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Biotransformation of aflatoxin B1 and aflatoxin G1 in peanut meal by anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus
International Journal of Food Microbiology 211 (2015) 1–5
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Biotransformation of aflatoxin B1 and aflatoxin G1 in peanut meal by anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus Yujie Chen a, Qing Kong a,⁎, Chen Chi a, Shihua Shan b, Bin Guan a a b
School of Food Science and Engineering, Ocean University of China, Qingdao, Shandong 266003, China Shandong Peanut Research Institute, Qingdao, Shandong 266110, China
a r t i c l e
i n f o
Article history: Received 21 February 2015 Received in revised form 9 June 2015 Accepted 24 June 2015 Available online 26 June 2015 Keywords: Aflatoxin B1 Aflatoxin G1 Anaerobic solid fermentation Heat treatment MTT assay
a b s t r a c t The purpose of this study was to explore the ability of anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to biotransform aflatoxins in peanut meal. The pH of the peanut meal was adjusted above 10, and then heated for 10 min at 100 °C, 115 °C and 121 °C. The S. thermophilus and L. delbrueckii subsp. bulgaricus were precultured together in MRS broth for 48 h at 37 °C. The heated peanut meal was mixed with precultured MRS broth containing 7.0 × 108 CFU/mL of S. thermophilus and 3.0 × 103 CFU/mL of L. delbrueckii subsp. bulgaricus with the ratio of 1 to 1 (weight to volume) and incubated in anaerobic jars at 37 °C for 3 days. The aflatoxin content in the peanut meal samples was determined by HPLC. The results showed that the peanut meal contained mainly aflatoxin B1 (AFB1) (10.5 ± 0.64 μg/kg) and aflatoxin G1 (AFG1) (18.7 ± 0.55 μg/kg). When heat treatment was combined with anaerobic solid fermentation, the biotransformation rate of aflatoxins in peanut meal could attain 100%. The cytotoxicity of fermented peanut meal to L929 mouse connective tissue fibroblast cells was determined by MTT assay and no significant toxicity was observed in the fermented peanut meal. Furthermore, heat treatment and anaerobic solid fermentation did not change the amino acid concentrations and profile in peanut meal. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxins are secondary metabolites produced mainly by some strains of Aspergillus flavus and Aspergillus parasiticus. They are highly toxic, mutagenic, carcinogenic and teratogenic compounds found in a wide variety of important agricultural products such as peanuts, corn, rice and cottonseed (Binder et al., 2007; Rustom, 1997). Aflatoxincontaminated diets have been reported to be linked with high liver cancer incidence (Saalia and Phillips, 2011). AFB1, the most toxic aflatoxin, is one of the most frequently detected contaminant compounds in food products and is the most potent naturally occurring mutagen and carcinogen (Teniola et al., 2005). Peanut meal is the high protein by-product remaining after commercial extraction of peanut oil (White et al., 2013), and is a common ingredient in livestock feeds in developed and developing countries. Peanuts are one of the most widely grown oil seeds in the world with 29 million metric tons produced every year (Zhang et al., 2011). A majority of peanuts are crushed to produce oil and this process generates a large amount of peanut meal as the coproduct (Reddy et al., 2013). However, the use of peanut meal is limited because of high levels of
⁎ Corresponding author. E-mail address: [email protected] (Q. Kong).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.021 0168-1605/© 2015 Elsevier B.V. All rights reserved.
contamination by aflatoxins, so a safe and efficient method to degrade aflatoxins in peanut meal is urgently required. Several strategies have been reported to degrade and remove aflatoxins in crops, including physical, chemical and biological methods. A variety of physical methods are widely adopted for the degradation of aflatoxins, such as heat, absorption, irradiation, extraction, gamma rays and ultraviolet light (Das and Mishra, 2000; Kabak et al., 2006). A number of chemical methods have been screened for their ability to alter the chemical structure of aflatoxins, for example, ozonization, ammoniation, treatment with formaldehyde and calcium hydroxide, or exposure to chlorine gas and hydrogen peroxide (Luo et al., 2014; Saalia and Phillips, 2011). Several biological methods also have been reported for the elimination or inactivation of aflatoxins in foods and feeds. These include bacterial detoxification of aflatoxins by lactic acid bacteria (Hernandez-Mendoza et al., 2009), Bacillus subtilis UTBSP1 (Farzaneh et al., 2012), Enterococcus faecium, Mycobacterium fluoranthenivorans and Corynebacterium rubrum (Samuel et al., 2014), and the enzymatic degradation of aflatoxins by horse radish peroxidase and laccase from several fungal species (Alberts et al., 2006; Das and Mishra, 2000). However, each treatment has its own limitations, such as certain nutrients may be destroyed in the process, expensive equipment may be required or there may be safety concerns. Heat treatment, as one of important physical methods, has been used to remove aflatoxins. Rustom (1997) summarized the results of
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some studies conducted in connection with the degradation of aflatoxins in foods by different heat treatments, and showed that higher temperature or longer time are necessary to attain partial destruction of the toxins because aflatoxins are very stable and not degraded up to 270 °C in dry conditions (Samuel et al., 2014). Obviously, traditional heat techniques have many limitations to remove aflatoxins in peanut meal such as loss of nutrition and required for expensive equipment. Biodegradation of aflatoxins using microorganisms or enzymes is a common method to reduce aflatoxin concentration in foods and feeds (Alberts et al., 2006; Das and Mishra, 2000; Farzaneh et al., 2012; Hernandez-Mendoza et al., 2009; Samuel et al., 2014). Many lactic acid bacteria, due in large part to their Generally Recognized as Safe (GRAS) status and usage as probiotics, are of particular interest for binding aflatoxins (Hernandez-Mendoza et al., 2009). The studies of ElNezami et al. (1998) showed that lactic acid bacteria have a significant effect in reducing levels of aflatoxins in liquid media. But up to now, there is no report that lactic acid bacteria have been used to reduce aflatoxins in solid media. In order to remove the aflatoxins in peanut meal, we used aerobic solid fermentation first, but the tested strains such as Bacillus and Candida had little effect on the aflatoxin levels in peanut meal. It is well known that some recalcitrant compounds can be easily degraded by anaerobic metabolism. So in this study we combined heat treatment and anaerobic solid fermentation to explore a new method to remove aflatoxins in peanut meal. Two lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were used in this study. S. thermophilus is a facultative anaerobe, and is able to deplete the residual oxygen in the anaerobic jars to facilitate the anaerobic solid fermentation. In addition, the effect of the treatment on content of amino acid in peanut meal was also determined.
