Food Bioprocess Technol (2013) 6:2230–2234 DOI 10.1007/s11947-011-0770-9
COMMUNICATION
Efficiency of Lactic Acid Bacteria Strains for Decontamination of Aflatoxin M1 in Phosphate Buffer Saline Solution and in Skimmed Milk Fernanda Bovo & Carlos H. Corassin & Roice E. Rosim & Carlos A. F. de Oliveira
Received: 27 October 2011 / Accepted: 26 December 2011 / Published online: 11 January 2012 # Springer Science+Business Media, LLC 2012
Abstract The aim of this study was to evaluate the ability of seven lactic acid bacteria (LAB) strains to remove aflatoxin M1 (AFM1) in phosphate buffer saline (PBS) and in skimmed milk samples. The mean AFM1 removal by LAB in PBS ranged from 5.60±0.45% to 45.67±1.65% (n03). Heat-killed cells showed AFM1 removal percentages significantly higher than viable cells in contact times of 15 min or 24 h, although there were no significant differences between those times. AFM1/ LAB complex resulted from tests with PBS was unstable, as 40.57±4.66% to 87.37±1.82% of AFM1 retained by bacteria were recovered in solution after washing with PBS. Heat-killed cells of Lactobacillus rhamnosus, Lactobacillus delbrueckii spp. bulgaricus, and Bifidobacterium lactis had the highest percentage (>33%) of AFM1 removal in PBS tests. In ultrahigh temperature (UHT) skimmed milk spiked with AFM1, the three selected LAB strains showed no significant differences in removing AFM1 at 37 °C, and only B. lactis had greater ability to remove AFM1 at 4 °C. Results demonstrated that AFM1 removal by LAB has a potential application to reduce toxin concentrations until safe levels in milk. Keywords Aflatoxin M1 . Binding . Lactic acid bacteria . Decontamination . UHT milk
Introduction Aflatoxin M1 (AFM1) is a hepatocarcinogen found in milk of animals that have consumed feeds contaminated with F. Bovo : C. H. Corassin : R. E. Rosim : C. A. F. de Oliveira (*) Departamento de Engenharia de Alimentos, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Av. Duque de Caxias—Norte, 225, CEP 13635-900 Pirassununga, Sao Paulo, Brazil e-mail:
[email protected]
aflatoxin B1 (AFB1), a secondary metabolite produced by Aspergillus species, particularly Aspergillus flavus and Aspergillus parasiticus (An 2005). Although AFM1 is about ten times less toxigenic than AFB1, its cytotoxic, genotoxic, and carcinogenic effects have been demonstrated in several species (Fallah 2010). The International Agency for Research on Cancer (IARC 2002) has classified AFM1 as belonging to group 1, carcinogenic for humans. Considering that high incidence of AFM1 in milk is a serious public health problem, several countries have legislation for aflatoxins (Van Egmond and Jonker 2004). Brazilian regulations establishes the maximum allowable limit of AFM1 in fluid milk is 0.5 μg/L (Agência Nacional de Vigilância Sanitária 2011), although the European Union considers 0.05 μg/kg as maximum level for AFM1 in raw milk, heattreated milk, and milk for the manufacture of dairy products (European Commission 2006). The best way to prevent aflatoxin contamination in the food chain is the adoption of improved agricultural practices and control of storage conditions. However, practical difficulties to prevent contamination have led to the development of decontamination methods that are safe, effective, environmentally friendly, and have a cost–benefit (Elgerbi et al. 2006). Lactic acid bacteria (LAB) including some probiotic species (Dalié et al. 2010) have been widely studied and some strains have shown great ability for binding of aflatoxin in contaminated medium (El-Nezami et al. 1998; Pierides et al. 2000; Haskard et al. 2001; Kabak and Var 2008). The objectives of this study were to determine the ability of seven LAB strains (six of them not analyzed previously) in removing the AFM1 in contaminated phosphate buffer saline (PBS), to evaluate the stability of LAB/AFM1 complex produced and the ability of AFM1 removal by the LAB strains in ultrahigh-temperature skimmed milk artificially contaminated with AFM1.
