Botany Research International 2 (4): 235-238, 2009
ISSN 2221-3635
© IDOSI Publications, 2009
Establishment of an Inexpensive Procedure to Reduce
the Microbial Load of Spices Used in Mayonnaise Industry
1
Samira Bahramian, 2Sabihe Soleimanian-Zad, 2Mahmood Sheikh-Zeinoddin and 2Gholam Hossein Kabir
Islamic Azad University - Sanandaj Branch, Kurdistan, Iran
Department of Food Science and Technology, Isfahan university of Technology, Isfahan, Iran
1
2
Abstract: In order to reduce the microbial load of spice formulation that was used for mayonnaise production,
its acidic suspensions were heat treated at six different temperature-time combinations (65, 70, 75, 80, 85 and
90°C for 5 minutes). Among all heat point tested, at temperatures above 75°C, significant reduction in bacterial
vegetative and spore was seen. Therefore, 5 types of mayonnaise by the same formulation but with spice
suspensions that were heat treated at different temperatures (unheated, 75, 80, 85 and 90 °C), were prepared.
These samples were incubated at 27 and 37 °C for 1, 15, 30 and 45 days. The microbiological examination in
order to determine the effect of heat treatment and incubation periods on total aerobic count and aerobic spore
count of mayonnaise was performed. The results indicated that the effect of incubation time at both
temperatures on reducing total aerobic count of mayonnaise is significant but by increasing the severity of heat
treatment on spice suspensions, total aerobic count of mayonnaise did not decrease significantly. The results
also showed that incubation time of mayonnaise had not significant effect (p>0.05) on reducing aerobic spore
count whereas increasing the severity of heat treatment especially at 90°C for 5 minutes was effective
significantly.
Key words: Spice
Mayonnaise
Heat treatment
pH
INTRODUCTION
Mayonnaise and other salad dressing spoil for a
variety of reasons: separation of emulsion, oxidation
and hydrolysis of the oils by strictly chemical
processes and from the growth of microorganisms [1].
They are resistant to most microbial spoilage and are
spoiled by only a few select groups of microorganisms.
In addition, properly prepared mayonnaise and salad
dressing will not support growth of pathogenic
bacteria [2]. The organisms most frequently found
and involved in spoilage are those that are able to
survive or grow at low pH values, high salt and/or
high sugar concentration of these products [2].
Baumgart (1965) reported that lactobacilli, yeast and
aerobic
spore-formers
were
the
groups
of
microorganisms most commonly found in freshly prepared
mayonnaise. The usual spoilage organisms of mayonnaise
and salad dressing are lactobacilli, bacilli and yeasts [2, 3].
The microorganisms in salad dressings come from
the ingredients, from manufacturing equipment and from
the air [1].
Aerobic spore count
Spices are one of the ingredients for production of
these kinds of products. They are classed as important
vehicle for various microorganisms implicating possible
health problems for consumers and shelf life problems for
foods [4].
It is likely that in early studies the high levels of
aerobic spore formers isolated from spoiled products
were a reflection of the microbiological content of the
raw materials, particularly spices, which can be highly
contaminated with members of the genus Bacillus. The
presence of these spore forming bacilli in the finished
product probably resulted from the resistance of the
spores rather than from growth of the vegetative
cells [5]. Spices contain phenolics and essential oils
which are inhibitory to microorganisms but the effect on
microorganisms may depend on the type as well as the
medium. For example, it was reported that fats and protein
bind or solubilise phenolic compounds thereby reducing
their availability for antimicrobial activity [6].
The aim of this study was to evaluate the effect of
heat treatment on microbiological quality of acidic spice
suspensions that are used in mayonnaise production and
Corresponding Author: Samira Bahramian, Islamic Azad University- Sanandaj Branch, Kurdistan, Iran
235
Bot. Res. Intl., 2 (4): 235-238, 2009
also to evaluate the time and temperature of incubation on
microbiological quality of final product.
The production of mayonnaise was carried out by
blending of all ingredients except oil and gums. Then oil
together with gums was added under controlled
conditions during continuous blending. The pH of the
mayonnaise after preparation was 3.3.
By considering the intensity of heat treatment on
acidic spice suspension, 5 types of mayonnaise were
prepared. All of them had the same formulation but in
preparation of mayonnaise number 1, unheated acidic
suspension was used and in mayonnaise number 2, 3, 4
and 5 respectively, heat treated acidic suspension at
temperatures 75, 80, 85 and 90°C for period of five minutes
were used.
