- Email: [email protected]
Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu, Malaysia
Accepted Manuscript Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu, Malaysia A.Farah Nadira, J. Rosita, M.E. Norhaizan, S.Mohd Redzwan PII:
S0956-7135(16)30421-2
DOI:
10.1016/j.foodcont.2016.08.004
Reference:
JFCO 5174
To appear in:
Food Control
Received Date: 1 April 2016 Revised Date:
9 July 2016
Accepted Date: 2 August 2016
Please cite this article as: Nadira A.F., Rosita J., Norhaizan M.E. & Redzwan S.M., Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu, Malaysia, Food Control (2016), doi: 10.1016/j.foodcont.2016.08.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Screening of aflatoxin M1 occurrence in selected milk and dairy products in
2
Terengganu, Malaysia. A. Farah Nadira, J. Rosita*, M. E. Norhaizan, S. Mohd Redzwan
4 5
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
RI PT
3
6 7
*
8
Assoc. Prof. Dr. Rosita Jamaluddin
9
Email: [email protected] Phone: +603 89472467
11
Fax: +603 89426769
M AN U
10
SC
Correspondence
12
Abstract
14 15 16 17 18 19 20 21 22 23 24 25 26 27
The study was conducted to screen the occurrence of aflatoxin M1 (AFM1) in 53 selected milk and dairy product samples (11 liquid milk, 12 powdered milk, 8 3-in-1 beverages, 6 condensed sweetened milk, 2 evaporated milk, 7 cultured milk drink, 5 yogurt and 2 cheese samples). These samples were purchased from selected markets in Terengganu, Malaysia in January 2014 based on a questionnaire survey among 212 respondents on the types and brands of milk and dairy products that were frequently consumed. Based on the responses, 53 milk and dairy products were purchased and the competitive enzyme-linked immune-absorbent assay (ELISA) method was used to determine the level of AFM1 in the samples. Of 53 samples, 19 samples were positive with AFM1 (35.8%) ranging from 3.5 to 100.5 ng/L. Although 4/53 (7.5%) of the tested samples had the contamination level greater than the European Commission (EC) limit ( > 50 ng/L), the contamination levels were still below the Malaysia Food Regulation 1985 limit (less than 500 ng/L). This study provided a pioneering data on the occurrence of AFM1 in milk and dairy products in Malaysia.
28
Keywords
29
Aflatoxin, Aflatoxin M1, milk and dairy products, Terengganu, Malaysia.
30
1.
31 32 33
Nowadays, globalization does not only develop in term of technology, but increase of harmful exposure of food contamination should also be a serious concern. Aflatoxins are toxic fungal metabolites produced by Aspergillus species, mainly by Aspergillus flavus and Aspergillus
AC C
EP
TE D
13
Introduction
1
ACCEPTED MANUSCRIPT
parasiticus (Groopman, Chain, & Kensler, 1988). There are 14 different types of aflatoxins identified; the naturally occurring aflatoxins are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) (Leong, Latif, Ahmad, & Rosma, 2012). In Malaysia, aflatoxin exposure has been reported in the literature. Based on recent studies using aflatoxin biomarkers detected in serum (Leong et al., 2012; Mohd Redzwan et al., 2014) and urine (Sabran, Jamaluddin, & Abdul Mutalib, 2012; Mohd Redzwan, Rosita, Mohd Sokhini, & Nurul Aqilah, 2012a; Mohd Redzwan et al., 2015), Malaysians are moderately exposed to aflatoxin compared to the population with high prevalence of aflatoxin contamination. In a review paper on aflatoxin exposure in Malaysia, Mohd Redzwan et al. (2013) explained that aflatoxins have been detected in foods such as nuts, cereals, spices and herbs that are commonly used in the preparation of many Malaysian delicacies.
73
Study location
74 75
This study was conducted in households in Kuala Terengganu and Marang, Terengganu, Malaysia.
76
Data and sample collection
SC
RI PT
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
M AN U
One of the metabolites of AFB1 that can be detected in foodstuffs is aflatoxin M1 (AFM1). AFM1 is a toxic metabolite of AFB1. Ingested AFB1 is metabolized into AFM1 by the hepatic microsomal cytochrome P450 in the liver which is then excreted in the milk (de Oliveira et al. 2013). At first, AFM1 was classified as an agent in Group 2B possible carcinogenic effect on humans (IARC, 1993). However due to its toxicity, this toxin was then reclassified as Group 1 carcinogenic agent (IARC, 2002). Due to this, the European Commission (EC) has established the AFM1 maximum admissible level in milk at 50 ng/L (Fallah, 2010) while the Malaysia Food Regulation 1985 stated that AFM1 in milk and dairy products should be less than 500 ng/L (FoSIM, 1985).
2.
AC C
EP
TE D
Since AFM1 is stable at high temperature and cannot be removed from milk by heating process, this toxin can be transferred into the milk products such as liquid milk, powdered milk, cheese, yogurt and other milk based products (Duarte, Pena, & Lino, 2013). Many countries had reported the occurrence of AFM1 in the milk products. For example, 38.1% of infant formula analyzed by Er, Demirhan, & Yentur (2014) had detectable level of AFM1 ranging from 55-201 ng/kg whereas all powdered milk samples collected in Argentina and Brazil had AFM1 ranged from 100 to 920 ng/kg (Garcia Londono et al., 2013). Besides, a study in Portugal also reported the detection of AFM1 in natural yogurt and yoghurt with strawberries ranging from 43 to 45 ng/kg and 19 to 68 ng/kg, respectively (Martins & Martins, 2004). In fact, to our best knowledge there is no scientific study on the occurrence of AFM1 in any milk and dairy products in Malaysia has been reported. Therefore, this is the first pioneer study to screen and measure AFM1 in milk and dairy products in this country.
Methods
2
ACCEPTED MANUSCRIPT
Prior to the samples collection, a questionnaire survey involving 212 subjects in Kuala Terengganu and Marang, Malaysia was conducted. The objective of this survey was to identify the types and brands of milk and dairy products which were commonly consumed on a weekly basis. Therefore, this was a pioneer study to screen AFM1 in selected and commonly consumed milk and dairy products in Kuala Terengganu and Marang, Malaysia and did not represent the AFM1 contamination levels in milk and dairy products sold in Malaysia.
102 103 104 105 106 107 108 109 110 111 112 113
The quantitative analysis of AFM1 in the milk samples was performed by competitive enzyme immunoassay using Helica Aflatoxin M1 ELISA Quantitative test kit (Helica Biosystem, Inc., Santa Ana, CA USA). According to the Helica test kit guidelines, the limit of detection was 2 ppt (ng/L). Each sample was analyzed in triplicates. For the sample preparation, 3-in-1 beverages were prepared according to the preparation of the powdered milk, while for condensed sweetened milk and yogurt, the samples were prepared by following steps, similar to cheese preparation as suggested by El Khoury, Atoui & Yaghi (2011).
114 115 116 117
Powdered milk / 3-in-1 beverages Approximately 10 g of sample was dissolved in 100 mL of deionized water. The solution was stirred with magnetic stirrer for 5 minutes and then centrifuged for 15 minutes at 3000 x g (Kubota Centrifuge Model 2810, Tokyo, Japan) to separate the fat layer.
RI PT
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
M AN U
SC
Based on the responses from the survey, 53 milk and dairy products were identified and they were purchased from retail shops in Kuala Terengganu and Marang, Malaysia for the analysis of AFM1 by employing simple random sampling method. Eleven samples were liquid milk, 12 samples were powdered milk, 8 samples were 3-in-1 beverages, 6 samples were condensed sweetened milk, 2 samples were evaporated milk, 7 samples were cultured milk drink, 5 samples were yogurts and 2 samples were cheese. The samples were selected by taking into consideration their manufactured date which were on July 2013. For each product, three samples from the same batch were purchased as recommended by the Malaysian Food Regulations 1985 (Part III – Regulation 4: Procedure on taking samples for physical and chemical analysis) (Food Regulation 1985, 2014) The samples were then transported to the Nutrition Laboratory of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia and stored at 4 ℃ until analysis. This study was a part of whole study (data not include here) where the association between the consumption of milk and dairy products with the occurrence of AFM1 in urine sample was determined among the subjects.
EP
TE D
Experimental analysis
AC C
Sample preparation
Liquid milk / Evaporated sweetened milk/ Cultured milk These samples were used directly in the assay.
3
ACCEPTED MANUSCRIPT
Cheese / Condensed sweetened milk / Yogurt Exactly one gram (1 g) of finely grated or otherwise macerated sample was mixed with 5 mL of HPLC-grade methanol (Merck, Darmstadt, Germany). Then, the mixture was vortexed for 5 minutes. Then, the mixture was evaporated to dryness using purified nitrogen gas. Approximately 0.5 mL of provided blank skimmed milk was added to the tube and vortexed vigorously for 1 minute. The tube was allowed to stand for another 5 minutes and 200 µL of this milk extract was used in the assay.
125 126 127 128 129 130 131 132 133 134 135
Assay procedure Initially, the standard/sample was diluted with distilled water at the ratio of 1:20. Then, 200 µL aliquots of standard and samples were dispended into appropriate wells in triplicates. The plate was covered with aluminum foil and incubated at ambient temperature (19℃– 25℃) for 2 hours. The content of the wells was washed with PBS- Tween 20®. The washing steps were repeated three times. Then, the wells were tapped face down on a layer of adsorbent paper to remove residual wash buffer. Hundred microliters (100 µL) of the conjugate was added to each well. The plate was re-sealed and incubated for 15 minutes. Hundred microliters (100 μL) of stop solution were added to stop the reaction. The optical density (OD) of each microwell was read with a microplate reader (SIRIO S Microplate Reader, RADIM, Italy) at 450 nm. Analyses were performed according to the test kits instructions.
136
Method validation
137
Linearity
138 139 140 141
A calibration graph of AFM1 was plotted at six standards of AFM1 in stabilized skimmed milk (0, 5, 10, 25, 50 and 100 ng/L). Each sample was analyzed in triplicates. The occurrence and level of AFM1 in milk sample tested were measured by interpolation from the standard curve plotted.