2. Materials and methods 2.1. Materials and cultural conditions Peanut meal was kindly donated by Luhua Corperation (Laiyang, Shandong Province, China). S. thermophilus, L. delbrueckii subsp. bulgaricus and L929 mouse connective tissue fibroblast cells were preserved in the School of Food Science and Engineering, Ocean University of China. AFB1 and AFG1 standards were purchased from Sigma Chemical (St. Louis, MO, USA) and stored at 4 °C. The S. thermophilus and L. delbrueckii subsp. bulgaricus used in this study were precultured together in Man Rogosa Sharpe broth (MRS broth) and incubated in anaerobic jars (Mitsubishi Gas Chemical Company, Tokyo, Japan) for 48 h at 37 °C. The concentration of lactic acid bacteria was determined by spread plate method, and genomic DNA was extracted from the colonies and used to identify the cultures by 16S rDNA sequence analysis. The L929 cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), at 37 °C in a humidified incubator (NuAire, Plymouth, Minnesota, USA) containing 5% CO2. All chemicals were of HPLC-grade.
2.2. Aflatoxin biotransformation assay NaOH (0.25 M; 100 mL) was firstly added to 100 g peanut meal. After homogeneous mixing, the samples were heated for 10 min at 100 °C, 115 °C and 121 °C. An unheated sample was retained as the control. Then 100 mL precultured MRS broth containing 7.0 × 108 CFU/mL of S. thermophilus and 3.0 × 103 CFU/mL of L. delbrueckii subsp. bulgaricus was added into the heated and control peanut meal and incubated in anaerobic jars (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C for 3 days. The aflatoxins remaining in the samples were analyzed quantitatively by HPLC (Agilent, Santa Clara, CA, USA). The experiment was repeated three times.
2.3. Aflatoxin determination in peanut meal The aflatoxins in peanut meal were extracted using immunoaffinity columns (AflaTest Columns, Cat. No. G1010, VICAM, Omaha, USA) and estimated by a HPLC-fluorescent detector with pre-column derivatization. A modification of the methods of Chiavaro et al. (2005) and Kong et al. (2014) was used as briefly mentioned below. Three grams of sample were homogenized and blended with 0.6 g of NaCl in 10 mL of methanol–water (60:40) for 3 min. After filtration through a paper filter, an aliquot of 5 mL of filtrate was diluted with 5 mL ultrapure water and passed through an immunoaffinity column. Aflatoxins were eluted with 1.0 ml of methanol in a glass vial and dried near to dryness under gentle stream of nitrogen. Pre-column derivatization was performed with trifluroacetic acid (TFA) and a 25 μL of the derivatized extract was injected into HPLC system equipped with a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm, Agilent, USA) and a 470 fluorescent detector (G1321A, Agilent, USA) (λexc 360 nm; λem 440 nm). The analysis was performed using a mobile phase solvent of water, methanol and acetonitrile (50:40:10) at a flow rate of 0.8 mL/min with a run time of 15 min. The aflatoxins were identified by their retention time, compared with those of aflatoxin standard solution under identical conditions.
2.4. MTT assay The MTT (3-(4, 5-dimethythiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) assay originally described by Mosmann (1983) is widely used for assessment of cytotoxicity. L929 cells, derived from an immortalized mouse fibroblast cell line, are internationally recognized cells that are routinely used in in vitro cytotoxicity assessments (Lopes et al., 2011). The aflatoxins in the untreated peanut meal (3 g, 6 g and 9 g) and the biotransformed derivatives in the fermented peanut meal (3 g, 6 g and 9 g) for the MTT assay were extracted by methanol– water (60:40). Then the extracts were dried by a vacuum dryer (Shanghai Jinghong Laboratory Instrument Co., Ltd., Shanghai, China). Ten microliters of dimethyl sulfoxide (DMSO) was added to dissolve the extracts and then the extracts were diluted by the DMEM to 1 mL. The cytotoxicity of untreated peanut meal, fermented peanut meal and control (DMEM containing 1% DMSO) to L929 cells was assessed by MTT cell proliferation and cytotoxicity assay kit (Sangon Biotech, Shanghai, China) and carried out according to the manufacturer's instruction. Briefly, 100 μL DMEM per well was added into 96-well culture plates (Corning Incorporated, Corning, New York, USA). Then 5.0 × 103 cells per well were seeded and exposed to 10 μL extracts of untreated peanut meal, fermented peanut meal and control for 24, 48 and 72 h, respectively. At the end of each test time, the cultural supernatants were removed and MTT reagent (component A) (0.5 mg/mL) was added to each well and the plates were incubated for 4 h at 37 °C. Then the MTT reagent was removed and Formazan Solubilization Solution (component B) was added to dissolve formazan crystals. The absorbance at 570 nm was read on a microplate reader (Thermo scientific, Grand Island, New York, USA). The percentage viability was calculated as (AT / AC) ∗ 100, where AT and AC were the optical densities of treated and control cells, respectively.
2.5. Amino acid analysis The content of amino acid in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal was analyzed by an amino acid analyzer (HITACHI L-8800, Hitachi Ltd., Tokyo, Japan).
Y. Chen et al. / International Journal of Food Microbiology 211 (2015) 1–5
The degradation rate of AFG1 (%)
3.2. MTT assay
unfermented peanut meal fermented peanut meal
120
Cytotoxic effects of untreated peanut meal (3 g, 6 g and 9 g) extracts and fermented peanut meal (3 g, 6 g and 9 g) extracts were analyzed in L929 cells by MTT assay. As shown in Fig. 3, the extracts from fermented peanut meal showed no significant cytotoxicity at any concentration and exposure time, whereas those from the untreated peanut meal showed noticeable cytotoxicity, especially at higher concentration and longer exposure time. When the extracts from 9 g untreated peanut meal were incubated with L929 cells for 72 h, ~63% loss of cell viability of L929 cells resulted. Under the same treatment conditions, the extracts from 9 g fermented peanut meal caused only ~6% loss of cell viability of L929 cells.
