Probiotics and Antimicrobial Proteins

https://doi.org/10.1007/s12602-023-10149-4

RESEARCH

Weissella confusa N17 Derived from Loach (Misgurnus anguillicaudatus)

Exhibits Promising for Further Applications in Loach Aquaculture
Bintong Yang1,2,3 · Haichao Song1 · Renge Hu1 · Luotao Tao3 · Zhenlin Liang1 · Wei Cong1 · Yuanhuan Kang1,3,4

Accepted: 22 August 2023

© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2023

Abstract
The application of probiotics, in aquaculture, is becoming increasingly widespread and have had positive application effects.
However, reports of loach-derived probiotics are quite limited. In this study, two representative strains of lactic acid bacteria
with excellent traits, namely, Weissella confusa N17 and Lactobacillus saniviri N19, were screened from the intestine of
healthy loaches. W. confusa N17 and L. saniviri N19 could inhibit different common various pathogenic bacteria, especially
Aeromonas spp., and were sensitive to the most common antibiotics. The survival rate of the two strains exceeded 50% after
4 h of incubation in 10% loach bile. Moreover, the two strains showed significant tolerance to trypsin. Their autoaggregation
capacity and hydrophobicity were greater than 30%. In addition, the aggregation ability of both strains was higher than 30%
for both A. veronii TH0426 and A. hydrophila TPS. The two strains had a high biofilm-forming ability and strong adhesion
to epithelioma papulosum cyprini (EPC) cells. Scanning electron microscopy results showed that the culture supernatants
of the two strains had a significantly destructive effect on A. veronii TH0426 and A. hydrophila TPS. Overall, the traits of
W. confusa N17 were better than those of L. saniviri N19. Genome sequencing and analysis demonstrated a lack of viru-
lence factor-related or drug resistance–related genes in genome N17. The diet supplemented with the W. confusa N17 strain
significantly improved the resistance of loaches to A. veronii infection, and the protection rate reached 57.1%. Therefore, W.
confusa N17 exhibits promising for further applications in loach aquaculture.

Keywords Loach (Misgurnus anguillicaudatus) · Weissella confusa · Antibacterial activity · Immune protection

Introduction in recent years. To meet market needs, artificial loach farm-

Loach (Misgurnus anguillicaudatus), belonging to the fam-
ily Cobitidae of the order Cypriniformes, is one of the most
popular delicacies for consumers because of its delicious
taste and rich nutrition [1]. Due to overfishing and environ-
mental pollution, natural populations have sharply decreased
* Yuanhuan Kang
kangyuanhuan@sdu.edu.cn
1
Marine College, Shandong University/Key Laboratory
of Modern Marine Ranching Technology of Weihai,
Weihai 264209, China
2
Shandong Fu Han Ocean Sci-Tech Co., Ltd, Haiyang 265100,
China
3
College of Veterinary Medicine/College of Animal
Science and Technology, Jilin Agricultural University,
Changchun 130118, China
4
Shandong Key Laboratory of Animal Microecological
Preparation, Tai’an 271000, China
ing is gradually emerging. However, excessive breeding
density has led to increasingly prominent disease problems,
and the reported disease pathogens include fungi, parasites,
bacteria, and viruses, among which bacterial pathogens are
the most harmful [1]. The more common diseases are caused
by Aeromonas veronii and Aeromonas hydrophila, which can
cause ulceration on the surface of the gill cover, ulceration
of subcutaneous muscles, and enlargement and congestion
of the liver, kidney, and spleen [2, 3]. Some diseased fish
even show clinical signs such as punctate, mass or diffuse
bleeding on the body surface, and ascites [4], which seri-
ously endanger the health of loach while affecting the pro-
duction efficiency of loach aquaculture. To prevent and con-
trol bacterial diseases, the long-term use of chemicals and
antibiotics leads to the development of resistance to aquatic
pathogens and suppression of the host’s immune system.
In the long run, the horizontal transfer of antibiotic resist-
ance genes, the harm to the ecological environment, and the
requirements for “reducing and limiting antibiotics” in the

