Binding ability of aflatoxin M 1 to yoghurt bacteria

Ankara Üniv Vet Fak Derg, 51, 2004
195
Ankara Üniv Vet Fak Derg, 51, 195-198, 2004


Binding ability of aflatoxin M1 to yoghurt bacteria

Belgin SARIMEHMETO?LU, Özlem KÜPLÜLÜ

Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, University of Ankara, 06110, Diskapi, Ankara,
Turkey


Summary: Aflatoxin M1 (AFM1) is a highly toxic compound found in milk. Several microorganisms have been previously
reported to bind or degrade AFM1 from liquid media. This study was performed to assess the binding of AFM1 in contaminated phos-
phate buffered saline (PBS). Lactobacillus delbrueckii subsp. bulgaricus CH-2 and Streptococcus thermophilus ST-36 were used for
this purpose. Removal activities of two strains were also assessed using contaminated reconstituted milk and contaminated yoghurt
made from reconstituted milk. ELISA procedure was used in this study Lactobacillus delbrueckii subsp. bulgaricus CH-2 bound in
PBS at 18.70% and in milk at 27.56% while Streptococcus thermophilus ST-36 bound in PBS at 29.42% and in milk at 39.16%.
AFM1 was bound at the level of merely 14.82% in yogurt. The results indicated that binding ability of Streptococcus thermophilus
ST-36 was higher than that of Lactobacillus delbrueckii subsp. bulgaricus CH-2 in both PBS and reconstituted milk. Both of micro-
organisms bound higher in milk than in PBS. Also, AFM1 binding levels were at least level in yoghurt (%14.82). These findings
supported that specific yoghurt bacteria used in this study can offer decontaminating AFM1 from milk.
Key words: Aflatoxin M1, binding, yoghurt bacteria
Aflatoksin M1’in yo?urt bakterilerine ba?lanma yetene?i
Özet: Bu çal??mada, kontamine PBS’de AFM1’in ba?lanma yetene?i ara?t?r?ld?. Bu amaçla, Lactobacillus delbrueckii subsp.
bulgaricus CH-2 ve Streptococcus thermophilus ST-36 bakterileri kullan?ld?. AFM1’in bu iki bakteriye ba?lanma yetene?i, ayn?
zamanda kontamine sütte ve kontamine sütten yap?lan yo?urtta da ara?t?r?ld?. Çal??mada, ELISA yöntemi kullan?ld?. Lactobacillus
delbrueckii subsp. bulgaricus CH-2’nin AFM1’i PBS’de % 18.70, sütte % 27.56; Streptococcus thermophilus ST-36’n?n ise PBS’de
% 29.42 ve sütte % 39.16 düzeylerinde ba?lama yetene?inde oldu?u belirlendi. Yo?urtta ise ba?lanma en dü?ük düzeyde saptand?(
% 14.82). Sonuç olarak, gerek PBS’de gerekse sütte Streptococcus thermophilus’un ba?lama yetene?i, Lactobacillus delbrueckii
subsp. bulgaricus CH-2’den daha yüksek bulundu. Ayn? zamanda her iki bakterinin de PBS’e göre sütte daha fazla ba?lama yetene?i
oldu?u saptand?. Elde edilen bulgulara göre, bu çal??mada kullan?lan spesifik yo?urt bakterilerinin AFM1’i sütten uzakla?t?rma yete-
ne?inde oldu?u belirlendi.
Anahtar sözcükler: Aflatoksin M1, ba?lanma, yo?urt bakterileri