142
Recovery
143 144 145 146 147
Recovery studies were performed by spiking skimmed milk sample with two different concentrations of AFM1 (10 ng/L and 25 ng/L). The concentration was chosen based on the manual of Helica Aflatoxin M1 ELISA Quantitative test kit and within the range of standards provided in the test kit (5-100 ng/L). The spiked samples were treated as described previously. Each concentration was repeated 4 times.
148
Data evaluation
149 150
The value of % absorbance of each sample was calculated based on the formula stated in Figure 1.
151
Figure 1: Formula for % Absorbance (Helica Aflatoxin M1 ELISA Quantitative test kit)
152 153 154 155
In order to obtain the exact value of AFM1 concentration, the values were further multiplied by the corresponding dilution factor. This was 1 for liquid milk, evaporated milk, cultured milk and 3-in-1 beverages while for cheese, yoghurt and condensed milk the dilution factor was 5. The data was analyzed by using Excel 2010. The levels of AFM1 were expressed as mean ± SD,
AC C
EP
TE D
M AN U
SC
RI PT
118 119 120 121 122 123 124
4
ACCEPTED MANUSCRIPT
156 157
percentages of positive samples and as the total percentage of samples exceeding EC and Malaysia limit.
158 159
3.
Results and Discussion
161
Data Validation
162 163 164 165 166
The correlation of coefficient, r2 of the standard curve was 0.994 confirmed the linearity of the graph and in agreement with the protocol provided in the ELISA kit. Besides, limit of detection (LOD) of the analysis was 2 ng/L (Based on manual). The results for the recovery experiment were expressed in Table 1. The mean recovery of two spiked concentrations was 84.13%. The CV were below 15% and in agreement with the protocol provided in the ELISA kit
167 168
Table 1: Recovery and coefficient of variance of two AFM1 spiked milk samples. AFM1 spiked (ng/L) (n = 4)a 10
CV (%)
Mean recovery ± SD (%)
2.11
88.13 ± 1.98
3.13
80.13 ± 5.12
TE D
25
M AN U
SC
RI PT
160
84.13 ± 3.65b
CV = Coefficient of variance SD = Standard deviation a Repetition number = 4 b Average recovery of two different spiked concentration
174
AFM1 identification
175 176 177 178 179 180 181 182 183 184 185
Data obtained from 212 subjects through a questionnaire survey were used to sample milk and dairy products in Terengganu, Malaysia. Based on the responses, 53 samples were analyzed and they were 11 samples of liquid milk, 12 samples of powdered milk, 8 samples of 3in-1 beverages, 6 samples were of condensed sweetened milk, 2 samples of evaporated milk, 7 samples of cultured milk drink, 5 samples of yogurts and 2 samples of cheese. The concentration of AFM1 in 53 samples is shown in Table 2. Of 53 samples, only 19 (35.8%) samples were contaminated with AFM1 ranging from 3.5 to 100.5 ng/L. Among these 8 groups of milk and dairy products, AFM1 was detected in all cheese samples (100%), whereas none of the evaporated milk had detectable level of AFM1. Of 19 positive samples, C5 had the highest AFM1 contamination level of 100.5 ± 15.3 ng/kg, while P9 had the lowest level of AFM1 detected (3.5 ± 1.4 ng/L).
AC C
EP
169 170 171 172 173
5
ACCEPTED MANUSCRIPT
186
Table 2: Occurrence and Level of AFM1 in Milk and Dairy Products (n = 53) Level of AFM1 No. of Positive Samples Positive sample (ng/L)a/ (ng/kg)b n (%) (Code) Positive mean ± SD 3/11 (27.3) 3.5 ± 1.4 P9 29.1 ± 5.0 P10 86.0 ± 4.8 P11 4/12 (33.3) 11.8 ± 3.1 L8 8.1 ± 2.4 L9 10.0 ± 1.7 L11 7.3 ± 2.4 L12 1/8 (12.5) B5 57.0 ± 3.7 3/6 (50) C1 15.6 ± 6.0
RI PT
Milk and Dairy Products Sample
SC
Powdered
Liquid
3-in-1 beverages Condensed sweetened milk
4/7 (57.1)
Cheese
2/2 (100)
C5 C6 T1 T2 T3 T4 Y1 Y2 S1 S2
Total 19/53 (35.8) a ng/L (for liquid, evaporated, cultured milk, 3-in-1 beverages) b ng/kg (for powdered, yogurt, cheese, condensed milk)
AC C
189 190 191 192 193 194 195 196 197 198 199 200
2/5 (40)
EP
Yogurt
TE D
Cultured milk drink
M AN U
187 188
6
100.5 ± 15.3 3.6 ± 4.5 3.7 ± .3.3 8.1 ± 2.4 4.6 ± 2.6 61.9 ± 1.2 25.4 ± 7.2 7.5 ± 1.6 4.6 ± 2.7 22.2 ± 4.2
ACCEPTED MANUSCRIPT
Table 3: Number of milk and dairy products exceeds the EC and Malaysia limit for AFM1
No. of Positive Samples n (%)
Powdered
11
(3) 27.3
1
Liquid
12
(4) 33.3
-
3-in-1 Beverages
8
(1) 12.5
Condensed sweetened milk
6
(3) 50.0
Evaporated milk
2
Cultured milk drink
7
Yogurt
5
Cheese
a
SC 1
-
-
-
-
-
(4) 57.1
1
-
(2) 40.0
-
-
2
(2) 100.00
-
-
53
(19) 35.8
4
-
EP
For milk, yogurt, the EC limit is 50 ng/L (50 pg/ml) ;For cheese EC limit is 250 ng/kg (250 pg/ml) b For all milk and dairy products, the Malaysia limit is 0.5μg/L (500 ng/L) A study at Minas Gerais, Brazil in 2009 reported the occurrence of AFM1 in 75 UHT liquid milk samples, where 21 (30.7%) samples were contaminated by AFM1 with the level ranging from 1000 to 4100 ng/L (de Oliveira et al., 2013). Moreover, in Thailand, Ruangwises & Saipan (2010) found AFM1 in all 240 raw milk samples ranging from 50 to 101 ng/L. Both studies in Brazil and Thailand reported higher value of AFM1 contamination in liquid milk compared to the present study (7.3-11.8 ng/L). Besides, it was also observable that the contamination level of AFM1 in samples of raw cow milk from Syria (Ghanem & Orfi, 2009), milk samples from Punjab, Pakistan (Sadia et al., 2012), and UHT milk from China (Zheng et al., 2013) were much higher than the present study, where the level of contamination from these three studies ranged from 2 to 794 ng/L. Galvano et al. (1996) had mentioned that the variation
AC C
202 203 204 205 206 207 208 209 210 211 212 213 214 215
-
0 (0)
TE D
Total
1
No. of Samples Exceed Malaysia Limitb ˃500 ng/L
RI PT
No. of Sample
No. of Samples Exceed EC Limita ˃50 ng/L
Milk and Dairy Products Sample
M AN U
201
7
ACCEPTED MANUSCRIPT
216 217
of AFM1 in milk and dairy products was normally affected by the geographical area, analytical method used and seasons variability.
218
RI PT
In term of powdered milk, one of the three positive samples in the present study had slightly lower AFM1 contamination level compared to a study in Syria, where the AFM1 contamination level of 12 ng/kg was detected in powdered milk (Ghanem & Orfi, 2009). However, when comparing to a study in India, the contamination level of AFM1 in infant formula ranged from 143 to 770 ng/kg (Rastogi et al., 2004), where these values were much higher than the values in powdered milk tested in the present study. Blanco et al. (1988) mentioned that the variations in the contamination level of AFM1 in this food product could be due to the seasonal changes and different processing techniques.
SC
219 220 221 222 223 224 225 226
M AN U
227
AFM1 was detected in 40% of the yogurt samples tested with, the levels ranging from 7.5 to 31 ng/kg. These observations indeed are comparable with a study by El-Khoury et al. (2011) as the authors found relatively lower levels of AFM1 in the Lebanese milk and yoghurt as compared to other regions around the world (Temamogullari & Kanici, 2014; Ertas et al., 2011; Mason et al., 2015). Conversely, Iha et al. (2011) had detected AFM1 as high as 529 ng/kg in yoghurt samples from Brazil. In addition, it was observed that the highest level of AFM1 contamination in yogurt samples tested from Pakistan reached the concentration of 615.8 ng/kg (Iqbal & Asi, 2013). By comparison to the data in the present study and the aforementioned studies (Iha et al., 2011; Iqbal & Asi, 2013), the level of AFM1 contamination of yogurt from India and Pakistan were considered higher than in Malaysia. As previously indicated, seasonal changes and different processing techniques could be attributable to the occurrence of AFM1 in milk products, and the reduction of AFM1 during yoghurt production could be due to several factors such as low pH, formation of organic acids or fermentation by-products and the presence of lactic acid bacteria (Govaris et al., 2002). Moreover, differences in extraction procedures, concentration of toxin, time elapsed before analysis, storage temperature, variability in composition of milk, or differences in the behavior of starter cultures used in preparing the yoghurt also become the factors of different level of AFM1 present in the yogurt. In particular, during the storage period, glucose might be oxidized by glucose oxidase and this reaction can produce hydrogen peroxide and gluconolactone in the yoghurt (Yousef & Marth, 1989). The latter will further undergo hydrolysis process that produces gluconic acid, which is capable to lower the pH (3.9) of the yoghurt and subsequently degrade the AFM1 (Yousef & Marth, 1989).
