INTRODUCTION
In the last few years, the contamination of milk is considered as one of the
main dangerous aspects. Milk can be contaminated by residues of organochlorine
(OC) and organophosphorous (OP) pesticides (Fontcuberta
et al., 2008), through a variety of sources. The major source of
(OC) residues is from fodder and soil, while for (OP) residues is mainly associated
with ingestion (through licking) of insecticide used for controlling of parasites
on animals (Snelson and Tuinstra, 1979; Waliszewski
et al., 1997). Pesticides are used for controlling weed, plant pests
and diseases. As a result, their residues are transferred to milk (Ismail
et al., 1987). Pesticides have played an important role in the dramatic
increases in agricultural productivity which has been achieved in the developed
world over the last few decades. Production and use of organochlorine pesticides
has been declining in recent years. This suggests that residue problems will
decrease but the persistence of these materials in the environment means that
some residues will be encountered for many years (Fries
et al., 1969). OC pesticides were widely used worldwide until restrictions
were introduced in the late seventies both in Europe and the USA, initially
for DDT (Fontcuberta et al., 2008). Some of these
pesticides are still widely used by farmers because of their effectiveness and
their broad spectrum activity (Amoah et al., 2006)
and also, are being extensively used in tropical countries in malaria control
programs and against livestock ectoparasites and agricultural pests (Curtis,
1994). Pesticide exposure independently or in synergism with modifiable
risk factors, is recognized as an important environmental risk factor associated
with hemopoetic cancers, cancers of the prostate, pancreas, liver and other
body systems (Jaga and Dharmani, 2005). No entirely
safe pesticides have been developed yet. It behooves us to emphasize that milk
and dairy products can be contaminated by pesticides by improper handling and
by feeding the animals on contaminated feeds. The misuse of any of such pesticides
in dairy farms and for dairy industries may endanger the health of the consumer
(Mitchell et al., 1986). Besides presence of
pesticides may lead to some technological problems. Many researchers studied
growth rate and behavior of some bacterial starters in different of media containing
insecticides. On the contrary, little information is available on the effect
of fungicides, herbicides and insecticides on pure cultures of bacterial used
in food and dairy industries. The present work was conducted to study the effect
of two fungicides, herbicides and insecticides commonly used in Egypt, on the
bacterial growth and lactic acid, acetaldehyde and diacetyl production of certain
important lactic acid bacteria which are used in dairy industry.
MATERIALS AND METHODS
Bacterial strains:Lyophilized culture of Streptococcus salivarius
subsp. thermophilus H, Lactobacillus acidophilus (type 145) and
Bifidobacterium spp. 420 were obtained from Laboratorium wiesby, (2007)
Niebull, Germany. Lactobacillus delbrueckii subsp. burglarious,
Lactobacillus casei subsp. casei, yoghurt and ABT cultures were
obtained from Chr. Hansen's Lab., Denmark (2008).
Milk and skimmed milk: Fresh buffaloes' milk were obtained from El-Serow Station for Animal Production Research and spray dried skim milk powder, low heat, of France origin was used during this study.
Pesticides: Six pesticides commonly used in Egypt were obtained from local market. Two fungicides, Anadol 80% WP {manganese ethylenebis (dithiocarbamate) polymeric complex with zinc salt} and Tazolen 72% WP {methyl N-(methoxyacetyl)-N-(2,6-xylyl)-DL-alaninate and manganese ethylenebis (dithiocarbamate) polymeric complex with zinc salt}, two herbicides, Round up 48% WSC {N-(phosphonomethyle) glycine, isopropylammonium} and Saturn 50% (5-4 chlorobenzyl diethylthiocarbamate) and two insecticides, Lannate 90% SP {5-methyl N-(methyl carbamoyloxy) thioacetimidate} and Reldan 50% EC {0-0 diethyl 0-( 3, 5, 6 trichloro-2-pyridyl)-phosphorothioate.
Preparation of solution pesticides: A stock solution for each pesticide in 5% (v/v) ethanol was prepared. Samples (media or milk) were artificially contaminated by adding sufficient stock solution, distilled water and by serial concentrations were used to give desired concentrations.