100 80 60 40 20 0
o
0C
o
o
100 C 115 C Heat Treatment
o
121 C
(A) 120
3.1. Aflatoxin biotransformation assay In this study, the aflatoxins in untreated peanut meal were confirmed as AFG1 (18.7 ± 0.55 μg/kg) and AFB1 (10.5 ± 0.64 μg/kg). The numbers of S. thermophilus and L. delbrueckii subsp. bulgaricus in the fermented peanut meal were 2.0 × 109 CFU/g and 1.0 × 108 CFU/g, respectively. The effects of heat treatment and anaerobic digestion on AFG1 and AFB1 elimination are showed in Figs. 1 and 2, respectively. After direct anaerobic fermentation, the biotransformation rates (%) of AFG1 and AFB1 were 11.71 ± 1.47 and 17.01 ± 5.04, respectively. This result showed the aflatoxin content was almost unchanged when the peanut meal was not heated but directly incubated in anaerobic jars for 3 days. When the peanut meal was heated at 100 °C, 115 °C and 121 °C, the biotransformation rates (%) of AFG1 were 37.54 ± 2.44, 50.37 ± 1.57 and 56.86 ± 1.26, respectively; and the biotransformation rates (%) of AFB1 were 43.91 ± 0.59, 63.15 ± 7.64 and 71.19 ± 1.01, respectively. With the rising temperature, the concentration of AFG1 and AFB1 in peanut meal reduced. However, when the peanut meal was heated first and then incubated with lactic acid bacteria for 3 days, no aflatoxin could be detected.
80 60 40 20 0 0
3
6
9
Peanut meal (g)
(B)
unfermented peanut meal
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0
fermented peanut meal
3
6
9
Peanut meal (g)
100
(C) 120
80 Viability (% of control)
The degradation rate of AFG1 (%)
100
Viability (% of control)
3. Results
Viability (% of control)
Fig. 1. Percentage of aflatoxin G1 biotransformed by heat treatment and anaerobic solid fermentation. Error bars show the standard deviations (SD).
60 40 20 0
3
o
0C
o
o
100 C 115 C Heat Treatment
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Fig. 2. Percentage of aflatoxin B1 biotransformed by heat treatment and anaerobic solid fermentation. Error bars show the standard deviations (SD).
100 80 60 40 20 0
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3 6 Peanut meal (g)
9
Fig. 3. Cytotoxic effects of untreated (▲) and fermented (■) peanut meal extracts on L929 cells at 24 h (A), 48 h (B) and 72 h (C). Error bars show the standard deviations (SD).
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amino acids (Lys, Phe, Met, Thr, Ile, Leu and Val), demonstrating that peanut meal contains enough nutrition to feed animals. The content of amino acids was similar in different treatments. This indicated that heat treatment and anaerobic solid fermentation did not break down amino acids in peanut meal.
Table 1 The concentration and profile of amino acids in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal. Amino acid
Asp Thr Ser Glu Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Pro Total content
Amino acid content in peanut meal (mean ± SD) Untreated
Heated
Anaerobic fermented
18.99 ± 0.12 5.43 ± 0.02 12.06 ± 0.03 16.36 ± 0.07 23.54 ± 0.12 1.95 ± 0.00 1.13 ± 0.00 8.33 ± 0.02 3.58 ± 0.01 5.33 ± 0.04 13.44 ± 0.08 8.73 ± 0.01 13.98 ± 0.05 4.95 ± 0.03 6.87 ± 0.03 21.15 ± 0.11 4.94 ± 0.01 170.76 ± 0.60
19.52 ± 0.01 5.81 ± 0.00 12.43 ± 0.01 17.29 ± 0.01 24.11 ± 0.03 2.12 ± 0.01 1.11 ± 0.01 8.74 ± 0.00 3.98 ± 0.02 5.54 ± 0.01 13.82 ± 0.01 8.88 ± 0.08 13.12 ± 0.09 4.52 ± 0.02 7.06 ± 0.00 20.64 ± 0.05 5.15 ± 0.07 173.84 ± 0.31
18.78 ± 0.02 5.7 ± 0.01 12.05 ± 0.01 16.15 ± 0.04 25.28 ± 0.10 2.34 ± 0.01 0.69 ± 0.01 8.53 ± 0.01 3.85 ± 0.03 5.45 ± 0.00 13.43 ± 0.03 8.65 ± 0.07 13 ± 0.09 4.67 ± 0.05 7.06 ± 0.04 20.35 ± 0.07 6.36 ± 0.01 172.32 ± 0.51
4. Discussion This study found that heat treatment combined with anaerobic digestion by S. thermophilus and L. delbrueckii subsp. bulgaricus could biotransform aflatoxins in peanut meal. Aflatoxins have high temperatures for destruction ranging from 237 °C to 306 °C. Moisture content and pH have a significant impact on the degradation of aflatoxins by heat treatment. It has been reported that the lactone ring plays an important role in toxicity and carcinogenicity of aflatoxins (NicolásVázquez et al., 2010). When the lactone ring was opened, the carcinogenic properties of aflatoxins were removed (Bren et al., 2007). The presence of water facilitates the opening of the lactone ring in aflatoxins to form a terminal carboxylic acid. This terminal acid group can be easily degraded (Coomes et al., 1966). Rustom et al. (1993) suggested that heat treatments at pH 10.2, 130 °C, 20 s and at pH 10.2, 121 °C, 15 min removed the AFB1 by 78% and 88%, respectively. The degradation of AFB1 was attributed to the opening of the lactone ring, which then yields aflatoxin D1, a nonfluorescent compound. It is reported that AFD1 is less toxic and mutagenic than AFB1 (Lee et al., 1981; Méndez-Albores et al., 2005; Samuel et al., 2014). In this study, the
3.3. Amino acid analysis The content of amino acids in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal is shown in Table 1. Peanut meal contained 17 amino acids including seven essential
O O
O
O O
O
O O O O O
(A)
O O
H3C
no heat treatment
O
H3C
O
O
O
anaerobic digestion
O
O O O
O O
CH3
O CH3
NaOH heat treatment
O
O
O
O
HO
O
TFA derivatization
O O
O
aflatoxin D1
+ CH3
+
O O
O
HO
HO
(B)
O
O
pH<3
O
aflatoxin B1
CH3
O O
O
CH3
aflatoxin B2a
O HO
CO2 +
anaerobic digestion
O O
TFA derivatization compound not aflatoxin B2a compound not aflatoxin B1 pH<3
Fig. 4. Hypothesized schematic presentation of aflatoxin B1 biotransformation process by anaerobic solid fermentation. (A) Peanut meal proteins firmly combine with AFB1 and most AFB1 couldn't be broken down by direct anaerobic solid fermentation. (B) Alkaline heat treatment could break the linkage of peanut meal proteins and AFB1. And then partial of AFB1 could be converted into AFD1; most of AFB1 could be biotransformed by anaerobic digestion. When the AFB1 is derived by trifluroacetic acid (TFA), the AFB1 is transformed into AFB2a which could be easily detected by HPLC with a fluorescent detector.
