Assiut University web-site: www.aun.edu.eg

REDUCTION OF SALMONELLA HAZARD IN CHICKEN FILLETS USING VARIOUS MICROBIAL INHIBITORS

GHADA M. MOHAMED¹ and HALA I. SCHARAWEE²

¹ Animal Health Research Institute, Assiut Regional Lab, Meat Hygein.

² Animal Health Research Institute, Serology Unit.

Received: 27 March 2016;       Accepted: 29 April 2016

INTRODUCTION

Poultry meat and its products are very popular food throughout the world and no wonder since they are delicious, nutritious and considered as a good and cheap source of protein with good flavor and easily digested. They ranked first or second in foods associated with disease in most of the countries all over the world (Ibrahim et al., 2014). There is no primary religious rescission on the consumption of poultry meat (Kim and Day, 2007).  Poultry  products

Corresponding author: Dr. GHADA M. MOHAMED

E-mail address: ghada02468@yahoo.com

Present address: Animal Health Research Institute, Assiut Regional Lab, Meat Hygein.

are highly perishable, and depending on the processing condition, their spoilage varies significantly even under refrigeration (Patsas et al., 2006). It can easily be contaminated with microorganisms because fresh meat is very suitable for microbial multiplication. Meat has high water activity, high in nutrients and readily utilizable low molecular weight substances and is a source of carbon and energy. As a result; fresh meat is a suitable substrate for bacterial multiplication (Hinton, 2000). The major bacterial contamination on chicken includes pathogens such as Salmonella spp. (Kim and Day, 2007).

Salmonellae are members of the Enterobacteriaceae family. They are gram-negative facultative anaerobic, non-spore-forming coccobacilli. Salmonellae are usually motile except S. Gallinarum and S. Pullorum. The genus Salmonella has been divided into two species, Salmonella enterica with 6 subgroups (I, II, IIIa, IIIb, IV and VI) and Salmonella bongori (formerly subsp. V) (Brenner et al., 2000).

Salmonellosis is a foodborne illness caused by infection with Salmonella spp http://kidshealth. org/parent/general/sick/germs.html. Not everyone who ingests Salmonella bacteria will become ill. Children, especially infants, are most likely to get sick from it (The Nemours Foundation, 2015). It depends upon the health status, age and immune system of the person (Mahajan et al., 2003). However, the infective dose of salmonellosis is variable. Eating food contaminated with approximately 10⁵ to 10⁶ cells per gram of food causes an illness (Fehlhaber and Janetschke, 1992). A Salmonella infection generally causes nausea, vomiting, abdominal cramps, diarrhea, fever, and headache. Finally, the infection can cause other health problems, like meningitis and pneumonia (The Nemours Foundation, 2015).

Salmonella is one of the most important causes of food borne illness of known etiology in the world. These pathogens are usually present in chicken flocks during production and transferred to non-infected broiler chickens during the subsequent stages of transportation to processing plants, processing and retail (Ajok et al., 2014). Poor hygienic condition during the sub stages of processing may be responsible for the significant increase in Salmonella. The sub stages include stunning, slaughter, scalding, defeathering, evisceration, washing, deboning and packing. Cross contamination was more likely to occur on contact with surfaces of the machine containing a higher bacterial load (Bada et al., 2006). In addition, it has been noted that there is a greater population of bacteria on the breast than other edible portions of the chicken carcass making this an important site to control the organism (Kotula and Davis, 1999).

An analysis of a PCR technique to validate sensitivity and specificity 0f culture techniques for detecting Salmonella contamination in retail poultry meat was conducted. In the last time with the purpose of declaring the virulence factor (s) some authors reported that virulence of Salmonella is encoded with certain genes which can be detected by polymerase chain reaction (PCR). The location of the genes on chromosomes or plasmids has controversy character in published investigations (Osman, 2015).

