Food and Chemical Toxicology 59 (2013) 748–753

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Food and Chemical Toxicology

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Effects of Bacillus subtilis ANSB060 on growth performance, meat quality

and aflatoxin residues in broilers fed moldy peanut meal naturally
contaminated with aflatoxins
Yu Fan a,1, Lihong Zhao a,1, Qiugang Ma a, Xiaoying Li a, Huiqin Shi a, Ting Zhou b, Jianyun Zhang a,
Cheng Ji a,⇑
a
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
b
Guelph Food Research Center, Agriculture and Agri-Food Canada, Guelph N1G 5C9, Canada

a r t i c l e i n f o a b s t r a c t

Article history: This study was conducted to investigate the toxic effects of aflatoxins and the efficacy of Bacillus subtilis
Received 26 March 2013 ANSB060 for the amelioration of aflatoxicosis in broiler chickens. Six replicates of ten broilers each were
Accepted 3 July 2013 assigned to one of seven dietary treatments, which were labeled C0 (basal diet); M0 (basal diet containing
Available online 17 July 2013
moldy peanut meal); C500 and C1000 (C0 + 500 or 1000 g/t aflatoxin biodegradation preparations, com-
posed mainly of ANSB060); and M500, M1000 and M2000 (M0 + 500, 1000 or 2000 g/t aflatoxin biodeg-
Keywords: radation preparations). The concentrations of aflatoxin B1, B2, G1 and G2 in the moldy diets (M0, M500,
Aflatoxin B1
M100 and M2000) fluctuated around 70.7 ± 1.3, 11.0 ± 1.5, 6.5 ± 0.8 and 2.0 ± 0.3 lg/kg, respectively.
Aflatoxin biodegradation preparation
Bacillus subtilis ANSB060
The results showed that the M0 diet caused a significant decrease in average daily weight gain and
Growth performance increased feed requirements, with a gain ratio increasing from d 8 to 42, deterioration in meat quality
Meat quality and aflatoxin residues in broilers’ livers as compared with the C0 diet. The addition of ANSB060 to the
Residue aflatoxin-contaminated diets offset these negative effects, leading to the conclusion that ANSB060 has
a protective effect on growth performance and meat quality while reducing the amount of aflatoxin res-
idues in the livers of broilers fed naturally moldy peanut meal.
Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction
Aflatoxins are secondary toxic metabolites mainly produced by
the fungi Aspergillus parasiticus, Aspergillus flavus, Aspergillus nomi-
us, Aspergillus tamari and Aspergillus pseudotamarii (Diener et al.,
1987; Goto et al., 1997; Ito et al., 2001; Kurtzman et al., 1987).
Since the discovery of aflatoxins in the 1960s, the various mycotox-
ins have elicited great public health concerns because of their
widespread occurrences in animal feeds and human foods (Miazzo
et al., 2000; Oueslati et al., 2012). The toxicities of aflatoxins have
been widely investigated for their teratogenic, carcinogenic, muta-
genic and immunosuppressive effects (Gao et al., 2011; Wangikar
et al., 2005). Among the four major isoforms of aflatoxins, namely
Abbreviations: ADFI, average daily feed intake; ADG, average daily weight gain;
AFB1, aflatoxin B1; AFB2, aflatoxin B2; AFG1, aflatoxin G1; AFG2, aflatoxin G2; AFM1,
aflatoxin M1; F:G, feed: gain ratio; GRAS, generally recognized as safe; HPLC, high
performance liquid chromatography; ND, not detected; SEM, standard error of the
mean.
⇑ Corresponding author. Tel./fax: +86 10 62731019.
E-mail address: jicheng@cau.edu.cn (C. Ji).
1
These authors contributed equally to this work.
B1, B2, G1, and G2, aflatoxin B1 (AFB1) is the most potent toxin and
carcinogen (Corcuera et al., 2012; Mishra and Das, 2003).
In addition to their toxic effects, consumption of aflatoxin-
contaminated feeds by poultry may lead to other consequences
such as decreases in growth performance and meat quality, poor
feed utilization and an increase in the incidence of disease in
poultry, all causing huge economic losses (Dersjant-Li et al.,
2003; Liu et al., 2011). A large number of studies have been per-
formed that have demonstrated the negative effects of high levels
(1–5 mg/kg) of aflatoxins on broilers (Edrington et al., 1997; Kub-
ena et al., 1998; Zhao et al., 2010). There has been, however, little
research concerning chronic aflatoxicosis in animals due to lower
levels of contamination (Bintvihok and Kositcharoenkul, 2006;
Dersjant-Li et al., 2003), though aflatoxin contamination of feed-
stuffs is often at low concentrations under field and storage condi-
tions. A survey study in Southeast Asia revealed an incidence of
AFB1 in animal feed and feed raw materials of 34% with a mean le-
vel of 38 lg/kg in 415 samples (Binder et al., 2007). Considering
that poultry meat color, pH value, shear force and cooking loss
are critical food quality attributes important both for consumers’
initial selection of raw meat products in the marketplace and for
their evaluation and ultimate acceptance of the cooked product