Y. Chen et al. / International Journal of Food Microbiology 211 (2015) 1–5
results from HPLC confirmed that some aflatoxins were degraded by heat treatment (Figs. 1 and 2). A number of studies have screened lactic acid bacteria for the ability to bind aflatoxins and have reported a wide range of genus, species and strain specific binding capacities (El-Nezami et al., 1998; Haskard et al., 2001; Hernandez-Mendoza et al., 2009; Lee et al., 2003; Peltonen et al., 2000, 2001; Shahin, 2007; Zinedine et al., 2005). It has been suggested that lactic acid bacteria can significantly bind AFB1 in aqueous solution and the bacterial ability to remove AFB1 is dependent on bacterial cell wall structure (El-Nezami et al., 1998; Lahtinen et al., 2004). However, researchers have demonstrated that AFB1 can also bind firmly to the peanut meal proteins (Monteiro et al., 1966). In this study, we observed that the direct incubation of peanut meal with S. thermophilus and L. delbrueckii subsp. bulgaricus did not significantly contribute to the disappearance of AFG1 and AFB1 (Figs. 1 and 2). In contrast, when the peanut meal was heated in advance, the AFG1 and AFB1 were not detected after anaerobic solid fermentation. It is interesting that neither heat treatment nor anaerobic solid digestion alone could completely biotransform aflatoxins in peanut meal, but the combination of heat treatment and anaerobic solid fermentation appears to thoroughly detoxify aflatoxins in peanut meal. It was possible that AFB1 and AFG1 were converted into an unknown compound by the heat treatment and anaerobic digestion. Megalla and Hafez (1982) concluded that the pH may contribute to the transformation of AFB1 to the non-toxic AFB2a in acidogenous yogurt. In the HPLC assay, pre-column derivatization was performed with trifluroacetic acid. In this case, the sample pH was low enough to hydrate the AFB1 and AFG1 to AFB2a and AFG2a. The results of the HPLC analysis showed that the derivant of the fermented peanut meal extracts was not AFB2a and AFG2a, indicating that AFB1 and AFG1 were biotransformed into another compound. We hypothesized that the AFB1 and AFG1 bound by peanut proteins were released by heat treatment and then biotransformed by S. thermophilus and L. delbrueckii subsp. bulgaricus in anaerobic solid fermentation (Fig. 4). Alkaline treatment also aids aflatoxin biotransformation. The MTT assay suggested the biotransformed aflatoxins almost have no cytotoxicity (Fig. 3), indicating that our methods to biotransform aflatoxins in peanut meal are feasible. However, the specific biotransformation mechanism by which S. thermophilus and L. delbrueckii subsp. bulgaricus detoxifies aflatoxins in peanut meal is unclear, and further studies are needed to solve this question. We hypothesize that other lactic acid bacteria also have the ability to biotransform aflatoxins in peanut meal by anaerobic solid fermentation. 5. Conclusions This work reports that the method of heat treatment combined with anaerobic solid fermentation of S. thermophilus and L. delbrueckii subsp. bulgaricus could significantly biotransform aflatoxins in peanut meal. It showed that biotransformation of aflatoxins by this method in peanut meal could reach 100%. In addition, the heating temperature was controlled at a relative low level (100 °C) and the heating time was also controlled in a relative short time (10 min). The fermented peanut meal extracts showed no significant cytotoxicity and the concentration and profile of amino acids in untreated peanut meal, heated peanut meal (100 °C), and anaerobic fermented peanut meal were similar. Acknowledgments This research was supported by the National Natural Science Foundation of China (31471657). References Alberts, J.F., Engelbrecht, Y., Steyn, P.S., Holzapfel, W.H., van Zyl, W.H., 2006. Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures. Int. J. Food Microbiol. 109, 121–126.
