LWT - Food Science and Technology 173 (2023) 114237

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LWT
journal homepage: www.elsevier.com/locate/lwt

Antibiofilm effects of berberine-loaded chitosan nanoparticles against

Candida albicans biofilm
Quan Lin a, Yanxin Li b, Maokun Sheng a, Jiaman Xu a, Xiaoyan Xu c, Jintae Lee d, Yulong Tan a, *
a
Special Food Research Institute, Qingdao Agricultural University, Qingdao, China
b
College of Food Science and Engineering, Qingdao Agricultural University, Qingdao, China
c
QingYue Dental Clinic, Qingdao, China
d
School of Chemical Engineering, Yeungnam University, Gyeongsan, South Korea

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

Keywords: In recent years, the incidence of Candida contamination caused by Candida mastitis has an upward trend, which
Nanoparticle has a great impact on the hygienic quality of raw milk and dairy products. Candida albicans biofilm plays an
Chitosan important role in fungal infection. It can protect C. albicans from host defense and drug attacks, increasing its
Biofilm
drug resistance in the process of infection. Therefore, it has become an urgent problem to find effective means to
Berberine
Candida albicans
reduce the biofilm produced by C. albicans and the drug resistance of the strain. In this study, berberine (BBR)
was loaded on chitosan nanoparticles (CSNPs) to explore its anti-biofilm effect on C. albicans, and the antifungal
mechanism of BBR released from BBR-CSNPs on C. albicans was explored. Results showed that the drug loading
content (LC) and the encapsulation efficiency (EE) were the highest when the ratio of CS to BBR was 3:1.
Scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM) showed that BBR-CSNPs
had better disruption effect on the biofilm of C. albicans than BBR. BBR released from BBR-CSNPs could
destroy the cellular structure of C. albicans to achieve antifungal effect.

1. Introduction gastrointestinal, respiratory and neurological infections (Burgain et al.,

The quality of dairy products depends to a large extent on the quality
of raw milk, the content of microorganisms, especially pathogenic bac­
teria, is an important factor affecting the quality of fresh milk. Once raw
milk is contaminated by pathogenic bacteria, it not only affects the
nutrition and sensory quality of dairy products, but also brings safety
risks to the health of producers and consumers. From the existing
literature, national and international studies on milk pathogenic bac­
teria have mostly focused on bacteria, but there are few reports on
fungal contamination. Studies have shown that dairy cows are often
contaminated with Candida in fresh milk due to Candida mastitis, this
infected milk is often mixed with normal milk, which increases the
contamination rate of Candida in raw milk. If not handled properly,
chlamydospores may be retained or produced in dairy products. The
pasteurization commonly used in dairy products can not eliminate
chlamydospores thus affecting the quality of the products (Pfaller &
Diekema, 2007). The main pathogen causing Candida mastitis in dairy
cows is Candida albicans, a deep conditioned pathogenic yeast common
in humans and animals that can cause mucosal, cutaneous,
2019; Su et al., 2020). C. albicans produces biofilm in the process of
proliferation. The biofilm formed by C. albicans is a dense network
system with complex three-dimensional structure, which is composed of
a large number of extracellular matrix, yeast cells, pseudohyphae and
hyphae (Fulaz, Vitale, Quinn, & Casey, 2019; Liang et al., 2019). The
biofilm structure can make C. albicans have stronger resistance to anti­
fungal drugs and host immune system than planktonic cells (Chandra
et al., 2001; Galdiero et al., 2021; Sharma, Misba, & Khan, 2019). It is
reported that the drug resistance of cells in biofilm is more than 1000
times higher than that of planktonic cells (Davies, 2003). The problem of
drug resistance caused by C. albicans biofilm makes clinical treatment
more difficult.
At present, the main antifungal drugs used to treat C. albicans
infection, including amphotericin B, 5-fluorocytosine, azoles and echi­
nocandins (Gray et al., 2012; Kathiravan et al., 2012; Newman & Cragg,
2012). However, these types of antifungal drugs have large side effects:
amphotericin B not only binds to ergocalciferol in fungal cell mem­
branes, but also binds to cholesterol in mammalian cell membranes,
causing toxic effects. Azoles have become resistant to an increasing

