1Department of Food Hygiene and control, Faculty of Veterinary Medicine, Benha University 3Department of Food Hygiene, Animal Health Research Institute, Tanta, Egypt. Agricultural Research Center (ARC)

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.

Nisin, Thyme oil, Bacillus cereus, Shelf life of chicken burger

Chickens are the most ubiquitous of all livestock species, and are 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 consumed (El Bayomi et al., 2018)[1]. 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 (Morshdy et al., 2021). 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 (Negi, 2012)[2]. 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 (Anita and Swaid, 2014)[3].

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 (Ehling -Schulz et al., 2006)[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 (Sateriale et al., 2023) [5] 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 (Patricia et al., 2001)[6]. 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.

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. (2003)[7].

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.,(2003) [8].

Hassan (1999)[9].

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 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, (1984) Bacteriological analyses: on Mannitol Egg Yolk Polymyxin (M.Y.P) media according to (Tallent et al., 2012)[10]. Statistical Analysis: Statistical Analysis: The obtained results were statistically evaluated by application of one way ANOVA test according to (Feldman et al., 2003)[11].

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 14 th 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.

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 (Darwish et al., 2022)[12] 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 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 Amani et al. (2018)[14] 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 Sasse et al., (2009)[15] 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 (Zhang and Guo, 2016)[16] 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 Hassan et al. (2019)[9] 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 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 Nutsuda Sumonsiri (2019)[17] 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 table. (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 table. (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 10 th day respectively, with reduction percentages 23.6%,54%,85.3% ,93.3% and 96.5 % respectively and spoiled after10 th 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 table. (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,12 th till 14th day respectively, spoiled after 14 th day, with 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 Gutierrez et al .2009)[18] 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 (Senter et al., 2000)[19] till the 12th day of storage. The obtained result of nisin 50 ppm were nearly similar to who ( Hassan et al. 2019)[9] 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 ( Mohamed et al . 2015)[20] 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. 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. 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 (Montville et al. 2006)[21].

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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