249 250 251 252 253
Of the two cheese samples tested in the present study, both had detectable level of AFM1. This finding was in agreement with a study in Iran where all of the cheese samples tested was contaminated by AFM1 (Ertas et al., 2011). The high detection of AFM1 in cheese samples was undoubted since many studies reported that approximately more than half of cheese samples
AC C
EP
TE D
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
8
ACCEPTED MANUSCRIPT
tested were contaminated by AFM1. For example, in Turkey 82.4% of white brined cheese were contaminated with AFM1 (Ardic et al., 2009). Although all cheese samples in the present study had detectable level of AFM1, the contamination level was very low compared to a study by Tekinsen & Eken (2008) as the authors found AFM1 contamination level of 50 to 690 ng/kg in Kashar cheese sold in Turkey. Since AFM1 is a water soluble compound, a high concentration of AFM1 in cheese sample is undoubted (Dosako et al., 1980). The variation of the cheese samples are generally affected by several factors such as geographical region, cheese-making procedures and conditions of cheese ripening (Fallah et al., 2009).
277 278 279 280 281 282 283 284 285 286 287
In term of 3-in-1 beverages, previous study by Khayoon, Saad, Lee, & Salleh (2013) reported that aflatoxins (AFG2, AFG1, AFB2 and AFB1) were absent in all samples of malt beverages and canned coffee. This finding was nearly similar to the present study where only 1 out of 8 samples were contaminated with AFM1. The 3-in-1 beverages only contained a small amount of dairy based products (creamer) and the additional ingredient might change the AFM1 composition in the samples and hence, 3-in-1 beverages were less likely to be contaminated by AFM1. However since the tested number of samples was small (n= 8), we cannot confidently justify that the food product is safe for the consumer as the analyzed sample was not representable of the whole samples. Besides, the level of AFM1 in the positive sample was over the permissible limit established by EC.
288 289 290 291 292
In this study, only evaporated milk sample did not show the occurrence of AFM1. Evaporated milk had lower fat content and required higher heat treatment compared to condensed sweetened milk production. This could be the reason AFM1 was not detected in the sample. Milk samples with greater fat content have greater but not significantly greater (= 0.067) levels of AFM1 compared to those of lower fat milk (Carvajal et al., 2003).
293 294
The culture milk drink, which normally has probiotic bacteria is potentiated to be used to prevent human dietary aflatoxin exposure (El-Nezami et al., 2006; Mohd Redzwan et al., 2016). The
RI PT
254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
AC C
EP
TE D
M AN U
SC
Among the 8 groups of milk and dairy products tested, condensed milk showed the highest level of AFM1 occurrence ranging from 3.6 to 100.5 ng/kg and one of the positive samples was above the EC limit. Since this is the first study to screen the occurrence of AFM1 in condensed sweetened milk, the result was not comparable to other findings. Nevertheless, Mohd Redzwan et al. (2012a) reported a borderline significant correlation between the consumption of condensed milk with AFM1 detected in urine samples, ( = ݎ-0.384; = 0.063). However, the correlation was negative which indicated that the higher the consumption of condensed milk, the lower the concentration of urinary AFM1. Generally, high ingestion of AFM1 would reflect the high level of AFM1 detection in urine sample, but not in the study conducted by Mohd Redzwan et al. (2012). Condensed milk is among the top 10 foods mostly consumed by Malaysians (Norimah et al., 2008). This statistic shows that the consumption of condensed milk by Malaysian is quite high compared to other types of milk. Since the present study found the highest level of AFM1 in condensed milk samples, further confirmation on the level of AFM1 by HPLC should be carried out.
9
ACCEPTED MANUSCRIPT
probiotic bacteria in the culture milk act as an adsorbent that binds aflatoxin. Despite that, it also could be contaminated by AFM1 if AFM1-contaminated milk is used in the fermentation process (Lin et al., 2004). Among 7 tested samples of cultured milk drink in the present study, 4 of them were contaminated with AFM1. In Taiwan, the incidence of AFM1 contamination in yoghurt drink (culture milk drink) was 12.5% as reported by Lin et al. (2004). Of 24 samples tested, Lin et al. (2004) found 3 samples contaminated by AFM1, with the concentrations of 7, 9 and 44 ng/L respectively. However, the level of AFM1 in the contaminated cultured drink in this study was slightly higher than in the study reported in Taiwan (Lin et al., 2004). Iha et al. (2011) reported that the concentration of AFM1 found in dairy drinks would be less than that of the milk being used for the process because many other food ingredients are added to the milk during preparation of the products, hence the lower level of AFM1 contamination in this kind of milk products were undoubted.
308 309 310 311 312 313 314 315 316 317 318 319 320
Although all the samples tested in the present study had AFM1 contamination level far below the Malaysian limit, we found four samples with AFM1 contamination level above the EC limit (Table 3). In the South-East Asian region, the study for the occurrence of AFM1 in the milk products had been conducted by our neighbor country, Indonesia, as of 113 fresh milk collected directly from five different areas in Yogyakarta, Indonesia in year 2006, 48 was below 5 ng/L (below LOD), 65 was within 5 ng/L to 25 ng/L and none of them was above 25 ng/L to 50 ng/L (EC limit) (Nuryono et al., 2009). Whereas in Thailand, of 90 samples of raw and pasteurized goat milk, 49 samples were contaminated by AFM1 and 7 of them were above the EC limit (Ruangwises, Saipan & Ruangwises, 2013). Hence, it can be postulated that milk and dairy product samples tested in the present study were more likely to get high level of AFM1 contamination compared to samples from Indonesia but less likely compared to samples from Thailand.
321
4.
322 323 324 325 326 327 328 329
Of 53 samples, only 19 samples were contaminated by AFM1 and 4 of them had AFM1 contamination level above the EC limit. Nevertheless, the contamination level was still far below the Malaysian limit of 500 ng/L. In this study, the highest contamination level of AFM1 was observed in the sweetened condensed milk samples with the contamination level of 100.5 ng/L. Since this study was a limited survey on the occurrence of AFM1 in milk and dairy products purchased in selected cities in Terengganu, Malaysia, further confirmation by using HPLC is warranted. Besides, a larger number of samples of milk and dairy products should be analyzed to represent the whole samples of milk and dairy products mostly consumed by Malaysian.
330
Acknowledgements
331 332 333 334
A. Farah Nadira would like to thank School of Graduate Studies, Universiti Putra Malaysia for the Graduate Research Fellow (GRF) scholarship and the Ministry of Higher Education
335 336
Ardic, M., Y, Karakaya., M, Atasever., & G, Adiguzel. (2009). Aflatoxin M1 levels of Turkish white brined cheese. Food Control, 20, 196-199.
TE D
M AN U
SC
RI PT
295 296 297 298 299 300 301 302 303 304 305 306 307
AC C
EP
Conclusion
References
10
ACCEPTED MANUSCRIPT
Bakirci, I. (2001). A study on the occurrence of aflatoxin M1 in milk and milk products produced in Van province of Turkey. Food Control, 12, 47-51.
339 340 341
Blanco, J., Domingueza, L; Gomez-Lucia, E., Garayzabal, J. F. F., Garcia, J. A., & Suarez, G. (1988). Presence of aflatoxin M1 in commercial ultra-high temperature treated milk. Applied Environmental Microbiology, 56, 1622-1623.
342 343 344
Carvajal, M., Bolonos, A., Rojo, F., & Mendez, I. (2003). Aflatoxin in pasteurized and ultrapasteurized milk with different fat content in Mexico. Journal of Food Protection, Ames, 66 (10), 1885-1892, ISSN 0362-028X
345 346 347
De Oliveria, C.P., N. de Fatima Ferreira Soares, T.V. de Oliveria, J.C. Baffa Junior & W. A. da Silva. (2013). Aflatoxin M1 occurrence in ultra-high temperature (UHT) treated fluid milk from Minas Gerais/ Brazil. Food Control, 30, 90-92.
348 349 350
Dosako, S., Kaminogawa, S., Taneya, S., & Yamauchi, K. (1980). Hydrophobic surface areas and net charges of ߙs1-, k-casein and ߙݏ1-casein: k-casein complex. Journal of Dairy Research, 4(7), 123-129.
351 352
Duarte, S. C., Pena., & A., Lino, C. M. (2011). Human ochratoxin A biomarkers from exposure to effect. Crit. Rev. Toxicol, 41, 187-212.
353 354 355
El Khoury, A., Atoui, A., & Yaghi, J. (2011). Analysis of Aflatoxin M1 in milk and yogurt and AFM1 reduction by lactic acid bacteria used in Lebanese industry. Food Control, 22, 1695-1699. Retrieved from http://dx.doi.org/10.1016/j.foodcont.2011.04.001 on January 14, 2015.
356 357 358 359
El-Nezami, HS, Polychronaki NN, Ma, J, Zhu, H, Ling, W, Salminen, EK, Juvonen RO, Salminen, SJ, Poussa, T, & Mykkänen, HM. (2006). Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China. The American Journal of Clinical Nutrition, 83, 1199-1203.
360 361 362
Er, B., Demirhan, B., & Yentur, G. (2014). Short communication: Investigation of aflatoxurkey. in M1 levels in infant follow-on milks and infants formulas sold in the market of Ankara, Turkey. American Dairy Science Association, 97(6), 3328-3331. 10.3168/jds.2013-7831.
363 364 365
Ertas, N., Gonulalan, Z., Yildirim, Y., & Karadal, F. (2011). A survey of concentration of aflatoxin M1 in dairy products marketed in Turkey. Food Control, 22(12), 1956–1959. doi:10.1016/j.foodcont.2011.05.009
366 367
Fallah, A. A., Jafari, T., Fallah, A., & Rahnama, M. (2009). Determination of aflatoxin M1 levels in Iranian white and cream cheese. Food and Chemical Toxicology, 47(8), 1872-1875.
368 369 370 371
Fallah, A. A. (2010). Assessment of aflatoxin M1 contamination in pasteurized and UHT milk marketed in central part of Iran. Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association, 48(3), 988–991. doi:10.1016/j.fct.2010.01.014
AC C
EP
TE D
M AN U
SC
RI PT
337 338
11
ACCEPTED MANUSCRIPT
FoSIM (Food Safety Information System of Malaysia) (1985). Food Safety and Quality Division, Department of Public Health, Ministry of Health Malaysia. Retrieved from http://fsis2.moh.gov.my on March 16, 2015.