* Corresponding author.
E-mail address: tanyulong@qau.edu.cn (Y. Tan).

https://doi.org/10.1016/j.lwt.2022.114237
Received 17 October 2022; Received in revised form 24 November 2022; Accepted 28 November 2022
Available online 1 December 2022
0023-6438/© 2022 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/).
Q. Lin et al.
number of pathogenic fungi, especially C. albicans, due to their extensive
clinical use. Therefore, it is urgent to develop new highly efficient and
low-toxic antifungal drugs.
Berberine (BBR) is an isoquinoline alkaloid. It naturally exists in
many plants, such as Annonaceae, Berberidaceae, Leguminosae and
Papaveraceae (Neag et al., 2018). BBR is also an important compound in
traditional Chinese medicine. It has a variety of physiological activities,
such as antibacterial, anti-tumor, anti-inflammatory, anti-diabetes,
cholesterol reduction and mitochondrial function damage repair (Chen
et al., 2016; Song, Luo, Wang, Almutairi, & Hong, 2019; Tong et al.,
2021). It has been reported that BBR could inhibit microbial adhesion
and biofilm formation (Tong et al., 2021). However, the low solubility
and bioavailability of BBR limit its application. Chitosan (CS) is a
copolymer composed of random repeating units of β-(1,4)-coupled
N-acetyl glucosamine. CS is widely used in the field of medical materials
and biomedicine because of its good biological activity (Sun et al.,
2017). In addition, CS is usually positively charged, while the lipid layer
on the surface of microorganisms is negatively charged, which makes
microorganisms easy to be electrostatically adsorbed by positively
charged CS particles, which also enhances the ability of drugs to pene­
trate cells (El-Newehy, Al-Deyab, Kenawy, & Abdel-Megeed, 2011). CS
can use tripolyphosphate (TPP) as crosslinking agent to form nano­
particles (NPs) (Algharib et al., 2022). Chitosan nanoparticles (CSNPs)
carrier has the advantages of smaller particle size, easy absorption, good
drug release, high drug loading and targeted delivery (Lima, Andrade,
Alves, de Morais, & Vieira, 2021; Manjunath, Reddy, & Venkateswarlu,
2005; Martinez et al., 2010). Therefore, in this study, CSNPs were pre­
pared by ionic gel method and BBR was loaded on CSNPs. The
anti-biofilm effect of BBR-CSNPs against C. albicans was explored, and
the antifungal mechanism of BBR released from BBR-CSNPs against
C. albicans was also studied.
2. Materials and methods
2.1. Materials
The standard strain of C. albicans DAY185 was stored in our labo­
ratory. C. albicans was cultured in Yeast Peptone Dextrose (YPD) me­
dium (Solarbio, China) at 30 ◦ C. CS (molecular weight = 30,000), BBR
and other reagents were all purchased from Shanghai Macklin
Biochemical Technology Co., Ltd (China).
2.2. Preparation of CSNPs
TPP was used as a cross-linking agent to form CSNPs. Briefly, CS was
dissolved in 1% acetic acid solution to form 1% CS solution and filtered
with 0.45 μm filter membrane. TPP was dissolved in double distilled
water (1 mg/mL), and then TPP was added to CS solution drop by drop.
The volume ratio of CS to TPP was 3:1, and then the solution was stirred
at the speed of 600 rpm/min for 2 h. The solution was centrifuged by
high-speed centrifuge (12,000 rpm, 30 min) to obtain CSNPs. The CSNPs
were stored at − 20 ◦ C after freeze-drying.
Preparation of BBR-CSNPs: 2048 μg/mL BBR was mixed with CS
solution (1 mg/mL) at different volume ratios (1:2, 1:3, 1:4, 1:5 and 1:6).
TPP was dissolved in double distilled water (1 mg/mL), and then TPP
was added to CS solution drop by drop. The volume ratio of CS to TPP
was 3:1, and then the solution was stirred at the speed of 600 rpm/min
for 2 h. After centrifugation at 12,000 rpm for 30 min, BBR-CSNPs were
obtained. BBR-CSNPs were freeze-dried and stored at − 20 ◦ C for sub­
sequent experiments.
2.3. Characterization of BBR-CSNPs
The surface charge of BBR-CSNPs were determined by Zeta Sizer
Nano ZS90 (Malvern Instruments, UK). The size and zeta potential of
BBR-CSNPs were analyzed by Dynamic Light Scattering (DLS) assays.
