R ES E A R C H Open Access
Aflatoxin B
1
, zearalenone and
deoxynivalenol in feed ingredients and
complete feed from different Province in
China
Li Wu
1†
, Jianjun Li
1†
, Yunhu Li
5†
, Tiejun Li
1
, Qinghua He
2
, Yulong Tang
1
, Hongnan Liu
1
, Yongteng Su
3,4
,
Yulong Yin
1*
and Peng Liao
1*
Abstract
Background: The current study was carried out to provide a reference for monitory of aflatoxin B
1
(AFB
1
),
zearalenone (ZEN) and deoxynivalenol (DON) contamination in feed ingredients and complete feeds were collected
from different Province in China from 2013 to 2015.
Methods: A total of 443 feed ingredients, including 220 corn, 24 wheat, 24 domestic distillers dried grains with
soluble (DDGS), 55 bran, 20 wheat shorts and red dog, 37 imported DDGS, 34 corn germ meal and 29 soybean
meal as well as 127 complete feeds including 25 pig complete feed (powder), 90 pig complete feed (pellet), six
duck complete feed and six cattle complete feed were randomly collected from different Province in China,
respectively, by high-performance chromatography in combined with UV or fluorescence analysis.
Results: The incidence rates of AFB
1
, ZEN and DON contamination of feed ingredients and complete feeds were
80.8, 92.3 and 93.9 %, respectively. The percentage of positive samples for DON ranged from 66.7 to 100 %.
Domestic DDGS and imported DDGS presented the most serious contamination AFB
1
, ZEN and DON contamination
levels of feeds ranged from 61.5 to 100 %, indicated that serious contamination over the studied 3-year period.
Conclusion: The current data provide clear evidence that AFB
1
, ZEN and DON contamination of feed ingredients
and complete feeds in different Province in China is serious and differs over past 3-year. The use of corn, domestic
DDGS, imported DDGS and corn germ meal, which may be contaminated with these three mycotoxins, as animal
feed may triggered a health risk for animal. Feeds are most contaminated with DON followed by ZEN and AFB
1
.
Mycotoxins contamination in feed ingredients and complete feeds should be monitored routinely in China.
Keywords: Aflatoxin B
1
(AFB
1
), Complete feed, Deoxynivalenol (DON), Feed ingredient, Zearalenone (ZEN)
Background
Mycotoxins are a large group of fungal secondary metabo-
lites mainly produced from different fungal species in
worldwide [1, 2]. Mycotoxin contamination is widespread
among food and feed ingredients and is known to pose
animal and human health risks in China, where aflatoxin
B
1
(AFB
1
), zearalenone (ZEN) and deoxynivalenol (DON)
are prevalent [2, 3]. Among the document reported of
more than 400 mycotoxins, and the most important my-
cotoxins in the contaminated feeds which is known to
cause several adverse effects in pigs are AFB
1
, ZEA and
DON, including negative effects on animal performance
such as a decrease in feed intake and impairment of the
immune system [3–11]. Considering that the gastrointes-
tinal tract and the immune system of pigs are not vastly
different that of humans, the pig can be regarded as a
good model that can be applied to humans [4, 5]. Add-
itionally, AFB
1
is the potent of the known hepatotoxic,
carcinogenic, mutagenic and teratogenic to animal and it
* Correspondence: yinyulong@isa.ac.cn; liaopeng@isa.ac.cn
†
Equal contributors
1
Institute of Subtropical Agriculture, Chinese Academy of Sciences, 644#
Yuandaer Road, Changsha 410125, China
Full list of author information is available at the end of the article
© The Author(s). 2016 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Wu et al. Journal of Animal Science and Biotechnology (2016) 7:63
DOI 10.1186/s40104-016-0122-8
usually triggered acute or chronic disease [4, 5]. Many
published papers show the toxic effects of DON on ani-
mals, mainly impairing the immune system and the health
status of the gastrointestinal tract and the brain [2, 4, 5,
7–11]. Ingestion of DON has also been associated with
gastroenteritis, as reflected by nausea, emesis, diarrhea,
anorexia and gastrointestinal hemorrhaging [4, 5, 11].
