Annals of Clinical and Medical
Case Reports
ISSN 2639-8109 Volume 12
Research Article
The Synergistic Effect of Two Different Types of Natural Additives on Bacillus Cereus
in Chicken Burger
Edris AM1, Haraz SM2, Arab WS1 and Radwa AL3*
1
Department of Food Hygiene and control, Faculty of Veterinary Medicine, Benha University, Egypt
2
Veterinary doctor in directorate Gabria government, Egypt
3
Department of Food Hygiene, Animal Health Research Institute, Tanta, Egypt. Agricultural Research Center, Egypt
Corresponding author:
*
Radwa A Lela,
Department of Food Hygiene, Animal Health
Research Institute, Tanta, Egypt. Agricultural
Research Center, Egypt
Received: 09 Nov 2023
Accepted: 05 Dec 2023
Published: 13 Dec 2023
J Short Name: ACMCR
Copyright:
©2023 Radwa AL. This is an open access article
distributed under the terms of the Creative Commons
Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially
Citation:
Keywords:
Nisin; Thyme oil; Bacillus cereus; Shelf life of chicken
burger
1. Abstract
Use of chemical preservatives is recently being considered by
customers due to concerns related to negative health. Natural preservatives are very vital for enhancing food safety and shelf life.
They are safe because they can limit microbial resistance and meet
consumers’ demands for healthier foods. There are types of natural preservatives. Plant-derived as Thyme and microbial metabolites as Nisin. Thyme has much attention due to its high content
phenolic compounds, antimicrobial, antioxidant properties also
influence food sensory properties including the flavor, taste, color,
texture, and acceptability of food and it is reasonably priced and
available for use. Nisin is bacitracin that has got FDA approval
for application as a food preservative. But it is very expensive and
hardly commercially available. The current study aimed to study
the effect of thyme, nisin, and their combination on Bacillus cereus inoculated in chicken burger. Found that combination of two
different types of preservatives (thyme and Nisin) have synergistic
effect as antimicrobial and enhance food sensory properties more
than thyme or nisin alone in different concentration. The results
showed that overall acceptability of the combination is to 14 th
day of storage. In contrast, control positive and control negative
showed overall acceptability till 4th, 6th day of storage respectively.
2. Introduction
Chickens are the most ubiquitous of all livestock species, and are
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Radwa AL, The Synergistic Effect of Two Different
Types of Natural Additives on Bacillus Cereus in Chicken Burger. Ann Clin Med Case Rep. 2023; V12(3): 1-6
found more or less everywhere inhabited by people. To enhance
human growth and health, chicken meat provides a considerable
part of microelements like as copper, iron, zinc, calcium, phosphorus, and cobalt. Besides, vitamins such as vitamin B group can
also be provided when meat chicken is consume [5]. Because they
are nutritious, delicious, and affordable, chicken meat and meat
products such chicken burgers are important sources of protein,
energy, vitamins, and minerals around the world [10]. The microbial safety of foods continues to be a major concern to consumers, food industries throughout the world. Many food preservation
strategies have been used traditionally for the control of microbial
spoilage in foods but the contamination of food and spoilage by
microorganisms is a problem yet to be controlled adequately. Although synthetic antimicrobials are approved in many countries,
the recent trend has been for use of natural preservatives, which
are alternative sources of safe, effective and acceptable natural
preservatives. Plants contain innumerable constituents and are valuable sources of new and biologically active molecules possessing
antimicrobial properties [12].
Bacillus cereus is one of the food-borne disease causing bacteria.
Bacillus Species and related genera have long been troublesome
to food producers on account of their resistant endospores. Bacillus cereus is well known as a cause of food poisoning, and much
more is known about the toxins produced by various strains of this
species [2].
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Bacillus cereus produces two types of toxins – emetic (vomiting)
and diarrheal causing two types of illness. The emetic syndrome
is occured by emetic toxin produced by the bacteria during the
growth phase in the food. The diarrhoeal syndrome is happened
by diarrheal toxins produced during growth of the bacteria in the
small intestine [4].
Thyme oil has an excellent antibacterial activity against important food pathogens such as Bacillus cereus. The bacteriostatic and
bactericidal activities of thyme is higher against the Gram-positive
pathogens than the Gram-negative ones. It was also active in counteracting the biofilm formation by Bacillus cereus [16].
Nisin is a preservative and antibacterial agent that is used to inhibit
the germination and outgrowth of spores; it alters cell properties
in bacteria to render it harmless. Nisin is currently recognized in
approximately 50 countries as a safe food preservative. Nisin is
anti-microbial agent, which is active against Gram positive bacteria including spore formers, but does not inhibit the majority of
Gram-negative bacteria. It is also stable under refrigerated conditions [14].