The use of antimicrobial agents allows products to be microbial safe with the enhanced ability to extend shelf life. Recently, acidified sodium chlorite (ASC) has been suggested as an effective in reducing microbial contamination and its use has been approved by the United States Food and Drug Administration (USFDA) for the application on various food products(Sexton et al., 2007 and kim et al., 2009). ASC is considered a broad spectrum oxidative antimicrobial effective on pathogenic bacteria. It works by forming Oxychlorus antimicrobial intermediates as it comes into contact with organic matter (Kross, 1984). The Oxychlorine compounds forming chlorite, chlorate and chlorine dioxide after contact with food surfaces. Chlorine dioxide is either evaporated or reduced without residual traces (FSAN, 2003). Candian Food Inspection Agency Meat Hygiene Directive (2001) listed ASC approved microbial control agent in range of 500-1200 ppm for use on Poultry.

In addition, chlorine is the most frequently used antimicrobial intervention in commercial poultry processing due to its availability, low cost, and efficacy (Northcutt and Jones, 2004). In general, chlorine compounds are effective against Gram-positive, Gram-negative, and acid fast bacteria. The potential antimicrobial action of chlorine summarized in the following: its molecules penetrate the bacterial cell wall and react with key enzymes to prevent normal respiration and carbohydrate metabolism (Fabrizio et al., 2002). Chlorine is permitted, the level do not normally exceed 50ppm, which results in a reduction in microbial load of around one log (Northcutt et al., 2005).

In general, it must be emphasized that, decontamination treatments are able to reduce the contamination level but do not completely eliminate pathogens. Their effectiveness depends on the initial microbial load and treatment conditions. Regarding treatment conditions, there are many factors affecting the efficacy of these antimicrobials including concentration of the substance, time of exposure, temperature, pH and hardness of water, strength of bacterial adhesion to the carcasses,(Lechevalier et al., 1988).

The purpose of the present investigation was designed to evaluate the prevalence of Salmonella spp. in chicken fillet, confirmation of the isolated strains by PCR with carrying out the virulence factor(s) in isolated organisms. Moreover, the effect of acidified sodium chlorite and chlorine in reducing the population of Salmonella with measurement their residues on chicken fillet were studied.

MATERIALS AND METHODS

1- Microbiological analysis of chicken fillet samples:

Collection and preparation of samples: (Jamshidi et al., 2008)

Eighty samples of chicken fillets were obtained from local markets with different sanitation levels in Assiut city. Samples were transported to the laboratory immediately after collection in an ice box. The frozen collected samples were left to be thawed overnight in refrigerator, then they were analyzed bacteriologically.

Isolation and Identification of Salmonella:

Twenty – five grams of each sample were put into a stomacher bag containing 225/ ml buffered peptone water and homogenized using a stomacher then incubated at 37°C for 18hrs. One ml was transferred to 10ml selenite cystine broth and incubated for 20-24hrs. at 37°c. Plating was carried on XLD agar and incubated at 37 for 24h. Then examined for typical colonies of Salmonella (red with black center). Presumptive Salmonella colonies were confirmed by biochemical test (Indole, Methyl red, Voges proskauer, citrate, urease, (TSI) and sugar fermentation) (APHA, 1992).

Serological Identification of suspected Salmonella isolates:

Using rapid agglutination technique as described by (Minor and popoff, 2000). Salmonella antisera were obtained from DENKA SEIKEN Co.Lid, Tokyo, Japan.

2- Molecular analysis:

DNA Extraction

This part was done by Benha University, Faculty of Veterinary Medicine, Food Analysis Center.

Using QIA amp. Kit according to (Shah et al., 2009) as shown in Table 1:

DNA amplification for the selected virulent genes:

As described by (Singh et al., 2013).

3- Studying the effect of Acidified Sodium Chlorite and Chlorine on survivial of Salmonella Enteritidis in chicken fillet:

Bacterial Cultures and Inculum:

Salmonella Enteritidis was cultured in tryptic soy broth and cultures were incubated at 37°C for 24h. After incubation the number of cfu/ml was determined according to (FAO, 1992).

Aseptically obtained chicken cubes:

Frozen chicken fillet was thawed by overnight refrigeration, and slicedaseptically into approximately   1 ´ 1 ´ 1 cm pieces (1g weigh pieces).