0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.fct.2013.07.010
 Y. Fan et al. / Food and Chemical Toxicology 59 (2013) 748–753 749

upon consumption (Fletcher, 1999), the possibility that mycotox- Table 1

ins may affect meat quality via oxidative stress is important (Shen Basal diet formulations and nutritional contents.

et al., 1994). However, the available literature remains scanty Ingredients Percentage Nutrition component Content
(Bonomi et al., 1993; Liu et al., 2011). Moreover, residues of afla- (%)
toxins and their metabolites may exist in the tissues, milk, and Maize 57.70 Crude protein (%) 21.48
eggs of animals receiving contaminated feeds to become a poten- Extruded-soybean 6.00 Metabolisable energy (MJ/ 12.60
tial human health hazard (Hussain et al., 2010). It has been demon- kg)
Soybean meal 8.20 Calcium (%) 0.99
strated that aflatoxin intake is associated with a high incidence of Peanut meal 21.00 Total phosphorus (%) 0.65
human liver cancer (Berry, 1988) and legislation restricting al- Limestone 1.37 Available phosphorus (%) 0.43
lowed levels in animal products such as milk has been established Calcium 1.80 Methionine (%) 0.62
in many countries. hydrophosphate
Salt 0.30 Methionine + cystine (%) 0.91
Numerous physical and chemical strategies for the elimination
Soybean oil 2.00 Lysine (%) 1.15
or inactivation of aflatoxins have been reported in the literature Lysine[98.5%] 0.47 Trytophan (%) 0.21
(Kabak et al., 2006; Parlat et al., 2001). Nevertheless, these meth- DL-Methionine 0.36 Threonine (%) 0.81
ods have some limitations such as losses in the nutritional value Threonine 0.19
and the palatability of feeds, as well as the expensive equipment Vitamin premixa 0.03
Choline chloride 0.10
required to implement these techniques, while none of these strat-
Mineral premixb 0.30
egies is sufficient to completely fulfill the necessary efficacy, safety, Zeolite powder 0.18
and cost requirements (CAST, 2007; Teniola et al., 2005). Therefore, Total 100.00
for its simplicity and affordability, researcher has focused on the a
Provided per kilogram of diet: vitamin A, 12,000 IU; cholecalciferol, 3,000 IU;
biological detoxification of mycotoxins using microorganisms or vitamin E, 7.5 IU; vitamin K3, 1.5 mg; thiamine, 0.6 mg; riboflavin, 4.8 mg; pyri-
enzymatic preparations (Taylor and Draughon, 2001). Some mi- doxine, 1.8 mg; vitamin B12, 9 lg; folic acid, 150 lg; niacin, 10.5 mg.
b
crobes, including fungal and bacterial isolates, have been reported Provided per kilogram of diet: calcium pantothenate, 7.5 mg; iron, 100 mg;
copper, 8 mg; manganese, 120 mg; zinc, 100 mg; selenium, 0.3 mg; iodine, 0.7 mg.
to possess various abilities regarding the degradation of aflatoxins,
such as, Armillariella tabescens (Liu et al., 2001), Rhodococcus ery-
thropolis (Teniola et al., 2005), Myxococcusfulvus (Zhao et al.,
All broilers were placed in wire-bottomed aluminum cages (100  130  59 cm,
2011), among others. However, most of these microorganisms length  width  height) and housed in an environmentally controlled house
are not officially allowed to be applied to foods or feeds under equipped with central heatings. The temperature was maintained at 30 °C for the
the US Food and Drug Administration (FDA), the Association of first week, and then was gradually decreased to 21 °C until the broilers had reached
American Feed Control Officials (AAFCO) and the Director of the 24 days of age and maintained thereafter. The relative humidity ranged from 65% to
70%. Broilers received 24 h of incandescent lighting for the first three days and 23 h
Ministry of Agriculture, People’s Republic of China.
of light and 1 h of darkness from four days of age onward. Ventilation was con-
In this regard, a strain of Bacillus subtilis ANSB060 possessing a trolled by negative pressure using fans and eight unpowered blast caps. The chicks
strong ability to detoxify aflatoxins has been screened from fish gut were offered feed and water ad libitum through tube feeders and nipple drinkers
by our group; this strain has also been shown to inhibit the growth throughout the experimental period. Body weight (BW), average daily weight gain
(ADG), average daily feed intake (ADFI) and feed: gain ratio (F:G) were measured on
of pathogens and to resist unfavorable conditions within simulated
a replicate basis at 21 and 42 days of age.
gut environments (Gao et al., 2011). Due to the generally recog-
nized as safe (GRAS) status of B. subtilis for nutritional and pharma-
2.2. Determination of mycotoxin content
ceutical use (Molnar et al., 2011), B. subtilis ANSB060 has been
considered as appropriate for application to animal diets for the The contents of the mycotoxins AFB1, AFB2, AFG1, AFG2, deoxynivalenol, zearal-
detoxification of aflatoxins (Ma et al., 2012). enone and ochratoxin A in the feed ingredients and formulated diets used in the
The aim of this research was to investigate the toxic effects of study were determined using the appropriate methods of HPLC (Binder et al.,
2007). The concentrations of AFB1, AFB2, AFG1, and AFG2 were 330.0, 80.1, 30.2
aflatoxins and efficacy of an aflatoxin biodegradation preparation and 7.1 lg/kg, respectively, in the moldy peanut meal used in this experiment while
(B. subtilis ANSB060) to reduce their effects on broiler chickens’ that of zearalenone was 15.1 lg/kg; the other mycotoxins were determined to be at
growth performance, meat quality and aflatoxin residue concen- concentrations below detection limits. Hardly any aflatoxins or other mycotoxins
trations when exposed to them at naturally-occuring levels of were found in the normal peanut meal (only 2 lg/kg AFB1) and other ingredients
in the basal diet. After seven diets had been prepared, each experimental diet
contamination.
was analyzed to confirm its final concentration of mycotoxins. The concentrations
of AFB1, AFB2, AFG1, AFG2 and zearalenone in groups M0, M500, M1000 and
M2000’s diets fluctuated around 70.7 ± 1.3 lg/kg, 11.0 ± 1.5 lg/kg, 6.5 ± 0.8 lg/kg,
2. Materials and methods 2.0 ± 0.3 lg/kg and 4.1 ± 0.3 lg/kg, respectively. The concentration of AFB1 was less
than 0.50 lg/kg in the diets of Groups C0, C500 and C1000.
2.1. Broiler chickens, diets, and management