Effect of pesticides residues on growth certain lactic acid bacteria:
This was determined by plate count according to Elliker
et al. (1956). Separately pesticides were added to selective media
immediately before inoculation with bacteria under study at concentrations 0.0,
1, 2, 3, 4, 5, 7 and 10 ppm.
Effect of pesticides residues on the acid production by certain lactic acid
bacteria a-Single strains of lactic acid tested bacteria: Reconstituted
skimmed milk powder (11%TS) was filled in 100 mL lots into 250 mL conical flasks
and autoclaved at 121°C/15 min. The pesticides (0.0, 2, 4, 6, 8 and 10 ppm)
were added under sterile conditions to each flask. High activity cultures of
bacteria under study were separately inculcated at level 2% into the flasks
containing pesticides to give initial appropriate counts. The All flasks were
incubated at 37-40°C with the exception of those Bifidobacterium
sp. was incubated anaerobically. During this period the titratable acidity was
determined as shown in Table 4-8.
In traditional and ABT yoghurt: Fresh buffaloes' milk (standardized 3%fat) was divided into thirty two 200 mL aliquots. Two served as a control, five 200 mL aliquots were contaminated with Anadol at levels 2, 4, 6, 8 and 10 ppm, this procedure has been pursued in the other pesticides (Tasolen, Round up, Saturn, Lannate and Reldan), all 32 (200-aliquots) were heated to 63°C for 30 min. and subsequently cooled to 40°C followed by addition 2% of traditional (zabady) culture. All treatments were incubated at 40°C. The same work was done in ABT yoghurt. Titratable acidity was determined as shown in Table 9 and 10, acetaldehyde and diacetyl were determined on the sixth day of cold storage.
Bacterial count: Bifidobacteria were enumerated according to Dave
and Shah (1996) using modified MRS agar supplemented with 0.05% L-cystein
and 0.3% lithium chloride. The plates anaerobically incubated at 37°C for
48 h.
Lctobacilli: L. acidophilus was enumerated according to Gilliland
and Walker (1990) using modified MRS agar supplement with 0.2% Oxagal. The
plates incubated at 37°C for 48 h. L. bulgaricus and L. casei
counts were determined using MRS agar according to De Man
et al. (1960) and the plates incubated at 40°C for 48 h.
St. theromophilus: St. theromophilus count was determined
using M17 agar (Terzaghi and Sandine, 1975). The plates
incubated at 37°C for 48 h.
Chemical analysis
Titratable acidity: Titratable acidity expressed as lactic acid (%) was
determined according to Ling (1963).
Determination of acetaldehyde:Acetaldehyde was estimated as given by
Lees and Jago (1969) using Conway micro diffusion-semicarbazide
method. One milliliter of micromole semicarbazide solution was pipetted in inner
wall of Conway micro diffusion cell. Three grams of the sample were rapidly
pipetted the outer and the cell was covered and placed in an incubator at 30°C
for 90 min. The solution in the inner wall transferred to 10 mL volumetric flask
and made up to volume. The absorption was measured at 224 nm using type CE 595
Double Beam Digital U.V. Spectro-photometer. The concentration of acetaldehyde
was calculated from standard curve of acetaldehyde solution ranging from 1 μmol
to 20 μmol/100 mL mixed with raw milk and treated as samples.
Determination of diacetyl: Diacety was estimated as described by Westerfeld
(1945) with some modification: five grams of samples were mixed with 15%
trichloroacetic acid solution to 50 mL; the solution was filtered then through
Whatman No. 42 filter paper. The filtrate was readjusted to the same pH as before,
then 1 mL filtrate was mixed with 4 mL distilled water, 1 mL of 0.5% creatine
and 1 mL 5% alpha-naphthol solution (in 2.5 N-NaOH) after 10 min at room temperature,
the optical density was measured at 540 nm after 10 min at room temperature.
The optical density was measured at 540 nm after 10 min using Double Beam Digital
U.V. Spectro photometer type (E595). The diacetycontent was estimated from a
standard calibrated curve for solution containing known concentration of pure
diacetyl.