The Antibacterial Agents: (Inatsu et al., 2010).

Acidified sodium chlorite solution (ASC):

Acidified sodium chlorite solution (ASC) was prepared by mixing 0.5g/log sodium chlorite and 1g/L citric acid and the solution pH was 2.62 then allowed to activate for 10 minute. Then diluted with sterile distilled water to form different dilutions of 400,600 and 900 part per million (ppm).

Chlorine solution: (Nassar et al., 1997).

This was prepared according to the instructions of the manufacturer: 14 g/m³ of water, giving 1 ppm chlorine. Three concentrations of chlorine in water were used in this experiment: 20 ppm, 30 ppm and 40ppm. The concentration of each solution was tested with a photometer to confirm the amount of chlorine. The various treatments were prepared just prior examination and placed in sterile containers.

Assessment of microbial growth:

Analysis was conducted on the artificially contaminated chicken fillet within determined time of analysis after bacterial inoculation and refrigeration (at 4^oC for4 hours). Counting of bacterial load was applied for S. Enteritidis according (FDA, 2011). For statistical analysis, average count of colonies on duplicate plates was transformed in to Log cfug-1.

4- Measurement of the residual ASC and chlorine:

ASC and chlorine treatment residue analysis by High Pressure Liquid Chromatography (HPLC) (NZFSA, 2003).

RESULTS

Table 2: Incidence of Salmonella spp. in the examined chicken fillet samples

P CR identification:

Fig. 1: Agarose gel electrophoresis of multiplex PCR of stn (260 bp), (260 bp), invA (284 bp) and fim H (1008 bp) virulent genes for identification and characterization of Salmonella spp.

Lane M: 100 bp ladder as molecular size DNA marker.

Lane 1: Control positive Salmonella strain for stn and inv A and fim H genes.

Lane 2: Control negative.

Lane 3: Positive S. Enteritidis strain for stn and inv A genes.

Lane 4: Positive S. Enteritidis strain for stn and inv A and fim H genes.

Lanes 5 & 6: Positive S. Typhimurium strains for stn and inv A and fim H genes.

Lane 7: Positive S. Virginiastrain for stn and inv A genes.

Lane 8: Positive S. Finkenworderstrain for inv A gene.

Table 3: Effect of different concentrations of Acidified Sodium Chlorite on the viable count of inoculated S. Enteritidis in chicken fillet:

*C: Count

**R: Reduction

Table 4: Effect of different concentrations of chlorineon the viable count of inoculated S. Enteritidisin chicken fillet:

Fig. 2: Effect of different concentration of ASC on S. Enteritidisin chicken fillet

Fig. 3: Effect of different concentration of chlorine on S. Enteritidisin chicken fillet

DISCUSSION

1- Incidence of Salmonella spp. in the examined chicken fillet:

The isolation of invasive Salmonella serotypes in our study indicates the public health significance of these serovars as contaminated chicken meat may pose health hazards. The arised risk may further be higher if chicken meat is consumed undercooked or due cross contamination in the kitchen with Salmonella during meal preparation (FSIS/USDA, 2013).

Results achieved in Table (2) indicated that Salmonella spp. were isolated from7.5% of examined chicken fillet. Salmonellae could be identified serologically as S. Enteritidis (2.5%), S. Typhimurium (2.5%), S. Virginia (1.25%) and S. Finken worder (1.25%).

The incidence of Salmonella spp. in examined chicken fillet samples in the present study showed marked lower than that recorded by Arafat et al. (2011); Saad et al. (2011); Lidia et al. (2006) and Ibrahim et al. (2014) where their findings were, (52%), (16%) and (15.39%) and (13%) respectively. Other studies of isolation of Salmonella spp. in chicken carcass which also were higher than the obtained result conducted by Paião et al. (2013) (45%), Alali et al. (2012) (27%), Chang (2000) (25.9%), Dhaher et al. (2011) (24.76%), Molla and Mesfin (2003) (15.4%), and Todd (1999) (13.3%). On the other hand, the incidence of this organism was nearly agreed with that results obtained by Straver et al. (2007) and Abdellah et al. (2009) who recorded 8.6 and 6.25% chicken fillets, respectively. Husnu and Walid (2015) cited 3.1% in chicken fillet which seem to be lower than this results. Moreover, Vaidya et al. (2005) and Bajaj et al. (2003) reported negligible prevalence as low as 5% of chicken carcasses.    