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LWT 173 (2023) 114237
The morphology of BBR-CSNPs were observed by Transmission Electron
Microscopy (TEM, HT7700, Japan, 80kv).
2.4. Determination of drug encapsulation of BBR-CSNPs
BBR and CSNPs with different volume ratios were prepared to obtain
an optimum ratio of drug loading. The BBR standard curve was quan­
titatively determined by UV-spectroscopy at 344 nm. The content of BBR
encapsulated in NPs was determined by centrifugation. After the prep­
aration of BBR-CSNPs, the solution was centrifuged immediately
(12,000 rpm, 30 min), and the supernatant was quantitatively analyzed
by UV-spectroscopy. The BBR encapsulation efficiency (EE) and loading
content (LC) were calculated as follows:
EE = (A-B)/A (1)
LC = (A-B)/C (2)
A = BBR added; B = Free non-loaded BBR; C = NPs weight
2.5. In-vitro release of BBR-CSNPs
The release behavior of BBR-CSNPs was measured with reference to
the previous literature and slightly modified (Shetta, Kegere, & Mam­
douh, 2019). The in-vitro release kinetics of BBR-CSNPs was assessed as
follows: 5 mL BBR-CSNPs (1 mg/mL) was added to the dialysis bag and
put into 200 mL PBS. The mixture was stirred at 100 rpm/min. A total of
5 mL PBS was removed at predetermined intervals and replaced with 5
mL fresh PBS. The BBR in PBS was determined by UV-spectroscopy at
344 nm.
2.6. The inhibitory effect of BBR-CSNPs or free BBR on C. albicans
biofilm
After overnight culture, C. albicans was diluted with medium to
OD600 of 0.01. C. albicans and different concentrations of BBR-CSNPs or
free BBR (64, 128, 256, 512, 1024 μg/mL) were added to 96-well plates,
and the plates were incubated at 30 ◦ C for 24 h. The inhibitory effect of
BBR-CSNPs or free BBR on C. albicans biofilm was measured quantita­
tively by crystal violet staining method. The biofilm was washed with
PBS for 3 times, stained with 100 μL 0.1% crystal violet solution for 30
min. After washing with distilled water for 3 times, the cells were treated
with 100 μL 30% acetic acid. The A570 of the extract was measured with
a microtitrate reader to determine the content of biofilm.
2.7. Disruption effects of BBR-CSNPs or free BBR on C. albicans biofilm
100 μL fungal solution and 100 μL YPD medium were added to 96-
well plates, and the plates were cultured for 48 h to form biofilm. The
supernatant was removed carefully and 100 μL fresh medium containing
BBR-CSNPs or free BBR (256, 512, 1024 and 2048 μg/mL) was added to
the formed biofilm and cultured for 24 h. Biofilms were quantified by
crystal violet staining method to evaluate the scavenging effect of BBR-
CSNPs or free BBR on formed biofilm.
2.8. Observation of C. albicans biofilm
The effect of free BBR or BBR-CSNPs on structural changes of
C. albicans biofilm were observed by scanning electron microscopy
(SEM, JEOL 7500F, Japan), confocal laser scanning microscopy (CLSM,
Zeiss, lsm900, GER) and fluorescence microscope (Nikon ECLIPSE Ti2).
Live or dead strains in biofilm were determined according to the in­
structions of Live & Dead Bacterial Staining Kit (40274 ES60, Yeasen).
The sterilized chip was placed in a 48-well plate. 1 mL of C. albicans
solution with OD600 of 0.01 was added on top of the chip and incubated
Q. Lin et al.
at 30 ◦ C for 48 h to form biofilm on the chip. The supernatant was
carefully taken out and rinsed with PBS for 3 times. BBR-CSNPs or free
BBR (512 μg/mL, 1 mL) was added 24 h, 1 mL of medium was used as the
control. The supernatant was sucked away and the chip was flushed with
PBS for 3 times. Afterwards, the chip was fixed in 2.5% (v/v) glutaral­
dehyde solution for 4 h, and then treated in 30%, 50%, 70%, 80%, 90%,
100% ethanol and 100% tert-butanol for 15 min. Finally, the C. albicans
biofilm was dried and sprayed with gold and observed by SEM (10 KV,
× 5000).
1 mL of C. albicans solution with OD600 of 0.01 was added to 48-well
plates and incubated at 30 ◦ C for 48 h to form biofilm. The supernatant
was carefully removed, rinsed 3 times with PBS, and treated with BBR-
CSNPs or free BBR (512 μg/mL, 1 mL) for 24 h, respectively. 1 mL of