ZEN is an endocrine disruptor which it can be bind to es-
trogen receptor leading to reproductive function disorders
and may have carcinogenic potential in humans [12].
Mycotoxins have a high toxicity for human and animal,
several countries has been document maximum levels
(MLs) and tolerance limits for these mycotoxins contam-
ination in food or feed [1, 6]. For example, the MLs stand-
ard set of AFB
1
for feed and completed feeding stuff from
5to20μg/kg and maximum guidance levels of 250 μg/kg
for ZEN and 900 μg/kg for DON in animal feeding prod-
ucts in the European Commission (Table 4) [3, 6, 13]. The
government of China has renewed set tolerance limits for
AFB
1
range from 10 to 50 μg/kg in feed, their set MLs of
ZEN is 500 μg/kg in complete feed, and their set the toler-
ance levels of DON from 1000 to 5000 μg/kg in complete
feed (Table 4) [3, 6].
Nowadays, China faces a feed source shortage issue.
Meanwhile, the high prices of protein source have lead-
ing to feed industrial use of alternative protein feed
sources such as distillers dried grains with soluble
(DDGS). In supplementation diet for DDGS for animal
feed must be considered and detection of mycotoxins
contaminationed, especially AFB
1
, ZEN and DON [14].
On the other hand, AFB
1
, ZEN and DON produce and
fungal growth depend on climate environment such as
high moisture and high temperature [3, 6, 14]. Corn,
wheat and soybean are the main cash crops, and the po-
tential hazards of the mycotoxin contamination of these
cash crops have a greater impact. The Yangtze River
basin and the Yellow River basin are one of the major
grain producing areas of China. In this area, maize and
wheat are the primary cash crops and are widely used in
animal feed [15, 16]. Li et al. reported 50.0 % of maize
samples collected from Beijing to be contaminated with
AFB
1
[3]. In another study in China, ZEN and DON
were found in 96.6 and 93.2 % corn samp les, with an
average contamination level of 289.7 and 1356.9 μg/kg,
respectively [14]. In the present study, a total of 443 feed
ingredients were randomly collected from Jiangsu,
Zhejiang, Shandong, Jiang xi, Inner Mongolia, Henan,
Guangdong, Jilin, Anhui, Hebei, Sichuan, Shanxi and
Fujian Province, and a total of 127 complete feeds were
randomly collected from Jiangsu, Shandong, Henan,
Zhejiang, Heb ei and Anhui Province, respectively.
Among of this regions, where the climate is warm and
humid and there is plenty of rainfall, which is suitable
for mould growth and mycotoxin formation. Ingram
et al. reported that temperature and rainfall are the key
climatic factors that influence plant pathogens and their
secondary metabolites [17]. Therefore, mycotoxin con-
tamination of feed ingredients is a serious problem in
this region. Cheng et al. reported that in this regions,
ZEN and DON contamination levels of wheat were 35.8
and 93.9 % in 2013, respectively [18]. Additionally,
93.75 % of maize was found to be contaminated by AFB
1
in 2005 [19]. AFB
1
, DON and ZEN are largely prevalent
in maize and wheat in this regions. Therefore, the con-
tamination of feed materials by mycotoxin will result in
increases in the mycotoxin levels of the feed product s.
The mycotoxins contamination in feed must be carefully
monitored from different region and different years.
This study was conducted to detection the AFB
1
, ZEN
and DON in feed contaminated from differ ent Province
in China. These results can serve as a reference for feed
industrial, animal farm and China government regula-
tory of feed and food safety issue in the future.
Methods
Samples collection and preparation
The protoco l for the analyses were reviewed and ap-
proved by the Institute Animal Care and Use Committed
at Institute of Subtropical Agriculture, Chinese Academy
of Sciences (Changsha, Hunan Province, China).
A total of 570 samples in our study were collected dir-
ectly animal farms and animal production company
from different location in China from 2013 to 2015. A
total of 443 feed ingredient samples, including 220 corn,
24 wheat, 24 domestic DDGS, 55 bran, 20 wheat shorts
and red dog, 37 imported DDGS, 34 corn germ meal
and 29 soybean meal. A total of 127 completed samples,
including 25 pig complete feed (powder), 90 pig
complete feed (pellet), six duck complete feed and six
cattle complete feed. All of the samples were undertaken
according to the method by European Regulation No.