The aim of the study is to throw alight on the effect of thyme and
nisin alone and their mixture on B.cereus in chicken burger.
3. Materials and Methods
3.1. Preparations of Inoculate
Bacillus cereus strain was obtained from Animal Health Research
Institute (AHRI), Dokki, with recommended dose (5.2 x 107 CFU/
ml) as recorded by McFarland’s nephelometer standards according
to Slabyj et al., [19].
3.2. Thyme Preparation
Thyme oil provided by (ARC) Agriculture Research Center, Egypt
‘’ by hydro-distillation method, Tween 80 added to essential oils
before applying in samples as diluent and therefore, easily distribution and dissolving According to Wilkinson et al., [21].
3.3. Nisin preparation was prepared at concentrations 50 and 100
ppm according to Hassan [8].
3.4. Preparation of Chicken Burger Samples
A total amount of 2100 gm of raw chicken burger was been purchased from a butcher shop from Tanta city, it taken and transferred directly to the laboratory under complete aseptic conditions
without undue delay. The sample was divided to 7 equal groups (3
x100 gm for each) first group was control negative (no treatment),
second group used as control positive, third group and fourth
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groups were treated with Thyme oil (1%-1.5%), respectively. Fifth
and sixth groups were treated with Nisin (50 ppm- 100 ppm), respectively, while the seventh groups treated with mixture of both
(thyme 1.5% and Nisin 100 ppm).
All the groups were packed in a separate sterile polyethylene bags
and stored in domestic refrigerator at nearly ±40c, each sample
was analyzed at zero, 2 nd, 4th, 6th, 8th, 10th and 12th days during
storage for presence of bacillus cereus and sensory properties. This
work was been conducted in triplicate.
Sensory examination: according to Pearson and Tauber [15].
Bacteriological analyses: on Mannitol Egg Yolk Polymyxin
(M.Y.P) media according to [20].
3.5. Statistical Analysis
The obtained results were statistically evaluated by application of
one-way ANOVA test according to [6].
4. Results
Table 1, illustrated the effects of various concentrations of thyme,
nisin and mixture of them on overall acceptability of groups.
Thyme (1% & 1.5%) showed accepted sensory properties extended to 6th, 8th day of storage respectively. Nisin (50 ppm & 100
ppm) showed overall acceptability till 10thday, 12th day of storage
respectively while mixture (thyme 1% ,nisin 100 ppm ) showed
overall acceptability to 14th day of storage. In contrast, control positive and control negative showed overall acceptability till 4th, 6th
day of storage respectively.
Table 2&3 and Figure 1, illustrated the antimicrobial effects and
reduction percentage of various concentrations of thyme, nisin
and mixture of them on counts of B. cereus artificially inoculated
into chicken burger. Thyme oil (1% &1.5%) decreased count of
B.cereus (cfu/g) from 5.2x107 (initial load) to 3.4x107and 8.5 x106
with reduction percentages 34% and 84% on 6thday and 8th day
of storage, respectively. Nisin (50 ppm and100 ppm) decreased
count of B. cereus (cfu/g) to 1.6 x106 and 2.4x105 with reduction
percentages 96.5% and 100 % on 10th day and 12th day of storage,
respectively. Mixture of (thyme and nisin) decreased count of B.
cereus (cfu/g) to 5.3 x 105 with reduction percentage100 % on 10th,
12th and 14th day of storage. In control positive group B. cereus
count increased from 5.2x107 (initial load) to 3.6x108 on 4th day
so, the differences between the effects of various concentrations of
thyme and nisin and their mixture on counts of B.Cereus (cfu/g)
artificially inoculated into chicken burger sample were significantly different.
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Volume 12 Issue 3 -2023
Research Article
Table 1: over all acceptability (sensory evaluation) of the examined chicken burger samples during cold storage at 40c.