Introduction
greatest demonstrated potential for introducing aflatoxins
Aflatoxins are the most potent toxic, mutagenic,
residues from foods of animal origin into the human diet
teratogenic and carcinogenic metabolites produced by the
and is also the main nutrient for infants and children, the
species of Aspergillus flavus, Aspergillus flavus subsp.
occurrence of aflatoxin M1 in milk and dairy products is
parasiticus and Aspergillus nomius on food and feed
a concern (9,10,12,21,22). The best way to control the
materials. There are four main toxins which have been
presence of AFB1 in foods and feeds is to prevent their
divided into B and G groups (B
formation. Various physical, chemical and biological
1, B2, G1 and G2). Of
these, aflatoxin B
agents have been used to detoxify aflatoxins from food
1 is most toxic and most carcinogenic.
Aflatoxin M
and feed materials (2,11,20). But there are currently no
1, a hydroxylated metabolite of aflatoxin B1
is an important toxin present in the milk of lactating
acceptable chemical, physical or biological methods to
animals fed with aflatoxin B
counteract the AFM
1 contaminated feeds. Pres-
1 problem in milk (24). Thus, a prac-
ence of aflatoxin M
tical and effective method is needed to be developed for
1 in milk is a public health hazard.
There is a general consensus that approximately 1-3% of
the detoxification of AFM1 contaminated milk.
the aflatoxin B
Some strains of lactic acid bacteria have been
1 initially present in the animal feedstuff
appears as aflatoxin M
reported to be effective in removing AFB
1 in milk (5,8,18,23). Evidence of
1 and AFM1
potential hazardous human exposure to AFM
from contaminated liquid media and milk(1, 14, 16, 17).
1 from dairy
products arises from many studies on the occurrence of
For this purpose, this study was carried out in order to
AFM
investigate the ability of Lactobacillus delbrueckii subsp.
1 in dairy products (15,27). Since milk has the

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196
Belgin Sar?mehmeto?lu - Özlem Küplülü
bulgaricus and Streptococcus thermophilus to remove
Bacterial pellets were collected as described earlier,
AFM1 from contaminated phosphate buffered saline
but bacterial pellets were suspended in contaminated
(PBS) and reconstituted skim milk. Removal activities of
nonfat reconstituted milks. Stock solution (1 µg AFM1/ 1
these strains were also assessed in fermented milk prod-
ml benzene/acetonitrile) was evaporated to dryness under
uct such as yoghurt because of symbiotic relationship.
a smooth N2 stream. The AFM1 residue was redissolved

in 1 ml methanol. A volume of 0.01 ml was transferred
Materials and Methods
from the contaminated methanol to 1.5 ml of reconsti-
tuted skim milk, resulting in milk containing 10 ng/ml
Standard of AFM1
AFM
Solid AFM
1. Bacterial pellets was suspended in reconstituted
1 (Sigma) was suspended in benzene-
milk contaminated with AFM
acetonitrile (97/3, vol/vol) to obtain an AFM
1 and incubated at 37°C for
1 concentra-
4 h. After incubation period, suspensions were centri-
tion of 1 µg/ml and 5 µg/ml.
fuged. Unbound AFM1 content in the supernatant was
Culture preparation
determined by ELISA (14). Each sample for the ELISA
Lactobacillus delbrueckii subsp. bulgaricus CH-2
analysis was diluted 1:125 in PBS.
and Streptococcus thermophilus ST-36 were originally
Cell-free reconstituted milk contaminated with
obtained from Chr. Hansen’s Lab (Denmark). Lactoba-
AFM1 was used for positive control. Bacteria suspended
cillus delbrueckii subsp. bulgaricus CH-2 was cultivated
in reconstituted milk were used for negative control. All
in 25 ml MRS broth (Oxoid CM 359) at 37°C for 24 h.
assays were performed at control groups, too. Procedure
Streptococcus thermophilus ST-36 was cultivated in 25
of contamination with AFM1 in reconstituted milk was
ml M17 broth (Oxoid CM817) at 37°C for 24 h. The
that of Pierides et al (17).
bacterial growth was determined at MRS agar (Oxoid
Contamination of reconstituted skim milk and
CM361) for Lactobacillus delbrueckii subsp. bulgaricus
yoghurt production
CH-2 and M17 agar (Oxoid CM785) for Streptococcus
Yoghurt was made from the reconstituted skim milk
thermophilus ST-36 after 24 hours incubation at 37°C
presented 12% nonfat dry matter. Prepared skim milk
using traditional plate counting. At the same time, culti-
was heated at 90°C for 5 min and then cooled to 42°C.
vation broths were centrifuged at 3500 x g for 15 min.
Stock solution (5 µg/ml AFM1 in ben-
The bacterial pellets were washed with PBS (Oxoid
zene/acetonitrile) was collected as described earlier. But
BR14a) twice.
AFM1 residue was redissolved in 2 ml methanol. A vol-
ume of 0.08 ml was transferred from the contaminated
Contamination with AFM1 in PBS
methanol to 20 ml of skim milk, resulting in milk con-
A solution of 10 ng AFM1/ml PBS was prepared for
taining 10 ng/ml AFM
the assay. The benzene/acetonitrile derived from the
1. After that, 20 ml milk was in-
oculated with 2% starter cultures.The ratio of Lactobacil-
stock was evaporated by heating in a water bath at 80°C.
lus delbrueckii subsp. bulgaricus CH-2 : Streptococcus
Bacterial pellets were suspended in 1.5 ml PBS contami-
thermophilus ST-36 was 1:1.
nated with AFM1 and incubated at 37°C for 4 h. Bacterial
Cell-free reconstituted milk contaminated with
suspensions were then centrifuged at 3500 x g for 10
AFM1 was used for positive control, yoghurt made from
min. Unbound AFM1 content in the supernatant was
reconstituted milk and uncontaminated as negative con-
determined by ELISA. Each sample for the ELISA
trol. All groups were incubated at 42°C for 4 h. Yoghurt
analysis was diluted 1:125 in PBS. ELISA procedure was
was centrifuged at the end of incubation and unbound
performed according to R-biopharm GmbH recommen-
AFM1 content in the supernatant was determined by
dations. Binding of AFM1 by Lactobacillus delbrueckii
ELISA. Each sample for the ELISA analysis was diluted
subsp. bulgaricus CH-2 and Streptococcus thermophilus
1:125 in PBS. ELISA procedure was performed accord-
ST-36 cells was analysed according to Pierides et al (17).
ing to R- biopharm GmbH recommendations.
Cell-free PBS contaminated with AFM1 was used for
In this study, all assays were performed five times
positive control. Bacteria suspended in PBS were used
and both positive and negative controls were included.
for negative control. All assays were performed at con-
trol groups, too.
Statistical analysis
The variance analysis (with two factors) was done
Milk contamination with AFM1
for determining the difference as binding amount of
Reconstituted milk with 12% nonfat dry matter was
aflatoxin M1 in two medium of two bacteria. In addition,
prepared from skim milk powder ( easy soluble skim
one-way ANOVA variance analysis was also done for
milk powder, PINAR) in distilled water. A portion of the
comparison of binding in yoghurt. DUNCAN test was
reconstituted milk was used for artificial AFM1 contami-
used for determining the different groups after the one-
nation. The rest was used for negative control.
way variance analysis.