375 376 377
Food Regulation 1985 (2014). Part III – Regulation 4: Procedure on taking samples for physical and chemical analysis Department of Public Health, Ministry of Health Malaysia. Retrieved http://fsq.moh.gov.my/v5/ms/food-regulations-1985-2 on April 7, 2016.
378 379
Galvano, F., Galofaro, V., & Galvano, G. (1996). Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. J. Food Prot. 59, 1079–1090.
380 381 382 383
Garcia Londoño, V. A., Boasso, A. C., de Paula, M. C. Z., Garcia, L. P., Scussel, V. M., Resnik, S., & Pacín, A. (2013). Aflatoxin M1 survey on randomly collected milk powder commercialized in Argentina and Brazil. Food Control, 34(2), 752–755. Retrieved from http://www.sciencedirect.com/science/article/pii/S0956713513003137
384 385 386
Ghanem, I., & Orfi, M. (2009). Aflatoxin M1 in raw, pasteurized and powdered milk available in the Syrian market. Food Control, 20(6), 603–605. Retrieved from http://www.sciencedirect.com/science/article/pii/S0956713508002429.
387 388 389
Govaris, A., Roussi, V., Koidis, P.A., & Botsoglou, N.A. (2002). Distribution and stability of aflatoxin M1 during production and storage of yogurt. Food Additives and Contaminant. 19, 1043-1050
390 391
Groopman, J. D., Cain, L.G., & Kensler, T. W. (1988). Aflatoxin exposure in human populations: measurements and relationship to cancer. Crit Rev Toxicol 19, 113–46.
392 393 394
IARC. (1993). Aflatoxin. In Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC monograph on the evaluation of carcinogenic risk to humans, 56, 245-395. Lyon: International Agency for Research on Cancer.
395 396
IARC. (2002). IARC monograph on the evaluation of carcinogenic risk to humans, 82, 171. Lyon: International Agency for Research on Cancer-World Health Organization.
397 398
Iha, M. H., Barbosa, C. B., Okada, I. A., & Trucksess, M. W. (2011). Occurrence of aflatoxin M1 in dairy products in Brazil. Food Control, 22, 1971-1974.
399 400
Iqbal, Z., & Asi, M. R. (2013). Assessment of aflatoxin M1 in milk and milk products from Punjab, Pakistan. Food Control, 30(1), 235–239. doi:10.1016/j.foodcont.2012.06.026.
401 402 403
Khayoon, W. S., Saad, B., Lee, T. P., & Salleh, B. (2012). High performance liquid chromatographic determination of aflatoxins in chili, peanut and rice using silica based monolithic column. Food Chem. 133, 489–496. doi: 10.1016/j.foodchem.2012.01.010.
404 405 406
Lee, J. E., Kwak, B.-M., Ahn, J.-H., & Jeon, T.-H. (2009). Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography. Food Control, 20(2), 136–138. doi:10.1016/j.foodcont.2008.03.002
AC C
EP
TE D
M AN U
SC
RI PT
372 373 374
12
ACCEPTED MANUSCRIPT
Leong, Y.-H., Latiff, A. a, Ahmad, N. I., & Rosma, A. (2012). Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review. Mycotoxin Research, 28(2), 79–87. doi:10.1007/s12550-012-0129-8
410 411
Lin, L., Liu, F., Fu, Y., & Shih, D. Y. (2004). Survey of Aflatoxin M1 Contamination of Dairy Products in Taiwan, 12(2), 154–160.
412 413 414
Mason, S., Arjmandtalab, S., Hajimohammadi, B., Arsanjani, A. K., Karami, S., Sayadi, M., & Oryan, A. (2015). Aflatoxin M 1 Contamination in Industrial and Traditional Yogurts Produced in Iran, 2, 11–14.
415 416 417
Marnissi, B. E., Belkhou, R., Morgavi, P. D., Bennani, L., & Boudra, H. (2012). Occurrence of aflatoxin M1 in raw milk collected from traditional dairies in Morocco. Food and Chemical Toxicology, 50, 2819-2821
418 419 420 421
Martins, M. L., & Martins, H. M. (2004). Aflatoxin M1 in yoghurts in Portugal International Journal of Food Microbiology, 91, 315–317 Mohammadi, H., & Alizadeh, M. (2010). A Study of the Occurrence of Aflatoxin M1 in Dairy Products Marketed in Urmia , Iran, 12, 579–583.
422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Nurul ‘Aqilah, A.R. (2012a) Association between aflatoxin M1 excreted in human urine samples with the consumption of milk and dairy products. Bulletin of Environmental Contamination and Toxicology, 89, 1115-1119.
438 439 440
Mohd Redzwan, S., Jamaluddin, R., Mohd Sokhini, A.M., Nurul ‘Aqilah, A.R., Zurain, A., Karimi, G., Parvaneh, K. (2015). Ultra-high performance liquid chromatographic determination of aflatoxin M1 in urine. World Mycotoxin Journal, 8, 405-413. 2014
441 442 443 444 445
Mohd Redzwan, S., Abd. Mutalib, M.S., Wang, J.S., Ahmad, Z., Kang, M.S., Abdul Rahman, N.A., Nikbakht Nasrabadi, E., & Jamaluddin, R. (2016). Effect of supplementation of fermented milk drink containing probiotic Lactobacillus casei Shirota on the concentrations of aflatoxin biomarkers among employees of Universiti Putra Malaysia: a randomized, double-blind, cross over, placebo-control study. British Journal of Nutrition, 115, 39-54
M AN U
SC
RI PT
407 408 409
TE D
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Nurul ‘Aqilah, A.R. (2012b) Sociodemographic and socio-economic determinants of adults’ knowledge on fungal and aflatoxin contamination in the diets. Asian Pacific Journal of Tropical Biomedicine, 2 (3), Supplement, S1835-S1841.
EP
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Zuraini, A. (2013). A mini review on aflatoxin exposure in Malaysia: past, present and future. Frontier in Microbiology, 4 (334), 1-8.
AC C
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., Nurul ‘Aqilah, A.R., Wang, J-S., & Kang, M-S., Zuraini, A. (2014) Detection of serum AFB1-lysine adduct in Malaysia and its association with liver and kidney functions. International Journal of Hygiene and Environmental Health, 217, 443-451.
13
ACCEPTED MANUSCRIPT
Norimah. A. K., Safiah, M., Jamal. K., Haslinda, S., Zuhaida, H., Rohida, S., Fatimah, S., Norazlin, S., Poh, B. K., Kandiah, M., Zalilah, M. S., Wan Manan, W. M., Fatimah, S., & Azmi, M. Y. (2008). Food Consumption Patterns: Findings from the Malaysian Adult Nutrition Survey(MANS). Mal J Nutr, 14 (1), 25 - 39.
450 451 452
Nuryono, N., Agus, a., Wedhastri, S., Maryudani, Y. B., Sigit Setyabudi, F. M. C., Böhm, J., & Razzazi-Fazeli, E. (2009). A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food Control, 20(8), 721–724. doi:10.1016/j.foodcont.2008.09.005
453 454 455
Rastogi, S., Dwivedi, P. D., Khanna, S. K., & Das, M. (2004). Detection of Aflatoxin M1 contamination in milk and infant milk products from Indian markets by ELISA. Food Control, 15(4), 287–290. doi:10.1016/S0956-7135(03)00078-1
456 457 458
Ruangwises, S., & Saipan, P. (2010). Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand. Aflatoxins - Recent Advances and Future Prospects. Retrived from http://dx.doi.org/10.5772/52723 February 17, 2014
459 460 461
Sabran, M.R., Jamaluddin, R., & Abdul Mutalib, M.S. (2012) Screening of aflatoxin M1, a metabolite of aflatoxin B1 in human urine samples in Malaysia: A preliminary study. Food Control, 28, 55-58.
462 463
Sadia, A., Jabbar, M. A., Deng, Y., Hussain, E. A, Riffat, S., & Naveed, S. (2012). A survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan. Food Control 26, 235–240
464 465
Temamogullari, F., & Kanici, A. (2014). Short communication: Aflatoxin M1 in dairy products sold in Şanlıurfa, Turkey. Journal of Dairy Science, 97(1), 162–5. doi:10.3168/jds.2012-6184
466 467
Tekinsen, K. K., & Eken, H. S. (2008). Aflatoxin M1 levels in UHT milk and Kashar cheese consumed in Turkey. Food and Chemical Toxicology, 46, 3287-3289.
468 469
Yousef, A.E., & Marth, E.H., (1989). Stability and degradation of aflatoxin M1. In: Van Egmond, H.P. (Ed.), Mycotoxins in Dairy Products. Elsevier Applied Science, London, 127–161.