medium was added as the control, and the supernatant was aspirated
and rinsed 3 times with PBS again. Biofilm was stained with Live & Dead
Bacterial Staining Kit. 1 volumetric DMAO and 2 volumetric EthD-III
were mixed in a microcentrifuge tube, then 8 volumetric 0.85% NaCl
solution were added to obtain 100 × dye solution. Then the solution was
incubated in the dark at room temperature for 15 min. The biofilm was
observed with CLSM and fluorescence microscope with FITC and Cy3
channels.
2.9. Antifungal mechanism of BBR released from BBR-CSNPs on
C. albicans
2.9.1. Determination of minimum inhibitory concentration (MIC) of BBR
The MIC of BBR against C. albicans was determined by microdilution
method. BBR solution with a concentration of 2048 μg/mL was prepared
and filtered with filter membrane. C. albicans was cultured in YPD me­
dium at 30 ◦ C for 12 h, and then diluted with the medium to OD600 of
0.01. 100 μL fungal solution and 100 μL drug solution were added to 96-
well plate to make the final concentration of BBR at 1024, 512, 128, 64,
32 and 16 μg/mL, with medium as control, and incubated at 30 ◦ C for 24
h. The MIC was recorded as the lowest concentration, which exhibited
complete inhibition of visible growth of test microorganism. The
experiment was repeated three times and the average value was taken.
2.9.2. Growth curve of C. albicans treated with BBR
C. albicans cultured to OD600 of 0.01 was added to a 24 well plate,
and then the BBR was added to the plates so that the total volume of
fungal solution was 1 mL. The fungal solution without BBR was used as
the control. The strain growth curve (Jie Ling, MicroScreen-HT) was
measured by cell growth curve analyzer. Instrument parameters: tem­
perature: 30 ◦ C, 800 rpm/min, measured every 1 h.
2.9.3. Determination of cell wall and cell membrane integrity of C. albicans
Alkaline phosphatase (AKP), nucleic acid and protein leakage were
detected to evaluate the integrity of C. albicans cell wall and cell mem­
brane. C. albicans was treated with BBR, and C. albicans without BBR was
used as control. The strain was incubated at 30 ◦ C in a 150 rpm/min
shaking table for 10 h, and its AKP was determined according to the
instructions of AKP activity determination kit (Nanjing Jiancheng,
China). After the fungal solution was centrifuged at 10,000 rpm/min at
4 ◦ C for 5 min, the absorbance of the supernatant was measured at 260
nm to determine the nucleic acid content (Lv, Liang, Yuan, & Li, 2011).
The content of protein in the supernatant was determined by Coomassie
brilliant blue quantitative method (Bradford, 1976).
2.9.4. Measurement of cell membrane permeability of C. albicans
1 MIC BBR was added into the suspension of C. albicans to make the
suspension concentration to OD600 of 0.01. The suspension was
cultured at 30 ◦ C at 150 rpm/min for 10 h. The distilled water was used
as the control. The concentration of K+ was measured by cell membrane
permeability (Nanjing Jiancheng, China), and the absorbance was
measured at 440 nm.
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LWT 173 (2023) 114237
2.9.5. Microstructure observation of C. albicans
The microstructure of C. albicans was observed by SEM (VEGA3,
TESCAN, China). After overnight culture, C. albicans was diluted with
medium to OD600 of 0.01. BBR was added to the experimental group so
that the final concentration of BBR was 512 μg/mL. The fungal solution
without BBR was used as the control. C. albicans was cultured for 24 h,
centrifuged at 4 ◦ C at 10000 rpm for 10 min and washed with PBS for 3
times. Then it was fixed with 2.5% (v/v) glutaraldehyde solution for 4 h,
and then treated in 30%, 50%, 70%, 80%, 90% 100% ethanol and 100%
tert-butanol for 15 min, respectively. Finally, the cells were dried and
sprayed with gold and observed by SEM.
2.10. Statistical analysis
All of the experimental results are expressed as the mean ± standard
deviation (n = 3). One-way analysis of variance (ANOVA) was per­
formed using SPSS 19 (SPSS Inc., USA). Statistical significance was
accepted at p < 0.05.
3. Results and discussion
