Ecotoxicology and Environmental Safety 194 (2020) 110420

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Compound probiotics alleviating aflatoxin B1 and zearalenone toxic effects T

on broiler production performance and gut microbiota
Juan Changa,1, Tao Wanga,1, Ping Wanga, Qingqiang Yina,∗, Chaoqi Liua, Qun Zhub, Fushan Luc,
Tianzeng Gaod
a
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450002, China
b
Henan Delin Biological Product Co., Ltd., Xinxiang, 453000, China
c
Henan Puai Feed Co., Ltd., Zhoukou, 466000, China
d
Henan Guangan Biotechnology Co., Ltd., Zhengzhou, 450001, China

ARTICLE INFO ABSTRACT

Keywords: In order to alleviate toxic effects of aflatoxins B1 (AFB1) and zearalenone (ZEA) on broiler production perfor-
Compound probiotics mance and gut microbiota, three kinds of compound probiotics (CP) were selected. The optimal ratios of Bacillus
Aflatoxin B1 subtilis, Lactobacillus casei and Candida utilis in broiler diets were 7, 5 and 6 log CFU/g for ZEA biodegradation
Zearalenone (CP1); 6, 7 and 7 log CFU/g for AFB1 biodegradation (CP2); 7, 6 and 7 log CFU/g for ZEA + AFB1 biode-
Detoxification
gradation (CP3). A total of 350 1-day-old Ross broilers were randomly divided into 7 groups. Group A was the
Microbiota
basal diet, group B-G contained ZEA, AFB1, ZEA + AFB1, ZEA + CP1, AFB1+CP2, ZEA + AFB1+CP3, re-
Broilers
spectively. The experiment showed that AFB1 or AFB1+ZEA significantly decreased broiler production perfor-
mance, damaged liver and jejunum, increased mycotoxin residues in broiler body; however, three kinds of
compound probiotics additions could alleviate mycotoxin negative effects on the above parameters (p < 0.05).
The gut microbiota analysis indicated that AFB1+ZEA increased jejunal microbial richness, but which were
decreased to almost the same level as the control group by CP3 addition. CP3 addition significantly increased
jejunal Firmicutes and Lactobacillus aviarius abundances. The correlative analysis showed that gut Lactobacillus
aviarius abundance was positively correlated with average daily gain (ADG) of broilers (p < 0.05), while
AFB1+ZEA addition decreased its relative abundance, indicating that CP3 addition increased broiler growth by
increasing Lactobacillus aviarius abundance. AFB1 and ZEA residues in broiler body were negatively correlated
with the gut beneficial bacterial abundances (p < 0.01), but positively correlated with the potentially harmful
bacterial abundances (p < 0.05), which inferred that CP3 addition could decrease mycotoxin residues through
positively regulating gut relative bacterial abundances. In conclusion, compound probiotics could keep gut
microbiota stable, degrade mycotoxins, alleviate histological lesions, increase production performance and re-
duce mycotoxin toxicity for broilers.

1. Introduction
Mycotoxins such as aflatoxins B1 (AFB1) and zearalenone (ZEA) are
toxic compounds mainly produced by Aspergillus and Fusarium species
(Luo et al., 2018). Mycotoxin contamination commonly found in feed
and food commodities, climate change and the improper storage in-
creases the pollution risk substantially (Dong et al., 2019). It is reported
that approximately 25% of crops in the world have been contaminated
by mycotoxins, leading to serious economic losses (Rice and Ross,
1994). Moreover, several kinds of mycotoxins can co-occur in food and
feeds to cause more serious toxic effect on human and animal health
(Freire and Sant’Ana, 2018). It has been reported that AFB1 and ZEA are
the common harmful mycotoxins in food and feed chemistry (Wu,
2004). AFB1 and ZEA usually contaminate some grains together
(Kosicki et al., 2016), which may have greater hepatotoxicity than
single mycotoxin (Sun et al., 2015); therefore, the effective co-de-
gradation of AFB1 and ZEA is necessary.
AFB1 is well known for its teratogenic, carcinogenic and im-
munosuppressive effects on human and animals, it is mainly produced
by Aspergillus flavus and Aspergillus parasiticus. Consumption of AFB1-

Corresponding author.
∗

E-mail addresses: changjuan2000@126.com (J. Chang), wt9587@126.com (T. Wang), wangping850516@163.com (P. Wang), qqy1964@126.com (Q. Yin),
15093389011@163.com (C. Liu), Zhuqun1991@126.com (Q. Zhu), lfshn@126.com (F. Lu), zheng196604@126.com (T. Gao).
1
Juan Chang and Tao Wang contribute equally to this work.