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Role of lactone ring in structural, electronic, and reactivity properties of aflatoxin B1: a theoretical study. Arch. Environ. Contam. Toxicol. 59, 393–406. Peltonen, K.D., El-Nezami, H.S., Salminen, S.J., Ahokas, J.T., 2000. Binding of aflatoxin B1 by probiotic bacteria. J. Sci. Food Agric. 80, 1942–1945. Peltonen, K., El-Nezami, H., Haskard, C., Ahokas, J., Salminen, S., 2001. Aflatoxin B1 binding by dairy strains of lactic acid bacteria and bifidobacteria. J. Dairy Sci. 84, 2152–2156. Reddy, N., Chen, L.H., Yang, Y.Q., 2013. Thermoplastic films from peanut proteins extracted from peanut meal. Ind. Crop. Prod. 43, 159–164. Rustom, I.Y.S., 1997. Aflatoxin in food and feed: occurrence, legislation and inactivation by physical methods. Food Chem. 59, 57–67. Rustom, I.Y.S., López-Leiva, M.H., Nair, B.M., 1993. Effect of pH and heat treatment on the mutagenic activity of peanut beverage contaminated with aflatoxin Bl. Food Chem. 46, 37–42. Saalia, F.K., Phillips, R.D., 2011. Reduction of aflatoxins in peanut meal by extrusion cooking in the presence of nucleophiles. LWT Food Sci. Technol. 6, 1511–1526. Samuel, M.S., Sivaramakrishna, A., Mehta, A., 2014. Degradation and detoxification of aflatoxin B1 by Pseudomonas putida. Int. Biodeterior. Biodegrad. 86, 202–209. Shahin, A.A.M., 2007. Removal of aflatoxin B1 from contaminated liquid media by dairy lactic acid bacteria. Int. J. Agric. Biol. 9, 71–75. Teniola, O.D., Addo, P.A., Brost, I.M., Färber, P., Jany, K.D., Alberts, J.F., van Zyl, W.H., Steyn, P.S., Holzapfel, W.H., 2005. Degradation of aflatoxin B1 by cell-free extracts of Rhodococcus erythropolis and Mycobacterium fluoranthenivorans sp. nov. DSM44556T. Int. J. Food Microbiol. 105, 111–117. White, B.L., Oakes, A.J., Shi, X.L., Price, K.M., Lamb, M.C., Sobolev, V.S., Sanders, T.H., Davis, J.P., 2013. Development of a pilot-scale to sequester aflatoxin and release bioactive peptides from highly contaminated peanut meal. LWT Food Sci. Technol. 51, 492–499. Zhang, S.B., Lu, Q.Y., Yang, H.S., Li, Y., Wang, S., 2011. Aqueous enzymatic extraction of oil and protein hydrolysates from roasted peanut seeds. J. Am. Oil Chem. Soc. 88, 727–732. Zinedine, A., Faid, M., Benlemlih, M., 2005. In vitro reduction of aflatoxin B1 by strains of lactic acid bacteria isolated from Moroccan sourdough bread. Int. J. Agric. Biol. 7, 67–70.
Contents lists available at ScienceDirect
International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro
Biotransformation of aflatoxin B1 and aflatoxin G1 in peanut meal by anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus Yujie Chen a, Qing Kong a,⁎, Chen Chi a, Shihua Shan b, Bin Guan a a b
School of Food Science and Engineering, Ocean University of China, Qingdao, Shandong 266003, China Shandong Peanut Research Institute, Qingdao, Shandong 266110, China
a r t i c l e
i n f o
Article history: Received 21 February 2015 Received in revised form 9 June 2015 Accepted 24 June 2015 Available online 26 June 2015 Keywords: Aflatoxin B1 Aflatoxin G1 Anaerobic solid fermentation Heat treatment MTT assay
a b s t r a c t The purpose of this study was to explore the ability of anaerobic solid fermentation of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to biotransform aflatoxins in peanut meal. The pH of the peanut meal was adjusted above 10, and then heated for 10 min at 100 °C, 115 °C and 121 °C. The S. thermophilus and L. delbrueckii subsp. bulgaricus were precultured together in MRS broth for 48 h at 37 °C. The heated peanut meal was mixed with precultured MRS broth containing 7.0 × 108 CFU/mL of S. thermophilus and 3.0 × 103 CFU/mL of L. delbrueckii subsp. bulgaricus with the ratio of 1 to 1 (weight to volume) and incubated in anaerobic jars at 37 °C for 3 days. The aflatoxin content in the peanut meal samples was determined by HPLC. The results showed that the peanut meal contained mainly aflatoxin B1 (AFB1) (10.5 ± 0.64 μg/kg) and aflatoxin G1 (AFG1) (18.7 ± 0.55 μg/kg). When heat treatment was combined with anaerobic solid fermentation, the biotransformation rate of aflatoxins in peanut meal could attain 100%. The cytotoxicity of fermented peanut meal to L929 mouse connective tissue fibroblast cells was determined by MTT assay and no significant toxicity was observed in the fermented peanut meal. Furthermore, heat treatment and anaerobic solid fermentation did not change the amino acid concentrations and profile in peanut meal. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Aflatoxins are secondary metabolites produced mainly by some strains of Aspergillus flavus and Aspergillus parasiticus. They are highly toxic, mutagenic, carcinogenic and teratogenic compounds found in a wide variety of important agricultural products such as peanuts, corn, rice and cottonseed (Binder et al., 2007; Rustom, 1997). Aflatoxincontaminated diets have been reported to be linked with high liver cancer incidence (Saalia and Phillips, 2011). AFB1, the most toxic aflatoxin, is one of the most frequently detected contaminant compounds in food products and is the most potent naturally occurring mutagen and carcinogen (Teniola et al., 2005). Peanut meal is the high protein by-product remaining after commercial extraction of peanut oil (White et al., 2013), and is a common ingredient in livestock feeds in developed and developing countries. Peanuts are one of the most widely grown oil seeds in the world with 29 million metric tons produced every year (Zhang et al., 2011). A majority of peanuts are crushed to produce oil and this process generates a large amount of peanut meal as the coproduct (Reddy et al., 2013). However, the use of peanut meal is limited because of high levels of
⁎ Corresponding author. E-mail address: [email protected] (Q. Kong).
http://dx.doi.org/10.1016/j.ijfoodmicro.2015.06.021 0168-1605/© 2015 Elsevier B.V. All rights reserved.
