.edu.cn (X. Xiao), youshengzhang@263.net (Y. Zhang).

https://doi.org/10.1016/j.lwt.2022.114387
Received 10 October 2022; Received in revised form 25 December 2022; Accepted 27 December 2022
Available online 28 December 2022
0023-6438/© 2023 The Authors. 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/).
H. Xu et al.
compounds have been prepared as solid dispersions to enhance their
solubility, dissolution rate, and biological activity. For example, the
solubility and bioavailability of ticagrelor and curcumin were signifi­
cantly improved when transformed into solid dispersions (Chuah et al.,
2014; Kim et al., 2019). Hydrophilic polymers have been widely
investigated as carrier substances for the preparation of solid dispersions
using the solvent evaporation technique. Among these, poly­
vinylpyrrolidone (PVP) K30 has attracted much attention as it has been
used as a safe and aesthetically pleasing packaging material in the food
industry (Li et al., 2021). Here, a solid dispersion of DMY (DMYSD) was
prepared by the solvent evaporation technique using PVP K30 as the
carrier, and characterized using spectroscopic, thermoanalytical, anti­
oxidant and bacteriostatic methods. The effectiveness of DMYSD as a
preservative for meat products was evaluated by studying the changes in
protein and lipid oxidation, and bacterial colony counts, of treated
sturgeon fillets stored at 4 ◦ C.
2. Materials and methods
2.1. Reagents and bacterial strains
DMY (98%) was purchased from Hubei Marvel-Bio Medicine Co.,
Ltd., (Hubei, China). Tryptic soy ager (TSA) and tryptic soy broth (TSB)
were from Guangdong Huankai Microbial Technology Co., Ltd.,
(Guangdong, China). Pseudomonas CFC Selective Agar, Iron Agar, and
Aeromonas Medium Base were obtained from Qingdao Hope Biotech
Co., Ltd., (Qingdao, China). PVP K30, DPPH, ABTS and other reagents
were sourced from Shanghai Macklin Biochemical Co., Ltd., (Shanghai,
China).
Shewanella putrefaciens ATCC 8071, Pseudomonas fluorescens ATCC
13525, and Aeromonas GDMCC 801340 were supplied by the Guang­
dong Microbial Species Preservation Center (Guangdong, China).
2.2. Preparation of DMYSD
The solvent evaporation method was used to make a solid dispersion
of DMY. DMY (10 g) was dissolved in anhydrous ethanol at 60 ◦ C while
PVP K30 was weighed and dissolved in anhydrous ethanol at 60 ◦ C with
a DMY-to-carrier weight ratio of 1:7, respectively. The DMY solution
was added slowly to the carrier solution under stirring prior to soni­
cation for 30 min to remove air bubbles. The solvent was then removed
on a rotary evaporator, and the mixture stored at − 20 ◦ C for 12 h before
drying at 45 ◦ C for 24 h in a vacuum oven. The resultant material was
reduced to a fine powder, passed through a 100-mesh sieve, and stored
in a desiccator until required.
2.3. Solubility measurements
The reserve solution was prepared by properly weighing 30.00 ±
0.01 mg of DMY standard and dissolving it in anhydrous ethanol at a
consistent volume of 100 mL. The reserve solution was diluted in ab­
solute ethanol to get 15, 30, 45, 60, and 75 μg/mL of the solution to be
tested. The absorbance was measured at 292 nm using a UV-1800
UV–vis spectrophotometer (Shimadzu Corporation Co., Ltd., Japan),
and a standard curve was established.
DMY or DMYSD (0.5 g) was dissolved in ultrapure water (5 mL) using
ultrasonication for 30 min at room temperature. The samples were then
maintained under oscillation (100 rpm) in a water bath oscillation at
room temperature for 12 h, i.e., until equilibrium was attained and
undissolved material had settled in the bottom of the vessel. The solu­
bility was calculated from the absorbance of the supernatant at 292 nm
and the DMY concentration/absorbance standard curve.
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LWT 174 (2023) 114387
2.4. Characterization of DMYSD
2.4.1. UV-VIS spectrum
Solutions of DMY, DMYSD, and PVP K30, prepared in ultrapure
water, were scanned over 200–600 nm on a UV-1800 UV-VIS spectro­
photometer (Shimadzu Corporation Co., Ltd., Kyoto, Japan).
2.4.2. Fourier transform infrared reflectance (FTIR) spectrum
Samples of DMY, PVP K30, DMYSD, and DMY-PVP K30 (a physical
mixture obtained by uniformly mixing DMY and PVP K30 without any
treatment) were prepared as thin slices using the KBr pellet prior to
analysis using attenuated total reflection-Fourier transform infrared
(FTIR) spectroscopy. Spectra were acquired over 400 and 4000 cm− 1 on
a VERTEX 70 FTIR (Bruker Optics Inc., Billerica, MA, USA).