13
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aquaculture industry make it necessary to find alternatives to
antibiotics as soon as possible. Probiotics are the most prom-
ising because they are nontoxic, nonresistant, and residue-
free and can improve the performance of farmed animals [5].
Probiotics can provide numerous health benefits, such
as promoting digestion, improving nutrient bioavailability,
strengthening intestinal barrier function, detoxifying toxic
compounds, and modulating the immune response [6]. Vari-
ous types of probiotics are used in aquaculture, including
nitrifying bacteria, photosynthetic bacteria, Bacillus subtilis,
and lactic acid bacteria (LAB) [7–9]. Among these, LAB is
found to be the most prominent, as it is a part of the natu-
ral intestinal microflora of healthy aquatic animals. LAB
usually has high acid and bile-salt tolerance and inhibit the
growth of a wide range of pathogens [10, 11].
Most of the probiotic strains currently used in aquaculture
are not derived from the animal itself [12]. The physiological
peculiarities and differences of every host and the signifi-
cant influences of environmental factors make it difficult to
develop probiotic microorganisms with universal applica-
tions. The source of candidate probiotic microorganisms
significantly contributes to their success of application [13].
Therefore, in recent years, there has been increasing atten-
tion on the use of the host-associated microbiota, that is, iso-
lates from host animals or from the feeding environment as a
source of probiotics [14–17]. Many studies have focused on
host-associated microorganisms, especially the microbiota
in the intestine, which is a crucial object of interest for bio-
prospecting [13]. The host-associated microbiota is naturally
established in the host defense system and exhibits many
probiotic characteristics that give host-associated probiotics
a natural advantage in adapting to the host intestinal envi-
ronment as well as in performing their functions [18]. The
current research on probiotic bacteria from loach sources is
limited. Considering their nutritional and economic value, it
is necessary to accelerate the pace of research and promote
the development of loach aquaculture in the direction of a
green and healthy culture.
This study aimed to isolate, identify, and characterize
LAB from the intestinal microbiota of loach; screen LAB
strains with potential probiotic properties; and evaluate their
safety as well as their protective effect on loach to lay the
foundation for the later application of loach-derived LAB.
Materials and Methods
Strains and Experimental Animals
Aeromonas hydrophila HA336, BSK, and TPS strains; Aer-
omonas veronii TH0426, CL8155, JL1021, AV75, NJ723,
AV115, ATCC35624, and QXF strains; Aeromonas caviae
ATCC15468 strain; Salmonella enterica serovar Enteritidis
13
Probiotics and Antimicrobial Proteins
CVCC3378 strain; Streptococcus agalactiae strain and Sal-
monella typhimurium strain used in the experiments are all
preserved in the Laboratory of Preventive Veterinary Medi-
cine, Jilin Agricultural University. Weissella confusa N17
and L. saniviri N19 strains were isolated in this study, and
preserved in the Laboratory of Preventive Veterinary Medi-
cine, Jilin Agricultural University.
Two hundred healthy loaches (45 ± 3 g) were purchased
from a fishery market in Changchun (China) for screening
LAB, animal safety experiments, and challenge experiments.
The loaches were placed in the aquarium temporarily and
continuously oxygenated, and the water temperature was
controlled at 25 ± 2 °C for 7 days without any abnormalities
and then used for the screening experiment. After 1 month
of feeding, the loaches were fasted for 7 days for the animal
safety and challenge experiments.
Isolation and Identification of LAB and Preliminary
Screening of Antagonistic Bacteria
Isolation and Biochemical Identification of LAB
Healthy loaches were anesthetized, disinfected, and dis-
sected, and then, the intestine was removed, washed with
phosphate-buffered saline (PBS), cut into pieces, and resus-
pended in 10 × PBS solution. The dilution solution was taken
and spread evenly on a MRS agar medium (Haibo, China)
plate (in triplicate) and cultured in an anaerobic jar at 30 °C
for 48 h. Independent colonies were picked and placed in
MRS broth medium (Solarbio, China) and cultured anaero-
bically for 24 h. An appropriate amount of bacterial liquid
was further dipped with an inoculation ring. Then, the grown
bacteria were spread on the MRS medium plate to culture
for 24 h. According to the above process, bacteria were har-
vested by centrifugation at 8000 rpm for 10 min and stored
in glycerol (16% final concentration).
The purified LABs were Gram stained and tested for cata-
lase. An appropriate amount of suspected LAB was placed
on a sterile glass slide with a drop of 3% hydrogen peroxide.
The strains producing bubbles were discarded. Then, the
bacterial solution was centrifuged at 5000 rpm for 10 min
and resuspended in sterile PBS. An appropriate amount of
bacterial solution was placed on a sterile glass slide, Gram
staining was performed, and the Gram-positive strains were
retained.
Preliminary Screening of Strains Inhibiting A. veronii and A.