Groups
Zero day
Control -
Excellent
Control +
2nd
very very good
4th
Medium
6th
8th
10th
12th
14th
Fair
Excellent
Medium
Fair
SP
SP
SP
SP
SP
1%
Excellent
very very good
Good
Medium
SP
SP
SP
SP
1.50%
Excellent
very very good
very very good
Good
Medium
SP
SP
SP
50ppm
Excellent
very very good
Very good
Good
Medium
Fair
SP
100ppm
Excellent
very very good
Very good
Good
Good
Medium
Fair
Mix
Excellent
very very good
very very good
Very good
Good
Good
Medium
Thyme oil
Nisin
Fair
Sp: spoiled
Table 2: The effects of various concentration of thyme oil and nisin on counts of B.cerus (cfu)
Thyme 1%
Thyme 1.5%
Nisin 50 ppm
Nisin 100 ppm
Mixture
5.2x107±
5.2x107±
5.2x107±
5.2x107±
5.2×107±
5.2×107±
0.84 x107
0.84 x107
0.84 x107
0.84 x107
0.84×107
0.84×107
7.3x107±
4.2x107±
3.8x107±
3.5×107±
2.7×107±
2.4×107±
0.52 x107
0.63X107
0.60 X107
0.51×107
0.14×107
0.32×107
3.6x108±
4.0x107±
2.7x107±
2.4×107±
8.5×106±
6.4×106±
0.12 x108
0.43 X107
0.20X107
0.87×107
0.23×106
0.47×106
3.4x107±
1.4x107±
7.7×106±
4.7×106±
1.8×106±
0.32X107
0.33X107
0.73×106
0.50×106
0.47×106
8.5×106±
3.5×106±
7.5×105±
5.3×105±
6
6
5
0.11×105
Control +
Zero
2nd
4th
6th
SP
8th
SP
SP
10th
SP
SP
SP
12th
SP
SP
14th
SP
SP
0.18×10
0.45×10
0.37×10
1.6×106±
2.4×105±
0.19×106
0.18×105
SP
SP
ND
ND
SP
SP
SP
ND
ND
ND: not detected
Figure 1: Reduction % in count of B. cereus count
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Research Article
Table 3: Reduction % in count of B.cereus count.
Mixture (1,5% +100 ppm)
Nisin 100 ppm
Nisin 50 ppm
Thyme 1,5%
Thyme 1%
Days
53.80%
48.10%
88%
83.60%
23.60%
26,3%
19.20%
2nd
54%
47.60%
25%
4th
96%
90.10%
85,3%
73.10%
34%
6th
99%
98.50%
93.30%
84%
spoiled
8th
100%
99.50%
96.50%
Spoiled
spoiled
10th
100%
100%
spoiled
Spoiled
spoiled
12th
100%
spoiled
spoiled
spoiled
spoiled
14th
5. Discussion
Symptoms of B. cereus foodborne toxicoinfection depend on the
type of the toxin produced as there are diarrheal or emetic forms.
The diarrheal form is characterized by the watery diarrhea and abdominal cramps with an incubation period of 6-15 hrs. While the
emetic form is characterized by vomiting, and nausea with an incubation period of 30 minutes to 6 h [3].
In table 1, revealed that the all groups have excellent score at zero
day of storage, while in 4th day the control positive group showed
signs of deterioration with bad odor and texture. At 6th day the
control negative group decomposed, while the other treated groups
have very good to good overall acceptability but treated with thyme
1.5% and nisin 100 ppm were very good acceptability. At 6th day
the control negative group decomposed, while thyme 1.5%, nisin
50ppm, 100 ppm had good over all acceptability while mixture
(1.5 %, 100 ppm) had very good score of over-all Acceptability. at
8th day thyme oil was decomposed while treated with thyme oil 1.5
ppm were medium, nisin 50 ppm was fair but mixture was good
overall acceptability. At 10th day of treatment thyme1.5%, nisin
50 ppm were decomposed, while nisin 100 ppm was medium but
mixture was good overall acceptability. At last the 12th day the 100
ppm group was decomposed while mixture treated samples was
fair acceptability.
These results agreed with those obtained by [1]. There was a
decline of acceptability began after the first day of storage with
marked reduction of odor, color, texture and overall acceptability
values in the control samples at the 4th day of storage.
These results Also agreed with [17], who reported that many herbs
and spices as thyme contain antioxidant components that improve
both color and flavor stability in meat.
The results were in line with the [23] who found that essential oils
enhanced the organoleptic character of chicken meat. The sample
containing thyme had a better sensory appearance than the control
sample.
The results of nisin agree with those obtained by [8], who used
the treated and control have excellent score at zero day and first
day of treatment while in second day the treated and control were
very very good with starting the decrease the quality of texture and
odor of control .meanwhile the third day the control score start to
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decrease with medium score while 30 ppm,50 ppm had very good
score of over-all acceptability at last the fifth day the 10ppm group
was decomposed while 30ppm treated samples was fair over all
acceptability otherwise 50ppm was good to medium.
This results of nisin Also agree with those obtained by [13] who
use nisin (25-75 ppm) the samples treated with 50 and 75 ppm nisin
had significantly lower aerobic microbial counts than the control
without affecting sensory acceptability. The treated samples also
had the significantly higher scores in overall acceptance than the
control sample. The results recorded in Table 2 and 3, indicated that
thyme (1%) reduced B. cereus count (cfu/g) artificially inoculated
into chicken Burger samples from 5.2×107± 0.84 x107to 4.2×107±
0.63 ×107, 4 ×107± 0.43 × 107 ,3.4 x107±0.32 x107 in 2nd day, 4th,
6th day respectively, with reduction percentages 19.2% and 25%
,34% respectively, spoiled after 6 th day. Meanwhile thyme (1.5%)
reduced B. cereus count (cfu/g) artificially inoculated into chicken
Burger samples from 5.2×107± 0.84 x107 to 3.8 x107± 0.60 ×107,
2.7×107± 0.20 ×107,1.4 x107±0.33 x107and 8.5×106± 0.18 ×106 in
2nd day, 4th day 6th day and 8th day respectively, with reduction
percentages 26.3%, 47.6% and 73.1% ,84% in 2nd day, 4th day 6th
day and 8th day respectively, spoiled after 8th day.