2.3. Sample collection

One-day-old male broiler chickens (Ross 308) were obtained from a commercial
hatchery and fed a commercial rearing diet for 7 days to allow them to adapt to
At 42 days of age, two birds close to the average weight were selected from each
their surroundings. On Day 8, 420 male broiler chickens were individually weighed
pen. Each chicken was killed by cervical dislocation, then plucked and manually
(BW of 162.0 ± 0.4 g) and randomly assigned to seven treatment groups with six
eviscerated to obtain breast and thigh muscles. The skin along with all subcutane-
replicates of ten birds each. The seven dietary treatment groups were arranged as
ous fats and visible connective tissues were stripped from the muscles before anal-
follows: Group C0 (the positive control) was fed the basal diet containing 21% nor-
ysis. Various sizes of samples from the pectoralis major and gastrocnemius muscles
mal peanut meal; Group M0 (the negative control) received the basal diet contain-
were removed from standardized regions of each bird’s left side and immediately
ing 21% moldy peanut meal replacing the normal peanut meal; Groups C500 and
packed individually in sealable plastic bags and stored at 4 °C for the measurement
C1000 were fed the basal diet supplemented with 500 g/t and 1000 g/t of an afla-
of meat quality (Zhang et al., 2008). The liver and muscle samples from the pecto-
toxin biodegradation preparation; and Groups M500, M1000 and M2000 received
ralis major of the right side of each bird (one bird from each replicate) were re-
an M0 diet supplemented respectively with 500 g/t, 1000 g/t and 2000 g/t of an
moved and frozen at 20 °C for analysis of aflatoxin residues.
aflatoxin biodegradation preparation. The moldy peanut meal mainly contains
330.0 lg/kg AFB1, 80.1 lg/kg AFB2, 30.2 lg/kg AFG1 and 7.1 lg/kg AFG2. In this
experiment, aflatoxin biodegradation preparations were composed mainly of B. sub- 2.4. Meat quality
tilis ANSB060 using certain industrial fermentation and dry-processing technolo-
gies. The viable count of B. subtilis ANSB060 constituted more than 1  109 CFU/g The HunterLab values (L, lightness; a, redness; b, yellowness) were measured
of the aflatoxin biodegradation preparation. The composition of the basal diet has 45 min postmortem on the raw muscles using a hand-held colorimeter (SC-80C,
been presented in Table 1. Kangguang Apparatus Co. Ltd., Beijing, China), with a D65 illuminant and 10 stan-
750 Y. Fan et al. / Food and Chemical Toxicology 59 (2013) 748–753