The findings outlined in the same Table declared that the incidence of S. Enteritidisin chicken fillet samples was (2.5%.(  This trend was lower than that obtained by Ibrahim et al. (2014) who recorded 7% in chicken fillet, Hee et al.(2007) (21.9%), Paião et al. (2013) (12%) and Mohammad et al. (2014) (5.8%) in chicken carcases, respectively. While Saad et al. (2011) failed to recover S. Enteritidis from the examined samples of chicken fillet.

Salmonela Typhimurium as presented in Table (1) was recovered from chicken fillet in a percentage of 2.5% which was lower than reported bySaad et al. (2011) (8%) and Ibrahim et al. (2014) (7%) in chicken fillet, Mohammed (2013) (44%) and Hee et al. (2007) (23.4%) in chicken carcass. While, this study showed some what higher findings than that reported by Mohamed et al. (2014) who could isolate S. Typhimurium from 1.2% of chicken fillet. The prevalence of S. Typhimuriumin the examined chicken carcass recovered by Paião et al. (2013) (3%) was nearly agreement to the obtained.

Regarding S. Virginia, the findings illustrated in Table (1) revealed that examined chicken fillet samples proved to harbor 1.25% S. Virginia. Hee et al. (2007) and Mohamed et al. (2014) recorded that the incidence of S. Virginia was 6.3 and 3.5%, respectively in chicken carcass which was higher than that obtained in this study. Furthermore, Chang (2000) could isolate S.Virginia from raw chicken carcasses.

It is worth to mention that in this study, we have identified S. Finkenworder (1.25%) which is uncommon invasion non typhoidal Salmonella. Its presence in the examined chicken fillet may be due to the hazard exportation chicken or poultry products. Most cases of Salmonellosis which arecaused by non typhoidal Salmonella are mild (WHO, 2013).

The presence of Salmonella in chicken meat may be attributed to poor hygiene conditions, regarding the temperature of storage, the equipment and the employees' personal hygiene and the cutting tables were seldom washed, These benches could therefore be reservoirs from which Salmonellae could spread to other equipment through flies or direct contact (Stevens et al., 2006).

The strains which identified biochemically and serologically as Salmonella spp. were subjected to PCR test. The result indicated that, two strains were S. Enteritidis, another two were S. Typhimurium, one strain was S. Virginia and the last strain was S. Finkenworder.

The virulence of any particular Salmonella organisms is determined by its invasiveness, which depend upon the attachment of the organism to the mucosal epithelium and production of enzymes and toxins that damage the epithelium and /or alter epithelial permeability and facilitate bacterial entry into the mucosal cells and infection of the lamina propria (Coburn et al., 2007). One of the earliest steps in the pathogenic cycle of the facultative intracellular pathogen Salmonella spp is the invasion of cells of intestinal epithelium. Gene Inv A ne is amember of Inv genes those allow Salmonella organisms to enter the epithelial cells. In the present study Inv A gene was detected in 100% of the examined isolates (fig1) the used primer of the gene multiply a region of 284 bp. Many authors as Jacopsen and Holben (2007), Abd Elfatah (2014) and Osman (2015) could detect the Inv A gene and explained that the gene was necessary for the invasion of the cell. During the course of this study, Stn, gene Salmonella enterotoxin recovered in all isolates was investigated by using PCR. The Stn gene was a heat labile Salmonella enterotoxin. All isolates (S. Typhimurium, S. Eenteritidis and S. Virginia) except S. Finkenworder were detected to harbor Stn gene that encoded on plasmid DNA and amplified region 260 bp (Fig 1).