https://doi.org/10.1016/j.ecoenv.2020.110420
Received 17 December 2019; Received in revised form 28 February 2020; Accepted 1 March 2020
Available online 06 March 2020
0147-6513/ © 2020 Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
J. Chang, et al.
contaminated feeds by poultry may decrease feed intake and growth
performance as well as increase intestinal damage and mortality
(Williams et al., 2011). In addition, AFB1 residue in animal edible tis-
sues and products is also a potential threat to human health.
ZEA is a regular worldwide contaminant of cereals and feeds, which
is mainly produced by Fusarium strains such as F. graminearum and F.
culmorum (Luo et al., 2018). ZEA is an estrogenic mycotoxin to cause
reproduction disorders such as early sexual maturity, miscarriage, un-
developed embryos, and so on (Green et al., 1990).
In order to degrade mycotoxins, many methods such as physical,
chemical and biological treatments have been used. The traditional
physical and chemical methods have some drawbacks such as nutrient
losses, high equipment cost and bio-safety risk. Biodegradation is a
promising method for mycotoxin decontamination. Lactobacillus plan-
tarum was reported to have AFB1-degrading ability (Khaniana et al.,
2019), and Bacillus pumilus presented effectiveness in degrading ZEA
(Wang et al., 2017). Streptomyces sp. was found to be able to degrade
both AFB1 and ZEA (Harkai et al., 2016). Bacillus subtilis was reported to
alleviate the negative effects of AFB1 and ZEA on egg production and
feed intake (Jia et al., 2016). The previous research in our laboratory
showed that the compound probiotics of Bacillus subtilis, Lactobacillus
casei and Candida utilis could synchronously degrade both AFB1 and
ZEA (Huang et al., 2018). However, few data have been reported about
the synchronous biodegradation of AFB1 and ZEA application in broi-
lers as well as the interactions among probiotics, mycotoxins and gut
microbiota.
In order to alleviate the toxic effects of AFB1, ZEA or AFB1+ZEA on
broiler growth and gut microbiota, the different kinds of compound
probiotics were prepared in this study to reduce or eliminate the ha-
zards of AFB1 and ZEA in broiler production.
2. Materials and methods
2.1. Microbes and preparation
After considering the beneficial microbes with high ability for de-
grading both AFB1 and ZEA, three species of microorganisms such as
Bacillus subtilis (CGMCC1.0504), Lactobacillus casei (CGMCC1.2884),
Candida utilis (CGMCC2.0615) were obtained from China General
Microbiological Culture Collection Center (CGMCC). They were in-
cubated according to the published protocols (Huang et al., 2018) to
make the powder by freeze drying. Based on the previous results ob-
tained with response surface regression design in our laboratory in vitro,
three kinds of compound probiotics (CP) were prepared. The optimal
ratios of Bacillus subtilis, Lactobacillus casei and Candida utilis in broiler
diets were 1 × 107, 1 × 105 and 1 × 106 colony-forming units (CFU)/g
for ZEA biodegradation (CP1); 1 × 106, 1 × 107 and 1 × 107 CFU/g
for AFB1 biodegradation (CP2); 1 × 107, 1 × 106 and 1 × 107 CFU/g
for ZEA + AFB1 biodegradation (CP3), respectively. The above three
kinds of compound probiotics were prepared and added in broiler diets
according to the suggested microbial counts (CFU/g diet).
2.2. AFB 1 and ZEA determination
The contents of AFB 1 and ZEA were determined by ELISA method
with RIDASCREEN® AFB1 and ZEA test kits (R-Biopharm, Darmstadt,
Germany). The preparing process of samples for mycotoxin determi-
nation was as the following: About 5 g sample was ground, mixed with
25 mL 70% methanol (v/v), shaken for 3 min in a vortex mixer, filtered
with Whatman No.1 filter paper, and then 1 mL filtrate was diluted with
1 mL purified water. AFB1 and ZEA standard solutions were used for
making the AFB1 and ZEA calibration curves (Zuo et al., 2013). The
optical density was finally measured at 450 nm using ELISA 96-well
plate reader (BioTek Instruments Inc. Winooski, USA). The natural corn
meal contaminated with 14 μg/kg AFB1 and 57 μg/kg ZEA was used to
prepare the basal diet, and the moldy corn meal contaminated with
2
Ecotoxicology and Environmental Safety 194 (2020) 110420
69 μg/kg AFB1 and 60 μg/kg ZEA was used to prepare the mycotoxin-
treated diets.
2.3. Animals and managements
A total of 350 1-day-old Ross broilers were randomly divided into 7
groups, 5 replicates for each group, 10 broilers (half male and half fe-
male) in each replicate. All animals used in this experiment were
managed according to the guidelines of Animal Care and Use Ethics
Committee in Henan Agricultural University (SKLAB-B-2010-003-01).
The feeding experiment was divided into 2 stages (1–21 d and 22–42 d).
The basal diets in both stages were prepared according to the re-
commended standard (NRC, 1994). Feed and water were given to the
birds ad libitum. During the experiment, body weight, feed intake,
mortality, diarrhea rates were recorded, and average daily feed intake
(ADFI) and average daily gain (ADG) were measured. The feeding ex-
periment was designed as follows:
Group A: Basal diet (14.45 μg/kg AFB1, 58.58 μg/kg ZEA).
Group B: Basal diet with 500 μg/kg ZEA.
Group C: Basal diet with 50 μg/kg AFB1
Group D: Basal diet with 500 μg/kg ZEA+50 μg/kg AFB1
Group E: Basal diet with 500 μg/kg ZEA plus CP1.
Group F: Basal diet with 50 μg/kg AFB1 plus CP2.
Group G: Basal diet with 500 μg/kg ZEA+50 μg/kg AFB1 plus CP3.
Note: AFB1 and ZEA used for adjusting mycotoxin concentrations in
the diets from group B to group G were purchased from Sigma (Sigma-
Aldrich, St. Luis, USA).
2.4. Determinations of nutrient metabolic rates
At the end of feeding experiment, metabolic experiment was per-
formed using total fecal collection method. Fresh excreta were collected
without contamination from 5 birds in each replicate for 3 d. The feed
intake and excreta were recorded daily. The fecal samples of each re-
plicate were dried and ground. Crude protein, crude fat, calcium and
phosphorus in feed and excreta were determined with Kjeldahl, ether
extract, potassium permanganate (KMnO4) and ammonium molybdate
((NH4)6Mo7O24), respectively (Jurgens, 1997). The AFB1 and ZEA re-
sidues in excreta were also measured. The calculation of apparent nu-
trient metabolic rates was made as follows: Nutrient apparent metabolic
rate (%) = 100 × (nutrient content in diet - nutrient content in ex-
creta)/nutrient content in diet.
2.5. AFB1 and ZEA residues analysis
At the end of feeding experiment, 3 broilers in each group were
selected to be sacrificed. The blood sample, small intestine, liver and
chest muscle were collected. The extraction method of AFB1 and ZEA in
excreta was the same as the above protocol. The tissues samples of
small intestine, liver and chest muscle for AFB1 and ZEA extraction
were prepared as the following: 5 g tissues were added with 25 mL 70%
methanol (v/v), homogenized by an Ultra Turrax (T10, IKA Instrument
Company, Staufen, Germany) at 10,000 rpm for 2 min (Ghali et al.,
2008). The following procedure was the same as the above protocol.
2.6. Hepatic and jejunal sample preparation for microscopic analyses
The hepatic internal lobes and the middle section of jejunum from
each group were taken, cut at 1 cm3, cleaned with physiological saline.
The samples were fixed in formalin, dehydrated in ethanol, embedded
in paraffin, cut at thickness of 0.6 μm, stained with hematoxylin and
eosin, and then observed with a microscope (Olympus Co., Ltd. Tokyo,
Japan).
J. Chang, et al. Ecotoxicology and Environmental Safety 194 (2020) 110420