dard observer. The colorimeter was calibrated against a white-and-black tile before
determination. Measurements were made vertically along the meat surface of each
muscle at three different locations free from color defects, bruises and hemor-
rhages, and the mean values of L, a and b from each muscle were calculated
(Fletcher, 1999).
The pH values of the breast and thigh muscles were measured at 45 min and
24 h after sacrifice, using a Testo 205 pH meter (Testo Instrument Co. Ltd., Ger-
many) equipped with an insertion electrode that was standardized by a 2-point
method against standard buffers of pH 4.0 and pH 7.0. Three measurement values
of pH(45min) and pH(24h) were recorded and averaged for each breast and thigh
muscle.
Shear force of the breast and thigh meat samples were analyzed at 24 h after
sacrifice. The muscles were cooked in a water bath at 80 °C until an internal tem-
perature of 75 °C was reached. After being cooled to room temperature, cylindrical
cores 12.7 mm in diameter were removed parallel to the fiber orientation from the
anterior end of each muscle with an attached sampler. Each core was sheared per-
pendicular to the longitudinal orientation of the muscle fibers using a Digital Meat
Tenderness Meter (Model C-LM3, Northeast Agricultural University, Harbin, China)
with a 25-kg load cell and a crosshead speed of 200 mm/min. Each cooked core was
sheared at 3 locations, and the average of the maximum forces needed to achieve
shearing was used for data analysis (Tang et al., 2007).
The cooking loss was determined in accordance with the methods described by
Kondaiah et al. (1985). The muscles were refrigerated overnight at 4 °C and cooked
in a vacuum bag in a water bath at 80 °C to achieve an internal temperature of
72 °C, after which they were allowed to cool to room temperature. The weight of
each sample was recorded before and after cooking to calculate cooking loss, which
was defined as the weight lost divided by the uncooked weight multiplied by 100.
2.5. Analysis of aflatoxin residues
3. Results
3.1. Growth performance
The effects of B. subtilis ANSB060 on average daily weight gain
(ADG), average daily feed intake (ADFI) and feed: gain ratio (F:G)
of broilers fed moldy peanut meal naturally contaminated with
aflatoxins have been presented in Table 2.
The ADG, ADFI and F:G of the birds were not significantly influ-
enced by dietary aflatoxins and B. subtilis ANSB060 at the initial
experimental phase between Days 8 and 21 (P > 0.05). In the late
experimental phase (22–42d), feeding diet containing aflatoxins
to broiler chickens significantly increased the F:G ratio, and the
addition of B. subtilis ANSB060 to diets contaminated with aflatox-
ins (M500, M1000 and M2000) markedly decreased the F:G ratio
with these broilers performing as well as those in Group C0.
Throughout the entire experimental period, the change tendency
for the F:G ratios of the broilers in all treatment groups was consis-
tent with those of the late experimental phase (22–42 d); the ADG
levels of broilers fed moldy peanut meal naturally contaminated
with aflatoxins (M0) was markedly lower than those of the birds
fed normal peanut meal (C0), 59.2 vs. 62.6 g, while ADG levels were
enhanced to 62.0, 62.1 and 63.1 g/bird d in Groups M500 (P < 0.05),
M1000 (P < 0.05) and M2000 (P < 0.05), respectively.