401/2006 [20]. All samples were grinded, mixed and
stored at 4 °C until to analyses.
High-performance liquid chromatography
AFB
1
was analysis according to the previously methods
[21]. Briefly, 20 g of ground feed was extracted with
100 mL of methanol:water (80:20, v/v), blended at high
speed for 3 min and then filtered b Mycosep® #226
(Romer Labs. Inc., Singapore). The extract was diluted
with a phosphate-buffered saline solution (PBS, pH 7.4),
mixed well and filtered through microfiber filter paper.
The immunoaffinity column (AokinImmunoClean CF
AFLA, Aokin AG, Berlin, Germany) was conditioned
with 1 mL of sodium azide, and 10 mL of the diluted fil-
trate was passed throu gh the column at a flow rate of
1 mL/min. The column was then washed with 10 mL of
a methanol: water solution (10:90, v/v) at a flow rate of
Wu et al. Journal of Animal Science and Biotechnology (2016) 7:63 Page 2 of 10
3 mL/min. The retained chemicals were then eluted with
1 mL of methanol at a flow rate of 1 mL/min . Subse-
quently, 20 μL of the clear eluate was injected directly
into an HPLC system. If the eluate was found not to be
clear, it was passed through an organic filter unit
(0.45 μm) before injection. The mobile phase utilized a
methanol:water solution (50:50, v/v) with the flow rate
set at 1 mL/min. Post-column derivatization was per-
formed with a photochemical reactor (AURA, Los
Angeles, CA). A C
18
column (4.6 mm × 250 mm, 5 μm,
Dikma, Shanghai, China) was employed with the LOD
set at 0.5 ppb and the LOQ at 1.5 ppb. A fluorescence
detector (SHIMADZU, Kyoto, Japan) was set for excita-
tion and emission wavelengths of 360 and 440 nm, re-
spectively. The retention time was 16.5 min. The
temperature of the column was set for 30 °C.
ZEN and DON analysis according to the methods of
GB/T 23504-2009 and GB/T 23503-2009 [6, 14]. For
ZEN analysis, samples were analyzed according to the
certified Chinese GB/T 23504–2009 method. Briefly,
25 g of feed was extracted with 100 mL of a methanol:-
water solution (60:40, v/v), blended at high speed for
3 min and then filtered through a sheet of Waterman
Filter Paper No. 4. The extract was dil uted with a
phosphate-buffered saline solution (PBS, pH 7.40),
mixed well and filtered through microfiber filter paper.
The immunoaffinity column (AokinImmuno-Clean CF
ZEA, Aokin AG, Berlin, Germany) was conditioned with
1 mL of sodium azide, and 10 mL of the diluted filtrate
were passed through the column by gravity at a flow rate
of 1 mL/min. The column was then washed with 10 mL
of a methanol:water solution (10:90, v/v) at a flow rate of
3 mL/min. The bound chemicals were then eluted with
3 mL of methanol at a flow rate of 1 mL/min . Subse-
quently, 20 μL of the clear eluate was injected directly
into an HPLC system. If the eluate was found not to be
clear, it was passed through an organic filter unit
(0.45 μm) befo re injection. The mobile phase utilized an
acetonitrile:water: methanol solution (46: 46: 8, v/v/v
with the flow rate set at 1 mL/min. A C
18
column
(4.6 mm × 150 mm, 5 μm, Dikma, Beijing, China) was
employed with the LOD set at 1.5 ppb and the LOQ at
4 ppb. A fluorescence detector (SHIMADZU, Kyoto,
Japan) was set for excitation and emission wavelengths
of 274 and 440 nm, respectively. The retention time was
7.3 min.
For DON analysis, samples were analyzed according to
the method of GB/T 23503–2009. Briefly, 25 g of ground
feed was extracted with 100 mL of a solution of metha-
nol:water (60:40, v/v), blended at high speed for 3 min and
then filtered through a sheet of Waterman Filter Paper
No. 4. The extract was diluted with a phosphate-buffered
saline solution (PBS, pH 7.4), mixed well and filtered
through microfiber filter paper. The immunoaffinity
column (AokinImmunoClean CF DON, Aokin AG, Berlin.