Food Bioprocess Technol (2013) 6:2230–2234
Materials and Methods The following LAB strains were used in order to test their ability of AFM1 binding in a contaminated liquid medium: Lactobacillus plantarum CTC368 (donated by TECNOLAT—Dairy Research and Development Center, F o o d Te c h n o l o g y I n s t i t u t e , C a m p i n a s , B r a z i l ) , Enterococcus avium CTC469 (TECNOLAT), Pediococcus pentosaceus TR570 (TECNOLAT), Lactobacillus delbrueckii spp. bulgaricus LB340 (donated by Danisco Ltd., Brazil), Lactobacillus rhamnosus HOWARU® (Danisco Ltd.), Bifidobacterium lactis FLORA-FIT BI07 (Danisco Ltd.), and Lactobacillus gasseri ATCC 33323. All strains, in a lyophilized form, were reactivated in MRS (de Man, Rogosa and Sharpe) broth (Acumedia®, Lansing, USA) at 37 °C and cultivated in these conditions repeatedly until they achieved at least 109 CFU/mL. Estimation of bacterial concentration of each strain was determined by turbidimetric method. Bacterial concentration curves were constructed correlating the results obtained for absorbance measured at 600 nm in spectrophotometer Spectrumlab 22PC (Shanghai Lengguang Technology Co. Ltd., Shanghai, China) and logarithm of bacterial concentration obtained by pour plate counting (Wehr and Frank 2004) after culture incubation in MRS agar (Acumedia®, Lansing, USA) at 37 °C for 24 h under anaerobic conditions. From these data, equations were generated to calculate bacterial concentration in the medium and the volume of culture broth needed to achieve 1× 1010 CFU. The equations have adapted perfectly to data as their coefficients of determination (R2) ranged between 0.997 and 0.999. AFM1 standard (Supelco™, Bellefonte, USA) was suspended in acetonitrile and spectrophotometrically calibrated (Scott 1990) in order to obtain a 3.0 μg/mL stock solution. A 0.15 μg/mL working solution was prepared in PBS (Laborclin Ltd., Pinhais, Brazil), pH 7.3, evaporating the acetonitrile by nitrogen injection and heating in a hot-water bath (45 °C) until visible acetonitrile droplets disappeared. The assay of AFM1 binding in PBS was performed as described by Pierides et al. (2000) with some modifications. After bacterial growth, which was stopped by placing the culture in an ice-water bath for 3 min, bacterial cell concentration was determined by turbidimetry. A volume of culture broth corresponding to 1 × 10 10 cells was centrifuged (Microcentrifuge CT-14000, Cientec, Piracicaba, Brazil) at 1,800×g for 15 min in order to form the bacterial pellets, which were washed with sterile Milli-Q water (Simplicity 185, Millipore®, Billerica, MA, USA) and resuspended in 1.5 mL of PBS contaminated with AFM1 and incubated at 37 °C for 15 min or 24 h. After the contact times, bacterial cells were centrifuged again at 1,800×g for 15 min and supernatant was analyzed for determination of AFM1. The remaining pellets were washed with 1.5 mL of PBS not
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spiked with AFM1 and centrifuged under the same conditions, being the supernatant also analyzed for determination of AFM1. All analyzes were performed in triplicate and for each bacterial strain, a negative (bacterial cells suspended in PBS), a positive (0.15 μg/mL AFM1 in PBS) and a neutral (only PBS) controls were incubated and analyzed. Heatkilled bacterial cells were also tested, being bacterial culture inactivated by boiling at 100 °C for 1 h. Then, it was carried out the same steps outlined above from the collection of a volume of culture broth of 1×1010 cells. Quantification of AFM1 in PBS solutions was performed by injection of supernatant in a high-performance liquid chromatograph (HPLC) Shimadzu® system (Tokyo, Japan), consisting of a fluorescence detector RF-10A XL (Shimadzu®) equipped with a Synergy Fusion column 4 μm C18 4.6×150 mm (Phenomenex®, Torrance, USA) and autosampler SIL10AF (Shimadzu®). A flow rate of 1 mL/min was used with a mobile phase containing water, acetonitrile, and methanol (60:20:20). Detection was made at an excitation wavelength of 366 nm and emission at 428 nm. Detection limit for AFM1 was 0.01 ng/mL, as considered by the minimum amount of AFM1 that could generate a chromatographic peak three times over the baseline standard deviation. Under these conditions, the retention time of AFM1 was 6.1 min. The equation below was used to quantify AFM1 in each sample. Letter A represents the AFM1 percentage bound by bacteria and B, C, and D are the areas of chromatographic peaks of positive, neutral, and negative controls, respectively. After calculations, it was selected three bacterial strains, which showed the most efficient AFM1 binding for further tests in UHT skimmed milk. A¼
½ðB C Þ ðD EÞ BC
100
LAB strains selected were subjected to the same procedures as described before but using UHT skimmed milk previously spiked with 0.5 μg/L of AFM1. Two temperature conditions (4 and 37 °C) and only one incubation time of 15 min were used. Bacteria cells were heat killed to avoid any possible milk fermentation. Bacterial strains were evaluated in triplicate and each one had also a positive (0.5 μg/L AFM1 in milk), negative (bacterial cells suspended in milk), and neutral (only milk) controls. Determination of AFM1 in UHT milk samples was performed as recommended by Oliveira et al. (2006), using immunoaffinity columns (Neogen Europe Ltd., Scotland, UK) for clean-up. Quantification of AFM1 was achieved by HPLC as previously described. Statistical analysis of AFM1 binding assays was carried out in the General Linear Model of SAS® (SAS 1992) by using the Tukey Test for significant differences between strains and conditions at P