2.7. Jejunal microbiota analysis
The jejunal contents of 3 broilers in group A, D and G were collected at
the age of 42 d, respectively; and put in liquid nitrogen for further mo-
crobiota analysis. The total genomic DNA of each sample was extracted by
the stool DNA Kit (Omega Biotek, Norcross, GA, USA) according to
manufacturer's instructions. The V3 and V4 variable regions of the bac-
terial 16S rRNA genes were amplified by the universal primer pairs: 338F (
5′-ACTCCTACGGGAGGCAGCA-3′) and 806R (5′-GGACTACHVGGGTWT-
CTAAT-3′) (Liang et al., 2015). Reaction conditions consisted of an initial
95 °C for 3 min followed by 27 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C
for 45 s, and a final extension of 72 °C for 10 min. PCR reactions were
conducted in 20 μL reaction mixtures containing 10 ng template DNA,
0.8 μL each primer (5 μM), 2 μL 2.5 mM dNTPs, 0.4 μL FastPfu poly-
merase, and 4 μL 5 × FastPfu buffer. The PCR products were extracted
from 2% agarose gels and purified using the AxyPrep DNA Extraction Kit
(Axygen Biosciences, CA, USA), and then quantified using QuantiFluor-ST
(Promega Corporation, Madison, USA). The purified amplifications were
pooled in equimolar and paired-end sequenced on an Illumina platform
(Biomarker Technology Co., Ltd. Beijing, China). The ACE and Chao1
indexes were used to indicate the abundances of bacterial community, and
the Shannon and Simpson indexes were used to reflect the diversity of
bacterial community.
2.8. Bioinformatics analysis
The high-quality tags were clustered into the operational taxonomic
units (OTUs) using UPARSE (version 7.1, http://drive5.com/uparse/)
with a similarity threshold of 0.97. The phylogenetic affiliation of each
16S rRNA gene sequence was analyzed using the RDP Classifier
(https://rdp.cme.msu.edu/) at 0.80 confidence level. The coverage
percentage was calculated by Good's method (1953). The abundance-
based coverage estimator (ACE), the Chao1 richness estimator and the
Shannon and Simpson indices were analyzed with the MOTHUR pro-
gram (Schloss et al., 2009).
2.9. Statistical analyses
Experimental data were represented as the means ± standard er-
rors. The data were analyzed using the SPSS software (SPSS 19.0, IBM,
IL, USA), one-way ANOVA was used to compare treatment means.
Differences were considered statistically significance at p < 0.05. The
correlations between gut microbiota and toxin residues or ADG were
assessed by Pearson's correlation test using GRAPHPAD PRISM version
5.00 (GRAPHPAD Software, CA, USA).
3. Results and discussion
3.1. Broiler production performance and nutrient metabolic rates influenced
by mycotoxins and compound probiotics
The effect of three kinds of compound probiotics on broiler
production performance was presented in Tables 1 and 2. During both
feeding stages, ADFI and ADG in group E, F and G containing three
kinds of compound probiotics and mycotoxins were better than that in
their corresponding groups (group B, C and D) with only mycotoxins
(p < 0.05). The diarrhea rates were the highest in group C and D
during both feeding stages (p < 0.05). Mortality was the highest in
group B and C during the first feeding stage (1–21 d), and in group C
and D during the second feeding stage (22–42 d). During the second
feeding stage, Table 3 showed that protein metabolic rates from high to
low were group B, E > group G > group A > group D, F > group C
(p < 0.05). Crude fat metabolic rate in group B was significantly
higher than that in the other groups (p < 0.05), calcium metabolic rate
in group E was significantly higher than that in group C (p < 0.05). In
general, three kinds of compound probiotics additions in broiler diets
could significantly alleviate mycotoxin hazard, increase growth per-
formance and nutrient metabolic rates, decease diarrhea rates and
mortality for broilers (P < 0.05).
AFB1 is one of the most predominant and highly toxic mycotoxins in
the world, which has great adverse effects on animal production (Peng
et al., 2018). The previous report showed that the applications of diets
contaminated with AFB1 significantly decreased ADG, ADFI and nu-
trient metabolic rates (Jahanian et al., 2016). In this study, the low
growth rates, high diarrhea rates and mortality induced by AFB1 or
AFB1+ZEA were found, in correspondence with the previous reports.
This research also indicated that AFB1 caused more serious damages
than ZEA for broiler growth, health and nutrient availability. The lower
ADG in the control group in the first-stage feeding experiment may be
due to a little high AFB1 concentration in the basal diet and high sen-
sitivity to AFB1 for the young broilers.
ZEA can competitively bind to estrogen receptors to cause re-
productive problems (Conkova et al., 2003). The previous report
showed that ZEA didn't influence growth performance of piglets (Marin
et al., 2013), but another research indicated that small doses of ZEA
stimulated body weight gain in pre-pubertal gilts when metabolic
processes were intensified (Rykaczewska et al., 2018). In this study,
ZEA addition alone increased young broiler growth, improved crude
protein and crude fat metabolic rates. The reason might be due to the
following two factors: (1) The broilers are more tolerant to ZEA (Fink-
Gremmels and Malekinejad, 2007); (2) ZEA is one kind of estrogen-like
mycotoxin, so it may be able to increase animal growth and feed
availability except for its negative effect on animal reproduction
(Rykaczewska et al., 2018).
The previous research showed that AFB1+ZEA had a synergistic
toxicity to reduce egg production and feed intake for layers, compared
with the single mycotoxin (Jia et al., 2016). It was also reported that the
coexistence of AFB1 and ZEA could be more hepatotoxic than the in-
dividual one (Sun et al., 2015). However, the synergistic toxicity of
AFB1+ZEA has not been found in this study, maybe due to the low ZEA
sensitiveness for broilers.
Several beneficial microorganisms have been used for reducing
toxic effects on animal production. The inclusion of different compound
microorganism preparations in AFB1-contaminated diet significantly