RESULTS AND DISCUSSION
Effect of pesticides on bacterial growth: Initially, growth all studied
lactic acid bacteria was inhibited by pesticides. The inhibition increased with
increasing concentration of pesticides. Table 1-3
explain that all studied strains were strongly affected by the presence of fungicides,
herbicides and insecticides pesticides. From Table 1 it can
be observed that L. acidophilus was more resistant to Anadol followed
L. casei, St. theromophilus, L. bulgaricus then Bifidobacterium
spp. which was the most inhibition of Anadol and Tasolen. However, L. bulgaricus
was more resistant than other cultures in the existence Tasolen. Generally,
the last fungicide was more inhibition of tested lactic acid bacteria than Anadol.
| Table 1: |
Count of lactic acid bacteria in the present fungicides |
 |
| B. sp.: Bifidobacterium sp.; L. a: Lactobacillus
acidophilus; L. b: Lactobacillus delbrueckii subsp. burglarious;
L. c: Lactobacillus casei subsp. casei; St. th: Streptococcus
salivarius subsp. thermophilus. These results the average of
three replicates |
| Table 2: |
Count of lactic acid bacteria in the present herbicides |
 |
| B. sp.: Bifidobacterium sp.; L. a: Lactobacillus
acidophilus; L. b: Lactobacillus delbrueckii subsp. Burglarious;
L. c: Lactobacillus casei subsp.casei; St. th: Streptococcus
salivarius subsp. thermophilus. These results the average of
three replicates |
| Table 3: |
Count of lactic acid bacteria in the present of insecticides |
 |
| B. spp: Bifidobacterium spp.; L.a: Lactobacillus
acidophilus; L.b: Lactobacillus delbrueckii subsp. Burglarious;
L.c: Lactobacillus casei subsp. casei; St.th: Streptococcus
salivarius subsp. Thermophilus; -: not detected. These results
the average of three replicates |
Dhanalakshmi et al. (1997) reported that
L. acidophilus was found the most sensitive to Lindane unlike our results
concerning L. acidophilus. Results of Ismail et
al. (1987) may explain our results; they mentioned that no morphological
changes in bacterial cell of L. acidophilus and L. casei were
observed post incubation with Aldicarb LC50 for 90 min.
All tested lactic acid bacteria were more sensitivity herbicides than fungicides
with the exception Bifidobacterium sp. On the other hand, round up was
more destructive to all tested strains than Saturn. However, St. theromophilus
was the most sensitive to herbicides followed by L. casei, Bifidobacterium
spp., L. bulgaricus then L. acidophilus which recorded the highest
resistance among the tested bacteria (Table 2). The obtained
results agreed with Zidan et al. (1990) mentioned
that St. theromophilus had the highest sensitivity, i.e., the lowest
growth in the presence of Fenvalerate and DDT.
According to data in Table 3, insecticides were the most
inhibition lactic acid bacteria compared to herbicides or fungicides. Lactobacilli
genera showed more resistant than other bacteria. Nevertheless L. bulgaricus
surpassed other bacteria in tolerance insecticides; on the contrary, Bifidobacterium
sp. exhibited excessive sensitive toward insecticides. Present results correspond
with Zidan et al. (1990) attested to that L.
bulgaricus appeared to be more tolerant to the organochlorine insecticides
(DDT). Moreover, L. helveticus was the most resistant strain to the pyrethroid
Fenvalerate. In that way Cabras et al. (1994) affirmed
that L. plantarum resisted effectiveness Dichlofluanid.
Effect of pesticides on acidity development by single strain of lactic acid
bacteria: Variation either in type or concentration of pesticides affected
the lactic acid production by most strains studied. Inhibition effect of lactic
acid production by lactic acid bacteria appeared at more than 2 ppm concentration
of Lindane and Endrin (Abdou et al., 1983). Therefore,
2, 4, 6, 8 and 10 ppm concentrations of pesticides were used in our study. Generally,
as the incubation period progressed, the Titratable Acidity (TA) increased in
all samples. Noteworthy, insecticides were the most obstruction on lactic acid
production by all organisms studied followed by herbicides then fungicides.