Analysis of AFB1, AFB2, AFG1, AFG2, and AFM1 residues in the liver and muscle
tissues were performed using the methods of the Association of Official Analytical
Chemists (AOAC, 2000) and partially purified using the method of (Chiavaro et al.
(2005).
Five g of NaCl were added to 25 g of the ground defrosted samples and blended
in 100 ml of methanol–water (80:20) for three min. After passing the mixture
through a paper filter, an aliquote of 10 mL of the filtrate was diluted with 40 mL
of PBS/0.1% in a Tween-20 Wash Buffer and applied to an immunoaffinity column.
Aflatoxins were eluted with 1.0 ml of methanol in a glass vial and dried to nearly
complete dryness under a gentle stream of nitrogen and dissolved in an HPLC mo-
bile phase. Levels of AFB1, AFB2, AFG1, and AFG2 were determined using an HPLC
system (Shimadzu LC-10 AT) equipped with a reverse phase column (DIKMA,
C18, 5 lm, 15 cm  4.6 cm ID), a post-column photochemical derivation (AURA,
USA) and a fluorescence monitor (Shimadzu RF-20A), with excitation at 360 nm
and emission at 440 nm, with methanol: water (45:55) as the mobile phase at a
flow rate of 1 ml/min. For the determination of AFM1, HPLC was performed with
a fluorescence monitor at 365 nm for excitation and 425 nm for emission, and
water: acetonitril: methanol (68:24:8) as the mobile phase at a flow rate of 1 ml/
min.
2.6. Statistical analyses
Data were analyzed using the general linear model procedure of SAS software
(Version 9; SAS Institute, Inc., Cary, NC) for a completely randomized experimental
design. Duncan’s multiple range tests were used for multiple comparisons when the
analysis indicated significant differences among treatments. All statements of sta-
tistical significance were based on a probability of P < 0.05.
3.2. Meat quality
The color parameters (L*, a* and b*) of the broilers are shown in
Table 3. As compared with Group M0, the addition of B. subtilis
ANSB060 to the aflatoxin-contaminated diets (Group M500,
M1000 and M2000) significantly increased the a* value for breast
muscles (P < 0.05). Meanwhile, the broilers in Group M0 had signif-
icantly higher b* value for breast muscles, in comparison to those in
Group C0 (P < 0.05). Supplementation of 1000 g/t or 2000 g/t of B.
subtilis ANSB060 to the aflatoxin-contaminated diets (Groups
M1000 and M2000) lowered the b* value for breast muscles, reach-
ing a value similar to that of the positive control, Group C0
(P > 0.05). There were no significant differences in the L* values
for breast and thigh muscles along with the values of a* and b*
for thigh muscles among all treatments (P > 0.05).
Data on the pH values of breast and thigh muscles are summa-
rized in Table 4. Diets containing aflatoxins or B. subtilis ANSB060
were comparable to those of Group C0 and did not affect the
pH(45min) and pH(24h) values of breast and thigh muscles. But sup-
plementing of aflatoxin-contaminated diets with B. subtilis
ANSB060 (Groups M500, M1000 and M2000) significantly im-
proved the pH(45min) values for thigh muscles (P < 0.05), in contrast

Table 2
Effects of B. subtilis ANSB060 on the growth performance of broilers fed moldy peanut meal naturally contaminated with aflatoxins.

Item C0 M0 C500 C1000 M500 M1000 M2000 SEM P-value

ADG (g/bird d)
8 to 21 d 48.2 47.8 47.8 47.3 47.9 46.1 49.1 1.56 0.090
22 to 42 d 73.7 67.8 70.9 72.3 71.5 72.7 72.4 3.11 0.061
8 to 42 d 62.6a 59.2b 61.7a 62.5a 62.0a 62.1a 63.1a 1.72 0.023
ADFI (g/bird d)
8 to 21 d 70.2 70.8 69.4 69.1 70.4 68.5 71.1 1.94 0.222
22 to 42 d 134.6 132.3 133.6 137.4 137.1 133.5 136.7 4.27 0.336
8 to 42 d 110.2 107.3 108.0 110.2 110.4 107.4 110.5 2.89 0.222
F:G (g of feed/g of gain)
8 to 21 d 1.46 1.48 1.45 1.46 1.47 1.49 1.45 0.034 0.417
22 to 42 d 1.86 cd 1.97a 1.89bcd 1.90bc 1.92b 1.84d 1.89bcd 0.041 <0.001
8 to 42 d 1.74c 1.82a 1.75bc 1.76bc 1.78b 1.75bc 1.74c 0.025 <0.001

Each value is a mean of duplicate assays. SEM = standard error of the mean.
Values with different superscripts in each row are significantly different (P < 0.05).
 Y. Fan et al. / Food and Chemical Toxicology 59 (2013) 748–753 751

Table 3
Effects of B. subtilis ANSB060 on the color of breast and thigh muscles from broilers fed moldy peanut meal naturally contaminated with aflatoxins.