2.4.3. X-ray diffraction
The crystallization behavior of DMY, PVP K30, DMYSD, and DMY-
PVP K30 was investigated by X-ray diffraction (XRD) with a D8
Advance diffractometer (Bruker Co., Ltd., Germany) using Cu K-α radi­
ation over a 2θ range of 3–60◦ (2θ = 2◦ /min).
2.4.4. Differential scanning calorimetry
Thermal analysis was performed using a TGA 4000 differential
scanning calorimeter (DSC; PerkinElmer Inc., USA): Samples of DMY,
PVP K30, DMYSD, and DMY-PVP K30 (10 mg) were weighed into
aluminum pans and heated from 30 to 300 ◦ C at 10 ◦ C/min under a dry
nitrogen atmosphere at a flow rate of 25 mL/min.
2.4.5. Scanning electron microscopy
Microstructures were examined by scanning electron microscopy
(SEM) using a S-3400N-II (HITACHI Co., Ltd., Japan): Samples of DMY,
PVP K30, DMYSD, and DMY-PVP K30 were distributed uniformly on
aluminum foil using conductive tape followed by gold sputtering for 30
s; images were obtained at 500 times magnification.
2.4.6. Antioxidant capacity
DPPH radical scavenging assay. The method of Shao (Shao et al.,
2014) was used with some modifications. Solutions of DMY, DMYSD and
L-ascorbic acid (1 mL in ethanol; L-ascorbic acid was used as a positive
control) representing a range of concentrations (2–10 μg/mL), were
combined with DPPH solution (1 mL of 0.2 mM in ethanol), mixed
thoroughly, and allowed to react for 30 min at room temperature in the
dark. The absorbance of the supernatant was then measured (517 nm)
after centrifugation at 650g for 5 min; the absorbances of sample solu­
tions (1 mL) mixed with ethanol (1 mL), and DPHH solution (1 mL)
mixed with deionized water (1 mL), were used as the blank and control
respectively. The DPPH free radical clearance rate was calculated from
Eqn (1):
( )
A1 − A2
Scavenging rate (%) = 1 − × 100% (1)
A3
where A1, A2, and A3 are the absorbance of the supernatant, blank and
control respectively.
ABTS radical scavenging assay. The procedure of Re (Re et al., 1999)
was used with some minor amendments. A working solution was pre­
pared from equal volumes of ABTS solution (7.4 mM) and potassium
persulfate solution (2.45 mM) incubated for 14 h at room temperature
without exposure to light. PBS solution (50 mM; pH 7.4; absorbance
value 0.70 ± 0.02 at 734 nm) was then added to the above working
solution (150 μL) before combining with sample solutions (DMY,
DMYSD, L-ascorbic acid; 50 μL) in a 96-well microporous plate. The
absorbance was measured at 734 nm after incubation for 30 min at 30 ◦ C
in the dark; PBS solution (50 μL) was used as the control. The ABTS
radical scavenging activity was calculated from Eqn (2):
H. Xu et al.
( ) ( )
Asample
Scavenging rate % = 1 − × 100% (2)
Acontrol
Hydroxyl radical scavenging assay. Hydroxyl radicals were gener­
ated via the Fenton reaction by combining FeSO4 (2 mL of 6 mM) and
H2O2 (2 mL of 24 mM). Samples (DMY, DMYSD, L-ascorbic acid; 4.0 mL)
were added and the volume of the solution was made up to 25 mL with
salicylic acid (8 mM in anhydrous ethanol). The absorbance was
measured at 510 nm after incubation at 37 ◦ C for 30 min; samples
prepared in the same way with distilled water instead of H2O2 were used
as the control (Ge et al., 2021). The scavenging activity was calculated
from Eqn (3):
Acontrol − Asample
Scavenging rate (%) = × 100% (3)
Acontrol
2.4.7. Minimum inhibitory and bactericidal concentrations
All bacterial strains were incubated in TSB at 30 ◦ C for 24 h to obtain
a stable phase prior to dilution (TSB) using the McGregor trigonometric
method to obtain a target bacterial concentration for the test of about
106 CFU/mL. Tetracycline was used as the positive control.
The Clinical and Laboratory Standards Institute (CLSI) recommended
doubling dilution technique (CLSI, 2006) was used to determine the
minimum inhibitory concentrations (MIC) of DMY and DMYSD. Briefly,
the bacterial suspension was combined with a continuous two-fold
dilution of the antibacterial agent solution in a liquid medium fol­
lowed by incubation for 24 h in a 96 microplate well. The MIC was taken
as the lowest concentration at which bacterial growth visibly ceased, i.
e., the bacterial solution was no longer cloudy.
