EFFECT OF SOME ORGANIC ACIDS ON SALMONELLA TYPHIMURIUM IN CHICKEN MEAT

There are several health problems and economic losses, which are caused by bacterial species on chicken meat. The obligation to reduce the initial load of bacteria should be taken into serious consideration. So, the aim of this work was to detect the incidence of Salmonella spp in chicken meat parts with detection of their virulence genes, which were 4%, 8% and 6% in chicken fillet, thigh and drumsticks respectively, with detection of virulence genes % in isolates. This study is an attempt to evaluate the antibacterial effect against Salmonella Typhimurium by soaking chicken breast in acetic acid, citric acid and fumaric acid (1% & 2%) solutions for 15 seconds, as well as on sensory properties. The treatments reduced Salmonella counts effectively. Logarithmic reduction values of Salmonella, for acetic acid 1%, acetic acid 2%, citric acid 1%, citric acid 2%, fumaric acid 1%, and fumaric acid 2% were 0.82, 1.88, 1.07, 2.19, 0.91, and 2.85 log CFU/g, respectively.


INTRODUCTION
Poultry meat and its products are particularly well-liked food all over the world, due to their deliciousness, nutritional surroundings, making it underling or unsuitable quality for people's consumption. A risk to the public's health could come from contaminated poultry and chicken products, Ahmed and Ismail (2010). Salmonellae is the predominant significant bacterium found in poultry meat and is responsible for many food-borne illnesses. (Ahmed, 2014).
Although ingestion of a variety of foods has been linked to Salmonella infection, animal products are a major source of infection (Castillo et al., 2008). Salmonella has historically been spread from animals to people, and meat, especially chicken meat, has a significant impact on the epidemiology of different serovars (Perin et al., 2020).
The two most commonly isolated serovars from food-borne outbreaks worldwide are Salmonella enterica serovar Typhimurium and Salmonella enterica serovar Enteritidis (Herikstad et al., 2002). There are several virulence genes specific to Salmonella that are significant contributors to pathogenicity (Baumler et al., 2000). At least 80 distinct virulence genes have been found in Typhimurium serovar. It is known that some genes play a role in invasiveness and adhesion, including: Sef (Clouthier et al., 1993), fliC (Jamshidi et al., 2009), another gene associated with production of toxin, as Stn, (Makino et al., 1999).
Due to the health problems and economic losses caused by different species of bacteria in poultry meat, the obligation to reduce the initial load of bacteria needs to be seriously considered. This is an attempt to investigate the antibacterial effects of common food additives. Chemical disinfectants made of organic acids are frequently employed in the food business. Their antimicrobial activity has been studied in a number of food matrices and it is generally regarded as safe for human consumption (Mani-López et al., 2012).

Salmonella
spp identification and isolation: Preparation of samples: A number of 150 raw poultry meat samples are divided into 50 pieces each of the drumstick, thigh, and fillet. These samples were collected from various supermarkets and retail stores in the El Sharkia Governorate. Under strict aseptic circumstances, the collected samples were taken and quickly transported in an insulated ice box to the lab for microbiological analysis.

Bacteriological
identification of Salmonella spp.: Salmonella identification was carried out according to ISO (6579:2017) as follows: Pre-enrichment non-selective media: Test portions (25gm) were inoculated into 225 ml of buffered peptone water and incubated at 34-38 C°, for 18 hours±2 hours.
Plating on selective media: From the previous enrichment, broth streaks a loopful onto the surface of Xylose Lysine Desoxychoclate agar medium (X.L.D) was previously prepared, and incubated it at 34-38°C for 24 hours. Suspected colonies were red with or without black centers. Sub cultured to the obtained suspected colonies onto nutrient agar plates and incubated at 37 C° ± 1 C° for 24 ± 3 hours. The obtained colonies (red with or without black centers on XLD media) were plated on nutrient agar media for purification.

Identification of Salmenolla Serologically:
The isolates that are biochemically proven to be Salmonella organisms were confirmed by serological identification using a rapid diagnostic Salmonella antiserum kit, according to the Kauffman-White scheme (Kauffman, 1974).

Detection of virulence pathogenic factors (stn, hilA, fliC, and sef) of Salmonella enteritidis and Salmonella typhimurium by using Polymerase Chain Reaction (PCR):
The QIAamp DNA Mini Kit provides silicamembrane-based nucleic acid purification from different types of samples. The spincolumn procedure does not require mechanical homogenization, so total handson preparation time is only 20 minutes. The multiplex PCR reactions for (Stn, hilA, fliC& sef) were performed with initial denaturation at 95°C for 5 minutes followed by 35 cycles of denaturation at 95°C for 30 seconds, primer annealing at 58°C for 1 minute, primer extension at 72°C for 1 minute, and the final extension at 72°C for 10 minutes.

Oligonucleotide primers used in cPCR (Metabion, Germany)
They have a specific sequence and amplify a specific product, as shown in Table (1). for 24 hours. Colonies were taken, put into brain-heart infusion broth, and then incubated for 24 hours at 37°C. The bacterial growth were centrifuged at 3000 rpm for 15 minutes, twice washing in 10 ml of 0.01 phosphate buffered saline (PBS) and then diluted to obtain 6 log10 CFU/ml for inoculation of the chicken fillet samples. S.
Typhimurium count was detected by tenfold serial dilution and subsequent enumeration on XLD agar (Govaris et al., 2010).
Sample preparation for bacterial inoculation: chicken breast fillets that were proved to be negative in part I of the study were aseptically divided into 7 sets for different additives including the control group, each group subdivided into 3 trials in which the treated samples weighing 25 g. The control, as well as the treated groups, were placed in separate sterile polyethylene bags, inoculated with S. Typhimurium (6 log10 CFU/ml in PBS), then mixed with organic acids of different concentrations after 15 minutes of inoculation.