With regard to acidity development by Bifidobacterium sp. in sterilized
skim milk containing different amount of pesticides, data presented in Table
4 represent that the effect of 2 ppm of all pesticides didn't clearly affect
on Acidity Development (AD) compared with control except Lannet. Concentrations
of all pesticides higher than 2 ppm influenced gradually on (TA) by Bifidobacterium
sp. (AD) was moderate In the presence of 4 and 6 ppm concentrations of fungicides
but 8 and 10 ppm concentrations were High Impact. The same trend appeared in
other treatments except 6, 8 and 10 ppm violently slowed (AD) especially in
the presence of (Lannet and Reldan). Finally, (A.D.) by Bifidobacterium
spp. was extremely slow compared to other strains. These results might be due
to the highly sensitivity of Bifidobacterium spp. toward presence of
tested pesticides.
In this regard, must not forget it is well Known, technological problems have
arisen with using the therapeutic bacteria (Bifidobacterium spp, L.
acidophilus and L. casei) in preparing fermented milks, particularly,
the relatively long time needed for obtaining a satisfactory yoghurt coagulum
(Mahmoud, 1999). Obviously from Table
5 both insecticides were more effect on (AD) by L. acidophilus than
fungicides or herbicides. The effect of inhibition extremely appeared at 8 and
10 ppm concentrations of fungicides or herbicides. However, in the presence
of (Lannet and Reldan) L. acidophilus was very slow on lactic acid production
after 4 ppm also. The effect of both fungicides on (A.D.) by L. acidophilus
was alike as did both herbicides, but Reldan had a less effect than Lannet in
the same way, these findings are in agreement with those found by Zidan
et al. (1990) who reported that presence of Fenvalerate, Malathion
and DDT redused acid production by L. helveticus by 17, 9 and 20% at
the end of incubation period.
| Table 4: |
Acidity development by Bifidobacterium spp. in sterilized
skimmed milk containing pesticides |
 |
| These results the average of three replicates. -: Did not
estimate |
The results presented in Table 6 clearly indicate that L.
bulgaricus behaved the same way L. acidophilus in the presence of
both fungicides, but Round up and Lannet were more delay on lactic acid production
than Reldan and Saturn. Interesting observation that differences in (TA) were
little at the end of fermentation period, As well, these results indicate that
the all tested pesticides had a slight effect in less than 4ppm concentration,
which confirms previous results. Though Fenvalerate increased acid production
by L. bulgaricus (17%) particularly after 72 h incubation and uptill
the end of the incubation period (120 h) on the contrary acid production by
L. bulgaricus reduced in the presence of DDT and Malathion by between
12 to 44% (Zidan et al., 1990).
From preceding results became plain that L. casei was mild resistant
for all tested pesticides, materialized that from results recorded in Table
7 whereas, the effect of the two fungicides on lactic acid production were
similar such as the effect of the two herbicides and both of insecticides approximately.
| Table 5: |
Acidity development by L .acidophilus in sterilized
skimmed milk containing pesticides |
 |
| These results the average of three replicates; -: Did not
estimated |
| Table 6: |
Acidity development by L. bulgaricus in sterilized skimmed
milk containing pesticides |
 |
| These results the average of three replicates; -: Did not
estimated |
| Table 7: |
Acidity development by L. casei in sterilized skimmed
milk containing pesticides |
 |
| These results the average of three replicates; -: Did not
estimated |
Another noteworthy observation that (AD) by L. casei was affected by
8 and 10 ppm concentrations of all pesticides more pronounced than others. These
findings conflicted with Kim and Harmon (1968) who confirmed
that growth and fermentative ability of L. casei not affected by Methoxychlor
in the milk.
In the case of St. thermophiles, Table 8 indicated
that the samples without pesticides (control) had 0.72% (TA) after 5 h of incubation,
this value gradually decreased with increasing of fungicides concentrations,
this strain produced adequate quantity of lactic acid after 5 h except in the
presence of 10 ppm of fungicides, this harmonious with bacterial growth experiment.