hydrophila
Aeromonas hydrophila TPS and A. veronii TH0426 strains
were incubated in LB broth medium (Haibo, China) at 30 °C
for 24 h. Then, the bacterial solutions were streaked onto RS
medium (Haibo, China) plates to perform strain screening.
Probiotics and Antimicrobial Proteins
Continuous single colonies were selected and placed in LB
agar medium, which was then incubated for 24 h. This pro-
cess was repeated twice; the colonies were counted. Based
on the count, the pathogenic bacteria were diluted to a con-
centration of 1 × ­106 CFU/mL with LB broth medium for
later use.
After the LAB strains were purified and activated, their
bacterial solutions were centrifuged at 5000 rpm for 30 min.
The supernatants were filtered separately with 0.22 μm fil-
ters to prepare the LAB cell-free supernatants (CFS) and
incubated on MRS plates to verify sterility. The superna-
tants of LAB were co-cultured with A. hydrophila TPS and
A. veronii TH0426, respectively. Finally, the effect of the
supernatants of LAB on the morphology of the pathogenic
bacteria was observed by scanning electron microscopy. The
strains with better antibacterial effects were screened and
kept by the agar plate punching method, and the test was
repeated three times.
Resistance Assays
The LAB strains were activated, and the bacterial liquid was
centrifuged at 5000 rpm for 10 min at 4 °C to collect the
bacterial cells. Then, the LAB suspension was prepared at
a ratio of 1:10. Each of the following tolerance tests was
independently repeated three times.
1. Acid resistance test: The acid resistance test was con-
ducted using the viable counts method with minor modi-
fications, as described in previous studies [19]. The LAB
suspensions (1 × ­106 CFU/mL) were inoculated into
MRS broth medium at pH = 2, pH = 4, and pH = 6, and
the MRS broth medium (pH = 7) was used as a control.
After anaerobic cultivation at 30 °C for 4 h, the samples
were diluted with PBS and spread on MRS plates. The
residual viable bacterial population was estimated by
plate counting on MRS agar after incubation for 24 h.
Relative survival rate (%) = CFUpH∶x ∕CFUordinary MRS × 100.
CFUpH: x is the total number of surviving bacteria after
standing for 4 h in MRS medium at pH = 2, pH = 4, and
pH = 6, and ­CFUordinary MRS is the total number of surviving
bacteria after being left in MRS medium for 4 h.
2. Antibiotic susceptibility test: The antibiotic susceptibil-
ity test was performed using the disk diffusion method
[20]. 200 μL activated LAB suspensions (1 × ­106 CFU/
mL) were dropped on MRS agar medium plates and
coated uniformly. After the bacterial liquid was absorbed
by the medium, a total of 30 commonly used antibiotics
(Hangzhou Microbial Reagent, China) were tested by
placing them on MRS medium (the names of the 30 anti-
biotics can be found in Supplementary Table S2). The
medium was then incubated in an anaerobic environment
at 30 °C for 24 h. The diameter of the inhibition zone of
the drug-sensitive tablet was measured and analyzed.
3. Bile resistance test: The bile resistance test was con-
ducted by referencing previous methods with slight
modifications [21]. The loach bile was collected asepti-
cally, and then, the LAB suspension (1 × ­106 CFU/mL)
was inoculated into PBS containing 10% bile, and sterile
PBS without bile was used as a control. After anaerobic
cultivation at 30 °C for 4 h, the samples were also spread
on MRS plates, and then, the relative survival rate was
calculated after incubation for 24 h as follows:
Relative survival rate (%) = CFU10% ∕CFUPBS × 100
where ­CFU10% is the total number of surviving bacteria
after standing in PBS containing 10% loach bile for 4 h,
and ­CFUPBS is the total number of surviving bacteria
after standing in PBS for 4 h.
4. Trypsin resistance test: Similar to the previous method
[22], trypsin solutions were utilized to assess the growth
of LAB under simulated intestinal fluid environmental
conditions. The LAB suspension (1 × ­106 CFU/mL) was
inoculated into PBS containing 1% trypsin, and sterile
PBS without trypsin was used as a control. They were
anaerobically cultured at 30 °C for 0 h, 1 h, 2 h, 3 h, and
4 h. Finally, the colonies were counted, and the relative
survival rate was calculated as follows:
Relative survival rate (%) = CFU1% ∕CFU0% × 100
where ­CFU1% is the total number of surviving bacteria
after standing in sterile PBS containing 1% trypsin for
4 h, and ­CFU0% is the total number of surviving bacteria
after standing for 4 h in sterile PBS.
Cell Surface Activity of LAB
1. Hydrophobicity assays: The hydrophobicity of LAB,
which is reflected by the affinity of LAB to xylene,
is measured by bacterial adhesion to hydrocarbons
(BATH). The prepared bacterial precipitate was adjusted
by measuring absorbance at optical density ­(OD600),
until it was 0.6. And xylene was added at a ratio of 1:3.
Then, it was mixed well and incubated for 1 h, and PBS
buffer was used as a control. After the two phases were
separated, the absorbance at O­ D600 of the aqueous phase
was measured. The experiment was performed in tripli-
cate. The cell surface hydrophobicity (%) of the isolate
adhering to the solvent was calculated according to the
following equation:
Hydrophobicity (%) = (A1 − A2 )∕A1 × 100,
13