470 471
Zheng, N., Sun, P.,Wang, J. Q., Zhen, Y. P., Han, R.W., & Xu, X. M. (2013). Occurrence of aflatoxinM1 in UHT milk and pasteurized milk in China market. Food Control, 29,198-201
472 473
Figure
477
SC
M AN U
TE D
EP
AC C
474 475 476
RI PT
446 447 448 449
% Absorbance = Mean Absorption Standard or Sample x 100 Absorption Zero Standard
Figure 1: Formula for % Absorbance (Helica Aflatoxin M1 ELISA Quantitative test kit)
14
ACCEPTED MANUSCRIPT
Highlights
EP
TE D
M AN U
SC
RI PT
35.8% samples were positive with AFM1 Range of contamination from 3.5 to 100.5 ng/L 7.5% of the samples higher than the European Commission limit No samples exceed the Malaysia Food Regulation 1985 limit
AC C
• • • •
S0956-7135(16)30421-2
DOI:
10.1016/j.foodcont.2016.08.004
Reference:
JFCO 5174
To appear in:
Food Control
Received Date: 1 April 2016 Revised Date:
9 July 2016
Accepted Date: 2 August 2016
Please cite this article as: Nadira A.F., Rosita J., Norhaizan M.E. & Redzwan S.M., Screening of aflatoxin M1 occurrence in selected milk and dairy products in Terengganu, Malaysia, Food Control (2016), doi: 10.1016/j.foodcont.2016.08.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
1
Screening of aflatoxin M1 occurrence in selected milk and dairy products in
2
Terengganu, Malaysia. A. Farah Nadira, J. Rosita*, M. E. Norhaizan, S. Mohd Redzwan
4 5
Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia
RI PT
3
6 7
*
8
Assoc. Prof. Dr. Rosita Jamaluddin
9
Email: [email protected] Phone: +603 89472467
11
Fax: +603 89426769
M AN U
10
SC
Correspondence
12
Abstract
14 15 16 17 18 19 20 21 22 23 24 25 26 27
The study was conducted to screen the occurrence of aflatoxin M1 (AFM1) in 53 selected milk and dairy product samples (11 liquid milk, 12 powdered milk, 8 3-in-1 beverages, 6 condensed sweetened milk, 2 evaporated milk, 7 cultured milk drink, 5 yogurt and 2 cheese samples). These samples were purchased from selected markets in Terengganu, Malaysia in January 2014 based on a questionnaire survey among 212 respondents on the types and brands of milk and dairy products that were frequently consumed. Based on the responses, 53 milk and dairy products were purchased and the competitive enzyme-linked immune-absorbent assay (ELISA) method was used to determine the level of AFM1 in the samples. Of 53 samples, 19 samples were positive with AFM1 (35.8%) ranging from 3.5 to 100.5 ng/L. Although 4/53 (7.5%) of the tested samples had the contamination level greater than the European Commission (EC) limit ( > 50 ng/L), the contamination levels were still below the Malaysia Food Regulation 1985 limit (less than 500 ng/L). This study provided a pioneering data on the occurrence of AFM1 in milk and dairy products in Malaysia.
28
Keywords
29
Aflatoxin, Aflatoxin M1, milk and dairy products, Terengganu, Malaysia.
30
1.
31 32 33
Nowadays, globalization does not only develop in term of technology, but increase of harmful exposure of food contamination should also be a serious concern. Aflatoxins are toxic fungal metabolites produced by Aspergillus species, mainly by Aspergillus flavus and Aspergillus
AC C
EP
TE D
13
Introduction
1
ACCEPTED MANUSCRIPT
parasiticus (Groopman, Chain, & Kensler, 1988). There are 14 different types of aflatoxins identified; the naturally occurring aflatoxins are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1) and G2 (AFG2) (Leong, Latif, Ahmad, & Rosma, 2012). In Malaysia, aflatoxin exposure has been reported in the literature. Based on recent studies using aflatoxin biomarkers detected in serum (Leong et al., 2012; Mohd Redzwan et al., 2014) and urine (Sabran, Jamaluddin, & Abdul Mutalib, 2012; Mohd Redzwan, Rosita, Mohd Sokhini, & Nurul Aqilah, 2012a; Mohd Redzwan et al., 2015), Malaysians are moderately exposed to aflatoxin compared to the population with high prevalence of aflatoxin contamination. In a review paper on aflatoxin exposure in Malaysia, Mohd Redzwan et al. (2013) explained that aflatoxins have been detected in foods such as nuts, cereals, spices and herbs that are commonly used in the preparation of many Malaysian delicacies.
73
Study location
74 75
This study was conducted in households in Kuala Terengganu and Marang, Terengganu, Malaysia.
76
Data and sample collection
SC
RI PT
34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72
M AN U
One of the metabolites of AFB1 that can be detected in foodstuffs is aflatoxin M1 (AFM1). AFM1 is a toxic metabolite of AFB1. Ingested AFB1 is metabolized into AFM1 by the hepatic microsomal cytochrome P450 in the liver which is then excreted in the milk (de Oliveira et al. 2013). At first, AFM1 was classified as an agent in Group 2B possible carcinogenic effect on humans (IARC, 1993). However due to its toxicity, this toxin was then reclassified as Group 1 carcinogenic agent (IARC, 2002). Due to this, the European Commission (EC) has established the AFM1 maximum admissible level in milk at 50 ng/L (Fallah, 2010) while the Malaysia Food Regulation 1985 stated that AFM1 in milk and dairy products should be less than 500 ng/L (FoSIM, 1985).
2.
AC C
EP
TE D
Since AFM1 is stable at high temperature and cannot be removed from milk by heating process, this toxin can be transferred into the milk products such as liquid milk, powdered milk, cheese, yogurt and other milk based products (Duarte, Pena, & Lino, 2013). Many countries had reported the occurrence of AFM1 in the milk products. For example, 38.1% of infant formula analyzed by Er, Demirhan, & Yentur (2014) had detectable level of AFM1 ranging from 55-201 ng/kg whereas all powdered milk samples collected in Argentina and Brazil had AFM1 ranged from 100 to 920 ng/kg (Garcia Londono et al., 2013). Besides, a study in Portugal also reported the detection of AFM1 in natural yogurt and yoghurt with strawberries ranging from 43 to 45 ng/kg and 19 to 68 ng/kg, respectively (Martins & Martins, 2004). In fact, to our best knowledge there is no scientific study on the occurrence of AFM1 in any milk and dairy products in Malaysia has been reported. Therefore, this is the first pioneer study to screen and measure AFM1 in milk and dairy products in this country.
Methods
2
ACCEPTED MANUSCRIPT
Prior to the samples collection, a questionnaire survey involving 212 subjects in Kuala Terengganu and Marang, Malaysia was conducted. The objective of this survey was to identify the types and brands of milk and dairy products which were commonly consumed on a weekly basis. Therefore, this was a pioneer study to screen AFM1 in selected and commonly consumed milk and dairy products in Kuala Terengganu and Marang, Malaysia and did not represent the AFM1 contamination levels in milk and dairy products sold in Malaysia.
102 103 104 105 106 107 108 109 110 111 112 113
The quantitative analysis of AFM1 in the milk samples was performed by competitive enzyme immunoassay using Helica Aflatoxin M1 ELISA Quantitative test kit (Helica Biosystem, Inc., Santa Ana, CA USA). According to the Helica test kit guidelines, the limit of detection was 2 ppt (ng/L). Each sample was analyzed in triplicates. For the sample preparation, 3-in-1 beverages were prepared according to the preparation of the powdered milk, while for condensed sweetened milk and yogurt, the samples were prepared by following steps, similar to cheese preparation as suggested by El Khoury, Atoui & Yaghi (2011).
114 115 116 117
Powdered milk / 3-in-1 beverages Approximately 10 g of sample was dissolved in 100 mL of deionized water. The solution was stirred with magnetic stirrer for 5 minutes and then centrifuged for 15 minutes at 3000 x g (Kubota Centrifuge Model 2810, Tokyo, Japan) to separate the fat layer.
RI PT
77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101
M AN U
SC
Based on the responses from the survey, 53 milk and dairy products were identified and they were purchased from retail shops in Kuala Terengganu and Marang, Malaysia for the analysis of AFM1 by employing simple random sampling method. Eleven samples were liquid milk, 12 samples were powdered milk, 8 samples were 3-in-1 beverages, 6 samples were condensed sweetened milk, 2 samples were evaporated milk, 7 samples were cultured milk drink, 5 samples were yogurts and 2 samples were cheese. The samples were selected by taking into consideration their manufactured date which were on July 2013. For each product, three samples from the same batch were purchased as recommended by the Malaysian Food Regulations 1985 (Part III – Regulation 4: Procedure on taking samples for physical and chemical analysis) (Food Regulation 1985, 2014) The samples were then transported to the Nutrition Laboratory of the Faculty of Medicine and Health Sciences, Universiti Putra Malaysia and stored at 4 ℃ until analysis. This study was a part of whole study (data not include here) where the association between the consumption of milk and dairy products with the occurrence of AFM1 in urine sample was determined among the subjects.
EP
TE D
Experimental analysis
AC C
Sample preparation
Liquid milk / Evaporated sweetened milk/ Cultured milk These samples were used directly in the assay.
3
ACCEPTED MANUSCRIPT
Cheese / Condensed sweetened milk / Yogurt Exactly one gram (1 g) of finely grated or otherwise macerated sample was mixed with 5 mL of HPLC-grade methanol (Merck, Darmstadt, Germany). Then, the mixture was vortexed for 5 minutes. Then, the mixture was evaporated to dryness using purified nitrogen gas. Approximately 0.5 mL of provided blank skimmed milk was added to the tube and vortexed vigorously for 1 minute. The tube was allowed to stand for another 5 minutes and 200 µL of this milk extract was used in the assay.
125 126 127 128 129 130 131 132 133 134 135
Assay procedure Initially, the standard/sample was diluted with distilled water at the ratio of 1:20. Then, 200 µL aliquots of standard and samples were dispended into appropriate wells in triplicates. The plate was covered with aluminum foil and incubated at ambient temperature (19℃– 25℃) for 2 hours. The content of the wells was washed with PBS- Tween 20®. The washing steps were repeated three times. Then, the wells were tapped face down on a layer of adsorbent paper to remove residual wash buffer. Hundred microliters (100 µL) of the conjugate was added to each well. The plate was re-sealed and incubated for 15 minutes. Hundred microliters (100 μL) of stop solution were added to stop the reaction. The optical density (OD) of each microwell was read with a microplate reader (SIRIO S Microplate Reader, RADIM, Italy) at 450 nm. Analyses were performed according to the test kits instructions.
136
Method validation
137
Linearity
138 139 140 141
A calibration graph of AFM1 was plotted at six standards of AFM1 in stabilized skimmed milk (0, 5, 10, 25, 50 and 100 ng/L). Each sample was analyzed in triplicates. The occurrence and level of AFM1 in milk sample tested were measured by interpolation from the standard curve plotted.
142
Recovery
143 144 145 146 147
Recovery studies were performed by spiking skimmed milk sample with two different concentrations of AFM1 (10 ng/L and 25 ng/L). The concentration was chosen based on the manual of Helica Aflatoxin M1 ELISA Quantitative test kit and within the range of standards provided in the test kit (5-100 ng/L). The spiked samples were treated as described previously. Each concentration was repeated 4 times.