3.1. Characterization of BBR-CSNPs
As shown in Table 1, the LC of BBR-CSNPs with different volume
ratios (CS: BBR = 6:1, 5:1, 4:1, 3:1 and 2:1) were (6.95 ± 0.35) %,
(14.83 ± 0.32) %, (17.46 ± 0.49) %, (32.06 ± 0.53) % and (26.94 ±
0.38) %. When the volume ratio of CS: BBR = 3:1, the drug LC and EE of
BBR-CSNPs reached the maximum, which were (32.06 ± 0.53) % and
(46.96 ± 0.78) %. However, when the initial volume ratio of CS: BBR
increased to 2:1, the EE and LC values decreased, which might be due to
the saturation of BBR wrapped in CSNPs. Similarly, Keawchaoon et al.
found that the EE and LC of carvacrol-loaded CSNPs increased first and
then decreased with the increase of the proportion of CS (Keawchaoon &
Yoksan, 2011).
The particle size distribution and zeta potential of BBR-CSNPs were
measured by DLS. The diameter of CSNPs was (169.90 ± 1.97) nm. After
loading BBR to CSNPs, the size was changed to (183.63 ± 5.78) nm.
The zeta potential indicates that the charge of the particle is related
to the surrounding environment. The surface charge of CSNPs had direct
effects on the interaction with the BBR and was closely related to the
bioavailability of the BBR. Consequently, zeta potential was used to
detect the magnitude of the surface charge and then to assess the sta­
bility of the particles in the suspension (Cai et al., 2022). The Zeta po­
tential of CSNPs was (+23.43 ± 2.65) mV. After loading BBR to CSNPs,
the Zeta potential of NPs changed to (+33.27 ± 2.57) mV.
As can be seen in Fig. 1, The shape and microstructure of CSNPs with
BBR was demonstrated by TEM. TEM images showed that the BBR-
CSNPs were uniform and their surface were spherical and smooth.
However, the average diameters of samples measured by TEM were
smaller than those determined by DLS because DLS measured the hy­
dration radius of particles which certainly showed a larger size than
those observed by TEM.
3.2. In-vitro release of BBR-CSNPs
The drug release process of CSNPs can be divided into two stages: the
Table 1
Drug loading content and encapsulation efficiency of BBR-CSNPs.
BBR: CS (v/v) LC (%) EE (%)
1:2 26.94 ± 0.38 26.31 ± 0.37
1:3 32.06 ± 0.53 46.96 ± 0.78
1:4 17.46 ± 0.49 34.09 ± 0.95
1:5 14.83 ± 0.32 36.20 ± 0.78
1:6 6.95 ± 0.35 20.35 ± 1.02
Q. Lin et al.
Fig. 1. TEM image of BBR-CSNPs.
release mechanism of the first stage is attributed to the release of BBR
adsorbed on the surface of NPs (Woranuch & Yoksan, 2013). The second
stage is controlled release, and the drug release was slow. Fig. 2 shows
the release curve of BBR-CSNPs in vitro. BBR-CSNPs showed sudden
release in the first 11 h, and 40.4% load BBR was released. It is possible
that BBR was coated on the surface of NPs and eventually diffused easily
through the surface of NPs. Subsequently, the release rate of the drug
slowed down. Within 48 h, the amount of BBR released from BBR-CSNPs
was 61.3%. This result could be that the release of BBR is diffused into
the NPs system through the medium. In this mode, the BBR on
BBR-CSNPs can be delivered to the interior of the biofilm by NPs, and
the solution can be slowly released in the biofilm structure to achieve a
slow fungicidal effect, reducing the drug resistance of high-dose solution
to the fungi in the biofilm.
3.3. Inhibitory effect of free BBR and BBR-CSNPs on C. albicans biofilm
formation
As shown in Fig. 3, free BBR and BBR-CSNPs had significant inhib­
itory effect on C. albicans biofilm, and the inhibitory effect on biofilm
was concentration-dependent. Free BBR even at the lowest concentra­
tion (64 μg/mL), about 40% of the C. albicans biofilm formation was
inhibited. When free BBR concentration was higher than 512 μg/mL,
almost no biofilm formation was observed. In the same concentration
range, BBR-CSNPs showed lower inhibitory activity on the biofilm of
C. albicans compared with free BBR. The reason may be that the BBR-
Fig. 2. In vitro release behavior of BBR-CSNPs.
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LWT 173 (2023) 114237
CSNPs could not release the same concentration of BBR immediately,