Table 1
Effects of compound probiotics on production performance of 1-21d broilers (n = 5).
Groups Initial weight (g) Final weight (g) ADG (g) ADFI (g) ADFI/ADG Diarrhea rate (%) Mortality (%)

a d d d ab d
A 50.55 ± 0.46 561.65 ± 6.79 24.34 ± 0.34 42.40 ± 0.04 1.74 ± 0.02 2.92 ± 0.22 0
B 50.54 ± 0.38a 585.97 ± 5.91b 25.50 ± 0.28b 44.98 ± 0.01b 1.76 ± 0.02a 2.90 ± 0.40d 4
C 50.55 ± 0.21a 546.74 ± 1.82e 23.63 ± 0.08e 41.34 ± 0.07e 1.75 ± 0.01a 5.83 ± 0.67a 4
D 50.33 ± 0.16a 570.25 ± 7.77c 24.76 ± 0.37c 42.41 ± 0.01d 1.71 ± 0.02c 5.22 ± 0.56b 2
E 50.67 ± 0.15a 618.58 ± 3.51a 27.04 ± 0.17a 46.50 ± 0.03a 1.72 ± 0.01bc 3.26 ± 0.27d 0
F 50.73 ± 0.18a 572.41 ± 5.97c 24.84 ± 0.28c 42.49 ± 0.02d 1.71 ± 0.02c 4.47 ± 0.25c 0
G 50.32 ± 0.24a 577.48 ± 5.68c 25.10 ± 0.28c 43.84 ± 0.02c 1.75 ± 0.02a 4.51 ± 0.26c 0

Note: The different letters in the same columns indicate significant difference (p < 0.05), while the same letters in the same columns indicate insignificant difference
(p > 0.05).