148
Data evaluation
149 150
The value of % absorbance of each sample was calculated based on the formula stated in Figure 1.
151
Figure 1: Formula for % Absorbance (Helica Aflatoxin M1 ELISA Quantitative test kit)
152 153 154 155
In order to obtain the exact value of AFM1 concentration, the values were further multiplied by the corresponding dilution factor. This was 1 for liquid milk, evaporated milk, cultured milk and 3-in-1 beverages while for cheese, yoghurt and condensed milk the dilution factor was 5. The data was analyzed by using Excel 2010. The levels of AFM1 were expressed as mean ± SD,
AC C
EP
TE D
M AN U
SC
RI PT
118 119 120 121 122 123 124
4
ACCEPTED MANUSCRIPT
156 157
percentages of positive samples and as the total percentage of samples exceeding EC and Malaysia limit.
158 159
3.
Results and Discussion
161
Data Validation
162 163 164 165 166
The correlation of coefficient, r2 of the standard curve was 0.994 confirmed the linearity of the graph and in agreement with the protocol provided in the ELISA kit. Besides, limit of detection (LOD) of the analysis was 2 ng/L (Based on manual). The results for the recovery experiment were expressed in Table 1. The mean recovery of two spiked concentrations was 84.13%. The CV were below 15% and in agreement with the protocol provided in the ELISA kit
167 168
Table 1: Recovery and coefficient of variance of two AFM1 spiked milk samples. AFM1 spiked (ng/L) (n = 4)a 10
CV (%)
Mean recovery ± SD (%)
2.11
88.13 ± 1.98
3.13
80.13 ± 5.12
TE D
25
M AN U
SC
RI PT
160
84.13 ± 3.65b
CV = Coefficient of variance SD = Standard deviation a Repetition number = 4 b Average recovery of two different spiked concentration
174
AFM1 identification
175 176 177 178 179 180 181 182 183 184 185
Data obtained from 212 subjects through a questionnaire survey were used to sample milk and dairy products in Terengganu, Malaysia. Based on the responses, 53 samples were analyzed and they were 11 samples of liquid milk, 12 samples of powdered milk, 8 samples of 3in-1 beverages, 6 samples were of condensed sweetened milk, 2 samples of evaporated milk, 7 samples of cultured milk drink, 5 samples of yogurts and 2 samples of cheese. The concentration of AFM1 in 53 samples is shown in Table 2. Of 53 samples, only 19 (35.8%) samples were contaminated with AFM1 ranging from 3.5 to 100.5 ng/L. Among these 8 groups of milk and dairy products, AFM1 was detected in all cheese samples (100%), whereas none of the evaporated milk had detectable level of AFM1. Of 19 positive samples, C5 had the highest AFM1 contamination level of 100.5 ± 15.3 ng/kg, while P9 had the lowest level of AFM1 detected (3.5 ± 1.4 ng/L).
AC C
EP
169 170 171 172 173
5
ACCEPTED MANUSCRIPT
186
Table 2: Occurrence and Level of AFM1 in Milk and Dairy Products (n = 53) Level of AFM1 No. of Positive Samples Positive sample (ng/L)a/ (ng/kg)b n (%) (Code) Positive mean ± SD 3/11 (27.3) 3.5 ± 1.4 P9 29.1 ± 5.0 P10 86.0 ± 4.8 P11 4/12 (33.3) 11.8 ± 3.1 L8 8.1 ± 2.4 L9 10.0 ± 1.7 L11 7.3 ± 2.4 L12 1/8 (12.5) B5 57.0 ± 3.7 3/6 (50) C1 15.6 ± 6.0
RI PT
Milk and Dairy Products Sample
SC
Powdered
Liquid
3-in-1 beverages Condensed sweetened milk
4/7 (57.1)
Cheese
2/2 (100)
C5 C6 T1 T2 T3 T4 Y1 Y2 S1 S2
Total 19/53 (35.8) a ng/L (for liquid, evaporated, cultured milk, 3-in-1 beverages) b ng/kg (for powdered, yogurt, cheese, condensed milk)
AC C
189 190 191 192 193 194 195 196 197 198 199 200
2/5 (40)
EP
Yogurt
TE D
Cultured milk drink
M AN U
187 188
6
100.5 ± 15.3 3.6 ± 4.5 3.7 ± .3.3 8.1 ± 2.4 4.6 ± 2.6 61.9 ± 1.2 25.4 ± 7.2 7.5 ± 1.6 4.6 ± 2.7 22.2 ± 4.2
ACCEPTED MANUSCRIPT
Table 3: Number of milk and dairy products exceeds the EC and Malaysia limit for AFM1
No. of Positive Samples n (%)
Powdered
11
(3) 27.3
1
Liquid
12
(4) 33.3
-
3-in-1 Beverages
8
(1) 12.5
Condensed sweetened milk
6
(3) 50.0
Evaporated milk
2
Cultured milk drink
7
Yogurt
5
Cheese
a
SC 1
-
-
-
-
-
(4) 57.1
1
-
(2) 40.0
-
-
2
(2) 100.00
-
-
53
(19) 35.8
4
-
EP
For milk, yogurt, the EC limit is 50 ng/L (50 pg/ml) ;For cheese EC limit is 250 ng/kg (250 pg/ml) b For all milk and dairy products, the Malaysia limit is 0.5μg/L (500 ng/L) A study at Minas Gerais, Brazil in 2009 reported the occurrence of AFM1 in 75 UHT liquid milk samples, where 21 (30.7%) samples were contaminated by AFM1 with the level ranging from 1000 to 4100 ng/L (de Oliveira et al., 2013). Moreover, in Thailand, Ruangwises & Saipan (2010) found AFM1 in all 240 raw milk samples ranging from 50 to 101 ng/L. Both studies in Brazil and Thailand reported higher value of AFM1 contamination in liquid milk compared to the present study (7.3-11.8 ng/L). Besides, it was also observable that the contamination level of AFM1 in samples of raw cow milk from Syria (Ghanem & Orfi, 2009), milk samples from Punjab, Pakistan (Sadia et al., 2012), and UHT milk from China (Zheng et al., 2013) were much higher than the present study, where the level of contamination from these three studies ranged from 2 to 794 ng/L. Galvano et al. (1996) had mentioned that the variation
AC C
202 203 204 205 206 207 208 209 210 211 212 213 214 215
-
0 (0)
TE D
Total
1
No. of Samples Exceed Malaysia Limitb ˃500 ng/L
RI PT
No. of Sample
No. of Samples Exceed EC Limita ˃50 ng/L
Milk and Dairy Products Sample
M AN U
201
7
ACCEPTED MANUSCRIPT
216 217
of AFM1 in milk and dairy products was normally affected by the geographical area, analytical method used and seasons variability.
218
RI PT
In term of powdered milk, one of the three positive samples in the present study had slightly lower AFM1 contamination level compared to a study in Syria, where the AFM1 contamination level of 12 ng/kg was detected in powdered milk (Ghanem & Orfi, 2009). However, when comparing to a study in India, the contamination level of AFM1 in infant formula ranged from 143 to 770 ng/kg (Rastogi et al., 2004), where these values were much higher than the values in powdered milk tested in the present study. Blanco et al. (1988) mentioned that the variations in the contamination level of AFM1 in this food product could be due to the seasonal changes and different processing techniques.
SC
219 220 221 222 223 224 225 226
M AN U
227
AFM1 was detected in 40% of the yogurt samples tested with, the levels ranging from 7.5 to 31 ng/kg. These observations indeed are comparable with a study by El-Khoury et al. (2011) as the authors found relatively lower levels of AFM1 in the Lebanese milk and yoghurt as compared to other regions around the world (Temamogullari & Kanici, 2014; Ertas et al., 2011; Mason et al., 2015). Conversely, Iha et al. (2011) had detected AFM1 as high as 529 ng/kg in yoghurt samples from Brazil. In addition, it was observed that the highest level of AFM1 contamination in yogurt samples tested from Pakistan reached the concentration of 615.8 ng/kg (Iqbal & Asi, 2013). By comparison to the data in the present study and the aforementioned studies (Iha et al., 2011; Iqbal & Asi, 2013), the level of AFM1 contamination of yogurt from India and Pakistan were considered higher than in Malaysia. As previously indicated, seasonal changes and different processing techniques could be attributable to the occurrence of AFM1 in milk products, and the reduction of AFM1 during yoghurt production could be due to several factors such as low pH, formation of organic acids or fermentation by-products and the presence of lactic acid bacteria (Govaris et al., 2002). Moreover, differences in extraction procedures, concentration of toxin, time elapsed before analysis, storage temperature, variability in composition of milk, or differences in the behavior of starter cultures used in preparing the yoghurt also become the factors of different level of AFM1 present in the yogurt. In particular, during the storage period, glucose might be oxidized by glucose oxidase and this reaction can produce hydrogen peroxide and gluconolactone in the yoghurt (Yousef & Marth, 1989). The latter will further undergo hydrolysis process that produces gluconic acid, which is capable to lower the pH (3.9) of the yoghurt and subsequently degrade the AFM1 (Yousef & Marth, 1989).
249 250 251 252 253
Of the two cheese samples tested in the present study, both had detectable level of AFM1. This finding was in agreement with a study in Iran where all of the cheese samples tested was contaminated by AFM1 (Ertas et al., 2011). The high detection of AFM1 in cheese samples was undoubted since many studies reported that approximately more than half of cheese samples
AC C
EP
TE D
228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248
8
ACCEPTED MANUSCRIPT
tested were contaminated by AFM1. For example, in Turkey 82.4% of white brined cheese were contaminated with AFM1 (Ardic et al., 2009). Although all cheese samples in the present study had detectable level of AFM1, the contamination level was very low compared to a study by Tekinsen & Eken (2008) as the authors found AFM1 contamination level of 50 to 690 ng/kg in Kashar cheese sold in Turkey. Since AFM1 is a water soluble compound, a high concentration of AFM1 in cheese sample is undoubted (Dosako et al., 1980). The variation of the cheese samples are generally affected by several factors such as geographical region, cheese-making procedures and conditions of cheese ripening (Fallah et al., 2009).