Itema C0 M0 C500 C1000 M500 M1000 M2000 SEM P-value

Breast muscle
L 47.25 46.82 48.89 49.28 49.33 48.81 47.27 2.726 0.115
a 5.26ab 4.77b 5.40ab 5.85a 6.07a 5.81a 6.02a 0.846 0.006
b 16.68b 18.91a 18.34ab 18.25ab 19.17a 18.22ab 17.48ab 1.836 0.034
Thigh muscle
L 53.82 54.61 54.96 54.64 55.56 54.48 54.41 1.904 0.472
a 6.85 6.07 7.24 7.44 7.05 7.08 6.91 1.668 0.098
b 17.64 19.20 16.23 18.58 19.31 19.56 19.66 2.760 0.060

Each value is a mean of 12 broilers per group. SEM = standard error of the mean.
Values with different superscripts in each row are significantly different (P < 0.05).
a
L = lightness; a = redness; b = yellowness.

Table 4
Effects of B. subtilis ANSB060 on the pH values of breast and thigh muscles from broilers fed moldy peanut meal naturally contaminated with aflatoxins.

Itema C0 M0 C500 C1000 M500 M1000 M2000 SEM P-value

Breast muscle
pH(45min) 6.42 6.47 6.31 6.40 6.45 6.49 6.50 0.213 0.458
pH(24h) 5.89 5.88 5.90 5.90 5.86 5.90 5.89 0.078 0.961
Thigh muscle
pH(45min) 6.48ab 6.29b 6.46ab 6.54a 6.57a 6.53a 6.54a 0.179 0.016
pH(24h) 6.20 6.08 6.21 6.19 6.19 6.15 6.20 0.107 0.180

Each value is a mean of 12 broilers per group. SEM = standard error of the mean.
Values with different superscripts in each row are significantly different (P < 0.05).
a
The muscle pH at 45 min and 24 h is described as pH(45min) and pH(24h), respectively.

Table 5
Effects of B. subtilis ANSB060 on aflatoxin residues (lg/kg) in the livers of broilers fed moldy peanut meal naturally contaminated with aflatoxins.

Item C0 M0 C500 C1000 M500 M1000 M2000 SEM P-value

AFB1 NDa 0.24a ND ND 0.14b 0.11c 0.09d 0.016 <0.001
AFB2 ND 0.03a ND ND 0.02b 0.01c 0.01c 0.005 <0.001
AFM1 ND 0.20a ND ND 0.12b 0.12b 0.12b 0.012 <0.001

Each value is a mean of 6 broilers per group. SEM = standard error of the mean.
Values with different superscripts in each row are significantly different (P < 0.05).
a
ND = Not detected.