For the measurement of minimum bactericidal concentrations
(MBC), culture medium (100 μL, without bacterial growth) was placed
over TSA on a 96-well microporous plate and cultivated at 30 ◦ C for 72
h. MBC was taken as the lowest concentration at which no bacterial
growth occurred.
2.5. Preservative effects
2.5.1. Preparation and sample treatment of sturgeon fillets
Fifteen live sturgeon (1000 ± 100 g each) were acquired from a local
supermarket (China Resources Vanguard, Guangzhou, China) and
transferred to the laboratory. After evisceration and removal of the skin
and bones, select the muscles from the same place on the back of the fish.
The muscles were rinsed with deionized water before being cut into
cuboid fillets measuring 3 x 2 × 1 cm. All fillets were allocated at
random to four groups for immersion soaking (250 mL, 30 min) in the
following solutions: a) Deionized water (control); b) PVP K30 solution
(4.48 mg/mL); c) DMY solution (0.64 mg/mL); d) DMYSD solution
(5.12 mg/mL; DMY solution (0.64 mg/mL). The results from the anti­
oxidant and antibacterial tests of DMY and DMYSD were considered in
the selection of the solution concentrations. After treatment, samples
were dried under a nitrogen stream on a sterile surface before sealing in
polyethylene bags and storing at 4 ◦ C for 12 days. Samples were taken at
0, 3, 6, 9, and 12 d, and three replicate analyses were conducted on each
sample.
2.5.2. Enumeration of microbial communities
The microbiological analysis of sturgeon fillets was conducted based
on the method reported by Jouki with little modifications (Jouki et al.,
2014). Fish fillet samples (5 g) were transferred from each group into
normal sterile saline (45 mL) and homogenized for 60 s at 7000 rpm. The
pour plate method was used to count bacteria in serially diluted ho­
mogenate. Plate counting agar, Pseudomonas CFC Selective Agar, Iron
Agar, and Aeromonas Medium Base were used for the total viable count
(TVC), Pseudomonas, H2S-producing bacteria, and Aeromonas counts,
respectively. Three appropriate dilution gradients were chosen giving
three parallel lines for each dilution. The plate was then inverted and
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LWT 174 (2023) 114387
cultured for 72 h in a biochemical incubator (30 ◦ C).
2.5.3. Measurement of pH
Sturgeon fillets (5.0 g) were homogenized in distilled water (45 mL)
for 60 s at 7000 rpm using an T25 digital homogenizer (IKA Co., Ltd.,
Germany), centrifuged at 10,000 g and 4 ◦ C for 5 min, and the super­
natants collected for measurement using a laboratory pH meter.
2.5.4. Total volatile basic nitrogen
The method of Zhai (Zhai et al., 2017) was used to measure the total
volatile basic nitrogen (TVB-N) values of the sturgeon fillets. Samples
(5.0 g) were homogenized with 45 mL of deionized, and an aliquot of the
filtrate (5.0 mL) transferred to a Kjeltec™ 8400 Kjeldahl analyzer (Foss,
Runcorn, Cheshire, UK); the volume of 10 mM hydrochloric acid solu­
tion used to titrate the distillate was used to determine TVB-N as mg
N/100 g sample.
2.5.5. Thiobarbituric acid reactive substances
Thiobarbituric acid reactive substances (TBARS) was determined by
the method of Kunyaboon (Kunyaboon et al., 2021). Samples (5.0 g)
were homogenized in trichloroacetic acid solution (45 mL of 7.5%
(w/v)), oscillated (180 rpm) at 50 ◦ C for 30 min. The filtrate was
collected, TBA solution (5 mL of 0.02 M) was added, and the mixture
was heated at 90 ◦ C for 30 min. The absorbance of the cooled solution
was measured at 532 nm and trichloroacetic acid solution was used as a
blank control. TBARS was expressed as mg of malondialdehyde (MDA)
per unit mass of sturgeon fillets (mg MDA/kg sample) obtained from a
standard curve of 1,1,3,3-tetraethoxypropane.
2.5.6. Carbonyl and sulfhydryl contents
The myofibrillar protein (MP) of sturgeon fillets was extracted by the
procedure of Walayat (Walayat et al., 2021), and its protein concen­
tration determined with the Biuret method using bovine serum albumin
as a standard.