277 278 279 280 281 282 283 284 285 286 287
In term of 3-in-1 beverages, previous study by Khayoon, Saad, Lee, & Salleh (2013) reported that aflatoxins (AFG2, AFG1, AFB2 and AFB1) were absent in all samples of malt beverages and canned coffee. This finding was nearly similar to the present study where only 1 out of 8 samples were contaminated with AFM1. The 3-in-1 beverages only contained a small amount of dairy based products (creamer) and the additional ingredient might change the AFM1 composition in the samples and hence, 3-in-1 beverages were less likely to be contaminated by AFM1. However since the tested number of samples was small (n= 8), we cannot confidently justify that the food product is safe for the consumer as the analyzed sample was not representable of the whole samples. Besides, the level of AFM1 in the positive sample was over the permissible limit established by EC.
288 289 290 291 292
In this study, only evaporated milk sample did not show the occurrence of AFM1. Evaporated milk had lower fat content and required higher heat treatment compared to condensed sweetened milk production. This could be the reason AFM1 was not detected in the sample. Milk samples with greater fat content have greater but not significantly greater (= 0.067) levels of AFM1 compared to those of lower fat milk (Carvajal et al., 2003).
293 294
The culture milk drink, which normally has probiotic bacteria is potentiated to be used to prevent human dietary aflatoxin exposure (El-Nezami et al., 2006; Mohd Redzwan et al., 2016). The
RI PT
254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276
AC C
EP
TE D
M AN U
SC
Among the 8 groups of milk and dairy products tested, condensed milk showed the highest level of AFM1 occurrence ranging from 3.6 to 100.5 ng/kg and one of the positive samples was above the EC limit. Since this is the first study to screen the occurrence of AFM1 in condensed sweetened milk, the result was not comparable to other findings. Nevertheless, Mohd Redzwan et al. (2012a) reported a borderline significant correlation between the consumption of condensed milk with AFM1 detected in urine samples, ( = ݎ-0.384; = 0.063). However, the correlation was negative which indicated that the higher the consumption of condensed milk, the lower the concentration of urinary AFM1. Generally, high ingestion of AFM1 would reflect the high level of AFM1 detection in urine sample, but not in the study conducted by Mohd Redzwan et al. (2012). Condensed milk is among the top 10 foods mostly consumed by Malaysians (Norimah et al., 2008). This statistic shows that the consumption of condensed milk by Malaysian is quite high compared to other types of milk. Since the present study found the highest level of AFM1 in condensed milk samples, further confirmation on the level of AFM1 by HPLC should be carried out.
9
ACCEPTED MANUSCRIPT
probiotic bacteria in the culture milk act as an adsorbent that binds aflatoxin. Despite that, it also could be contaminated by AFM1 if AFM1-contaminated milk is used in the fermentation process (Lin et al., 2004). Among 7 tested samples of cultured milk drink in the present study, 4 of them were contaminated with AFM1. In Taiwan, the incidence of AFM1 contamination in yoghurt drink (culture milk drink) was 12.5% as reported by Lin et al. (2004). Of 24 samples tested, Lin et al. (2004) found 3 samples contaminated by AFM1, with the concentrations of 7, 9 and 44 ng/L respectively. However, the level of AFM1 in the contaminated cultured drink in this study was slightly higher than in the study reported in Taiwan (Lin et al., 2004). Iha et al. (2011) reported that the concentration of AFM1 found in dairy drinks would be less than that of the milk being used for the process because many other food ingredients are added to the milk during preparation of the products, hence the lower level of AFM1 contamination in this kind of milk products were undoubted.
308 309 310 311 312 313 314 315 316 317 318 319 320
Although all the samples tested in the present study had AFM1 contamination level far below the Malaysian limit, we found four samples with AFM1 contamination level above the EC limit (Table 3). In the South-East Asian region, the study for the occurrence of AFM1 in the milk products had been conducted by our neighbor country, Indonesia, as of 113 fresh milk collected directly from five different areas in Yogyakarta, Indonesia in year 2006, 48 was below 5 ng/L (below LOD), 65 was within 5 ng/L to 25 ng/L and none of them was above 25 ng/L to 50 ng/L (EC limit) (Nuryono et al., 2009). Whereas in Thailand, of 90 samples of raw and pasteurized goat milk, 49 samples were contaminated by AFM1 and 7 of them were above the EC limit (Ruangwises, Saipan & Ruangwises, 2013). Hence, it can be postulated that milk and dairy product samples tested in the present study were more likely to get high level of AFM1 contamination compared to samples from Indonesia but less likely compared to samples from Thailand.
321
4.
322 323 324 325 326 327 328 329
Of 53 samples, only 19 samples were contaminated by AFM1 and 4 of them had AFM1 contamination level above the EC limit. Nevertheless, the contamination level was still far below the Malaysian limit of 500 ng/L. In this study, the highest contamination level of AFM1 was observed in the sweetened condensed milk samples with the contamination level of 100.5 ng/L. Since this study was a limited survey on the occurrence of AFM1 in milk and dairy products purchased in selected cities in Terengganu, Malaysia, further confirmation by using HPLC is warranted. Besides, a larger number of samples of milk and dairy products should be analyzed to represent the whole samples of milk and dairy products mostly consumed by Malaysian.
330
Acknowledgements
331 332 333 334
A. Farah Nadira would like to thank School of Graduate Studies, Universiti Putra Malaysia for the Graduate Research Fellow (GRF) scholarship and the Ministry of Higher Education
335 336
Ardic, M., Y, Karakaya., M, Atasever., & G, Adiguzel. (2009). Aflatoxin M1 levels of Turkish white brined cheese. Food Control, 20, 196-199.
TE D
M AN U
SC
RI PT
295 296 297 298 299 300 301 302 303 304 305 306 307
AC C
EP
Conclusion
References
10
ACCEPTED MANUSCRIPT
Bakirci, I. (2001). A study on the occurrence of aflatoxin M1 in milk and milk products produced in Van province of Turkey. Food Control, 12, 47-51.
339 340 341
Blanco, J., Domingueza, L; Gomez-Lucia, E., Garayzabal, J. F. F., Garcia, J. A., & Suarez, G. (1988). Presence of aflatoxin M1 in commercial ultra-high temperature treated milk. Applied Environmental Microbiology, 56, 1622-1623.
342 343 344
Carvajal, M., Bolonos, A., Rojo, F., & Mendez, I. (2003). Aflatoxin in pasteurized and ultrapasteurized milk with different fat content in Mexico. Journal of Food Protection, Ames, 66 (10), 1885-1892, ISSN 0362-028X
345 346 347
De Oliveria, C.P., N. de Fatima Ferreira Soares, T.V. de Oliveria, J.C. Baffa Junior & W. A. da Silva. (2013). Aflatoxin M1 occurrence in ultra-high temperature (UHT) treated fluid milk from Minas Gerais/ Brazil. Food Control, 30, 90-92.
348 349 350
Dosako, S., Kaminogawa, S., Taneya, S., & Yamauchi, K. (1980). Hydrophobic surface areas and net charges of ߙs1-, k-casein and ߙݏ1-casein: k-casein complex. Journal of Dairy Research, 4(7), 123-129.
351 352
Duarte, S. C., Pena., & A., Lino, C. M. (2011). Human ochratoxin A biomarkers from exposure to effect. Crit. Rev. Toxicol, 41, 187-212.
353 354 355
El Khoury, A., Atoui, A., & Yaghi, J. (2011). Analysis of Aflatoxin M1 in milk and yogurt and AFM1 reduction by lactic acid bacteria used in Lebanese industry. Food Control, 22, 1695-1699. Retrieved from http://dx.doi.org/10.1016/j.foodcont.2011.04.001 on January 14, 2015.
356 357 358 359
El-Nezami, HS, Polychronaki NN, Ma, J, Zhu, H, Ling, W, Salminen, EK, Juvonen RO, Salminen, SJ, Poussa, T, & Mykkänen, HM. (2006). Probiotic supplementation reduces a biomarker for increased risk of liver cancer in young men from Southern China. The American Journal of Clinical Nutrition, 83, 1199-1203.
360 361 362
Er, B., Demirhan, B., & Yentur, G. (2014). Short communication: Investigation of aflatoxurkey. in M1 levels in infant follow-on milks and infants formulas sold in the market of Ankara, Turkey. American Dairy Science Association, 97(6), 3328-3331. 10.3168/jds.2013-7831.
363 364 365
Ertas, N., Gonulalan, Z., Yildirim, Y., & Karadal, F. (2011). A survey of concentration of aflatoxin M1 in dairy products marketed in Turkey. Food Control, 22(12), 1956–1959. doi:10.1016/j.foodcont.2011.05.009
366 367
Fallah, A. A., Jafari, T., Fallah, A., & Rahnama, M. (2009). Determination of aflatoxin M1 levels in Iranian white and cream cheese. Food and Chemical Toxicology, 47(8), 1872-1875.
368 369 370 371
Fallah, A. A. (2010). Assessment of aflatoxin M1 contamination in pasteurized and UHT milk marketed in central part of Iran. Food and Chemical Toxicology : An International Journal Published for the British Industrial Biological Research Association, 48(3), 988–991. doi:10.1016/j.fct.2010.01.014
AC C
EP
TE D
M AN U
SC
RI PT
337 338
11
ACCEPTED MANUSCRIPT
FoSIM (Food Safety Information System of Malaysia) (1985). Food Safety and Quality Division, Department of Public Health, Ministry of Health Malaysia. Retrieved from http://fsis2.moh.gov.my on March 16, 2015.
375 376 377
Food Regulation 1985 (2014). Part III – Regulation 4: Procedure on taking samples for physical and chemical analysis Department of Public Health, Ministry of Health Malaysia. Retrieved http://fsq.moh.gov.my/v5/ms/food-regulations-1985-2 on April 7, 2016.