where A1 is the absorbance value measured after the
LAB suspension is mixed, and A2 is the absorbance
value of the suspension with LAB and xylene standing
for 1 h. Surface hydrophobicity greater than 50% rep-
resents highly hydrophobic, between 20 and 50% rep-
resents moderately hydrophobic, and lower than 20%
represents low hydrophobic.
2. Autoaggregation assays: The specific interactions
between LAB cells were determined by the autoaggre-
gation assay. Three milliliters of the suspension of LAB
(absorbance at O
­ D600 was 0.6) were placed in an Eppen-
dorf (EP) tube and placed at room temperature for 2 h.
Then, the upper layer solution was taken to determine
the ­OD600. The test was repeated three times. The cal-
culation formula is:
Autoaggregation (%) = (1 − A2 ∕A1 ) × 100
where A1 represents the initial absorbance of the LAB
suspension, and A2 represents the absorbance measured
after the LAB suspension stands for 2 h.
Molecular Biology Identification of LAB
The genomic DNA of isolates was extracted according to
the instructions of the kit (TIANGEN, China), and then, 16S
rDNA genes were amplified by PCR using universal primers
27F and 1492R. The primer sequences are as follows:
27F: 5′-AGA​GTT​TGATCMTGG​CTC​AG-3′
1492R: 5′-TAC​GGY​TAC​CTT​GTT​ACG​ACTT-3′
The PCR step consisted of initial denaturation at 94 °C
for 5 min, followed by 30 cycles of denaturation at 94 °C
for 1 min, annealing at 55 °C for 1 min, and extension at
72 °C for 1 min. The final extension step was performed at
72 °C for 10 min. The PCR products were then detected and
purified with 1% agarose gels. The purified PCR products
were sequenced using the Sanger sequencing method by
Sangon Biotech-Shanghai, China. The nucleotide sequences
were compared with the available sequence of LAB spe-
cies in the National Center for Biotechnology Information
(NCBI) database using the Basic Local Alignment Search
Tool (BLAST) program [23]. The phylogenetic analysis was
conducted using the neighbor-joining method in MEGA 7.0
software.
Detection of Isolates’ Biological Characteristics
Growth Curve and Acid Production Test
The growth curve of the LAB isolates was obtained by spec-
trophotometer. The activated LAB solution was inoculated into
13
Probiotics and Antimicrobial Proteins
MRS broth medium at a ratio of 5% and cultivated at 30 °C
from 0 to 13 h. The absorbance at ­OD600 was measured at each
time point. The experiment was repeated three times. At the
same time, the pH values of the LAB solution were measured
using a pH meter at 12 h, 24 h, 48 h, and 72 h to determine the
acid production ability of isolated strains. This experiment was
repeated three times independently.
Biofilm Formation Capability Test
The biofilm formation capability test was performed according
to the microplate detection method [24]. First, the concentra-
tion of activated LAB was adjusted to 1 × ­106 CFU/mL. Then,
190 μL of MRS medium and 10 μL of bacterial solution were
added into the experimental wells and PBS in the same propor-
tion as a control. Nine replicate wells were set for each sample
(including the control group), and the 96‐well flat‐bottom plate
(Corning, Beijing, China) was sealed with parafilm. Then, the
plates were anaerobically cultured at 30 °C for 24 h. After
incubation, the bacterial liquid was discarded, and all 96‐well
flat‐bottom plates were washed with PBS solution. This step
was repeated twice. Then, 99% methanol was added to each
well to fix the bacteria and stained with 0.1% crystal violet
solution. Next, the dye solution was discarded, and acetic acid
at a concentration of 33% was added to dissolve the crystal
violet. Finally, the plate was incubated for 5 min, and the
absorbance at O ­ D595 was measured with a microplate reader.
This experiment was independently repeated three times.
Coaggregation Test
Aeromonas hydrophila TPS and A. veronii TH0426 were used
as indicator bacteria for the preparation of the bacterial suspen-
sion of indicator bacteria and LAB, referring to the method as
described above. The suspension containing LAB was mixed
with equal volumes of indicator bacteria in a 4 mL EP tube.
After vortexing and mixing, the test was scored as positive
if sedimentation of the cells was visible within 2 h at room
temperature. The upper layer solution was taken to measure
the absorbance at ­OD600. This experiment was repeated three
times independently. The coaggregation rate was calculated
as follows:
Coaggregation rate (%) = (A1 − A2 )∕A1 × 100
where A1 is the absorbance value measured after mixing
the suspension of LAB and indicator bacteria and A2 is the
absorbance value measured after standing for 2 h.
In Vitro Adhesion Assay
The adhesion capability of LAB was evaluated by the adhe-
sion strength to epithelioma papulosum cyprini (EPC) cells
Probiotics and Antimicrobial Proteins
(ATCC, DC, USA, http://​www.​atcc.​org/). The method was
described in a previous report and suitably improved [25].
EPC cells were cultured in M199 cell culture medium con-
taining 10% sterile fetal bovine serum and 1% (v/v) peni-
cillin/streptomycin at 25 °C until the cells were confluent.
The EPC cells were resuspended in trypsin and distributed
into 6-well flat-bottom cell culture plates (Corning, Beijing,
China) to ensure that there were no less than 1 × ­105 cells/
mL per well. When the bottom of the well was covered with
a layer of cells, the cells were gently washed three times
with cell culture medium without antibiotics and serum.
Additionally, the LAB were resuspended in cell culture
medium without antibiotics and serum to a final density of
1 × ­108 CFU/mL. They were added to a 6-well flat-bottom
cell culture plate (Corning, Beijing, China) at a ratio of 10:1
(bacteria: cells) and incubated at 25 °C for 1 h. Then, the
adherent cells in the wells were washed with sterile PBS to
remove the LAB that did not adhere to the cells. EPC cells
were lysed with 1% Triton-X100, and then, the mixture was
dropped onto MRS agar medium for culture. The adhesion
capability of LAB was assessed by counting the number
of LAB on MRS agar medium. Lactobacillus rhamnosus
(ATCC53103) was used as a positive control since it had
good adhesion capability [26]. The experiments were inde-
pendently repeated three times.
Antimicrobial Spectrum Analyses
The LAB and 15 strains of indicator bacteria (A. hydrophila
HA336, BSK, TPS strains; A. veronii TH0426, CL8155,
JL1021, AV75, NJ723, AV115, ATCC35624, QXF strains;
S. agalactiae; A. caviae ATCC15468; S. enterica serovar
Enteritidis CVCC3378; and S. typhimurium) were activated
according to the method as described above, and the cell-
free supernatant of LAB was prepared. Then, the antibacte-
rial spectrum was determined according to the agar plate
punching method as described above. The experiments were
repeated three times independently.
In Vitro Inhibition of Isolates on A. hydrophila and A. veronii
The activated indicator bacteria (A. hydrophila TPS and
A. veronii TH0426) and LAB cell-free supernatant were
prepared according to the method as described above. The
indicator bacteria were diluted to 1 × ­103 CFU/mL and added
to the wells of a 6-well flat-bottom cell plate (Corning, Bei-
jing, China). And the glass slides were placed in the wells
incubated with 0.1% polylysine in advance. Each indica-
tor bacteria was performed for three replicates. Then, the
cell-free supernatant of LAB was added to the cell plate
at a ratio of 1:7 (LAB: indicator bacteria) without adding
supernatant as the control. All 6-well flat-bottom cell plates
(Corning, Beijing, China) were incubated at 30 °C for 12 h.
After incubation, the bacterial solution was discarded, and
the slides were washed three times with sterile PBS. The
slides were fixed with 2.5% glutaraldehyde and sent to the
electron microscope studio of Jilin University to observe
the interaction between LAB and indicator bacteria. The
slide processing and scanning electron microscope (SEM)
observation were completed with the support of the Electron
Microscope Studio of Jilin University.
Safety Evaluation
After the screening of the above experiments, the W. con-
fusa N17 strain with good properties was finally selected for
safety evaluation.
DNA Extraction, Whole‑Genome Sequencing, and Analysis
of the N17 Strain
The genomic DNA of the activated W. confusa N17 strain
was extracted, and the integrity, concentration, and purity
­(OD260/OD280 = 1.8 ~ 2.0) were assessed. Determination and
analysis of the whole-genome sequencing of the N17 strain
were performed in cooperation with Changchun Berrygen-
etech Co., Ltd. An Illumina-PE library with an insert size
of 300–500 bp and a PacBio library with an insert size of
15–20 kb were constructed. The Circos v0.64 software was
used to draw the N17 genome circle diagram. In addition,
virulence factor-related genes in the N17 genome and plas-
mids were predicted using the VFDB database (http://​www.​
mgc.a​ c.c​ n/c​ gi-b​ in/V
​ Fs/v​ 5/m
​ ain.c​ gi). The resistance genes in
the genome and plasmid gene sequences were predicted and
analyzed by the CARD online software (https://c​ ard.m ​ cmast​ er.
​ca/​analy​ze).
Animal Safety Test
Based on the reference [20], six healthy loaches were
divided into two groups (1 experimental and 1 control), with
each group consisting of three loaches. The cultivated LAB
strains were centrifuged at 5000 rpm for 10 min at 4 °C,
washed twice with PBS, and obtained the LAB resuspen-
sion. Cell density was determined through viable counts.
In the experimental group, each loach was intraperito-
neally injected with 200 μL of activated W. confusa N17
(1 × ­108 CFU/mL). The control group was injected intra-
peritoneally with an equal volume of sterile PBS solution.
The health status of the loaches was observed twice a day
for 7 days. On the 8th day, the loaches were anesthetized
and dissected to observe whether there was visceral injury.
Based on this, the safety of W. confusa N17 strain was ini-
tially evaluated.
13