The carbonyl content of sturgeon fillets was measured by the reac­
tion of its soluble protein with 2,4-dinitrophenylhydrazine (DNPH)
(Dalle-Donne et al., 2003). The sturgeon MP solution was added to
DNPH (10 mM in 2 M HCl) in the ratio 1:1 (v/v) and incubated in the
dark at room temperature for 1 h. An equal volume of 20% trichloro­
acetic acid (w/v) was added to terminate the reaction, the mixture was
centrifuged at 4500 g for 5 min, and the precipitate was washed three
times with ethanol/ethyl acetate solution (1:1, v/v) to remove the
unreacted reagent. The final precipitate was then dissolved in guanidine
hydrochloride (6 M in 20 mM sodium phosphate buffer) and incubated
at 37 ◦ C for 15 min. The absorbance of the solution was measured 370
nm, and the carbonyl content calculated using the molar absorbance
coefficient (22000 M− 1 cm− 1) and expressed as nmol/mg MP.
Ellman’s reagent (5,5 ′ -dithiobis-(2-nitrobenzoic acid)) was used to
quantify the free sulfhydryl groups in sturgeon MP solution (Shi et al.,
2014). MP sample solution was mixed with Ellman’s reagent (10 mM)
and the absorbance value was measured (412 nm) after 30 min. The free
sulfhydryl content was calculated from the molar absorbance coefficient
(13600 M− 1 cm− 1) and expressed as nmol/mg MP.
2.6. Statistical analysis
All data were reported as the mean of three replicate measurements
and shown as the mean ± standard deviation. Differences and compar­
isons of means were evaluated by ANOVA and Duncan’s multiple
comparisons test using SPSS Statistics for Windows, Version 26.0 (IBM
Corp Armonk, NY USA). The differences between the means were
considered significant at p < 0.05.
H. Xu et al.
3. Results and discussion
3.1. Characterization of DMYSD
3.1.1. Solubilities of DMY and DMYSD
When DMY was prepared as a solid dispersion at a weight ratio of 1:7
on PVP K30, its solubility in water at room temperature increased from
0.69 to 64.51 mg/mL. PVP K30 is a highly water-soluble amorphous
polymer. During the preparation of the solid dispersion system, micro­
scopic crystals of DMY are incorporated into the molecular gaps of PVP
K30. Under this condition, the crystals cannot aggregate, their crystal­
linity is reduced, and hence the solubility of DMY in water increases
(Alves et al., 2014).
3.1.2. UV-VIS spectrum of DMYSD
Fig. 1A shows the UV-VIS spectra of DMY and DMYDS. Both DMY
and PVP K30 exhibited single absorption peaks at 292 nm and 213 nm
respectively, and the presence of both chromophores in DMYSD indi­
cated that successful complexation of the flavonoid with the carrier had
occurred and no new substances were formed during preparation. This
result agreed with observations for similar dispersant systems reported
elsewhere (Li et al., 2021; Zhang et al., 2005).
3.1.3. FTIR spectrum of DMYSD
The FTIR spectra of DMY, PVP K30, DMYSD, and DMY-PVP K30 are
shown in Fig. 1B. Peaks characteristic of DMY were observed at 3359
cm− 1 (broad, –OH stretching vibration), 1550, 1475 and 1456 cm− 1
(aromatic ring stretching vibrations). Peaks characteristic of PVP K30
were observed at 3475 cm− 1 (-OH stretching vibration of water absor­
bed by the hydrophilic carrier), 2951 cm− 1 (C–H stretching vibration),
1648 cm− 1 (C=O stretching vibration), 1494 and 1463 cm− 1 (C=C
stretching vibrations), and 1289 cm− 1 (C–N stretching vibration).
DMYSD showed a broader –OH peak, suggesting that DMY and PVP K30
had established a hydrogen bond, and the absence of new peaks
confirmed that no new compounds were generated during preparation
Fig. 1. (A) UV-VIS spectrum; (B) FTIR spectrum; (C) XRD spectrum; (D) DSC spectrum. In the figure, the black line is for the DMY group, the red line is for the PVP
K30 group, the blue line is for the DMYSD group, and the green line is for the PVP K30 group. (For interpretation of the references to colour in this figure legend, the
reader is referred to the Web version of this article.)
4
LWT 174 (2023) 114387
and DMY remained stable. The spectral peaks of DMY and PVP K30
could be superimposed on those of the DMY-PVP K30 system suggesting
that physical mixing of the flavonoid and carrier resulted in no molec­
ular interactions.
The carbonyl oxygen and nitrogen atoms of PVP K30 readily accept
protons, which facilitates hydrogen bonding (Frizon et al., 2013). The
formation of hydrogen bonds between DMY and PVP K30 in the solid
dispersion prevents recrystallization of the flavonoid, which inhibits its
aggregation. As a result, the crystallinity of DMY in the solid dispersion
is lower than that of the DMY monomer, and its solubility increases.