therefore BBR-CSNPs was not as effective as free BBR in inhibiting the
formation of C. albicans biofilm.
3.4. Scavenging effect of free BBR and BBR-CSNPs on mature biofilm of
C. albicans
Fig. 4 shows the scavenging effects of free BBR and BBR-CSNPs on
the formed mature biofilm of C. albicans at different concentrations. It
can be seen from the figure that the clearance rate of biofilm by BBR-
CSNPs was higher than that of free BBR. The clearance rate of biofilm
by the highest concentration of BBR-CSNPs was more than 70%, while
the clearance rate of biofilm by free BBR was 42%. At the lowest con­
centration of 256 μg/mL, the clearance rate of BBR-CSNPs was 36%
higher than that of free BBR. The results showed that the biofilm
disruption effect of BBR-CSNPs was superior to that of BBR. There are
several possible reasons for this result. Firstly, CS not only has good
hydrophilicity, biocompatibility and biodegradability, but also contains
amino groups, which is the only positively charged polysaccharide (Tan,
Leonhard, Moser, & Schneider-Stickler, 2016). The nanostructures and
positive charge of CSNPs make them have good permeability and easy to
combine with the cells on the surface and inside the biofilm, causing the
change of cell surface charge, increasing the sensitivity of cells to drugs,
and then affecting the formation of biofilm (Lrl, Fka, Dra, Smdm, & Rsv,
2021). Moreover, the nanostructure and positive charge of BBR-CSNPs
had good permeability and were easy to be combined with microbial
cells. Secondly, CS can also form NPs with polyanion TPP through
electrostatic interaction in aqueous solution. CSNPs have small particle
size and large specific surface area (Tan et al., 2022). They can deliver
drugs to the biofilm matrix and have slow-release behavior. At last, BBR
continues to be released from BBR-CSNPs until 48 h. The BBR released
from BBR-CSNPs could be transported to the biofilm through CSNPs,
thus killing C. albicans cells in the biofilm. Compared with direct
fungicidal effect of high concentration BBR on C. albicans, BBR-CSNPs
could achieve fungicidal effect at low concentration, so as to effec­
tively reduce the drug resistance of strains. Therefore, BBR-CSNPs
release drugs in the biofilm to achieve sustained-release and long-term
antifungal effects.
3.5. Effects of BBR and BBR-CSNPs on C. albicans biofilm observed by
fluorescence microscope
Fig. 5 shows the living and dead C. albicans cells in the biofilm
observed by fluorescence microscope. When C. albicans was stained with
Live & Dead Bacterial Staining Kit, living cells show green fluorescence,
and damaged or dead cells show yellow-green or red fluorescence.
Fig. 5A shows the untreated C. albicans, and as can be seen from the
figure, almost all of which are green fluorescence, which indicates that
almost all of the fungi in the control group are viable. While Fig. 5B
shows C. albicans treated with BBR, some red fluorescence appears in the
picture, indicating that BBR has antifungal activity against C. albicans
and kills some fungi. Fig. 5C shows the effect of BBR-CSNPs on
C. albicans biofilm. It can be seen from the figure that red fluorescence
accounts for the majority of the figure, indicating that BBR-CSNPs have
stronger killing ability against C. albicans.
Fig. 6 shows the three-dimensional structural changes of C. albicans
biofilm after treatment with free BBR and BBR-CSNPs. When C. albicans
was stained with Live & Dead Bacterial Staining Kit, living cells show
green fluorescence, and damaged or dead cells show yellow-green or red
fluorescence. Fig. 6A shows the control group, and it can be seen from
the figure that the biofilm produced by C. albicans has strong green
fluorescence, the biofilm was thick and had compact structure. After
BBR treatment, the red fluorescence increased and the biofilm thickness
decreased (Fig. 6B); Fig. 6C shows the C. albicans treated with BBR-
CSNPs. It can be seen from the figure that the biofilm produced by the
strain has strong red fluorescence and large holes in the biofilm,
Q. Lin et al. LWT 173 (2023) 114237