MATERIALS AND METHODS
Preparation of Acidic Solution: To prepare the acidic
spice sample, acidic solution was made by combining
39 g of 5.2% acetic acid solution, 60 g water and 0.91 g
citric acid in crystal form. The pH of this solution before
adding to the spices was 2.35.
Preparation of Spice Suspension: Spice formulation for
mayonnaise production consists of cinnamon, mustard,
cloves and garlic powder. In order to prepare spice
suspension ten grams of this formulation were weighed
under aseptic conditions into glass bottle containing 90ml
of sterile acidic solution. Before making dilutions, the
suspension was vigorously shaked for 1 min in a 1-ft arc.
Mayonnaise Incubation: All types of mayonnaise were
aseptically weighed in 20g aliquots and dispensed into
sterile containers with aluminum foil lid. Then in order to
do microbiological examination, they were incubated at
27 and 37°C incubator for periods of 1, 15, 30 and 45 days.
Heat Treatment: After preparation of spice acidic
suspension, heat treatment was performed for 5 minutes
at different temperatures (65, 70, 75, 80, 85, 90°C) in
water bath. Suspensions were then immediately cooled
in iced-water to bellow 4°C.
Microbiological Examination of Mayonnaise: After
incubation periods at 27 and 37°C, microbiological
examination in order to determine total aerobic count and
aerobic spore count were performed.
Preparation of Dilutions: A 10 g sample was placed in
90 ml of sterile phosphate buffer and mixed. Subsequent
higher dilutions with phosphate buffer were made from
this mixture [1].
Microbiological Analysis
Preparation of Dilutions: Serial decimal dilutions were
made in eppendorf tubes using Butterfield Buffer
Phosphate (pH 7.2) as diluent. For this purpose 900 µl of
the diluent was poured in eppendorfs and 100 µl of each
sample suspension was added to the first eppendorf. Each
dilution was vortexed for 1 minute. From this eppendorf,
100 µl was transferred to the second one and so on.
Total Aerobic Count: Plate count agar (PCA) with 100 ìg
of cycloheximide per ml for inhibiting fungi was used to
determine the number of aerobic bacteria. Plates were then
incubated for 72 hr at 32°C [1].
Total Aerobic Count: To obtain the total aerobic bacterial
count of spice acidic suspension before and after
heat treatment, dilutions ranging of 10 1 to 10 1 were
plated on plate count agar (PCA) and the plates were then
incubated at 32°C for 18 hr.
Aerobic Spore Count: Aerobic spore count was
performed according to APHA standard method [1].
Statistical Analysis: An analysis of variance (ANOVA)
for the obtained results was investigated and the mean
value was calculated from the data obtained from three
replicates. Means were compared using LSD test in
MSTATC Software (version 1.42).
Aerobic Spore Count: Aerobic spore count was
performed on spice suspension according to the APHA
standard method. Trypton glucose extract (TGE) agar was
used as the selective medium [1].
RESULTS AND DISCUSSION
Preparation of Mayonnaise: The ingredients for
mayonnaise production were: retail sunflower oil, liquid
egg, acetic acid (wine vinegar, 5.2% acidity), sucrose and
NaCl, citric acid (crystal form), ground mixed spices,
xanthan and CMC.
Table 1 shows total aerobic count of heat-treated
acidic spice suspensions and an untreated one.
The same results were seen in spore count reduction.
Table 2 shows the effect of heat treatment on the number
of aerobic spore former.
236
Bot. Res. Intl., 2 (4): 235-238, 2009
Table 1: Comparison of total aerobic count of acidic spice suspensions,
Temperature (°C)
Untreated 65
70
75
80
85
No. of viable
cells (log cfu/g)
before and after heat treatments
90
Total Aerobic Count
(log cfu/g)
6.04
4.54 3.86
a
b
3.99
ab
3.17
c
1.2
d
0.00
e
The same mark on the values is an indication of the insignificant difference
5
4
3
2
1
0
1
(p>0.05) between the values presented
2
3
4
45
5
mayonnaise type
Table 2: Comparison of spore load of acidic spice suspensions, before and
30
15
1
incubation time
(day)
after heat treatments
Temperature (°C)
Untreated 65
70
75
80
85
Fig. 3: The effect of heat treatment of spice acidic
suspensions and incubation time of mayonnaise
on aerobic spore count of mayonnaise at 27°C
incubator
90
Aerobic spore count
(log cfu/g)
5.60
5.56a 5.14b 4.94b 4.22c 3.27d 0.00e
The same mark on the values is an indication of the insignificant difference
(p>0.05) between the values presented
No. of viable
cells (log
cfu/g)
No. of
viable cells
(log cfu/g)
5
5
4
3
2
1
0
3
2
1
0
1
1
1
15
30
incubation
45
time (day)
2
3
4
mayonnaise type
5
3
2
1
0
3
mayonnaise type
4
5
30
45
15
4
5
45
30
15
1
incubation
time (day)
The decrease in heat resistance of native, fully
mineralized spores when heated in acidic media has been
attributed to an acid wash of the minerals of the spores. It
has also been proposed that a protonization of the cortex
could be responsible for the decrease in heat resistance in
acidic media [7].