On the completely contrary in the presence of herbicides whereas it was the
most sensitive in the bacterial growth experiment, herbicides clearly delayed
acid production by St. thermophiles. Presence of insecticides delayed
acid production by that strain though it was more capability in acid production
than other studied strains at high concentrations. Worth mentioning (AD) by
St. thermophiles slightly affected by Tasolen, Round up and Lannet more
than others. These results may be due to the different in the growth media (Synthetic
growth medium M17 or milk). The results agreed with (Zidan
et al., 1990) they reported that Fenvalerate, DDT and Malathion decreased
acid production by between 12 to 31% by St. lactis and St. thermophiles,
also our results compatible with Ismet et al. (2004)
who announced that the spores of the bacteria are sensitive to chemical pesticides.
| Table 8: |
Acidity development by St. thermophiles in sterilized
skimmed milk containing pesticides |
 |
| These results the average of three replicates; -: Did not
estimated |
Behavior yoghurt and ABT culture in the presence of pesticides: In the
terminal section of this study, yoghurt and ABT yoghurt were made in the presence
of (0.0, 2, 4, 6, 8 and 10 ppm) concentrations of pesticides (AD) and flavor
compounds (acetaldehyde and diacetyle) were determined. From Table
9 progressive increase in (AD) occurred with time of incubation in all samples.
Also, it is interesting to note that insecticides (Lannet and Reldan) delayed
the acid production by yoghurt culture more than herbicides and fungicides,
quaint that (TA) was higher in the presence of herbicide (Saturn) than other
pesticides contrary to Lannet in the end of incubation period. Presence 2 ppm
of all pesticides didn't retard acid production except Lannet, Presence 8 and
10 ppm of all pesticides had a violent effect. These findings correspond with
Abdou et al. (1983), they attested to Lindane
retarded (AD) at each stage. Lindane and Endrin caused little effect until their
concentration reached 0.5 ppm.
Regarding ABT culture, control treatment reached 0.65% (TA) after 6 h Table 10, this quantity gradually decreased with incubation time or increasing pesticides concentration in the presence all studied pesticides but after 8ppm caused a sharp decreased after 7 h. Anther noteworthy observation, the variations between two fungicides, herbicides and insecticides were negligible. Furthermore ABT culture affected by presence tested pesticides more than yoghurt culture.
From The results of experiments of acidity development by the single strain
and these findings in the current point appeared that pasteurization had not
apparent impact on pesticides destroyed, these results agreed with Dhanalakshmi
et al. (1997).
| Table 9: |
Acidity development by yoghurt culture in milk (standardized
3%fat) in the presence of pesticides |
 |
| |
| Table 10: |
Acidity development by ABT yoghurt culture in milk (standardized
3%fat) in the presence of pesticides |
 |
 |
| Table 11: |
Values of acetaldehyde and diacetyl by yoghurt culture in
the presence of pesticides on the 6th day from cold storing at 6-8°C |
 |
| Table 12: |
Values of acetaldehyde and diacetyl by ABT culture in the
presence of pesticides on the 6th day from cold storing at 6-8°C |
 |
Acetaldehyde and diacetyl are considered as important constituents of the flavor of fermented milks. Data presented in Table 11 indicated that the highest acetaldehyde and diacetyl on the sixth day were attained with yoghurt free pesticides (control) samples and decreased with increasing of pesticides. The 2 ppm of all pesticides studied slightly delayed the flavor compounds production, the high concentrations 8 and 10 ppm decreased sharply of acetaldehyde and diacetyl. Worth mentioning, Lannet was the most effect in disable the acetaldehyde and diacetyl production followed by Reldan, Round up, Saturn, Tasolen then Anadol.
As for the Acetaldehyde and diacetyl production by ABT culture, it took the same direction with some differences Table 12, the studied pesticides strongly affected on flavor compounds production more than they did in the yoghurt culture, the difference between the two fungicides, herbicides or insecticides was very slightly, However, presence of insecticides delayed acetaldehyde and diacetyl production by ABT followed herbicides then fungicides which had the lowest effect in that way. These observations might be due to harmful effect of pesticides on the cells metabolism.
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