3.1.4. Crystallinity of DMYSD
The XRD spectra of DMY, PVP K30, DMYSD, and DMY-PVP K30 are
given in Fig. 1C. The crystalline nature of each material was represented
as sharp peaks in their diffraction spectra. The main peaks in the spec­
trum of DMY were observed at 8.01◦ , 10.23◦ , 11.2◦ , 13.18◦ , 16.09◦ ,
18.42◦ , 21.81◦ , 23.91◦ , while those at 26.05◦ , and 27.06◦ were repre­
sentative of its typical crystal nature. PVP gave a poorly defined peak
between 11.13◦ and 20.6◦ demonstrating lower crystallinity, and the
absence of representative diffraction peaks in the solid dispersion indi­
cated complete complexation of DMY into PVP K30. In comparison, all
representative diffraction peaks of DMY and PVP K30 were present in
the physically mixed preparation. DMYSD and DMY-PVP K30 both
showed no additional peaks, suggesting that the stability of the original
composition was maintained, which agreed with the FTIR results.
3.1.5. Thermal performance of DMYSD by DSC
The DSC curves of DMY, PVP K30, DMYSD, and DMY-PVP K30 are
shown in Fig. 1D. DMY exhibited three endothermic peaks at 121.63,
166.02, and 250.09 ◦ C, corresponding to the melting point, recrystalli­
zation, and water loss, respectively; the single exothermic peak at
257.71 ◦ C was due to oxidation. The characteristic endothermic peak for
PVP K30 broadened and moved to a higher temperature in DMYSD,
while the melting point peak for DMY also broadened but moved to a
lower temperature. Similar observations were recorded for DMY-PVP
H. Xu et al.
K30. However, the distinctive endothermic melting peak of DMY in
DMYSD was broader than that in the physical mixture and moved to a
lower temperature, and this could be ascribed to its reduced crystal­
linity. After the solvent completely evaporates, the drug finally disperses
in the polymer matrix without forming a crystal lattice (the amorphous
feature of random arrangement). Some related studies have reported
similar results, that is, that PVP K30 causes the drug to form an amor­
phous structure in the solid dispersion system (Kaewnopparat et al.,
2009). Comparison of the differential scanning patterns of the solid
dispersion and physical mixture showed no significant differences,
implying preservation of the starting materials during preparation of the
solid dispersion system. The results also agreed with those of FTIR and
XRD.
3.1.6. Microstructure of DMYSD examined by SEM
The SEM microstructure images of DMY, PVP K30, DMYSD, and
DMY-PVP K30 are shown in Fig. 2. The surface morphology of DMY
presented as a rod-shaped crystalline powder of uniform size, whereas
PVP K30 appeared as particulate material with small holes. Irregular
block structures were observed in DMYSD, which was also devoid of the
crystalline and particulate materials observed in the individual com­
ponents. In comparison, DMY-PVP K30 comprised many PVP K30 par­
ticles surrounded by rod-shaped DMY crystals. This also supported the
formation of a complex between DMY and PVP K30 in DMYSD, and the
subsequent decrease in crystallinity of DMY in the solid dispersion.
3.1.7. Antioxidant properties
Because free radicals have a negative impact on food and biological
systems, the radical scavenging activity of a preservative is a crucial
component of the system. Among the methods available for antioxidant
activity estimation, DPPH and ABTS have been used extensively for a
wide range of matrices due to their simplicity, speed, sensitivity and
generation of stabile radicals(Gülçin et al., 2012; Mishra et al., 2012).
DPPH radical scavenging activity. Under test, DMYSD had a greater
DPPH radical scavenging capacity than either DMY or ascorbic acid (p <
0.05) (Fig. 3A). Their DPPH scavenging rate increases with the increase
of concentration. When the concentration reaches 10 μg/mL, the solid
dispersion has the highest scavenging capacity on DPPH free radicals,
Fig. 2. SEM of: (A) DMY; (B) PVP K30; (C) DMYSD; (D) DMY-PVP K30.
5
LWT 174 (2023) 114387
with the scavenging rate reaching 84.76%.
ABTS free radical scavenging ability of DMYSD. The ABTS free
radical scavenging rates of DMY, DMYSD, and the positive control are
given as a function of concentration in Fig. 3B: Between concentrations
of 2 and 4 μg/mL, the ABTS radical scavenging rate of DMY was slightly
higher than that of DMYSD; thereafter, DMYSD reached a maximum
radical scavenging rate of 57.03% at 10 μg/mL, which was significantly
higher than that of DMY (p < 0.05).