Fig. 3. Inhibitory effects of BBR and BBR-CSNPs on C. albicans biofilm. A: C. albicans biofilms treated with BBR; B: C. albicans biofilms treated with BBR-CSNPs. Data
are expressed as means ± standard deviations (n = 3), *p < 0.05, **p < 0.01 versus BBR control group.

Fig. 4. Scavenging effect of BBR and BBR-CSNPs on C. albicans mature biofilm. A: C. albicans biofilms treated with BBR; B: C. albicans biofilms treated with BBR-
CSNPs. Data are expressed as means ± standard deviations (n = 3), *p < 0.05, **p < 0.01 versus BBR control group.

Fig. 5. Effect of BBR and BBR-CSNPs on C. albicans biofilms observed by fluorescence microscope. A: Control; B: C. albicans biofilms treated with BBR; C: C. albicans
biofilms treated with BBR-CSNPs.

indicating that the structure of the biofilm was destroyed and the
C. albicans were killed after treated with BBR-CSNPs. The results also
indicated that BBR-CSNPs was easier to penetrate the biofilm and kill the
cells in the biofilm of C. albicans.
Fig. 7 shows the effect of BBR and BBR-CSNPs on C. albicans biofilm
by SEM. It can be seen from the figure that C. albicans in the control
group formed dense three-dimensional structures of biofilm (Fig. 7A).
After treated with free BBR and BBR-CSNPs, the biofilm structure dis­
integrated and cells detachment (Fig. 7B and C). In particular, the bio­
film of the strain changed significantly after treating with BBR-CSNPs,
the situation of biofilm structure disintegration and cell shedding was
even more severe. The results of SEM were consistent with those of
fluorescence microscope and CLSM.