Solutions
of organic acids: the concentration of (1 and 2% w/v) of the tested organic acids (acetic acid, citric acid and fumaric acid) was prepared according to manufacturer's instructions. Fumaric acid was reserved at a 50˚C from preparation and during the experiment time to evade its precipitation. Treatment trials were done under strict aseptic precautions. Samples were dipped for 15 seconds in acetic, citric and fumaric acid (1&2%) separately. After treatment, samples were aseptically homogenized with 225 ml of 0.1 % sterile peptone water to about 2 minutes at 3000 rpm using sterile homogenizer. Tenfold serial dilution was prepared. Salmonella Typhimurium counts were performed on XLD, as selective media. The values of log reduction were estimated by subtracting counts of samples treated with the different organic acids from counts of control samples, according to (Fernández et al., 2020).

Statistical analysis and experimental design:
Experiments were done in seven groups separately with three trials in each one. The antimicrobial effectiveness of the three organic acids with different concentrations on Salmonella typhimurium count was compared using a one-way ANOVA analysis (Adhikari et al., 2020).

DISCUSSION
Food safety threats posed by food-borne infections, such as Salmonella, continue to be of a great concern for the industry of food. Salmonella disease is a major public health issue and more important worldwide in developing countries (Wang et al., 2008). The frequency of Salmonella serotypes found by isolation from chicken flesh samples tested in Table 2 was 4%, 8%, and 6% in fillets, thigh, and drumsticks, respectively. As shown in Table 3., serologically identified salmonellae were Salmonella wingrove, Salmonella kentucky, Salmonella enteritidis, Salmonella Typhimurium, Salmonella apeyeme and Salmonella tsevie. The incidence of Salmonella in poultry meat was resulted from contamination within manual handling, slaughter and by workers (Carraminana et al., 1997). Organic matter dispersed on the bird surface provides a harbor for salmonella, acts as a contamination source to scalding boiling tanks and facilitates cross-infection between chickens. The plucker's rubber fingers have multiple cracks that allow organic matter to migrate and cause cross-contamination between chickens. In addition, crosscontamination can occur during the evisceration stage due to leakage of intestinal contents (Berrang et al., 2011). Control Acetic acid 1%

Fumaric acid 2%
Mean ±SE Log Reduction PCR findings declared in Table (4) and Figures (1-4) showed a total of 3 Salmonella serotypes (2 S. Typhimurium & 1 S. enteritidis) were tested using multiplex PCR for the presence of virulence genes, including hilA, stn, fliC and seF. A complex phenomenon, diarrhea was caused by Salmonella, and was resulted by multiple pathogenic mechanisms, like production of enterotoxin. The production of enterotoxin is activated by stn and it plays an important role in developing gastroenteritis through the enterotoxin production (Chopra et al., 1987).
In our study, stn gene was found in 2 of 3 isolates (66 %). Comparable results obtained by Ammar et al. (2016) and Eldesouky et al. (2016), who found stn gene in 58.82% and 41.17% of isolates, respectively. The hilA gene is a key component of Salmonella pathogenicity, because it's required for colonization of bacteria of the host intestine extracellular space, (Murray and Lee, 2000). Based on our results, hilA was detected in 3 of 3 isolates ( et al., 2006). As evidenced by PCR results, two S. Typhimurium isolates (100%) generated an identical band of 613 bp, the predicted size for the fliC gene.
Fimbrial antigen of S. enteritidis (sefA) which is specific for the discovery of salmonella entritidis serovar, was detected, and this agreed with Chagas et al. (2013).
These indicate the need to find a suitable approach to prevent salmonella in chicken meat, and show the necessity to increase cleanliness and hygienic principles in poultry slaughter houses.
Concerning of the tested acids effect on the intended pathogen, as shown in Table (5) and Figure (1), it has been cleared that the three additives proved their effectiveness (fumaric 2%> citric 2%> acetic 2% > citric acid 1%). On the other hand, acetic acid 1% and fumaric acid 1% has little significant effect.
Near finding obtained by (Cutter and Betancourt, 2000), they recorded 2 log reduction of Salmonella Typhimiurium in beef meat after treatment by 2% acetic acid, furthermore, Cosansu and Ayhan (2012) recorded that 1% and 2% acetic acid solutions reduced Salmonella enteritidis count in chicken leg meat by 0.85 and 0.95 log, respectively from 4-5 log initial number : while the same concentration of the citric acid reduced the same initial Salmonella enteritidis count in breast chicken meat by 0.95 and 1.58 log, respectively, Madushanka et al. (2018) concluded that the used acids have an antibacterial effect on S. Typhimurium in chicken meat, and Fernandez et al. (2021) found that count of Salmonellae in fumaric acid treated samples were significantly differ than control samples.
Ph range from 6.5 to 7.5 were preferred by Salmonella (Huiying, 2011), so their biological function may be lost under extreme pH levels and possible suppression. The three acids used in our study are having quite acidic pH. At room temperature acetic, citric and fumaric acids are shown following pH values 3.51, 3.91 and 2.48 that are antagonistic to the growth of Salmonella spp. The used acids not only cause the cytoplasm to become acidified and the difficulties in performing the essential metabolic functions in the bacterial cell but also cause an energy loss because ATP is used to actively export protons, (Davidson et al., 2013)

CONCLUSION
Approaches to diminish Salmonellae count on raw chicken meat should be assumed to increase the safety of chicken meat products and decrease the frequency of human salmonellosis from its consumption, It can be concluded that organic acids (acetic, citric and fumaric) have an antimicrobial effect on S. Typhimurium and, so, they can be used for the refinement process of chicken meat.