contamination by aflatoxins, so a safe and efficient method to degrade aflatoxins in peanut meal is urgently required. Several strategies have been reported to degrade and remove aflatoxins in crops, including physical, chemical and biological methods. A variety of physical methods are widely adopted for the degradation of aflatoxins, such as heat, absorption, irradiation, extraction, gamma rays and ultraviolet light (Das and Mishra, 2000; Kabak et al., 2006). A number of chemical methods have been screened for their ability to alter the chemical structure of aflatoxins, for example, ozonization, ammoniation, treatment with formaldehyde and calcium hydroxide, or exposure to chlorine gas and hydrogen peroxide (Luo et al., 2014; Saalia and Phillips, 2011). Several biological methods also have been reported for the elimination or inactivation of aflatoxins in foods and feeds. These include bacterial detoxification of aflatoxins by lactic acid bacteria (Hernandez-Mendoza et al., 2009), Bacillus subtilis UTBSP1 (Farzaneh et al., 2012), Enterococcus faecium, Mycobacterium fluoranthenivorans and Corynebacterium rubrum (Samuel et al., 2014), and the enzymatic degradation of aflatoxins by horse radish peroxidase and laccase from several fungal species (Alberts et al., 2006; Das and Mishra, 2000). However, each treatment has its own limitations, such as certain nutrients may be destroyed in the process, expensive equipment may be required or there may be safety concerns. Heat treatment, as one of important physical methods, has been used to remove aflatoxins. Rustom (1997) summarized the results of
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some studies conducted in connection with the degradation of aflatoxins in foods by different heat treatments, and showed that higher temperature or longer time are necessary to attain partial destruction of the toxins because aflatoxins are very stable and not degraded up to 270 °C in dry conditions (Samuel et al., 2014). Obviously, traditional heat techniques have many limitations to remove aflatoxins in peanut meal such as loss of nutrition and required for expensive equipment. Biodegradation of aflatoxins using microorganisms or enzymes is a common method to reduce aflatoxin concentration in foods and feeds (Alberts et al., 2006; Das and Mishra, 2000; Farzaneh et al., 2012; Hernandez-Mendoza et al., 2009; Samuel et al., 2014). Many lactic acid bacteria, due in large part to their Generally Recognized as Safe (GRAS) status and usage as probiotics, are of particular interest for binding aflatoxins (Hernandez-Mendoza et al., 2009). The studies of ElNezami et al. (1998) showed that lactic acid bacteria have a significant effect in reducing levels of aflatoxins in liquid media. But up to now, there is no report that lactic acid bacteria have been used to reduce aflatoxins in solid media. In order to remove the aflatoxins in peanut meal, we used aerobic solid fermentation first, but the tested strains such as Bacillus and Candida had little effect on the aflatoxin levels in peanut meal. It is well known that some recalcitrant compounds can be easily degraded by anaerobic metabolism. So in this study we combined heat treatment and anaerobic solid fermentation to explore a new method to remove aflatoxins in peanut meal. Two lactic acid bacteria Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus were used in this study. S. thermophilus is a facultative anaerobe, and is able to deplete the residual oxygen in the anaerobic jars to facilitate the anaerobic solid fermentation. In addition, the effect of the treatment on content of amino acid in peanut meal was also determined.
2. Materials and methods 2.1. Materials and cultural conditions Peanut meal was kindly donated by Luhua Corperation (Laiyang, Shandong Province, China). S. thermophilus, L. delbrueckii subsp. bulgaricus and L929 mouse connective tissue fibroblast cells were preserved in the School of Food Science and Engineering, Ocean University of China. AFB1 and AFG1 standards were purchased from Sigma Chemical (St. Louis, MO, USA) and stored at 4 °C. The S. thermophilus and L. delbrueckii subsp. bulgaricus used in this study were precultured together in Man Rogosa Sharpe broth (MRS broth) and incubated in anaerobic jars (Mitsubishi Gas Chemical Company, Tokyo, Japan) for 48 h at 37 °C. The concentration of lactic acid bacteria was determined by spread plate method, and genomic DNA was extracted from the colonies and used to identify the cultures by 16S rDNA sequence analysis. The L929 cells were cultured in Dulbecco's modified eagle medium (DMEM), supplemented with 10% fetal bovine serum (FBS), at 37 °C in a humidified incubator (NuAire, Plymouth, Minnesota, USA) containing 5% CO2. All chemicals were of HPLC-grade.
2.2. Aflatoxin biotransformation assay NaOH (0.25 M; 100 mL) was firstly added to 100 g peanut meal. After homogeneous mixing, the samples were heated for 10 min at 100 °C, 115 °C and 121 °C. An unheated sample was retained as the control. Then 100 mL precultured MRS broth containing 7.0 × 108 CFU/mL of S. thermophilus and 3.0 × 103 CFU/mL of L. delbrueckii subsp. bulgaricus was added into the heated and control peanut meal and incubated in anaerobic jars (Mitsubishi Gas Chemical Company, Tokyo, Japan) at 37 °C for 3 days. The aflatoxins remaining in the samples were analyzed quantitatively by HPLC (Agilent, Santa Clara, CA, USA). The experiment was repeated three times.
2.3. Aflatoxin determination in peanut meal The aflatoxins in peanut meal were extracted using immunoaffinity columns (AflaTest Columns, Cat. No. G1010, VICAM, Omaha, USA) and estimated by a HPLC-fluorescent detector with pre-column derivatization. A modification of the methods of Chiavaro et al. (2005) and Kong et al. (2014) was used as briefly mentioned below. Three grams of sample were homogenized and blended with 0.6 g of NaCl in 10 mL of methanol–water (60:40) for 3 min. After filtration through a paper filter, an aliquot of 5 mL of filtrate was diluted with 5 mL ultrapure water and passed through an immunoaffinity column. Aflatoxins were eluted with 1.0 ml of methanol in a glass vial and dried near to dryness under gentle stream of nitrogen. Pre-column derivatization was performed with trifluroacetic acid (TFA) and a 25 μL of the derivatized extract was injected into HPLC system equipped with a ZORBAX SB-C18 column (4.6 × 250 mm, 5 μm, Agilent, USA) and a 470 fluorescent detector (G1321A, Agilent, USA) (λexc 360 nm; λem 440 nm). The analysis was performed using a mobile phase solvent of water, methanol and acetonitrile (50:40:10) at a flow rate of 0.8 mL/min with a run time of 15 min. The aflatoxins were identified by their retention time, compared with those of aflatoxin standard solution under identical conditions.