to the diet of Group M0. There were no marked differences in
pH(24h) values for either breast or thigh muscles or pH(45min) values
for breast muscles among all treatment groups (P > 0.05).
No significant differences (P > 0.05) for cooking loss or shear
force of breast and thigh muscles were observed among the treat-
ment groups (data not shown).
3.3. Aflatoxin residues
In this study, no AFB1, AFB2, AFG1, AFG2 or AFM1 residues were
observed in the muscles (data not shown). The aflatoxin residues
found in the livers are given in Table 5.There were no detectable
aflatoxin residues in the livers of broilers consuming the diets
fed Groups C0, C500 and C1000. AFB1, AFB2 and AFM1 residues
were the highest in Group M0 at 0.24, 0.03 and 0.20 ng/g, respec-
tively. Contaminated diets supplemented with B. subtilis ANSB060
(Groups M500, M1000 and M2000) showed significant decreases in
residues of AFB1 (0.14, 0.11 and 0.09 ng/g), AFB2 (0.02, 0.01 and
0.01 ng/g) and AFM1 (0.12, 0.12 and 0.12 ng/g) when compared
with the mold-contaminated Group M0 (P < 0.05). No AFG1 or
AFG2 residues were detected in livers during the treatments (data
not shown).
4. Discussion
Aflatoxins pose serious health and economic hazards for both
animals and humans because of their frequent occurrence, toxicity
and the presence of their residues in animal products. Controlling
and reducing aflatoxin contamination in food and feedstuffs re-
mains a major problem, creating a great demand for an effective
decontamination technology. B. subtilis ANSB060 shows a strong
ability to detoxify aflatoxins in vitro, with degradation by this
strain of aflatoxin B1, M1, and G1 showing reductions of 81.5%,
60%, and 80.7%, respectively (Gao et al., 2011). Moreover, its pro-
tective effects on the performance and antioxidation of laying hens
have also been verified, which suggests that B. subtilis ANSB060 can
enhance eggshell strength, the activity of SOD and GSH-Px, and the
recovery of protein synthesis in the liver, as well ameliorating the
damage to the liver and kidney tissue caused by aflatoxin (Ma
et al., 2012).
In this study, diets naturally contaminated with aflatoxins at a
level of 70 lg/kg (M0) markedly decreased the ADG of broilers
between Days 22 to 42 and Days 8 to 42 as compared with the
control group (C0) (P < 0.05), in correspondence with previous
studies (Kubena et al., 1998; Oguz et al., 2000). Nevertheless, del-
eterious effects on the birds’ growth were not significant during
the initial trial period between Days 8 to 21. The adverse effects
of aflatoxins on birds’ growth performance may be a result of an-
orexia, reluctance and an inhibition of protein synthesis and lipo-
genesis (Oguz and Kurtoglu, 2000). This delay of growth
inhibition in broilers suggests that the length of exposure to afla-
toxins as well as their level of concentration can influence an ani-
mal’s responsein terms of performance (Yunus et al., 2011). It also
indicates that prolonged exposure to aflatoxins at low levels can
752 Y. Fan et al. / Food and Chemical Toxicology 59 (2013) 748–753
have detrimental effects on broilers’ productivity (reflected in a
lower F:G).
In the current study, supplementing a contaminated diet with B.
subtilis ANSB060 significantly ameliorated the toxic effects of afla-
toxins on broilers’ growth performance. The basic mechanism
seems to be that spores of B. subtilis ANSB060 germinate in ani-
mals’ intestinal tract and secrete the active substance which de-
grades aflatoxins, thus alleviating the effects of aflatoxicosis.
Some studies have suggested that microbial enzymes are responsi-
ble for cleaving the lactone or difuran ring of the AFB1 molecule
in vitro, thus reducing its toxicity (Guan et al., 2010; Liu et al.,
1998). However, the specific biotransformation mechanism by
which B. subtilis ANSB060 detoxifies aflatoxins in animals’ intesti-
nal tract is unclear, and further studies are needed to explore this
question. Researchers have reported that many kinds of bacteria
can reduce the amount of aflatoxins in feed and food. It has been
demonstrated that Enterococcus faecium can remove 45% of AFB1
throughout a 48 h incubation period (Topcu et al., 2010). The strain
Myxococcus fulvus shows a great ability in degrading AFB1 (71.89%),
AFG1 (68.13%) and AFM1 (63.82%) after 48 h of incubation (Zhao
et al., 2011). The Lactobacillus casei strain is able to bind 49.2% of
available aflatoxin (4.6 lg/ml) after 4 h of incubation (Hernan-
dez-Mendoza et al., 2009). The differences among the results
may be due to the variations in bacteria species, their degrading
mechanisms, variations in the concentrations of aflatoxins, and
incubation time.
In recent years, the quality of poultry meat has become a gen-
eral concern of consumers. Many factors including breed, sex,
age, stress, the presence of mycotoxins, and other processing fac-
tors all affect meat quality (Owens et al., 2000). Meat color is an
important parameter affecting a consumer’s acceptance of the
product (Barbut, 1997), and pH is one of most important factors
determining meat color, tenderness, and water-holding capacity.
In this research, broiler chickens’ consumption of aflatoxin-con-
taminated diets resulted in a slight decreased in the a *value for
breast muscles and pH(45min) values for thigh muscles (P > 0.05),
while significantly increasing the b⁄ value for breast muscles