The effect of heat treatment of spice acidic
suspensions (at 75, 80, 85 and 90 °C for 5 min) and time of
mayonnaise incubation (1, 15, 30 and 45 days) at 27°C on
total aerobic count of mayonnaise is shown in Figure 1
and in Figure 2 the same treatment on total aerobic count
of mayonnaise that was incubated at 37°C, is defined.
It can be seen that by increasing the severity of heat
treatment on spice suspensions, total aerobic count of
mayonnaise was reduced but not significantly. It indicates
that in addition to spices, other ingredients that were used
to mayonnaise production have influenced on total
aerobic count of mayonnaise. But as shown in Figure 1
and 2, the time of mayonnaise incubation (especially
after 45 days) at both temperatures, on reducing total
aerobic count of mayonnaise, is significant.
4
2
3
Fig. 4: The effect of heat treatment of spice acidic
suspensions and incubation time of mayonnaise
on aerobic spore count of mayonnaise at 37°C
incubator
5
1
2
mayonnaise type
Fig. 1: The effect of heat treatment of spice acidic
suspensions and incubation time of mayonnaise
on total aerobic count of mayonnaise at 27°C
incubator.
No. of viable
cells (log
cfu/g)
4
1
incubation
time (day)
Fig. 2: The effect of heat treatment of spice acidic
suspensions and incubation time of mayonnaise
on total aerobic count of mayonnaise at 37°C
incubator.
It can be seen that, by increasing the severity of heat
treatment at acidic solution, the number of total aerobic
count and spore count have decreased significantly.
The data also indicate that the effect of pH is greater at
the higher temperatures and by heat treatment at 90°C for
5 minutes, 6 log cycle reductions was occurred.
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Bot. Res. Intl., 2 (4): 235-238, 2009
ACKNOWLEDGMENT
Figures 3 and 4 show the effect of heat treatment of
spice acidic suspensions and incubation time of
mayonnaise (at 27 and 37 °C for 1, 15, 30 and 45 days) on
the number of aerobic spore count.
As it is seen, at both incubation temperatures the
effect of time of incubation on the number of spores is
not significant whereas by increasing the severity of
heat treatment on spice suspensions especially at 90°C,
the number of spores in mayonnaise were reduced
significantly.
The reason for this behavior may be that the large
number of spores in mayonnaise can inoculated by
spices and spice heat treatment significantly reduces the
number of spores in mayonnaise but the effect of other
mayonnaise ingredients on spore count are minute.
The most important antimicrobial preservative
substances found in mayonnaise and salad dressing is
acetic and/or citric acid. Undissociated acid molecules
were responsible for the toxic properties of organic acid to
bacteria [2]. In Radford’s investigation (1993) it was
concluded that all vegetative cells died in acetic acid
mayonnaise pH<4.4. Clostridium spore numbers were
virtually unaffected by any acidity [8]. According to
Smittle (1977), the vegetative cells of microorganisms
are rapidly killed primarily by the acetic acid whereas
endospore of the spore formers remains viable, but in the
event that germination occurs, cells will not grow. The
extent of growth inhibition or loss of viability was
dependent on acetic acid concentration or pH, storage
temperature and type and number of microorganisms
present [2].
We thanks to laboratory of Food Science and
Technology of Isfahan University for their support to
the project.
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CONCLUSION
In this study we observed that incubation of
mayonnaise can reduce some microbial contamination
especially not spore formers but in order to reduce
bacterial spore count, it is better to heat treatment the
acidic spice suspension because spices may contain
different types and amounts of spore formers.
8.
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