This result agrees with ElEbeedy (2011)who reported that Stn gene is widely distributed among Salmonella irrespective to their Serovars and source of isolation. The Stn gene was encoded mainly on plasmid DNA and hence on total DNA. Several workers have corroborated this findings (Rahman, 1999; Rahman et al., 2000 and El Ebeedy, 2011).

Characterization of the fim H gene encoding the fimbrial adhesions indicated two allelic variants. fim H is responsible for mediating binding to eukaryotic cells and mutants unable to produce fim H have been shown to lack adhesive ability (Hancox et al.,1998).Consequently, the fim H polypeptide is considered to be the adhesion molecule of type 1 fimbriae and confers the binding specificity upon the fimbriae. Jennifer et al. (2002)detected fim H gene is S. Typhimuriumserovars these results agree with our results. Other serovars didn't harbor fim Hgene (S. Enteritidies, S. Virginia and S. Finkenworder) except one S. Enteritidis serovar harbor fim H gene.

2- Effect of acidified sodium chlorite (ASC) on Salmonella Enteritidis:

Acidified Sodium chlorite is activated when pH values in the range 2.5-2.9 Candian Food Inspection Agency, 2001). ASC is Known as Generally Recognized as Safe Acid (GRAS), (Shireen and Abdelmonem, 2014).

In present study the cubes of the chicken fillet witch inoculated with10⁶ log cfu/g, of S. Enteritidis were dipped in different concentrations of ASC (400,600 and 900 ppm) and left to drain (leaving non treated sample as a control) then counted at the 0, 1, 2, and 4 hours of refrigeration storage for the viable cells of S. Enteritidis. Meanwhile, the initial count of control samples in zero time was 5.6 log cfu/g while the number differed in the different dipped samples with different ASC concentrations. The maximum reduction in the count of Salmonella to the inoculated chicken cubes (4.1 log cfu/g, with reduction value 1.5log cfu/g compared to control) was immediately occur after treatment (0hr) with concentration 900 ppm, as shown in (Fig2). As it was 4.5, and 4.3 in the concentration 400 and 600 ppm, whereas the corresponding values of reduction were 1.1 and 1.3 log cfu/g respectively. The cidal effect of acid decontamination of fresh meat surfaces depends principally on the immediate lethality (Dickson and Anderson, 1992).

The immediate and short-term bactericidal effect of ASC on Salmonella on chicken samples has been recorded by Sexton et al. (2006) who observed immediate reduction of 0.05 log cfu/g for Salmonella after 20 seconds dip in ASC (900-1000 ppm), Salmonella prevalence was reduced from 90% to 10%, which was initially very low.

During storage at 4ºC the counts of control samples after the first hour was 3.8 log cfu/g, while when examining samples dipped in 400,600 and 900 ppm ASC gave S. Enteritidis number as 3.2, 2.9 and 2.6 log cfu/g, with reduction values 0.6, 0.9 and1.2 log cfu/g respectively.

The control sample showed a slight decrease in the second hours of experiment 3.5 log cfu/g, while it was 3.1, 2.8 and 2.5 log cfu/g for samples dipped in ASC of the three concentrations, the corresponding values for reduction were 0.4, 0.7 and 1.0 log cfu/g respectively.

In the fourth hour of storage, S. Enteritidis in control samples, decreased to be 3.2 log cfu/g. while when examination of samples which dipped in 400,600 and 900 ppm ASC concentrations become 3, 2.7 and 2.3 log cfu/g with reduction values 0.2, 0.5 and 0.9 log cfu/g respectively.

From the mentioned results, the inoculated control samples with S. Enteritidis showed obvious decrease in its bacterial count during refrigeration storage, which approves that low temperature treatments can inactivate the living cell by the attribution of ice nucleation and dehydration (Brunnberg et al., 2009).