3
J. Chang, et al. Ecotoxicology and Environmental Safety 194 (2020) 110420

Table 2
Effects of compound probiotics on production performance of 22–42 d broilers (n = 5).
Groups Initial weight (g) Final weight (g) ADG (g) ADFI (g) ADFI/ADG Diarrhea rate (%) Mortality (%)

a ab a ab ab d
A 560.00 ± 9.35 1723.00 ± 61.81 55.38 ± 2.60 116.58 ± 2.93 2.11 ± 0.06 2.29 ± 0.22 2
B 568.00 ± 7.58a 1741.00 ± 66.65a 55.86 ± 3.15a 116.48 ± 7.26ab 2.09 ± 0.06b 2.60 ± 0.37d 0
C 564.00 ± 8.22a 1644.00 ± 55.27b 51.43 ± 2.28b 112.20 ± 5.97b 2.18 ± 0.05a 3.83 ± 0.52a 8
D 566.00 ± 12.45a 1645.00 ± 95.07b 51.38 ± 4.31b 109.94 ± 9.01b 2.14 ± 0.04ab 3.86 ± 0.42a 4
E 568.00 ± 10.37a 1777.00 ± 72.33a 57.57 ± 3.17a 122.44 ± 6.68a 2.13 ± 0.02ab 2.70 ± 0.27cd 0
F 570.00 ± 6.12a 1770.00 ± 28.28a 57.14 ± 1.50a 121.06 ± 4.76a 2.12 ± 0.03ab 3.07 ± 0.26bc 0
G 557.00 ± 5.70a 1788.00 ± 61.60a 58.62 ± 3.13a 124.72 ± 5.36a 2.13 ± 0.05ab 3.47 ± 0.30ab 0

Table 3
Effect of compound probiotics on nutrient metabolic rates of broilers (%, n = 5).
Groups Crude protein Crude fat Calcium Phosphorus

A 53.67 ± 0.68cd 73.67 ± 0.01b 30.40 ± 0.83ab 39.24 ± 0.50a

B 55.17 ± 0.75ab 74.30 ± 0.60a 31.27 ± 0.41ab 39.76 ± 0.31a
C 50.38 ± 0.80e 73.51 ± 0.45b 30.05 ± 1.65b 39.52 ± 0.77a
D 52.65 ± 0.52d 73.24 ± 0.11b 30.16 ± 0.26ab 39.16 ± 0.96a
E 56.39 ± 1.22a 73.78 ± 0.29b 31.87 ± 0.88a 40.26 ± 0.66a
F 52.82 ± 0.61d 73.62 ± 0.06b 30.87 ± 0.72ab 40.47 ± 0.85a
G 54.02 ± 0.81bcd 73.32 ± 0.20b 31.04 ± 0.19ab 40.36 ± 1.16a

Note: The different letters in the same columns indicate significant difference (p < 0.05), while the same letters in the same columns indicate insignificant difference
(p > 0.05).

increased broiler growth (Zuo et al., 2013; Liu et al., 2018). Jia et al.
(2016) reported that the supplementation of Bacillus subtilis in AFB1 and
ZEA-contaminated diet could significantly alleviate their negative ef-
fects on feed intake and egg production of layers. In this study, the
additions of three kinds of compound probiotics significantly relieved
the toxic effects of AFB1 and ZEA on broiler production performance,
indicating their effective detoxification for broilers.
3.2. Effect of compound probiotics on AFB1 and ZEA residues for broilers
Table 4 showed that AFB1 residues in serum, excreta and various
tissues in group C and D were significantly higher than that in group A
(p < 0.05), indicating that the contamination of AFB1 or AFB1+ZEA in
diets increased AFB1 residues in broilers. However, the supplement of
CP2 or CP3 in AFB1-contaminated diet in group F or G significantly
decreased the AFB1 residues in liver, chest muscle and small intestine,
compared with group C and D (p < 0.05).
Table 5 indicated that ZEA was not detected in chest muscle of
broiler in all the groups, but ZEA residues in serum, excreta and other
tissues significantly increased in group B and D, compared with group A
(p < 0.05), indicating that the contamination of ZEA or ZEA + AFB1
in diets increased ZEA residues in broilers. In addition, ZEA residues in
all parts of group D were significantly higher than that of group B
(p < 0.05), inferring that the addition of AFB1 increased ZEA residues
in serum, liver and small intestine. Nevertheless, CP1 or CP3 supple-
ment in ZEA-contaminated diets in group E or G significantly decreased
ZEA residues in serum, liver, small intestine and excreta, compared
with the corresponding group B or D (p < 0.05). It could be concluded
that three kinds of compound probiotics additions in broiler diets were
able to reduce ZEA and AFB1 residues in broiler body.
AFB1 and ZEA can transmit through farm animals to human food
chain, possessing a potential threat to human health. In this study, AFB1
and ZEA contamination increased the corresponding mycotoxin re-
sidues in broilers. Moreover, AFB1+ZEA exerted synergistic effects on
ZEA residues. Three kinds of compound probiotics additions to the ZEA
and AFB1 contaminated diets were effective in reducing mycotoxin
residues. It was reported that AFB1 and ZEA were absorbed from the
gastrointestinal tract and extensively penetrated into various tissues
(Buranatragool et al., 2015). Bacillus subtilis was reported to have the
ability to germinate in animal intestinal tract, reduce AFB1 absorption
and residues in the internal organs of broilers (Salem et al., 2018). The
compound probiotics of L. casei, B. subtilis and Pichia anomala were
shown to decrease AFB1 residues in serum, liver, and muscle of broilers
(Zuo et al., 2013). In this study, three kinds of compound probiotics
additions in broiler diets significantly decreased both AFB1 and ZEA
residues in small intestine and liver, AFB1 in chest muscle, and ZEA in
serum and excreta. It is inferred that low AFB1 and ZEA residues by
three kinds of compound probiotics additions are probably attributed to
mycotoxin absorption and biodegradation by the beneficial bacteria
(Salem et al., 2018; Liu et al., 2019).
3.3. Hepatic and jejunal villus tissue damages induced by AFB1 and ZEA
and their restoration by CP3 addition
The hepatic cell cords disorder, hepatocyte vacuolization and he-
patic sinusoid hypertrophy were induced by AFB1+ZEA addition. The
massive atrophy and degeneration of microvilli, low crypt depth and
thin intestinal smooth muscle were observed by AFB1+ZEA addition.