Germany) was conditioned with 1 mL of sodium azide,
and 10 mL of the diluted filtrate was passed through the
column by gravity at a flow rate of 1 mL/min. The column
was then washed with a 10 mL solution of methanol:water
(10:90, v/v) at a flow rate of 3 mL/min. The bound com-
pounds were then eluted with 3 mL of methanol at a flow
rate of 1 mL/min. The purified samples were dried under
a stream of nitrogen gas at 50 °C. For the mobile phase,
the residue was dissolved in 200 μL after evaporation.
Subsequently, 20 μL of the eluate was injected into an
HPLC system. The mobile phase utilized a methanol:
water solution (30: 70, v/v) with the flow rate set at 1 mL/
min. A C
18
column (4.6 mm × 150 mm, 5 μm, Agilent,
Santa Clara, CA) was employed with the LOD set at
0.02 ppm and the LOQ at 0.06 ppm. The absorption UV
wavelength (SHIMADZU, Kyoto, Japan) was set at
218 nm. The retention time was 5.6 min.
Data analysis
All data were calculated using Microsoft Excel 2007 and
are exp ressed as percentages or means, median and
maximum.
Results and discussion
Mycotoxin occurrence
All of AFB
1
, ZEN and DON occurrence data are showed
in Tables 3 and 4. In summary, detected feed ingredients
and complete feeds were mostly contaminated by DON,
followed by ZEN and AFB
1
. A total of 573 samples in-
cluding 443 samples of feed ingredients and 130 samples
complete feeds were analyzed to determination AFB
1
,
ZEN an d DON. Of these samples analyze for DON,
93.9 % contained this mycotoxin, with le vels ranging
from 0 to 4402.7 μg/kg. The occurrence rate of ZEN and
AFB
1
were 92.3 and 80.8 %, respectively.
AFB
1
in feed ingredients and complete feeds
The result s of AFB
1
were shown in Table 1 from 2013 to
2015. AFB
1
was analysis in corn samples in 2013, with
levels ranging from 0 to 15.9 μg/kg, and in 67.6 and
82.4 % of the corn samples in 2013 and 2015, respect-
ively. From 2013 to 2015, all of the corn samples were
not exceeding to 50.0 μg/kg regulatory limits in China.
Moreover, in 2005, Gao et al. analysis 279 corn sam-
ples collected in China and found A FB
1
contaminated
in 74.6 % in detection s amples , with an average value
for 39.64 μg/kg [19]. A similar s tudy wa s shown in
Bhat et al. document , he analysis 2074 samples of
corn collected from rural and urban areas o f 11 states
for AFB
1
contamination and analysis a median value from
<5 to 35 μg/kg [22]. The prevalence of AFB
1
contamin-
ation in wheat was almost the same in the 3 year investi-
gated and was significantly lower than that observed in
Wu et al. Journal of Animal Science and Biotechnology (2016) 7:63 Page 3 of 10
Table 1 Analyses of AFB
1
(μg/kg) in feed ingredients and complete feeds
Item Growing years Numbers of samples Positive samples Numbers of samples in the range, μg/kg
%
a
Mean Median Maximum <0.5 0.5–10 10–50 50–500 >500
Feed ingredients
Corn 2013 74 67.6 3.5 1.4 15.9 24 44 6 0 0
2014 55 94.6 4.3 3.7 11.1 3 50 2 0 0
2015 91 82.4 3.9 4.4 25.5 16 73 2 0 0
Wheat 2013 11 27.2 0.4 0 1.8 8 3 0 0 0