Hydroxyl radical scavenging activity. The occurrence of highly
reactive hydroxyl radicals in biological systems can result in cell mem­
brane damage, destruction of DNA base sequences, induced disintegra­
tion of the double-helix structure, and eventually cell death and
mutations (Al-Mamary et al., 2014). The results shown in Fig. 3C
demonstrated that the hydroxyl radical scavenging capacity of the solid
dispersion was greater than that of DMY and the positive control (p <
0.05), which agreed with the findings of the DPPH and ABTS radical
scavenging assays.
3.1.8. MIC and MBC
The MIC and MBC of DMY, DMYSD and Tetracycline towards the
three different spoilage bacteria are shown in Table 1. Due to the fact
that tetracycline was an antibiotic with broad-spectrum antibacterial
activity and had a good antibacterial effect on both Gram-negative and
Gram-positive bacteria (Grossman, 2016), it inhibited and sterilized the
three bacteria more efficiently than DMY and DMYSD. The MIC range of
DMYSD for the three strains (0.08–0.64 mg/mL) was lower than that of
DMY (0.16–1.28 mg/mL). DMYSD showed a stronger antibacterial effect
towards P. fluorescens and S. putrefaciens compared with DMY, and its
MIC was about half that of DMY, indicating that the bioactivity of DMY
was enhanced during the preparation of the solid dispersion. Variations
in the subcellular microstructures of different bacteria can result in
variable inhibition responses towards the same active substance. Natu­
ral preservatives tend to have different antimicrobial profiles and may
have very different inhibitory effects towards the same bacteria (Wu
et al., 2019). DMY and DMYSD both exhibited good bactericidal effects
towards Aeromonas but not P. fluorescens and S. putrefaciens.
H. Xu et al. LWT 174 (2023) 114387

Fig. 3. Antioxidant performance of DMYSD. (A)

DPPH free radical scavenging activity; (B) ABTS free
radical scavenging activity; (C) Hydroxyl radical
scavenging ability. The concentration of DMYSD is
shown as the concentration of DMY in the DMYSD
solution. The bars indicated standard deviations (n =
3). Different letters in the columns indicate signifi­
cantly different (p < 0.05), uppercase letters are intra-
group comparisons, and lowercase letters are inter-
group comparisons. In the figure, the black histo­
gram is for the DMY group, the red histogram is for
the DMYSD group, the blue histogram is for the L-
ascorbic acid group. (For interpretation of the refer­
ences to colour in this figure legend, the reader is
referred to the Web version of this article.)

antibacterial effects towards the three different of bacteria types in the

Table 1
laboratory media and fish matrix.
The MICs and MBCs of DMY, DMYSD and tetracycline on three strains.
MIC (mg/mL) DMY DMYSD Tetracycline 3.2.2. Changes in pH values
Pseudomonas fluorescens 0.16 0.08 0.004 As shown in Fig. 5A, the pH values of the four groups of sturgeon
Shewanella putrefaciens 1.28 0.64 0.002 fillets initially decreased and then increased, which agreed the studies
Aeromonas 0.32 0.32 0.004 elsewhere (Sun et al., 2019; Yu et al., 2017). Maximum and minimum
pH variation occurred in the CK and DMYSD groups (p < 0.05), indi­
MBC (mg/mL) DMY DMYSD Tetracycline cating that sturgeon fillets treated with the solid dispersion passed
Pseudomonas fluorescens >5 >5 1.28 through the post-mortem stages of rigor mortis, loss of stiffness, and
Shewanella putrefaciens >5 >5 0.32 autolysis. As respiration ceases and ATP and creatine levels fall, the
Aeromonas 0.64 0.64 0.016 tissue enters rigor mortis, and formation of some organic acids by
Notes: The concentration of DMYSD is shown as the concentration of DMY in the glycolysis lower the pH. The muscle tissue then loses stiffness, and
DMYSD solution. DMY and DMYSD represent dihydromyricetin and dihy­ autolysis releases amino acids and small molecular peptides from the
dromyricetin solid dispersion, respectively. proteins. These are then utilized by microorganisms, and the alkaline
substances produced raise the pH. Under the influence of endogenous
3.2. Preservative effects of DMYSD on sturgeon fillets enzymes and those generated by microorganisms on the surface of the