Many investigators evaluated the effectiveness of ASC as a processing aid for dipping or spraying to control Salmonella increasing the safety of chicken carcasses. Kemp et al. (2000) cited that the using of ASC at concentrations (500ppm, 850 ppm) is an effective method for reducing microbial contamination on chicken carcasses, while the maximum antimicrobial activity of ASC when carcasses were dip in 1200 ppm for 5 seconds. While Kemp et al. (2001) applied an "ASC spray system" in reducing Salmonella levels on contaminated poultry carcasses, could report that the incidence of Salmonella was 10%. Moreover,Wang and Zhou (2014) evaluated the effects of ASC (concentrations ranging from 0 to 1 g/L) the results showed that bacterial numbers were significantly reduced with increasing concentrations of ASC reaching maximum reductions of 2.2 log cfu/g for Salmonella.

It is clearly evident from the previous results that the action of ASC on Salmonella occurred immediately after the cells were exposed to the treatment and minimal or delayed effects were noticed during storage. This is explained by Anderson and Marshall (1989) who recorded that ASC decontamination causes death and sub-lethal injury to microorganisms but in the favorable environment on meat surface after decontamination, injured cells on meat can repair and then grow normally.

The use of ASC can lead to substantial reductions in both the prevalence and concentration of important foodborne pathogens. The degree of reduction, if achieved commercially, is likely to substantially reduce consumer exposure while providing potential product quality benefits (FSANZ, 2003).

3- Effect of chlorine on Salmonella Enteritidis:

The decontaminating efficacy of chlorine depends on the concentration of chorine. Therefore, it is necessary to know its concentration in the treated solution, and a determination should be routinely done as quality control for decontamination (Fenner, 2005).

From the summarized results given in Table 3 it is evident that the cubes of the chicken fillet which inoculated with 10⁶ cfu/g, of S. Enteritidis were dipped in different concentrations of chlorine (20, 30 and 40 ppm) and left to drain (leaving non treated sample as a control) then counted in the 0, 1, 2 and 4 hours of refrigeration storage for the viable cells of S. Enteritidis. The count of control samples in zero time was 5.6 log cfu/g. The maximum reduction in the count of Salmonella to the inoculated chicken cubes (2.6 log cfu/g, with reduction values 3 log cfu/ gcompared to control) was immediately occurred after treatment (0h) with concentration 40ppm, as shown in (Fig3). However, the count was 3.2, and 2.7 log cfu/g at concentration 20 and 30 ppm, with reduction values 2.4 and 2.9log cfu/g respectively.

Nassar et al. (1997) recorded that the chicken carcasses  when subjected to 20 ppm and 50 ppm, of chlorine was no reduction in the number of Salmonella but when Northcutt et al. (2005) used 50 ppm of chlorine, recorded 3.1 log10 reduction of Salmonella which similar  to this result., On contrary, the presence of Salmonella reported by Whyte et al. (2001), Fabrizio et al. (2002) and Brunnberg et al. (2009) was lower than that obtained in our study, their reduction values were 1.04, 0.86and 0.75 log cfu/g respectively, treated by 20 and 25 ppm. While A higher reduction (4 log cuf/g) was recorded by Saad et al. (2015) when used 50 ppm.

The number of Salmonella in the control samples after the first hour of refrigeration storage was 3.8 log cfu/g, while when examining samples dipped in 20,30 and 40 ppm chlorine gave S. Enteritidis number as  2.3, 2.1 and 2 log cfu/g, with reduction values 1.5, 1.7 and1.8 % respectively. Chlorine is not available for a longer period; after one hour in this study we noticed that the effect of chlorine already was lowered because of the unstable chlorine.

In the second hour of experiment, the control sample was 3.5 log cfu/g, while it was 2.1, 1.9 and 1.8 log cfu/g for samples dipped in chlorine of the three concentrations respectively, the corresponding values for reduction were 1.4, 1.6 and 1.7 log cfu/ grespectively.

In addition, after 4 hours of storage, the control samples was 3.2 log cfu/g, where as the populations of S. Enteritidis for the samples treated with 20,30 and 40 ppm of chlorine was 2, 1.8 and 1.7 log cfu/g, with reduction values, 1.2 ,1.4 and 1.5 log cfu/g respectively. Our result was nearly agreement to that recovered by Saad et al. (2015) who recorded1.3 log cfu\g when used 30 ppm of chlorine.