Table 4
AFB1 residues in serum, liver, chest muscle, small intestine and excreta of broilers (μg/kg, n = 3).
Groups Serum Liver Chest muscle Small intestine Excreta

b c c d
A 1.81 ± 0.27 1.14 ± 0.37 0.91 ± 0.11 1.12 ± 0.26 2.30 ± 0.26c
B 1.83 ± 0.15b 1.13 ± 0.12c 0.80 ± 0.04c 1.12 ± 0.12d 2.36 ± 0.08c
C 2.85 ± 0.39a 9.50 ± 0.46a 3.05 ± 0.09ab 7.38 ± 0.66ab 10.01 ± 0.63ab
D 2.45 ± 0.89ab 9.13 ± 0.44ab 3.29 ± 0.19a 7.96 ± 0.40a 10.93 ± 0.65a
E 1.76 ± 0.10b 1.22 ± 0.16c 0.99 ± 0.11c 1.22 ± 0.19d 2.32 ± 0.20c
F 2.20 ± 0.43ab 8.86 ± 0.19b 3.03 ± 0.17b 6.69 ± 0.46c 9.14 ± 0.64b
G 1.71 ± 0.23b 8.85 ± 0.11b 3.02 ± 0.15b 7.02 ± 0.15bc 11.07 ± 0.97a

4
J. Chang, et al. Ecotoxicology and Environmental Safety 194 (2020) 110420

Table 5
ZEA residues in serum, liver, chest muscle, small intestine and excreta of broilers (μg/kg, n = 3).
Groups Serum Liver Chest muscle Small intestine Excreta

a d e
A ND 3.68 ± 0.31 ND 1.88 ± 0.57 38.75 ± 3.07d
B 8.72 ± 0.51b 8.72 ± 0.51bc ND 20.60 ± 0.66b 178.97 ± 7.09a
C ND 3.52 ± 0.35d ND 3.70 ± 0.43d 62.00 ± 1.35c
D 10.35 ± 0.24a 12.78 ± 1.97a ND 22.97 ± 2.31a 174.06 ± 5.70a
E ND 7.92 ± 0.29c ND 19.80 ± 0.50b 130.40 ± 9.90b
F ND 3.78 ± 0.14d ND 3.58 ± 0.40d 61.67 ± 0.48c
G 8.15 ± 0.49c 9.59 ± 0.60b ND 12.59 ± 0.60c 131.85 ± 8.38b

a
ND = Not detected.