2.4. MTT assay The MTT (3-(4, 5-dimethythiazol-2-yl) 2, 5-diphenyl tetrazolium bromide) assay originally described by Mosmann (1983) is widely used for assessment of cytotoxicity. L929 cells, derived from an immortalized mouse fibroblast cell line, are internationally recognized cells that are routinely used in in vitro cytotoxicity assessments (Lopes et al., 2011). The aflatoxins in the untreated peanut meal (3 g, 6 g and 9 g) and the biotransformed derivatives in the fermented peanut meal (3 g, 6 g and 9 g) for the MTT assay were extracted by methanol– water (60:40). Then the extracts were dried by a vacuum dryer (Shanghai Jinghong Laboratory Instrument Co., Ltd., Shanghai, China). Ten microliters of dimethyl sulfoxide (DMSO) was added to dissolve the extracts and then the extracts were diluted by the DMEM to 1 mL. The cytotoxicity of untreated peanut meal, fermented peanut meal and control (DMEM containing 1% DMSO) to L929 cells was assessed by MTT cell proliferation and cytotoxicity assay kit (Sangon Biotech, Shanghai, China) and carried out according to the manufacturer's instruction. Briefly, 100 μL DMEM per well was added into 96-well culture plates (Corning Incorporated, Corning, New York, USA). Then 5.0 × 103 cells per well were seeded and exposed to 10 μL extracts of untreated peanut meal, fermented peanut meal and control for 24, 48 and 72 h, respectively. At the end of each test time, the cultural supernatants were removed and MTT reagent (component A) (0.5 mg/mL) was added to each well and the plates were incubated for 4 h at 37 °C. Then the MTT reagent was removed and Formazan Solubilization Solution (component B) was added to dissolve formazan crystals. The absorbance at 570 nm was read on a microplate reader (Thermo scientific, Grand Island, New York, USA). The percentage viability was calculated as (AT / AC) ∗ 100, where AT and AC were the optical densities of treated and control cells, respectively.
2.5. Amino acid analysis The content of amino acid in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal was analyzed by an amino acid analyzer (HITACHI L-8800, Hitachi Ltd., Tokyo, Japan).
Y. Chen et al. / International Journal of Food Microbiology 211 (2015) 1–5
The degradation rate of AFG1 (%)
3.2. MTT assay
unfermented peanut meal fermented peanut meal
120
Cytotoxic effects of untreated peanut meal (3 g, 6 g and 9 g) extracts and fermented peanut meal (3 g, 6 g and 9 g) extracts were analyzed in L929 cells by MTT assay. As shown in Fig. 3, the extracts from fermented peanut meal showed no significant cytotoxicity at any concentration and exposure time, whereas those from the untreated peanut meal showed noticeable cytotoxicity, especially at higher concentration and longer exposure time. When the extracts from 9 g untreated peanut meal were incubated with L929 cells for 72 h, ~63% loss of cell viability of L929 cells resulted. Under the same treatment conditions, the extracts from 9 g fermented peanut meal caused only ~6% loss of cell viability of L929 cells.
100 80 60 40 20 0
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3.1. Aflatoxin biotransformation assay In this study, the aflatoxins in untreated peanut meal were confirmed as AFG1 (18.7 ± 0.55 μg/kg) and AFB1 (10.5 ± 0.64 μg/kg). The numbers of S. thermophilus and L. delbrueckii subsp. bulgaricus in the fermented peanut meal were 2.0 × 109 CFU/g and 1.0 × 108 CFU/g, respectively. The effects of heat treatment and anaerobic digestion on AFG1 and AFB1 elimination are showed in Figs. 1 and 2, respectively. After direct anaerobic fermentation, the biotransformation rates (%) of AFG1 and AFB1 were 11.71 ± 1.47 and 17.01 ± 5.04, respectively. This result showed the aflatoxin content was almost unchanged when the peanut meal was not heated but directly incubated in anaerobic jars for 3 days. When the peanut meal was heated at 100 °C, 115 °C and 121 °C, the biotransformation rates (%) of AFG1 were 37.54 ± 2.44, 50.37 ± 1.57 and 56.86 ± 1.26, respectively; and the biotransformation rates (%) of AFB1 were 43.91 ± 0.59, 63.15 ± 7.64 and 71.19 ± 1.01, respectively. With the rising temperature, the concentration of AFG1 and AFB1 in peanut meal reduced. However, when the peanut meal was heated first and then incubated with lactic acid bacteria for 3 days, no aflatoxin could be detected.
80 60 40 20 0 0
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The degradation rate of AFG1 (%)
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3. Results
Viability (% of control)
Fig. 1. Percentage of aflatoxin G1 biotransformed by heat treatment and anaerobic solid fermentation. Error bars show the standard deviations (SD).
60 40 20 0
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Fig. 2. Percentage of aflatoxin B1 biotransformed by heat treatment and anaerobic solid fermentation. Error bars show the standard deviations (SD).
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Fig. 3. Cytotoxic effects of untreated (▲) and fermented (■) peanut meal extracts on L929 cells at 24 h (A), 48 h (B) and 72 h (C). Error bars show the standard deviations (SD).
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amino acids (Lys, Phe, Met, Thr, Ile, Leu and Val), demonstrating that peanut meal contains enough nutrition to feed animals. The content of amino acids was similar in different treatments. This indicated that heat treatment and anaerobic solid fermentation did not break down amino acids in peanut meal.