2014 8 62.5 1.1 1.0 4.0 3 5 0 0 0
2015 5 80.0 1.5 1.3 3.0 1 4 0 0 0
Domestic DDGS 2013 6 100 10.5 9.7 13.1 0 4 2 0 0
2014 11 100 10.0 10.2 13.6 0 3 8 0 0
2015 7 100 11.0 10.9 12.7 0 1 6 0 0
Bran 2013 27 59.3 3.1 1.3 10.9 11 14 2 0 0
2014 10 90 2.4 2.3 4.5 1 9 0 0 0
2015 18 83.3 2.1 2.4 3.8 3 15 0 0 0
Wheat shorts and red dog 2013 7 100 6.0 8.1 9.3 0 7 0 0 0
2014 3 100 4.3 1.5 10.5 0 2 1 0 0
2015 10 80 5.0 6.2 8.0 2 8 0 0 0
Imported DDGS 2013 7 100 9.0 9.1 13.7 0 4 3 0 0
2014 17 100 10.5 10.4 15.2 0 7 10 0 0
2015 13 61.5 7.0 10.0 13.8 5 2 6 0 0
Corn germ meal 2013 6 83.3 4.2 1.8 10.2 1 3 2 0 0
2014 9 88.9 9.3 10.6 13.5 1 2 6 0 0
2015 19 68.4 7.5 10.2 14.1 6 3 10 0 0
Soybean meal 2013 13 92.3 4.5 3.1 9.8 1 12 0 0 0
2014 3 100 6.4 6.4 7.1 0 3 0 0 0
2015 13 84.6 2.6 2.2 6.2 2 11 0 0 0
Total - 443 - - - - - - - - -
Complete feeds
Pig complete feed (powder) 2013 10 90 6.4 7.9 5.4 1 8 1 0 0
2014 2 100 6.3 7.9 5.2 0 2 0 0 0
2015 13 100 19.9 8.2 9.1 0 13 0 0 0
Pig complete feed (pellet) 2013 19 68.4 4.6 5.2 11.1 6 13 0 0 0
2014 33 100 8.0 7.3 18.1 0 26 7 0 0
2015 38 63.2 3.4 2.5 9.6 14 23 1 0 0
Duck complete feed 2013 - - - - - - - - - -
2014 - - - - - - - - - -
2015 6 100 6.44 3.97 8.84 0 6 0 0 0
Cattle complete feed 2013 - - - - - - - - - -
2014 - - - - - - - - - -
2015 6 100 4.5 3.9 8.3 0 6 0 0 0
Total - 127 - - - - - - - - -
a
Positive samples are defined as those with aflatxoin B
1
≥ 0.5 μg/kg (LOD)
Wu et al. Journal of Animal Science and Biotechnology (2016) 7:63 Page 4 of 10
corn. The domestic DDGS and imported DDGS were
more seriously contaminated by AFB
1
than bran and
wheat shorts and red dog. It was highly contaminated in
2013 and 2014, with 100 and 100 % of the domestic
DDGS and imported DDGS samples contaminated by
AFB
1
, respectively. AFB
1
detection results showed that
it’s to exceeded regulatory limits, with an average levels
of 10.0 (Table 4). AFB
1
detection rate and concentrations
were higher than the previously study by Li et al. [3].
AFB
1
contaminated in corn germ meal and soybean
meal results were lower than those found in the corn,
domestic DDGS and imported DDGS and did exceeding
the maximum limits set in China (Table 4). Li et al.
document results showed that 6 % of the DDGS con-
tained concentrations of AFB
1
that exceeded regulatory
limits, with an average content of 9.8 μ g/kg in 2011.
From 2014 to 2015, all of the domestic DDGS samples
were exceeding to 9.8 μg/kg regulatory limits in China ,
with an average content of 10.2 and 10 .9 μg/kg. From
2014 to 2015, all of the imported DDGS samples were
exceeding to 9.8 μg/kg regulatory limits in Ch ina, with
an average content of 10.4 and 10.0 μg/kg, respe ctively.
These result was not agreement with the results of a sur-
vey in China occurrence of mycotoxins in feedstuff and
feed conducted by Li et al. [3]. Possible reason was that
feed mill and farmers pay more attention to storage condi-
tions because of the highly prices of soybean meal as a
protein source feed.