Challenge Test
Sixty healthy loaches were randomly divided into three
groups (i.e., PBS group, W. confusa N17 + A. veronii
TH0426 group, and A. veronii TH0426 group), and each
group included 20 loaches. The PBS group and A. veronii
TH0426 group were fed commercial feed. The W. confusa
N17 + A. veronii TH0426 group was fed commercial feed
mixed with an average of 1 × ­109 CFU/g activated W. con-
fusa N17. The loaches were fed for 1 month, and then, a
challenge experiment was carried out. For the A. veronii
TH0426 strain, the median lethal concentration (­ LC50) was
about 1 × ­106 CFU/mL. The concentration of activated A.
veronii TH0426 was adjusted to 4 × ­10 6 CFU/mL. Each
loach was intraperitoneally injected with 200 μL A. veronii
TH0426 bacterial solution for the W. confusa N17 + A.
veronii TH0426 group and A. veronii TH0426 group, and
the PBS group was injected with an equal amount of ster-
ile PBS. The loaches were observed twice daily, and the
number of deaths was recorded over seven days. Finally,
the survival rate was calculated for each group.
Statistical Analyses
Statistical analyses were conducted using the SPSS 26.0
software, while graphics were created using the Graph-
Pad Prism v8.4.0 software. The data are reported as the
mean ± standard error (SE) of three replicates. One-way
analysis of variance (ANOVA) was employed for variance
analysis between treatments. For comparison among multi-
ple groups, Tukey’s test was applied. When calculating the
relative survival rates, hydrophobicity, and autoaggrega-
tion rates, different letters were used to indicate significant
differences between treatments (P < 0.05). And when com-
paring biofilm formation capability, coaggregation rates,
and adhesion capability, the symbol “*” was used to indi-
cate a significant difference compared to the control group.
Specifically, “*,” “**,”and “***” represent significance
levels of P < 0.05, P < 0.01, and P < 0.001, respectively.
Results
Isolation, Screening, and Identification of LAB
from Loaches
Preliminary Results of the Isolated Strains
A total of 228 suspected LAB strains were isolated from
the intestines of loaches. A total of 117 strains were
13
Probiotics and Antimicrobial Proteins
identified as both Gram-positive and catalase-negative
bacteria. Part of the Gram staining results are shown in
Fig. S1.
Screening Results of Strains Inhibiting A. veronii and A.
hydrophila
There were 19 suspected LAB strains with inhibitory effects
on A. veronii TH0426 and A. hydrophila TPS. The results
are shown in Table S1.
Tolerance Analysis of LAB
1. Acid tolerance: The results demonstrated that N1, N14,
N15, N17, N18, and N19 strains exhibited a progres-
sively faster growth rate within the pH range of 2.0–4.0.
These strains displayed relatively good acid tolerance
compared to the other 13 strains (Fig. 1A–C) (P < 0.05).
At pH 4, the survival rates after 4 h of exposure ranged
from 31 to 56%. Consequently, these six isolates were
chosen for further experiments.
2. Antibiotic susceptibility: Drug susceptibility was
assessed in the N1, N14, N15, N17, N18, and N19
strains. As shown in Table S2, the N1 isolate strain
showed multidrug resistance and was discarded. In addi-
tion, it was found that some LAB had already devel-
oped resistance to broad-spectrum antibiotics such as
polymyxin B and norfloxacin, suggesting that antibiot-
ics should be reduced or banned as soon as possible to
prevent the transfer of resistance genes.
3. Bile tolerance: To determine the survival potential of
the N14, N15, N17, N18, and N19 strains in the host
gastrointestinal tract, the strains with bile-salt treatments
were exposed for 4 h. All the tested isolates were able
to survive in PBS containing 10% loach bile. However,
in comparison to the survival rates of the other strains,
which were below 20%, the relative survival rates of N17
and N19 strains were significantly different, reaching
65.15% and 53.51%, respectively (P < 0.05) (Fig. 1D).
4. Trypsin resistance: Trypsin had little effect on the sur-
vival rates of strains N14, N15, N17, N18, and N19,
which could be ignored. There were no significant dif-
ferences between the strains (P > 0.05) (Fig. S2).
Cell Surface Hydrophobicity and Cellular Autoaggregation
Analysis
The hydrophobicity of the LAB was evaluated by examin-
ing the adhesion of N14, N15, N17, N18, and N19 strains
to xylene. The results demonstrated that the cell surface
hydrophobicity of all these strains exceeded 50%, indicating
a high level of hydrophobicity. Notably, the N18 strain dis-
played the highest hydrophobicity, which was significantly
Probiotics and Antimicrobial Proteins
Fig. 1  Tolerance test results of LAB isolates. A–C Acid tolerance
result. D Bile tolerance result. E Cell surface hydrophobicity of LAB
isolates. F Autoaggregation ability of LAB isolates. The values were
different from N15, N17, and N19 strains (P < 0.05), but not
significantly different from N14 strain (P > 0.05) (Fig. 1E).
In contrast, the autoaggregation rates of N17 and N19 strains
were 42.5% and 39.9%, respectively, which were signifi-
cantly higher compared to the other strains (P < 0.05). This
suggests that N17 and N19 strains have a relatively stronger
autoaggregation property, as depicted in Fig. 1F. Based on
these findings, it can be inferred that strains N17 and N19
may possess an enhanced capability to adhere to epithelial
cells and mucosal surfaces.
Identification by 16S rRNA Sequencing and Phylogenetic
Analysis
The ClustalW method was used for homologous sequence
analysis and alignment with known gene sequences. The
phylogenetic tree was constructed using the neighbor-joining
expressed as the means ± SE (n = 3), and different letters represent
significant differences among the strains (P < 0.05)
method in the MEGA 7.0 software. As shown in Fig. 2A,
the N17 strain and W. confusa ML26 (LC274616.1) were
clustered into a branch, indicating that they were closely
related, and the N17 strain was identified as W. confusa.
However, the N19 strain has high sequence homology with
L. saniviri, which was identified as L. saniviri (Fig. 2B). And
the N17 strain was identified as W. confusa (99.2% sequence
identity). However, the N19 strain has high sequence homol-
ogy with L. saniviri (99.5% sequence identity) (Fig. 2B).
Biological Characterization of the N17 and N19
Strains
Growth Rate Analysis
The growth curves of the N17 and N19 strains were drawn
to determine their growth ability. As shown in Fig. 3A, both
13