5
Q. Lin et al.
Fig. 6. Effect of BBR and BBR-CSNPs on C. albicans biofilms observed by CLSM. A: Control; B: C. albicans biofilms treated with BBR; C: C. albicans biofilms treated
with BBR-CSNPs.
Fig. 7. Effect of BBR and BBR-CSNPs on C. albicans biofilms observed by SEM. A: Control; B: C. albicans biofilms treated with BBR; C: C. albicans biofilms treated with
BBR-CSNPs.
3.6. Antifungal mechanism of BBR released from BBR-CSNPs on
C. albicans
The results showed that the MIC of BBR to C. albicans was 512 μg/
mL. C. albicans was treated with 1 MIC, and the untreated strain was
used as the control. The growth curve of the strain was shown in Fig. 8A.
It can be seen from the figure that the untreated strain showed an S-
shaped growth curve with stable fungal growth, logarithmic growth
period and stable growth period. while the growth curve decreased
significantly after BBR treatment, indicating that BBR had a significant
inhibitory effect on the growth of C. albicans, and the inhibition rate
reached 93%.
Many natural medicine solutions inhibit microorganisms by inter­
fering with cell membrane structure and enzyme system. The cell wall
maintains the inherent shape of the cell and protect the cell against the
hypotonic environment, thus acting as a barrier. AKP is an enzyme that
exists between cell wall and cell membrane. Normally, AKP cannot be
detected outside the cell, but AKP will leak out of the cell when the cell
wall or cell membrane is damaged. The integrity of cell wall can be
reflected by detecting the changes of extracellular AKP (Diao et al.,
2018; Hara & Yamakawa, 1995; Sharma, Bajpai, & Baek, 2013). The cell
membrane, also known as the plasma membrane, is not only the
boundary of the cell structure to make cells have a stable internal
environment, but also plays a decisive role in the exchange of matter,
energy and information between the cell and the environment. Inoue
et al. found that K+ leakage is one of the direct evidence of membrane
damage (Inoue et al., 2004). By studying the bacteriostatic mechanism
of tea polyphenols against Pseudomonas aeruginosa, Long et al. have
shown that biomolecules such as nucleic acid and protein, which run
through the whole cell membrane and cytoplasm, are important com­
ponents of cells. When the cell membrane is destroyed, macromolecules
such as nucleic acid and protein will leak out of the cell (Long et al.,
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LWT 173 (2023) 114237
2014). In this study, the effects of BBR on the cell wall and cell mem­
brane integrity of C. albicans are shown in Fig. 8B-E. As can be seen from
the figure, the content of extracellular nucleic acid, AKP, protein and K+
increased significantly after the treatment of BBR, indicating that BBR
could break the cell wall and cell membrane of C. albicans.
The microscopic changes on the surface of C. albicans cells treated
with BBR and without BBR were observed by SEM. It can be seen from
Fig. 9 that the untreated C. albicans shows the normal form of the strain,
the cell was oval and the surface was smooth. After BBR treatment, the
surface of the strain appeared depression, the surface roughness of the
strain increased, and the phenomena of cytoplasmic leakage and cell
adhesion and aggregation appeared. The results of SEM were consistent
with those in the determination of extracellular nucleic acid, AKP,
protein and K+.
4. Conclusions
In this study, the prepared BBR-CSNPs had positive charge, spherical
shape, uniform particle size, high entrapment efficiency and drug
loading, and good sustained-release behavior. BBR-CSNPs had a good
inhibitory effect on the biofilm of C. albicans. BBR released from BBR-
CSNPs could inhibit the growth of C. albicans and destroy the integrity
of cell wall and cell membrane of C. albicans. BBR-CSNPs has great
application potential in inhibiting C. albicans biofilm infection.
CRediT authorship contribution statement
Quan Lin: Writing – original draft, carried out the experiments and
wrote the manuscript. Yanxin Li: Writing – original draft, did a lot of
work on the writing and revision of the article in the later period.
Maokun Sheng: assisted in the experiment and checked the format of
the article. Jiaman Xu: guided and helped the experiment. Xiaoyan Xu:
Q. Lin et al. LWT 173 (2023) 114237

Fig. 8. Inhibitory effect of BBR on the growth, cell wall and cell membrane integrity of C. albicans. A: Growth curve; B: Nucleic acid content; C: Protein content; D: K+
content; E: AKP content.

7
Q. Lin et al.
Fig. 9. The morphological differences of C. albicans treated (A) and untreated (B) with BBR observed by SEM.
Writing – original draft, has made changes to the writing and format of
the article. Jintae Lee: Writing – original draft, put forward his own
valuable suggestions in the structure and writing of the article. Yulong
Tan: provided the experimental ideas and guided the revision of the
article.
Declaration of competing interest
All authors declare no conflict of interest.
Data availability
Data will be made available on request.
Acknowledgements
The research was financially supported by the Young Taishan
Scholars Program of Shandong Province (tsqn202103094) and Talent
Research Foundation of Qingdao Agricultural University (665/
1121020).
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