Table 1 The concentration and profile of amino acids in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal. Amino acid
Asp Thr Ser Glu Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Pro Total content
Amino acid content in peanut meal (mean ± SD) Untreated
Heated
Anaerobic fermented
18.99 ± 0.12 5.43 ± 0.02 12.06 ± 0.03 16.36 ± 0.07 23.54 ± 0.12 1.95 ± 0.00 1.13 ± 0.00 8.33 ± 0.02 3.58 ± 0.01 5.33 ± 0.04 13.44 ± 0.08 8.73 ± 0.01 13.98 ± 0.05 4.95 ± 0.03 6.87 ± 0.03 21.15 ± 0.11 4.94 ± 0.01 170.76 ± 0.60
19.52 ± 0.01 5.81 ± 0.00 12.43 ± 0.01 17.29 ± 0.01 24.11 ± 0.03 2.12 ± 0.01 1.11 ± 0.01 8.74 ± 0.00 3.98 ± 0.02 5.54 ± 0.01 13.82 ± 0.01 8.88 ± 0.08 13.12 ± 0.09 4.52 ± 0.02 7.06 ± 0.00 20.64 ± 0.05 5.15 ± 0.07 173.84 ± 0.31
18.78 ± 0.02 5.7 ± 0.01 12.05 ± 0.01 16.15 ± 0.04 25.28 ± 0.10 2.34 ± 0.01 0.69 ± 0.01 8.53 ± 0.01 3.85 ± 0.03 5.45 ± 0.00 13.43 ± 0.03 8.65 ± 0.07 13 ± 0.09 4.67 ± 0.05 7.06 ± 0.04 20.35 ± 0.07 6.36 ± 0.01 172.32 ± 0.51
4. Discussion This study found that heat treatment combined with anaerobic digestion by S. thermophilus and L. delbrueckii subsp. bulgaricus could biotransform aflatoxins in peanut meal. Aflatoxins have high temperatures for destruction ranging from 237 °C to 306 °C. Moisture content and pH have a significant impact on the degradation of aflatoxins by heat treatment. It has been reported that the lactone ring plays an important role in toxicity and carcinogenicity of aflatoxins (NicolásVázquez et al., 2010). When the lactone ring was opened, the carcinogenic properties of aflatoxins were removed (Bren et al., 2007). The presence of water facilitates the opening of the lactone ring in aflatoxins to form a terminal carboxylic acid. This terminal acid group can be easily degraded (Coomes et al., 1966). Rustom et al. (1993) suggested that heat treatments at pH 10.2, 130 °C, 20 s and at pH 10.2, 121 °C, 15 min removed the AFB1 by 78% and 88%, respectively. The degradation of AFB1 was attributed to the opening of the lactone ring, which then yields aflatoxin D1, a nonfluorescent compound. It is reported that AFD1 is less toxic and mutagenic than AFB1 (Lee et al., 1981; Méndez-Albores et al., 2005; Samuel et al., 2014). In this study, the
3.3. Amino acid analysis The content of amino acids in untreated peanut meal, heated peanut meal (100 °C) and anaerobic fermented peanut meal is shown in Table 1. Peanut meal contained 17 amino acids including seven essential
O O
O
O O
O
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O O
H3C
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O O
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aflatoxin B2a
O HO
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anaerobic digestion
O O
TFA derivatization compound not aflatoxin B2a compound not aflatoxin B1 pH<3
Fig. 4. Hypothesized schematic presentation of aflatoxin B1 biotransformation process by anaerobic solid fermentation. (A) Peanut meal proteins firmly combine with AFB1 and most AFB1 couldn't be broken down by direct anaerobic solid fermentation. (B) Alkaline heat treatment could break the linkage of peanut meal proteins and AFB1. And then partial of AFB1 could be converted into AFD1; most of AFB1 could be biotransformed by anaerobic digestion. When the AFB1 is derived by trifluroacetic acid (TFA), the AFB1 is transformed into AFB2a which could be easily detected by HPLC with a fluorescent detector.
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results from HPLC confirmed that some aflatoxins were degraded by heat treatment (Figs. 1 and 2). A number of studies have screened lactic acid bacteria for the ability to bind aflatoxins and have reported a wide range of genus, species and strain specific binding capacities (El-Nezami et al., 1998; Haskard et al., 2001; Hernandez-Mendoza et al., 2009; Lee et al., 2003; Peltonen et al., 2000, 2001; Shahin, 2007; Zinedine et al., 2005). It has been suggested that lactic acid bacteria can significantly bind AFB1 in aqueous solution and the bacterial ability to remove AFB1 is dependent on bacterial cell wall structure (El-Nezami et al., 1998; Lahtinen et al., 2004). However, researchers have demonstrated that AFB1 can also bind firmly to the peanut meal proteins (Monteiro et al., 1966). In this study, we observed that the direct incubation of peanut meal with S. thermophilus and L. delbrueckii subsp. bulgaricus did not significantly contribute to the disappearance of AFG1 and AFB1 (Figs. 1 and 2). In contrast, when the peanut meal was heated in advance, the AFG1 and AFB1 were not detected after anaerobic solid fermentation. It is interesting that neither heat treatment nor anaerobic solid digestion alone could completely biotransform aflatoxins in peanut meal, but the combination of heat treatment and anaerobic solid fermentation appears to thoroughly detoxify aflatoxins in peanut meal. It was possible that AFB1 and AFG1 were converted into an unknown compound by the heat treatment and anaerobic digestion. Megalla and Hafez (1982) concluded that the pH may contribute to the transformation of AFB1 to the non-toxic AFB2a in acidogenous yogurt. In the HPLC assay, pre-column derivatization was performed with trifluroacetic acid. In this case, the sample pH was low enough to hydrate the AFB1 and AFG1 to AFB2a and AFG2a. The results of the HPLC analysis showed that the derivant of the fermented peanut meal extracts was not AFB2a and AFG2a, indicating that AFB1 and AFG1 were biotransformed into another compound. We hypothesized that the AFB1 and AFG1 bound by peanut proteins were released by heat treatment and then biotransformed by S. thermophilus and L. delbrueckii subsp. bulgaricus in anaerobic solid fermentation (Fig. 4). Alkaline treatment also aids aflatoxin biotransformation. The MTT assay suggested the biotransformed aflatoxins almost have no cytotoxicity (Fig. 3), indicating that our methods to biotransform aflatoxins in peanut meal are feasible. However, the specific biotransformation mechanism by which S. thermophilus and L. delbrueckii subsp. bulgaricus detoxifies aflatoxins in peanut meal is unclear, and further studies are needed to solve this question. We hypothesize that other lactic acid bacteria also have the ability to biotransform aflatoxins in peanut meal by anaerobic solid fermentation. 5. Conclusions This work reports that the method of heat treatment combined with anaerobic solid fermentation of S. thermophilus and L. delbrueckii subsp. bulgaricus could significantly biotransform aflatoxins in peanut meal. It showed that biotransformation of aflatoxins by this method in peanut meal could reach 100%. In addition, the heating temperature was controlled at a relative low level (100 °C) and the heating time was also controlled in a relative short time (10 min). The fermented peanut meal extracts showed no significant cytotoxicity and the concentration and profile of amino acids in untreated peanut meal, heated peanut meal (100 °C), and anaerobic fermented peanut meal were similar. Acknowledgments This research was supported by the National Natural Science Foundation of China (31471657). References Alberts, J.F., Engelbrecht, Y., Steyn, P.S., Holzapfel, W.H., van Zyl, W.H., 2006. Biological degradation of aflatoxin B1 by Rhodococcus erythropolis cultures. Int. J. Food Microbiol. 109, 121–126.
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