Fig. 2  Phylogenetic tree based on 16S rRNA nucleotide sequences. A Phylogenetic tree of the N17 strain; B phylogenetic tree of the N19 strain
of them entered the logarithmic phase at approximately 3 h,
but N17 grew faster than N19.
Analysis of Acid Production Capability
We tested the acid-producing capability of the N17 and N19
strains, and the results indicated that the pH values of N17
and N19 decreased rapidly from 0 to 12 h, and the decline
rate of N17 was faster than that of N19 (Fig. 3B). After that,
the pH values gradually stabilized, and the final values were
approximately 4.075 and 4.145.
Analysis of Biofilm Formation Capability
According to the results obtained from the 96‐well flat‐bot-
tom plate method, N17 strain exhibited a significantly higher
­OD595 value of 0.628 compared to N19 strain, which had a
value of 0.189 (P < 0.01) (Fig. 3C). This finding indicated
that N17 strain possesses a stronger ability to form biofilms
in comparison to N19 strain.
Analysis of Coaggregation Capability
As shown in Fig. 3D, both N17 and N19 strains demon-
strated the ability to co-aggregate with A. hydrophila TPS
and A. veronii TH0426. The co-aggregation rate of N19 with
A. hydrophila TPS (44.7%) was significantly higher than that
of N17 with A. hydrophila TPS (39.1%) (P < 0.01). How-
ever, the co-aggregation rate of N17 with A. veronii TH0426
(45.9%) indicated extremely significant difference compared
with N19 (36.8%) (P < 0.001).
Adhesion Effect on EPCs
According to the adherence results presented in Fig. 3E,
it was observed that the number of cells adhered by N17
strain was significantly higher than that of L. rhamnosus
13
Probiotics and Antimicrobial Proteins
ATCC53103 (P < 0.01). However, the number of cells
adhered by N19 strain did not show a significant difference
compared to the control (P > 0.05). These findings suggest
that both N17 and N19 strains have the ability to adhere
in vitro. However, N17 strain demonstrated a superior ability
to adhere compared to strain N19.
Antimicrobial Activity
To further understand the inhibition spectrum of the N17
and N19 strains, we expanded the range of pathogenic bac-
teria (Table 1). The results indicated that N17 has a strong
inhibitory effect on A. hydrophila (HA336, TPS and BSK
strains) and A. veronii (CL8155, TH0426, NJ723, AV75, and
ATCC35624 strains). N19 had a strong inhibitory effect on
A. hydrophila (HA336 and TPS strains), A. veronii (JL1021,
TH0426, AV115, ATCC35624, and QXF strains), and the A.
caviae ATCC15468 strain. However, the two strains did not
show any activity against S. enteritidis CVCC3378 and S.
typhimurium. Thus, the N17 and N19 strains showed signifi-
cant inhibition of most pathogens of the genus Aeromonas.
Inhibitory Effect of N17 and N19 Strains on A. veronii and A.
hydrophila
The antimicrobial capability of the N17 and N19 strains
was further confirmed by a cocultivation assay. As shown
in Fig. 4A–C, the extent of division and fragmentation of A.
veronii TH0426 caused by cocultivation with N17 superna-
tant was more serious than that caused by coculturing with
N19 supernatant. Intact bacteria could not be observed in
the coculture. The cocultivation assays with A. hydrophila
TPS revealed that when cocultured with N17 supernatant,
the surface of the bacteria became rough, flocculent debris
appeared, and the cells were broken, while the cells coc-
ultivated with N19 supernatant were sunken and broken
(Fig. 4D–F).
Probiotics and Antimicrobial Proteins