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Ankara Üniv Vet Fak Derg, 51, 2004
197
Results
ability of 12 Lactobacillus, 5 Bifidobacterium and 3
In this study, in vitro binding ability of AFM
Lactococcus in PBS. In their study, Lactobacillus strains
1 to
Lactobacillus delbrueckii subsp. bulgaricus CH-2 and
bound by 17.3-59.7% AFB1, Bifidobacterium strains by
Streptococcus thermophilus ST-36 was investigated in
18.0-48.7% and Lactococcus strains by 5.6-41.1% AFB1.
the liquid medium (PBS), reconstituted milk and yoghurt
El-Nezami et al (7) observed that L. rhamnosus GG and
comparatively.
L. rhamnosus LC 705 bound to AFB1 by 80% in 24 h.
Comparing two strains for statistical analysis, Strep-
These studies suggested that significantly different bind-
tococcus thermophilus ST-36 showed significantly high
ing abilities of lactic acid bacteria were due to different
(p< 0.01) percentage of AFM
cell-wall structure. Thus, in this study binding ability of
1 binding ability according
to Lactobacillus delbrueckii subsp. bulgaricus CH-2 in
yogurt cultures examined were found different. Also,
PBS and milk (Table 1). On the other hand the percent-
Pierides et al (17) reported that L. rhamnosus 1/3 pre-
age of removal activity of both Lactobacillus delbrueckii
sented a less binding ability than L. rhamnosus GG in
subsp. bulgaricus CH-2 and Streptococcus thermophilus
spite of the same genetic structure, and they presumed
ST-36 in PBS showed significant differences (p< 0.01)
that this was caused by different biological activities of
according to milk. At the same time the differences be-
the strain.
tween milk and yoghurt were found statistically impor-
When the binding ability of yoghurt cultures in PBS
tant (P< 0.01) (Table 1).
and reconstituted milk were compared, the binding was

much greater in milk (Table 1). The principal reason of
Table 1. Comparison of strains-media and yoghurt
that may be due to the binding properties of aflatoxin to
Tablo 1. Mikroorganizma-ortam ve yo?urdun kar??la?t?r?lmas?
casein. So, Brackett and Marth (3) reported that an aver-