378 379
Galvano, F., Galofaro, V., & Galvano, G. (1996). Occurrence and stability of aflatoxin M1 in milk and milk products: a worldwide review. J. Food Prot. 59, 1079–1090.
380 381 382 383
Garcia Londoño, V. A., Boasso, A. C., de Paula, M. C. Z., Garcia, L. P., Scussel, V. M., Resnik, S., & Pacín, A. (2013). Aflatoxin M1 survey on randomly collected milk powder commercialized in Argentina and Brazil. Food Control, 34(2), 752–755. Retrieved from http://www.sciencedirect.com/science/article/pii/S0956713513003137
384 385 386
Ghanem, I., & Orfi, M. (2009). Aflatoxin M1 in raw, pasteurized and powdered milk available in the Syrian market. Food Control, 20(6), 603–605. Retrieved from http://www.sciencedirect.com/science/article/pii/S0956713508002429.
387 388 389
Govaris, A., Roussi, V., Koidis, P.A., & Botsoglou, N.A. (2002). Distribution and stability of aflatoxin M1 during production and storage of yogurt. Food Additives and Contaminant. 19, 1043-1050
390 391
Groopman, J. D., Cain, L.G., & Kensler, T. W. (1988). Aflatoxin exposure in human populations: measurements and relationship to cancer. Crit Rev Toxicol 19, 113–46.
392 393 394
IARC. (1993). Aflatoxin. In Some naturally occurring substances: Food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC monograph on the evaluation of carcinogenic risk to humans, 56, 245-395. Lyon: International Agency for Research on Cancer.
395 396
IARC. (2002). IARC monograph on the evaluation of carcinogenic risk to humans, 82, 171. Lyon: International Agency for Research on Cancer-World Health Organization.
397 398
Iha, M. H., Barbosa, C. B., Okada, I. A., & Trucksess, M. W. (2011). Occurrence of aflatoxin M1 in dairy products in Brazil. Food Control, 22, 1971-1974.
399 400
Iqbal, Z., & Asi, M. R. (2013). Assessment of aflatoxin M1 in milk and milk products from Punjab, Pakistan. Food Control, 30(1), 235–239. doi:10.1016/j.foodcont.2012.06.026.
401 402 403
Khayoon, W. S., Saad, B., Lee, T. P., & Salleh, B. (2012). High performance liquid chromatographic determination of aflatoxins in chili, peanut and rice using silica based monolithic column. Food Chem. 133, 489–496. doi: 10.1016/j.foodchem.2012.01.010.
404 405 406
Lee, J. E., Kwak, B.-M., Ahn, J.-H., & Jeon, T.-H. (2009). Occurrence of aflatoxin M1 in raw milk in South Korea using an immunoaffinity column and liquid chromatography. Food Control, 20(2), 136–138. doi:10.1016/j.foodcont.2008.03.002
AC C
EP
TE D
M AN U
SC
RI PT
372 373 374
12
ACCEPTED MANUSCRIPT
Leong, Y.-H., Latiff, A. a, Ahmad, N. I., & Rosma, A. (2012). Exposure measurement of aflatoxins and aflatoxin metabolites in human body fluids. A short review. Mycotoxin Research, 28(2), 79–87. doi:10.1007/s12550-012-0129-8
410 411
Lin, L., Liu, F., Fu, Y., & Shih, D. Y. (2004). Survey of Aflatoxin M1 Contamination of Dairy Products in Taiwan, 12(2), 154–160.
412 413 414
Mason, S., Arjmandtalab, S., Hajimohammadi, B., Arsanjani, A. K., Karami, S., Sayadi, M., & Oryan, A. (2015). Aflatoxin M 1 Contamination in Industrial and Traditional Yogurts Produced in Iran, 2, 11–14.
415 416 417
Marnissi, B. E., Belkhou, R., Morgavi, P. D., Bennani, L., & Boudra, H. (2012). Occurrence of aflatoxin M1 in raw milk collected from traditional dairies in Morocco. Food and Chemical Toxicology, 50, 2819-2821
418 419 420 421
Martins, M. L., & Martins, H. M. (2004). Aflatoxin M1 in yoghurts in Portugal International Journal of Food Microbiology, 91, 315–317 Mohammadi, H., & Alizadeh, M. (2010). A Study of the Occurrence of Aflatoxin M1 in Dairy Products Marketed in Urmia , Iran, 12, 579–583.
422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Nurul ‘Aqilah, A.R. (2012a) Association between aflatoxin M1 excreted in human urine samples with the consumption of milk and dairy products. Bulletin of Environmental Contamination and Toxicology, 89, 1115-1119.
438 439 440
Mohd Redzwan, S., Jamaluddin, R., Mohd Sokhini, A.M., Nurul ‘Aqilah, A.R., Zurain, A., Karimi, G., Parvaneh, K. (2015). Ultra-high performance liquid chromatographic determination of aflatoxin M1 in urine. World Mycotoxin Journal, 8, 405-413. 2014
441 442 443 444 445
Mohd Redzwan, S., Abd. Mutalib, M.S., Wang, J.S., Ahmad, Z., Kang, M.S., Abdul Rahman, N.A., Nikbakht Nasrabadi, E., & Jamaluddin, R. (2016). Effect of supplementation of fermented milk drink containing probiotic Lactobacillus casei Shirota on the concentrations of aflatoxin biomarkers among employees of Universiti Putra Malaysia: a randomized, double-blind, cross over, placebo-control study. British Journal of Nutrition, 115, 39-54
M AN U
SC
RI PT
407 408 409
TE D
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Nurul ‘Aqilah, A.R. (2012b) Sociodemographic and socio-economic determinants of adults’ knowledge on fungal and aflatoxin contamination in the diets. Asian Pacific Journal of Tropical Biomedicine, 2 (3), Supplement, S1835-S1841.
EP
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., & Zuraini, A. (2013). A mini review on aflatoxin exposure in Malaysia: past, present and future. Frontier in Microbiology, 4 (334), 1-8.
AC C
Mohd Redzwan, S., Rosita, J., Mohd Sokhini, A.M., Nurul ‘Aqilah, A.R., Wang, J-S., & Kang, M-S., Zuraini, A. (2014) Detection of serum AFB1-lysine adduct in Malaysia and its association with liver and kidney functions. International Journal of Hygiene and Environmental Health, 217, 443-451.
13
ACCEPTED MANUSCRIPT
Norimah. A. K., Safiah, M., Jamal. K., Haslinda, S., Zuhaida, H., Rohida, S., Fatimah, S., Norazlin, S., Poh, B. K., Kandiah, M., Zalilah, M. S., Wan Manan, W. M., Fatimah, S., & Azmi, M. Y. (2008). Food Consumption Patterns: Findings from the Malaysian Adult Nutrition Survey(MANS). Mal J Nutr, 14 (1), 25 - 39.
450 451 452
Nuryono, N., Agus, a., Wedhastri, S., Maryudani, Y. B., Sigit Setyabudi, F. M. C., Böhm, J., & Razzazi-Fazeli, E. (2009). A limited survey of aflatoxin M1 in milk from Indonesia by ELISA. Food Control, 20(8), 721–724. doi:10.1016/j.foodcont.2008.09.005
453 454 455
Rastogi, S., Dwivedi, P. D., Khanna, S. K., & Das, M. (2004). Detection of Aflatoxin M1 contamination in milk and infant milk products from Indian markets by ELISA. Food Control, 15(4), 287–290. doi:10.1016/S0956-7135(03)00078-1
456 457 458
Ruangwises, S., & Saipan, P. (2010). Occurrence of Aflatoxin M1 in Raw and Pasteurized Goat Milk in Thailand. Aflatoxins - Recent Advances and Future Prospects. Retrived from http://dx.doi.org/10.5772/52723 February 17, 2014
459 460 461
Sabran, M.R., Jamaluddin, R., & Abdul Mutalib, M.S. (2012) Screening of aflatoxin M1, a metabolite of aflatoxin B1 in human urine samples in Malaysia: A preliminary study. Food Control, 28, 55-58.
462 463
Sadia, A., Jabbar, M. A., Deng, Y., Hussain, E. A, Riffat, S., & Naveed, S. (2012). A survey of aflatoxin M1 in milk and sweets of Punjab, Pakistan. Food Control 26, 235–240
464 465
Temamogullari, F., & Kanici, A. (2014). Short communication: Aflatoxin M1 in dairy products sold in Şanlıurfa, Turkey. Journal of Dairy Science, 97(1), 162–5. doi:10.3168/jds.2012-6184
466 467
Tekinsen, K. K., & Eken, H. S. (2008). Aflatoxin M1 levels in UHT milk and Kashar cheese consumed in Turkey. Food and Chemical Toxicology, 46, 3287-3289.
468 469
Yousef, A.E., & Marth, E.H., (1989). Stability and degradation of aflatoxin M1. In: Van Egmond, H.P. (Ed.), Mycotoxins in Dairy Products. Elsevier Applied Science, London, 127–161.
470 471
Zheng, N., Sun, P.,Wang, J. Q., Zhen, Y. P., Han, R.W., & Xu, X. M. (2013). Occurrence of aflatoxinM1 in UHT milk and pasteurized milk in China market. Food Control, 29,198-201
472 473
Figure
477
SC
M AN U
TE D
EP
AC C
474 475 476
RI PT
446 447 448 449
% Absorbance = Mean Absorption Standard or Sample x 100 Absorption Zero Standard
Figure 1: Formula for % Absorbance (Helica Aflatoxin M1 ELISA Quantitative test kit)
14
ACCEPTED MANUSCRIPT
Highlights
EP
TE D
M AN U
SC
RI PT
35.8% samples were positive with AFM1 Range of contamination from 3.5 to 100.5 ng/L 7.5% of the samples higher than the European Commission limit No samples exceed the Malaysia Food Regulation 1985 limit
AC C
• • • •