Fig. 3  Biological characterizations of the N17 and N19 strains. A were used to indicate a significant difference compared to the control
Growth curve analysis; B acid production capability analysis; C group. Specifically, “*,” “**,”and “***” represent significance levels
biofilm formation capability analysis; D coaggregation capability of P < 0.05, P < 0.01, and P < 0.001, respectively
analysis; E adhesion capability in vitro analysis. The symbols “*”

13
 Probiotics and Antimicrobial Proteins

Table 1  Antibacterial spectrum of N17 and N19 strains Safety Analysis

Pathogenic strains N17 N19
Based on the above experimental results, the N17 strain with
Aeromonas hydrophila TPS ++ ++ better characteristics was selected for safety analysis.
Aeromonas hydrophila HA336 ++ ++
Aeromonas hydrophila BSK ++ + Whole Genome Sequencing and Analysis of the N17 Strain
Aeromonas veronii TH0426 +++ ++
Aeromonas veronii CL8155 ++ + Sequencing and analysis of the W. confusa N17 genome
Aeromonas veronii JL1021 + ++ showed that the size of the genome was 2,279,677 bp (Gen-
Aeromonas veronii NJ723 ++ + Bank accession number: CP049097.1). In addition, three
Aeromonas veronii AV75 ++ + plasmids were also present, and their sizes were 31,622 bp
Aeromonas veronii AV115 + ++ (GenBank accession number: CP049098.1), 17,351 bp (Gen-
Aeromonas veronii ATCC35624 +++ ++ Bank accession number: CP049099.1), and 3,741 bp (Gen-
Aeromonas veronii QXF + ++ Bank accession number: CP04909100.1). The genome and
Aeromonas caviae ATCC15468 + ++ plasmid features of the W. confusa N17 strain are shown in
Streptococcus agalactiae + + Fig. 5. In addition, further analysis showed that there were
Salmonella enteritidis CVCC3378 - - no apparent virulence factor–related genes, and drug resist-
Salmonella typhimurium - - ance–related genes were found in the genome and plasmids,
The diameter of the bacteriostatic region, “-” means less than 8 mm; indicating that the W. confusa N17 strain did not produce
“+” means between 8.00–12.00 mm; “+ +” means between12.00 and toxins and might not be pathogenic. There is also no risk of
16.00 mm; “+ + +” means between 16.00 and 20.00 mm drug resistance gene transfer.

Fig. 4  Inhibitory effect of N17 and N19 strains on A. veronii TH0426 A. veronii TH0426 and supernatant of N19; D A. hydrophila TPS
and A. hydrophila TPS by SEM. A TH0426 strain; B coculture result strain; E coculture result of A. hydrophila TPS and supernatant of
of A. veronii TH0426 and supernatant of N17; C coculture result of N17; F coculture result of A. hydrophila TPS and supernatant of N19

13
Probiotics and Antimicrobial Proteins
Fig. 5  The genomic map of W. confusa N17. A The genome features of the N17 strain. B, C, and D The three plasmid features of the N17 strain
Animal Safety Assay
In the in vivo animal safety assay, the loaches that were
continuously fed and observed for 7 days were all alive.
Three loaches were randomly selected from each experi-
mental group for anatomy, and none of the groups showed
any pathological clinical signs on the body surface or
internal organs (Fig. S3). Therefore, it could be prelimi-
narily determined that the N17 strain was safe for loaches.
Fig. 6  Survival analysis of
loaches after A. veronii TH0426
challenge
Immunoprotective Effect of the N17 Strain
The immune protection effect of the N17 strain was evalu-
ated by determining the cumulative survival of loaches
after a challenge with A. veronii TH0426. The survival
rate of loaches fed common feed (TH0426 group) obvi-
ously declined after injection with A. veronii TH0426, and
finally, the loaches all died. The loaches fed the N17 strain
(N17 + TH0426 group) inhibited the process, with a survival
rate of 57.1% (Fig. 6). In contrast, no deaths occurred in the
PBS group after 7 days. The results suggested that a diet
13