fish, the continued degradation of nitrogen compounds produces more
3.2.1. Changes in microbial communities alkaline substances, which also raises the pH (Wang et al., 2017).
Changes in the TVC of sturgeon fillets during storage are shown in
Fig. 4A: On day zero, the TVC of sturgeon fillets in all groups was 3.2.3. Changes in TVB-N
approximately 3 log CFU/g; on day six, the TVC of the CK, PVP K30, and TVB-N is an indicator of the freshness of meat products, and its
DMY groups reached 7.35, 7.26, and 7.12 log CFU/g, respectively, i.e. in values typically rise with increasing storage time (Chaparro-Hernandez
excess of ng the maximum limit of 7 log CFU/g set by International et al., 2015), signaling the onset of spoilage. An upper TVB-N value
Commission for the Microbiological Specifications of Foods (Carroll & upper limit of 20 mg N/100 g has been proposed for sturgeon (Chen, Cai,
Paulson, 1986); on day nine, the TVC of the DMYSD group reached 6.92 et al., 2020). The TVB-N levels of all sturgeon groups showed a constant
log CFU/g, indicating that its shelf life could be extended at 4 ◦ C. Hence, upward trend (Fig. 5B): On day six, the TVB-N of the CK group reached
the results demonstrated that DMYSD treatment had the potential to 22.35 mg N/100 g (i.e., above the proposed limit) while the remaining
delay microbial contamination of sturgeon fillets during storage at 4 ◦ C. groups were below 20 mg N/100 g; on day nine, the PVP K30 and DMY
Pseudomonas and H2S-producing bacteria are the primary microbes treated fillets exceeded the proposed limit, while the DMYSD treated
responsible for fish spoilage during refrigeration (Wang et al., 2021); group remained below the limit (17.88 mg N/100 g). The results showed
Aeromonas is one of the main spoilage bacteria in sturgeon (Cai et al., that all treatment gave fresh-keeping benefits for sturgeon fillets. Among
2021). Pseudomonas and H2S-producing bacteria showed similar trends these, DMYSD treatment exhibited the best preservative effects.
to TVC during storage, and accounted for a significant proportion of the
total spoilage bacteria during refrigerated storage (Fig. 4B and C). 3.2.4. Changes in TBARS
Fig. 4D shows that both DMY and DMYSD had good inhibitory effects on The TBARS index is a valuable tool for assessing lipid oxidation in
Pseudomonas in sturgeon fillets. These findings corresponded to the in meat products, and measures aldehyde formation as an indicator of lipid
vitro antibacterial results, confirming that DMYSD exhibited stability. As shown in Fig. 5C, TBARS increased with increasing storage

6
H. Xu et al.
time in all four groups, with the CK group exhibiting the maximum
variation. Maximum and minimum TBARS values were observed in the
CK and DMYSD groups respectively. The increase in TBARS can be
explained by the accumulation of lipid hydroperoxides and peroxides,
which are formed via the oxidation of unsaturated fatty acids (Lu et al.,
2009; Sun et al., 2017). Hence the results showed that lipid oxidation of
the sturgeon flesh was slowed by all treatments, of which DMYSD
exhibited the optimum effect.
7
LWT 174 (2023) 114387
Fig. 4. Changes in (A) TVC, (B) Pseudomonas, (C)
H2S-producing bacteria and (D) Aeromonas of stur­
geon chunks during storage at 4 ◦ C. The bars indi­
cated standard deviations (n = 3). Different letters in
the columns indicate significantly different (p <
0.05), uppercase letters are intra-group comparisons,
and lowercase letters are inter-group comparisons.
(CK: immersed in deionized water; PVP K30: sub­
merged in 4.48 mg/mL PVP K30 solution; DMY:
immersed in 0.64 mg/mL DMY solution; immersed in
5.12 mg/mL DMYSD solution). In the figure, the
black histogram is for the CK group, the red histo­
gram is for the PVP K30 group, the blue histogram is
for the DMY group, and the green histogram is for the
DMYSD group. (For interpretation of the references to
colour in this figure legend, the reader is referred to
the Web version of this article.)
Fig. 5. Changes in (A) pH, (B) TVB-N, (C) TBARS of
sturgeon chunks during storage at 4 ◦ C. The bars
indicated standard deviations (n = 3). Different let­
ters in the columns indicate significantly different (p
< 0.05), uppercase letters are intra-group compari­
sons, and lowercase letters are inter-group compari­
sons. Sample grouping is the same as Fig. 4. In the
figure, the black line is for the CK group, the red line
is for the PVP K30 group, the blue line is for the DMY
group, and the green line is for the DMYSD group.
(For interpretation of the references to colour in this
figure legend, the reader is referred to the Web
version of this article.)
3.2.5. Changes in carbonyl and sulfhydryl contents
The most conclusive indicator of protein oxidation is believed to be
the generation of protein carbonyls, and the degree of oxidation in­
creases with carbonyl concentration. Proteins are naturally deficient in
carbon; hence oxidation is a means of introducing carbonyl bonds into
the polymer (Lund et al., 2011). Fig. 6A shows that there was an overall
rising trend in the MP carbonyl content of each group during storage.