(P < 0.05). All changes of these characteristics are regarded as being
related to the deterioration of meat quality in broilers (Boulianne
and King, 1995; El Rammouz et al., 2004). There are partially in
agreement with previous reports indicating that a mold-contami-
nated diet results in a significant increase in the b value while
decreasing the L value (Liu et al., 2011). In the current trial, B. sub-
tilis ANSB060 significantly improve meat quality of the broilers ex-
posed to aflatoxins. In the aflatoxins-B. subtilis ANSB060
supplemented diet groups, the a value for breast muscles and
pH(45min) values for thigh muscles of the broilers were increa-
sed(P < 0.05), and the b* value for breast muscles was decreased
(P < 0.05), when compared to those of the broilers in the aflatoxins
treated group. The rate of meat discoloration is related to the effi-
ciency of oxidation processes and enzymatic reducing systems in
regulating metmyoglobin levels in meat (Faustman and Cassens,
1989). Some studies have also indicated that AFB1 can form ad-
ducts with DNA, inducing cellular oxidative damage and lipid per-
oxidation (Imlay and Linn, 1988; Shen et al., 1994), which probably
results in deterioration of meat quality. Nevertheless, there has
been little research about the alleviative efficacy of aflatoxin bio-
degradation agents on meat quality againstaflatoxins. B. subtilis
ANSB060 can effectively degrade aflatoxins in intestine tract and
decrease the content of aflatoxins, and then diminish the adverse
effect of aflatoxins on meat quality. Thus, further studies on the
improvement of the meat quality of broilers fed diets naturally
contaminated with aflatoxins are needed.
However, it is the residue of aflatoxins in animal products that
is of the most concern to people. Among the metabolites of AFB1,
the toxicity of AFM1 in animals seems to be comparable to or
slightly less than that of AFB1. In our study, low levels (0.24, 0.03
and 0.20 lg/kg) of AFB1, AFB2 and AFM1 were retained in the livers
of broilers fed the moldy diets (AFB1 = 70.75 lg/kg) for 35 days.
Denli et al. (2009) found 0.166 lg/kg AFB1 in the liver tissue of
broilers following the feeding of 1000 lg/kg AFB1 for 42 days. Res-
idues of AFB1 (0.05 and 0.13 lg/kg) and AFM1 (0.10 and 0.32 lg/kg)
were also observed in the livers of broilers given 50 and 100 lg of
AFB1/kg of feed for 42 days (Bintvihok and Kositcharoenkul, 2006).
Differences in the type of bird and its diet, the concentrations of
AFB1 and duration of exposure may be reasons behind these vari-
ations. In many countries, the maximum tolerance level for AFB1
in human food products is 2 lg/kg. Although a small amount of
aflatoxin residues in broilers used for food might constitute a very
low risk to consumers’ health, it is necessary for the quality control
of poultry products to analyze aflatoxin residues in various tissues
of birds to safeguard public health. The data on aflatoxin residues
in the present study is consistent with previous reports that levels
of AFB1 and AFM1 are higher in the liver than in muscle tissues
(Bintvihok et al., 1998). In these studies neither G1 nor G2 were de-
tected in broiler tissue samples, which is consistent with the re-
ports of data indicating no carry-over of G1 or G2 in the tissues of
pigs (Furtado et al., 1979). Results have indicated that the aflatox-
ins G1 and G2 are readily metabolized into metabolites that are
either eliminated or else deposited in the tissues as unidentified
or unextractable compounds (Furtado et al., 1979). Residual levels
decreased with the incorporation of B. subtilis ANSB060 in the diet
during the period of exposure to aflatoxins. This result corrobo-
rates the hypothesis that the protective effects of B. subtilis
ANSB060 against aflatoxins might be due to its capability of effect-
ing a specific biotransformation of aflatoxins in animals’ intestinal
tract. Since the amount of aflatoxins absorbed by the intestinal
tract is reduced, meat quality is consequently improved while
the concentrations of aflatoxin residues in tissues are decreased.
In conclusion, this study clearly indicates that the presence of
aflatoxins in diets at a level of 70 lg/kg results in depressed growth
performance and meat quality, along with the deposition of afla-
toxin residues in the livers of broilers. Adding the aflatoxin biodeg-
radation preparation B. subtilis ANSB060 to moldy diets
significantly counteracts the adverse effects of aflatoxins on birds’
growth performance, effectively improving meat quality to some
extent and reducing the accumulation of aflatoxin residues in the
liver. Therefore, as a feed additive for biodegradation of aflatoxins,
the addition of B. subtilis ANSB060 to animal diets is believed to be
a viable potential approach to the biodetoxification of aflatoxins
naturally occurring in moldy feedstuffs.
Conflict of Interest
The authors declared that there are no conflicts of interest.
Acknowledgments
This study was supported by the Special Fund from National
Key Technology Research and Development Program of the Minis-
try of Science and Technology of China (Grant No. 2013BAD10B01
and 2013BAD10B02), National Natural Science Foundation of China
(Grant No. 31072063), and the Beijing Municipal Natural Science
Foundation (Grant No. 6132021).
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