However, CP3 addition could alleviate the toxic damages to make the It was reported that Bacillus subtilis significantly improved the pro-
hepatic and intestinal structure recover to the same status as the control portion of Proteobacteria, Bacteroidetes, Actinobacteria and Acidobacteria
group (Supplementary Fig. 1). in the jejunum of young broilers, and decreased Bacteroidetes propor-
Liver is the main target of mycotoxins. Histopathological examina- tion during growing phase (Li et al., 2019a); while Lactobacillus reuteri
tion can be used as an effective method to judge the toxic effect could increase Actinobacteria and Corynebacterium abundances, and
(Ellakany et al., 2011). Previous study showed that AFB1 caused hepatic decrease Proteobacteria and Campylobacterales abundances in broiler
architecture enlargement, bileduct hyperplasia, periportal fibrosis, he- ileum (Nakphaichit et al., 2011). The other report showed that ZEA
patocytic vacuolation and necrosis (He et al., 2013). Another report decreased the abundance of Actinobacteria and increased the abundance
showed that mycotoxin challenge had toxic effect on intestinal mor- of Cyanobacteria, Synergistetes and Proteobacteria in rabbit caecum (Li
phology with shorter and thinner villi (Jahanian et al., 2016). However, et al., 2018). It can be concluded that gut bacterial abundances at
the beneficial microbes and mycotoxin-degradation enzyme added in phylum level may be regulated by probiotics, mycotoxins, animal ages,
broiler diet could alleviate hepatic damage induced by AFB1 (Zuo et al., animal breeds, gut segments or their interactions.
2013), in agreement with this research. The reason why the compound At bacterial species level, the top 10 species of bacteria were pre-
probitotics treatment can alleviate the alteration of hepatic and jejunal sented in Fig. 1. The predominant bacterium in broiler jejunum among
histology induced by AFB1 and ZEA may be due to the low mycotoxin three groups was Lactobacillus aviarius. The relative abundance of Lac-
residues in tissues caused by CP3 addition. tobacillus aviarius in group G was higher than that in group D (p < 0.05)
and group A (p > 0.05), indicating that CP3 addition was able to in-
crease the relative abundance of Lactobacillus aviarius. The contamination
3.4. Gut microbial community influenced by compound probiotics and
of AFB1+ZEA significantly changed the relative abundances of
mycotoxins
uncultured_bacterium_g_Romboutsia, uncultured_bacterium_g_Terrisporobacter,
Clostridium_sensu_stricto_1, uncultured_bacterium_g_Lactobacillus, unculture-
The microbial abundance and diversity indices in Supplementary
d_bacterium_g_Turicibacter, bacterium_GC452011 (p < 0.05); however,
Table 1 showed that the microbial abundance (ACE and Chao1) in
CP3 addition significantly increased uncultured_bacterium_g_Lactobacillus
group D was higher than that in group A (p < 0.05); however, CP3
abundance.
addition in group G made the microbial abundance recover to the same
Previous study showed that AFB1 could induce dramatic changes in
level as group A (p > 0.05), indicating that CP3 addition could regain
the composition and metabolisms of intestinal microbiota (Wang et al.,
the normal gut microbiota disturbed by mycotoxins to keep broiler
2018). ZEA was also reported to affect the biodiversity of micro-
health. The previous research showed that ZEA could increase caecal
organisms, increase the Shannon's diversity index of gut microbiota in
microbial abundance for rabbits (Li et al., 2018). The other reports
porcine (Piotrowska et al., 2014), and decrease caecal Lactobacillus
indicated that probiotic Lactobacillus sp. added in swine and broiler
abundance of rabbits (Li et al., 2018). However, probiotics addition
diets increased microbial richness (ACE and Chao1) (Nakphaichit et al.,
could establish lactobacilli-enriched microbiota in broiler ileum and
2011; Shin et al., 2019). After considering the different results, it can be
swine feces (Nakphaichit et al., 2011; Shin et al., 2019), in agreement
concluded that microbial abundances increased by mycotoxins will be
with this research. Generally, supplementation with probiotics is con-
decreased by probiotics. In addition, Shannon and Simpson indexes
sidered as an effective method to treat the disorders of gut microbiota.
were not influenced by probiotics and mycotoxin supplements, in-
In this study, the high abundance of beneficial bacteria such as Lacto-
dicating that both of them had no effect on the diversity of bacterial
bacillus aviarius triggered by CP3 addition in group G may play an
community in broiler jejunum.
The relative abundances of bacteria at phylum level in
Supplementary Fig. 2 showed that Firmicutes, Bacteroidetes, Proteo-
bacteria, Actinobacteria and Campylobacterota were the main phyla in
broiler jejunum, accounting for over 90% of the total bacterial 16S
rDNA gene sequences in the samples. The relative abundance of Fir-
micutes in group G was significantly higher than that in group A and D
(p < 0.05), indicating that Firmicutes was easily influenced by the
external probiotics intervention. The previous researches showed that
probiotics addition could increase Firmicutes abundance (Li et al.,
2019a; Shin et al., 2019), corresponding with this study. The relative
abundances of Bacteroidetes, Proteobacteria and Actinobacteria in group
G were significantly lower than that in group D (p < 0.05). The re-
lative abundances of Actinobacteria, Fusobacteria, Cyanobacteria, Chlor-
oflexi, Acidobacteria and Gemmatimonadetes in group D were sig-
nificantly lower than that in group A (p < 0.05), while the relative
abundances of Fusobacteria, Cyanobacteria and Chloroflexi were regu-
lated to the same levels as group A by CP3 addition in group G. Fig. 1. Relative abundances of microbiota in broiler jejunum at species level.