AFB
1
detection results in complete feed samp les were
shown in Table 1. The AFB
1
contamination in complete
feed increased from 2013 to 2015. Pig completed feed
(powder) and pig complete feed (pellet) contamination
value were significantly higher those in duck and cattle
complete feed in our study. Pig complete feed (powder)
and pig complete feed (pellet) were the feed with most
serious contamination by AFB
1
, with value ranging from
63.2 to 100 %, followed by duck complete feed (50–
100 %) and cattle complete feed (0–100 %). These re-
sults not agreement with the ma ximum and average
values reported by previously survey [3, 6, 14]. A possi-
bility explanation for discrepancies between previously
study and our present study could be that the different
climate environment and detection methods for collec-
tion samples.
ZEN in feed ingredients and complete feeds
Over the studied 3-years period, the ZEN contamination
levels of corn, wheat, domestic DDGS, bran, wheat
shorts and red dog, imported DDGS , corn germ meal,
soybean meal and complete feeds (except for cattle
complete feed) were greater than 50 % and mean values
detection for corn, wheat, domestic DDGS, bran, wheat
shorts and red dog, imported DDGS , corn germ meal,
soybean meal, pig complete feed(including powder and
pellet) were exceeded 150 μg/kg in 2013 to 2015
(Table 2). In 2015, 99 % of the corn samples were con-
taminated, with levels ranging from 0 to 1442.5 μg/kg
and the ma ximum level (1442.5 μg/kg) was high than
those detectioned in 2013 (780.2 μg/kg) and
2014(833.9 μg/kg). Meanwhile, in 2015, 89.5 % of the
corn germ meal samples were contaminated, with levels
ranging from 0 to 1518.2 μg/kg and the maximum level
(1518.2 μg/kg) was high than those detectioned in 2013
(581.1 μg/kg) and 2014 (582.1 μg/kg). Additionally, the
highest ZEN contaminated levels of pig complete feed
(pellet) occurred in 2015 (94.7 %), but the highest max-
imum (1296.5 μg/kg), average (375.0 μg/kg) and median
value (283.2 μg/kg) in 2015. Rodrigues et al. showed
contaminated percentages and ZEN value s in corn sam-
ples from Middle East and African countries of 16 %
(maximum 310 μg/kg) [23]. Li et al. reported for central
China 84.1 % ZEN contaminated in corn samples at
values ranging from 10.1 to 1613.7 μg/kg [3]. Another
detection study in China, ZEN was found in 96.6 % of
corn samples, with an average contamination values of
289.7 μg/kg to 1894 μg/kg [14]. Most of survey findings
are consist with our study results, showing the serious-
ness of ZEN contamination in corn and pig complete
feed, it must be more carefully used in feed mill.
DON in feed ingredients and complete feeds
The detection results of DON in feed ingredient and
complete feeds are shown in Table 3. In 2014 and 2015,
corn, bran, imported DDGS, pig complete feed (includ-
ing powder and pellet) and duck complete feed were
highly contaminated with DON, as maximum, average
and median values occurred. Corn, cron germ meal and
pig complete feed (pellet) present the highest contamin-
ation rate with the percentage of DON-contaminated
corn germ mea l and pig complete feed (pelle t) samples
were 100, 89.5 and 84.2 % in 2015, whereas DON was
detectioned in only 88.9 and 66.7 % of corn germ meal
and pig complete feed (pellet) samples in 2014, respect-
ively. All of results in our study showed that DON con-
tamination values are first to those of ZEN. Guan et al.
showed that 93.2 % of corn sample collected from China
in 2009 were contaminated with DON, with average
values of 1357 μg/kg and maximum levels of 5150 μg/
kg. Meanwhile, Binder et al showed that 70 % of corn
samples in Asia were contaminated with DON, with a
maximum values of 10,626 μg/kg, which is similar to
that measured in Li et al. study [3, 24]. Moreover, Cui et al.
showed that DON was found in 89.3 % of wheat samples
harvested from Jiangsu and Anhui Province at values
ranging from 259 to 4957 μg/kg, which is consist with
our study results (4402.7 μg/kg in corn germ meal) [25].
Corn is an energy ingredient frequently used in animal
feeds in China, in the years in which corn and its by-
Wu et al. Journal of Animal Science and Biotechnology (2016) 7:63 Page 5 of 10