supplemented with the N17 strain improved the ability of
loaches to combat A. veronii infections.
Discussion
Loach is a widely distributed small freshwater fish in China,
known as “water ginseng,” which is a famous tonic food but
also one of the main aquatic product exports in China [1].
Artificial farming of loach in China has proliferated due to
high domestic and international demand. Although loaches
are highly adaptable and tolerant of low oxygen, various
diseases can occur during high-density artificial culture [1].
Antibiotic therapy is the most commonly used animal dis-
ease treatment, but this strategy is limited by its side effects.
Therefore, probiotics have received attention as a potential
replacement for antibiotics. As a safe and environmentally
friendly method, various types of probiotics are now used
in aquaculture for environmental improvement, disease
control, and growth promotion of aquatic organisms [7–9,
12]. Among these, LAB is widely used and highly effective
in improving growth performance, immunity, and disease
resistance [27]. However, there is also the problem of degra-
dation of strain characteristics. New strains with good traits
need to be explored continuously.
Weissella sp. is the dominant microorganism found in
the food and intestinal walls of humans and animals; from a
taxonomic point of view, it belongs to the order Lactobacil-
lales and family Leuconostocaceae [28]. Weissella sp. was
first isolated from fermented sausage in 1993, and it was a
relatively new genus compared to other LAB. Recently, the
probiotic potential of strains of Weissella sp. used in the
farming, food, and pharmaceutical industries has become
a research hotspot [29]. Strains of the Weissella sp. were
found to have the ability to produce antibacterial compounds
such as bacteriocins, hydrogen peroxide, exopolysaccharides
(EPS), and diacetyl, as well as to survive in the gastroin-
testinal tract (GIT) and even grow in the intestine [30–33].
Furthermore, most strains of the Weissella sp. with these
health-promoting properties have been shown to be safe due
to the absence or rare occurrence of virulence or antibiotic
resistance genes [34–38]. These attributes suggest that Weis-
sella sp. have potential as beneficial probiotics [39–41].
In our study, 117 strains of suspected LAB were obtained
by isolation and identification from the intestinal tract of
healthy loaches, and then, a total of 19 strains of suspected
LAB with good bacteriostatic effects were screened out
through bacteriostatic tests. Although the trypsin resistance
of each strain performed excellently, in PBS solution con-
taining 10% bile, the relative survival rates of the N17 and
N19 strains were 65.15% and 53.51%, respectively, and the
remaining strains were lower than 20%. The results sug-
gest that both N17 and N19 isolates can survive under low
13
Probiotics and Antimicrobial Proteins
pH conditions and high bile-salt concentrations. Surpris-
ingly, Xiong et al. found that the potential probiotic strain
W. confusa BSP201703 can survive at pH 2 and 0.3% (w/v)
bile-salt [42]. Therefore, we finally selected the N17 and
N19 strains, both strains of LAB with excellent traits, for
subsequent analysis of probiotic characteristics.
The strength of the adhesion ability of probiotics is the
precondition for their probiotic effect, and it is also the key
factor in whether they can colonize the animal intestine. The
hydrophobicity of cells determines the adhesion ability of
bacteria and is also one of the factors affecting the auto-
aggregation ability [43]. On the one hand, probiotics can
adhere to the intestinal surface of the host through autoag-
gregation to form biofilms; on the other hand, probiotics
can form a barrier to prevent the adhesion and invasion of
pathogens by coaggregation. In this study, the N17 and N19
strains had high hydrophobicity and strong autoaggregation
ability, indicating that the N17 and N19 strains had a good
adherence capacity. In addition, the present study confirmed
the strong adhesion of N17 and N19 to EPC cells. It was
reported that the ability to adhere was considered to be an
important property for the effective colonization of the intes-
tine by bacterial strains used as probiotics [44]. Therefore,
these results suggest, to some extent, that the N17 and N19
strains have a strong ability to colonize the intestine.
In addition to good adhesion properties to intestinal cells,
probiotics should have the ability to competitively exclude
or inhibit pathogens that adhere to host intestinal cells. Zhu
et al. found that the potential probiotic bacteria Weissella
cibaria C-10 screened from the intestine of crucian carp
showed different inhibitory effects on a variety of patho-
genic bacteria of fish. In particular, it had the most obvious
inhibitory effect on the pathogens Aeromonas sp. [45]. The
present study was similar, and the N17 and N19 strains could
produce certain inhibitory effects on most Aeromonas sp.
pathogens. Additionally, the coaggregation assays suggest
that the N17 and N19 strains can effectively coaggregate
with TPS and TH0426, respectively, indicating that they
have good adhesion inhibition ability to the two pathogens.
We further observed that the culture supernatants of the N17
and N19 strains could damage the cell structures of A. vero-
nii TH0426 and A. hydrophila TPS to varying degrees. Com-
pared with N19, N17 damaged pathogenic bacteria more
seriously, which confirmed that N17 had stronger inhibition
of A. veronii TH0426 and A. hydrophila TPS. Therefore, the
two strains have a certain probiotic ability to improve the
resistance of loach to the invasion of pathogenic bacteria.
In addition, the biofilm formed by probiotics after autoag-
gregation not only allows them to improve their viability in
the intestinal tract but also improves their ability to compete
with pathogenic bacteria for growth, thus resisting their inva-
sion [46]. Compared with the N19 strain, the N17 strain had
a stronger biofilm formation ability in the study, which may
Probiotics and Antimicrobial Proteins
give the N17 strain an advantage in colonizing the intestine
and resisting the invasion of pathogenic microorganisms.
After a series of tests, W. confusa N17 was found to have
good probiotic properties, colonization ability, and bacterio-
static effect, and therefore, it was selected for further evalua-
tion. The selected candidate strains should be nonpathogenic
to the host, which is an important precondition for their
application as probiotics [47]. In this study, intraperitoneal
injection of W. confusa N17 did not result in lesions or death
of loaches. Then, genome sequencing and analysis of W.
confusa N17 was further performed, and no significant viru-
lence factor–related and drug resistance–related genes were
found. These results suggest that W. confusa N17 may be
safe for loaches. Certainly, this remains to be further verified
experimentally. Similar findings have been found in other
studies, which were that W. confusa can be used as a direct-
feeding microbial product [34, 35].
In vitro test, the loaches fed W. confusa N17 showed a
high survival rate under infection with A. veronii at four
times the ­LC50 dose, indicating that W. confusa N17 could
significantly improve the resistance of the loach. We hypoth-
esized that this protective effect might be due to the direct
inhibition of A. veronii by W. confusa N17, or it was also
possible that W. confusa N17 enhanced the immune func-
tion of the loach, thereby generating resistance to A. veronii
infection. Currently, there are reports on the probiotic poten-
tial of W. confusa [42, 48]; however, reports on its ability
to enhance immune function and resistance to pathogenic
bacterial infections in animals are limited. Our study dem-
onstrated for the first time that W. confusa N17 significantly
enhanced the resistance of loach to A. veronii infection.
In summary, we isolated a strain of LAB with excellent
traits from the intestine of healthy loaches, namely, W.
confusa N17, which had good probiotic properties, coloni-
zation ability, bacteriostatic effects, protective effects, and
safety. It has potential as a candidate probiotic for loach
farming. However, the specific probiotic and antibacte-
rial mechanisms of W. confusa N17 need to be further
explored. The results of this study provide an important
reference for the future application of W. confusa in loach
and even in other aquatic animal cultures.
Supplementary Information The online version contains supplemen-
tary material available at https://d​ oi.o​ rg/1​ 0.1​ 007/s​ 12602-0​ 23-1​ 0149-4.
Author Contribution Conceived and designed the experiments: YHK
and WC. Performed the experiments: BTY, LTT, and HCS. Analyzed
the data: BTY, LTT, and RGH. Contributed analysis tools: HCS and
RGH. Wrote the paper: BTY. Reviewed and edited the paper: ZLL,
YHK, and WC.
Funding The Shandong Natural Science Foundation Youth Fund Pro-
ject (ZR2022QC079, ZR2023QD024) and the Jilin Provincial Natural
Science Foundation Project (20210101050JC) provided funding for
this work.
Data Availability The data that support the findings of this study are
available from the corresponding author upon reasonable request.
Declarations
Ethics Approval This study was conducted under the permission of the
Animal Care and Use Committee of Jilin Agricultural University. All
animal experimental procedures in the present study were conducted
in strict accordance with the Regulations for Animal Experimentation
of Jilin Agricultural University and the National Institutes of Health
Guide for the Care and Use of Laboratory Animals (NIH Publications
No.143 8023).
Conflict of Interest The authors declare no competing interests.
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