5
J. Chang, et al.
important role in re-establishing a new healthy microbial community
for alleviating mycotoxin toxicity in broilers.
3.5. Gut microbial community analysis and its correlation with broiler
growth and mycotoxin residues
In order to evaluate the correlation between jejunal microbiota (at
the species level) and mycotoxin residues or ADG, a Pearson's correla-
tion matrix was performed by calculating the Pearson's correlation
coefficient (Fig. 2). This research showed that jejunal Lactobacillus
aviarius abundance was positively correlated with ADG (p < 0.05), and
negatively correlated with AFB1 residue in serum, illustrating that
Lactobacillus aviarius plays an important role in promoting broiler
growth and decreasing AFB1 residue. Lactobacillus aviarius has been
found to be correlated with broiler growth (Torok et al., 2011), in
agreement with this study. The high abundance of lactic acid bacteria
has previously been used as an indicator of the healthy gut (Merrifield
et al., 2010). The other research showed that Lactobacillus had the
ability to produce bacteriocins and organic acids to inhibit pathogens
and inflammatory reactions for increasing body weight gain (Yang
et al., 2016).
AFB1 and ZEA contamination significantly decreased the relative
abundances of uncultured_bacterium_g_Lactobacillus and bacterium_GC452011
(p < 0.05), and both of them were negatively correlated with AFB1 and
ZEA residues in serum, chest muscle and small intestine (except for AFB1 in
serum) (p < 0.01); however, CP3 addition could enhance both bacterial
abundances. It can be speculated that the low mycotoxin residues caused by
CP3 addition may be due to its function of increasing both bacterial
abundances for mycotoxin biodegradation. Some strains of Lactobacillus
have been reported to have the ability of binding and removing AFB1 via
surface protein (Haskard et al., 2001), supporting this speculation.
The correlation analysis also revealed that Clostridium_sensu_
stricto_1, uncultured_bacterium_g_Turicibacter and uncultured_bacterium_g_
Terrisporobacter abundances were positively correlated with the AFB1 and
ZEA residues in serum, chest muscle and small intestine (except for AFB1
in serum) (p < 0.05). The Clostridium_sensu_stricto_1 and uncultured_
bacterium_g_Terrisporobacter abundances were found to be negatively cor-
related with ADG (p < 0.05), which was contrary to Lactobacillus aviarius.
Clostridium_sensu_stricto_1 belongs to genus Clostridium, which was reported
to be associated with intestinal disease and soft-tissue infections (Swartz,
2002). Turicibacter sp. was reported as a potential pathogen to have a
negative impact on gastrointestinal health and physiological function
(Rettedal et al., 2009). Terrisporobacter was shown to be positively asso-
ciated with oxidative stress and inflammation for human being (Cai et al.,
2019). However, the addition of CP3 could significantly decrease the re-
lative abundances of these three potential harmful species, which will help
to alleviate mycotoxin toxicity for broilers.
It was reported that the low ratio between Bacteroidetes and
Firmicutes was beneficial to fat deposition (Turnbaugh et al., 2006), so it
6
Ecotoxicology and Environmental Safety 194 (2020) 110420
Fig. 2. Correlations between jejunal microbial
abundances and mycotoxin residues or ADG. Note:
The different rectangles are colored based on the
Pearson correlation coefficients between microbial
abundances and mycotoxin residues or ADG. The
intensity of color represents the degree of correla-
tion, gray represents positive correlation, red re-
presents negative correlation. * indicates significant
correlation (p < 0.05), ** indicates highly sig-
nificant correlation (p < 0.01). AFB1-1: AFB1 re-
sidue in serum, AFB1-2: AFB1 residue in chest
muscle, AFB1-3: AFB1 residue in small intestine, ZEA-
1: ZEA residue in serum, ZEA-2: ZEA residue in small
intestine, ADG: average daily gain. . (For inter-
pretation of the references to color in this figure le-
gend, the reader is referred to the Web version of this
article.)
can be inferred that CP3 addition in group G contributes to broiler body
weight gain by decreasing the ratio. Another report showed that Acti-
nobacteria and Proteobacteria could influence nutrient metabolism and
disturb the normal microbial environment (Li et al., 2019b); therefore,
both bacterial abundances decreased by CP3 addition may help to es-
tablish the healthy microbiota, which will be beneficial to broiler
growth and nutrient availability.
In general, the intestinal microbiota plays an important role in an-
imal health and production performance. An optimal microbiota pre-
vents colonisation of the intestinal epithelium by pathogens, modulates
the gut-associated systemic immunity, and influences gastrointestinal
development (Mills et al., 2012). It can be inferred that compound
probiotics additions are able to decrease AFB1 and ZEA residues and
increase broiler growth by increasing the proliferations of beneficial
bacteria such as Lactobacillus species and decreasing the proliferations
of detrimental bacteria such as Clostridium and Terrisporobacter.
4. Conclusions
This study showed that AFB1 or AFB1+ZEA significantly decreased
broiler production performance, induced hepatic and jejunal tissue
damages, disturbed the balance of gut microbiota. However, the com-
pound probiotics added in broiler diets could positively regulate gut
microbiota, decrease residues and cytotoxicity of AFB1 and ZEA, alle-
viate mycotoxin negative effects on broiler production performance. It
was also found that gut microbiota was closely correlated with broiler
growth, mycotoxin metabolism and residues. This research will lay a
theoretical foundation for probiotic application in detoxification and
animal health protection.
CRediT authorship contribution statement
Juan Chang: Project administration, Investigation, Methodology,
Writing - original draft. Tao Wang: Methodology, Investigation. Ping
Wang: Methodology, Investigation. Qingqiang Yin: Funding acquisi-
tion, Project administration, Methodology, Investigation, Writing - re-
view & editing. Qun Zhu: Methodology, Investigation. Fushan Lu:
Data curation, Formal analysis. Tianzeng Gao: Data curation, Formal
analysis.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
This research was funded by the Natural Science Foundation of
J. Chang, et al. Ecotoxicology and Environmental Safety 194 (2020) 110420

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