age of 30.7% more AFM
Strain-Media
X± SX
1 was found in milk treated with
proteolytic enzyme than in untreated milk and they sug-
Lactobacillus delbrueckii subsp. bulgaricus
C18.7± 0.582
gested that AFM
CH-2
1 is bound by milk protein. Also, the
PBS
same authors (4) reported that AFM1 did not display a
Lactobacillus delbrueckii subsp. bulgaricus
B27.56±0.699
homogeneous distribution in milk and a part of AFM1
CH-2
could not be extracted from milk. Tabata et al (25) re-
Milk
ported that milk concentrations had an effect on AFM1.
Streptococcus thermophilus ST-36
B29.42± 0.601
Pierides et al (17) reported that contrarly to this study,
PBS
Streptococcus thermophilus ST-36
A39.16± 0.459
binding ability of AFM1 to L. rhamnosus GG and L.
Milk
rhamnosus LC-705 was less in milk.
Yoghurt
D14.82± 0.558
It was seen that the binding after yoghurt manufac-


turing was less than that in milk, examined separately
Discussion
(Table 1). This may be caused by fermentation, which is
It was determined that Streptococcus thermophilus
greater in yogurt than in milk and by the fact that AFM1,
ST-36 has a more binding ability in comparison Lacto-
which is bound to casein is extracted better than
bacillus delbrueckii subsp. bulgaricus CH-2 both in PBS
milk(19). Van Egmond et al (26) found AFM1 was re-
and reconstituted milk (Table 1).
covered in slightly greater amounts from yoghurt than
Lactic acid bacteria are known to bind aflatoxins.
from the original milk. They believe the increased AFM1
Recently, heat-killed bifidobacteria have been reported to
content in yoghurt possibly results from a more complete
bind aflatoxin B
recovery of AFM
1 in PBS (14). Lactobacillus rhamnosus
1 from yoghurt than milk. Munksgaard
GG, Bifidobacterium bifidum BGN4, Bifidobacterium sp.
et al (13) found the level of AFM1 during production of
JO3, Bifidobacterium longum JR 20 and Bifidobacterium
yoghurt to be increased on average by 9%. They ex-
sp. CH4 bound to AFB
plained that AFM
1 by 37±1%, 46±4%, 41±3%,
1 is extracted better from cultured prod-
37±3% and 37±1%, respectively. Pierides et al (17) re-
ucts. At the same time, the binding abilities may be de-
ported that viable L. rhamnosus GG bound to AFM
creased because of synergetic reproduction in yoghurt,
1 by
77±0.4%, L. rhamnosus LC-705 bound by 75.2±1.2%
although it is reported by El-Nezami (6) that the binding
and L. gasseri (ATCC 33323) bound by 51.4±1.9% after
abilities increased in acid treatment in PBS experimen-
4 h incubation in PBS. However the binding ability of
tally . In fact the binding determined in yoghurt, was
heat-killed same bacteria was determined as 57.8±3.3%,
found to be even less than the bindings determined sepa-
51.6±3.0% and 61.5±0.7%, respectively after the 15-16 h
rately in PBS.
incubation in PBS. In the same study, binding of AFM
In this study it was determined that both Lactobacil-
1
to both viable and heat-killed L. rhamnosus GG was
lus delbrueckii subsp. bulgaricus CH-2 and Streptococ-
reported as 18.8±1.9% and 26.6±3.2%; L rhamnosus LC-
cus thermophilus ST-36 have binding abilities during
705 was reported as 69.6±0.9% and 27.4±4.8% in skim
yoghurt manufacturing. Thus, it could be suggested that
milk. Peltonen et al (16) investigated the AFB
yoghurt cultures could be used in the removal of AFM
1 binding
1

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198
Belgin Sar?mehmeto?lu - Özlem Küplülü
from food and feed. Still, more research is required, e.g.
15. Oruc H, Sonal S (2001): Determination of aflatoxin M1
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amounts and dry-matter amounts. Conducting more ex-
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16. Peltonen K, El-Nezami H, Haskard C, Ahokas J,
in the protection from aflatoxins, a major public health
Salminen S (2001): Aflatoxin B1 binding by dairy strains
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2152-2156.
17. Pierides M, El-Nezami H, Peltonen K, Salminen S,
properties of bacteria.
Ahokas J (2000): Ability of dairy strains of lactic acid

bacteria to bind aflatoxin M
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