Compared with the other three groups, the DMYSD treated group had
consistently lower carbonyl values (p < 0.05), suggesting that the
H. Xu et al.
treatment could effectively inhibit protein oxidation during storage. The
carbonyl value of DMYSD treated sturgeon fillets was 8.28 nmol/mg on
day 12, which was similar to that of the CK group on day six. The gradual
rise in carbonyl contents of the CK and PVP K30 groups in the later
stages of storage may have been caused by unstable protein carbonyl
breakdown (Li et al., 2021).
During storage, sulfhydryl is readily oxidized to disulfides by disul­
fide bond formation (Chen et al., 2022). Changes in the MP sulfhydryl
content of all samples are shown in Fig. 6B. The sulfhydryl content of all
groups increased during the first 3 days, reducing thereafter. The initial
increase was probably due to structural modifications induced by pro­
tein oxidation and exposure to the sulfhydryl groups. With the pro­
gression of storage time, the number of sulfhydryl groups decreases due
to their exposure and eventual disulfide bond or sulfonic acid formation
(Chaijan et al., 2022). The rate at which the sulfhydryl groups decreased
in the DMYSD group was always slower than that of the other three
groups. After 12 d of storage, the sulfhydryl content of the DMYSD group
was 45.81 nmol/mg, which was significantly higher than that of the CK
group (23.74 nmol/mg; p < 0.05). This demonstrated that DMYSD had a
strong preventative effect towards oxidation of MP sulfhydryl groups.
4. Conclusion
The solid dispersion system significantly improved the solubility of
DMY by preventing crystal aggregation and reducing its crystallinity.
The incorporation of DMY into PVP K30, and absence of extraneous
compounds during preparation, confirmed maintenance of the stability
and biological activity of the active ingredient. The results demonstrated
that the solid dispersion of DMY enhanced its antioxidant and antibac­
terial functions. DMYSD also suppressed protein and lipid oxidation, and
bacterial growth, in sturgeon fillets stored at 4 ◦ C, thereby extending
their shelf life by at least 3 d compared with pure DMY. DMYSD has good
potential for the preservation of fresh fish.
Author declaration
We wish to confirm that there are no known conflicts of interest
associated with this publication and there has been no significant
financial support for this work that could have influenced its outcome.
We confirm that the manuscript has been read and approved by all
named authors and that there are no other persons who satisfied the
criteria for authorship but are not listed. We further confirm that the
order of authors listed in the manuscript has been approved by all of us.
We confirm that we have given due consideration to the protection of
intellectual property associated with this work and that there are no
impediments to publication, including the timing of publication, with
respect to intellectual property. In so doing we confirm that we have
followed the regulations of our institutions concerning intellectual
property.
We understand that the Corresponding Author is the sole contact for
the Editorial process. He is responsible for communicating with the
8
LWT 174 (2023) 114387
Fig. 6. Changes in (A) Carbonyl, (B) Sulfhydryl of
sturgeon chunks during storage at 4 ◦ C. The bars
indicated standard deviations (n = 3). Different let­
ters in the columns indicate significantly different (p
< 0.05), uppercase letters are intra-group compari­
sons, and lowercase letters are inter-group compari­
sons. Sample grouping is the same as Fig. 4. In the
figure, the black line is for the CK group, the red line
is for the PVP K30 group, the blue line is for the DMY
group, and the green line is for the DMYSD group.
(For interpretation of the references to colour in this
figure legend, the reader is referred to the Web
version of this article.)
other authors about progress, submissions of revisions and final
approval of proofs. We confirm that we have provided a current, correct
email address which is accessible by the Corresponding Author and
which has been configured to accept email from (youshengzhang@263.
net).
CRediT authorship contribution statement
Hao Xu: Conceptualization, Data curation, Investigation, Writing –
original draft. Tiantian Zhao: Formal analysis, Investigation. Fengsong
Liu: Formal analysis, Investigation. Yan Zhang: Investigation, Formal
analysis. Yijia Xie: Formal analysis. Xinglong Xiao: Conceptualization,
Writing – review & editing, Supervision, Funding acquisition. Yousheng
Zhang: Formal analysis, Funding acquisition.
Declaration of competing interest
The authors declare no conflict of interest.
Data availability
No data was used for the research described in the article.
Acknowledgements
This work was supported by National Natural Science Foundation of
China [No. 32172320]; Guangdong Provincial Agricultural Science and
Technology Innovation and Promotion Project [No. 2022KJ101];
Guangzhou Science and Technology Plan Project [No. 202206010079];
Open Funding Project of Key Laboratory of Functional Foods, Ministry of
Agriculture and Rural Affairs/Guangdong Key Laboratory of Agricul­
tural Products Processing [No. 202111].
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