The results recorded Table 2 and 3, indicated that nisin (50 ppm)
reduced B. cereus count (cfu/g) artificially inoculated into chicken
Burger samples from 5.2×107± 0.84 x107 to 3.5×107± 0.51×107, 2.4
×107± 0.87×107,7.7 x106± 0.73 x106,3.5x106±0.45 x106 in 2nd day,
4th day ,6th day, 8th and 10th day respectively, with reduction percentages 23.6%,54%,85.3% ,93.3% and 96.5 % respectively and
spoiled after10th day. Meanwhile nisin (100 ppm) reduced B.cereus count (cfu/g) artificially inoculated into chicken Burger samples
from 5.2×107± 0.84 x107 to 2.7 x107± 0.14 x107, 8.5 x106± 0.23
x106 , 4.7 x106±0.50 x106, 7.5 x105±.37x105,2.4x105±0.18x105 in
2nd day, 4th day and 6th day ,8th day and 10th day, respectively, with
reduction percentages 48.1%,83.6 % ,90.1 %,98.5%, 99.5% and
100% respectively, spoiled after 12th day.
The results recorded Table 1 and 2, indicated that mixture
(1.5%,100 ppm) reduced B.cereus count (cfu/g) artificially inoculated into chicken Burger samples from 4.9×5.2×107± 0.84 x107,to 2.4 x107±0.32 x107, 6.4 x106±0.47 x106, 1.8 x106±0.40x106,5.3
x105± 0.1 1 x105, ND after 2nd day , 4th day and 6th day ,8th day and
10th day, 12th till 14th day respectively, spoiled after 14th day, with
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Volume 12 Issue 3 -2023
reduction percentages 53.8%,88% ,96%,99%,98.5% and 100% respectively .
The observed reduction in microbial counts can be attributed to the
inhibitory effect of thyme on spoilage bacteria [7]. However, samples were considered spoiled if total bacterial counts were above
7 log CFU/g, In the current study chicken samples treated with
thyme did not exceed the value of 7.0 log CFU/g for TBC, which
was considered as the upper acceptability limit for fresh meat [18]
till the 12th day of storage.
The obtained result of nisin 50 ppm were nearly similar to who
[8] reported that Nisin at (50 ppm) reduced B. cereus count (cfu/g)
artificially inoculated into minced meat samples from 5.0×107±
0.82×107 to 9.74×106± 2.03×106 ,7.36×105± 1.15×105, ND and
ND after 1st day, 2nd day, 3rd day, 4th day and 5th day, respectively, with reduction percentages 80.5%, 98.5%, 99.9%, 100% and
100%, respectively.
The obtained result of nisin were nearly similar to [9] reported
that B.cereus more than the use of Nisin 100g/ton alone. The use
of Nisin 300g/ton decreased the count of B.cereus to 2.68 log cfu
/g, the use of Nisin 200g/ton decrease the count of B.cereus to
2.86 log cfu /g, the use of Nisin 100 g/ton decreased the count of
B.cereus to 3.22 log cfu /g and also said that the addition of 3 concentrations of nisin (100, 200 and 300 g/ton) reduced log the count
of inoculated B. cereus by 3-5 log cycles; similar results were recorded by Roberts and Hoover (1996) found that B.cereus initial
count was reduced by three log cycles when Nisin concentration
was 1.0 I.U. / ml.
Nisin also inhibits the outgrowth of germinated bacterial spores,
Organisms from several families of bacteria, including the Bacilli
, form small endospores in nutrient-deprived conditions, allowing
survival over extended periods of time, which would not be possible as vegetative cells [11].
The recent study revealed the best concentration of Thyme And
nisin controlling B.cereus is (1.5% - 100 ppm) reduced B. cereus
count (cfu/g) artificially inoculated into chicken burger samples
with reduction percentages reached to100% with sensory characteristics very good score of over-all acceptability over all the experimental time Therefore, It is recommended to improve safety of
the chicken burger.
5. Conclusion
The results of the current study represented that mixture of thyme
oil and nisin (1.5%, 100 ppm) improve the quality and sensory
characteristics of chicken burger under chilled storage (4oC) for
the economic and public health importance viewpoint.
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