From the qualitative observations, there was no detectable bleaching of the treated product with ASC and chlorine however, they were impossible to detect which carcasses had been treated. There was also no detectable odor difference between the control and treated samples either initially or throughout the shelf-life. Our results are in agreement to that recovered by FSANZ (2003) who reported that ASC treatment solution, after contact with food surfaces is either evaporated or reduced without any residual traces. While, Northcutt et al. (2005) observed that the use of chlorine did report no or only minor changes on poultry carcasses which nearly agreed with our obtained results.

However, when Izat et al. (1989) and Nassar et al. (1997) used high level of chlorine as100 ppm reduced the number of Salmonella organisms was reduced (70-75%) respectively, but had a yellowish appearance and a strong chlorine smell compared to the non-treated controls.

Levels of chlorine which used in the processing plants of Saudi Arabia are approximately 20 ppm to 50 ppm, since poultry processors believe that higher chlorine concentrations produce an undesirable tainting of and color in the carcass (Nassar et al., 1997).

Therefore, these results clearly indicate that chlorine treatment is more efficient way of inactivating of S. Enteritidis than the use of ASC in the chicken fillet.

Although the use of antimicrobial agents is only one step in the process of pathogen reduction it would suggest that it is worth trialing a commercial system and following product through further processing stages (such as boning, portioning and marinating) to determine if the significant reduction in pathogens can be achieved on further processed products Sexton et al. (2006).

4- Measurement of the residual ASC and chlorine:

Measurement of the residual ASC:

Chlorite and chlorate residues in carcasses dipped in Acidified sodium chlorite (ASC) was assumed as a behaviour of the decontaminant chemicals similar to the decontamination process applying ASC (Alcide, 2002).

In present study, residual ASC were detected immediately after treatments of chicken fillet samples by 400, 600 and 900 ppm for 5 s and dip in rinse water. The residual were (0.03 μg/cm2 of meat surface) which below the estimated detection limit for chlorite and chlorate.

The residual concentrations of chlorite and chlorate as reported in the data submitted to the Joint Food and Agriculture Organization of the United Nations (FAO)/ World Health Organization (WHO) Expert Committee on Food Additives (JECFA) (WHO, 2008) for raw meat and meat products, including poultry that had been treated with ASC solution were 0.1 mg/kg for both chlorite and chlorate.

Residues of chlorite and chlorate were reported by SCVPH (2003) for poultry carcasses immersed in a 150 mg/l ASC solution at pH 2.8 and 5 ^oC for 1 h, then drained for 5 min and rinsed for 5 min in clean water. The residue levels were lower than the detection limit (chlorate <19 μg/kg) or became so after 2 h (chlorite <16 μg/kg).

Measurement of the residual chlorine:

Hypochlorous acid and the hypochlorite ion together constitute aqueous chlorine. Aqueous chlorine is widely used throughout the food processing industry (McIntyre et al., 2008).

In this trial, Residual chlorine was detected immediately after treatments of chicken fillet samples by 20, 30 and 40 ppm for 5s and dip in rinse water. The residue levels of chlorine could not be detected by using (HPLC) and suggested that residues were below measurability.

WHO Working Party has established a tolerable daily intake (TDI) for chlorine of 150 μg/kg body weight/day (International Programme on Chemical Safety, 2000). No information was found on chlorine residues in chicken flesh following treatment with aqueous chlorine. While there is some evidence for formation of chlorinated compounds in chicken treated with aqueous chlorine, oxidation reactions appear to predominate for chlorine dioxide and acidified sodium chlorite (Hoenicke et al., 2004).

On the other hand, Robinson et al. (1981) could detect Chloroform in chicken treated with aqueous chlorine due to absorption of chloroform from chiller water, rather than direct formation in the chicken flesh. The highest average concentration of chloroform (177 μg/kg) was observed in muscle meat following dipping in a solution of 50 mg/L chlorine for 5 minutes at 15-16ºC. Thus, poultry (Scientific Committee on Veterinary Measures Relating to Public Health, 2003) chilled in chlorinated water contributes only 0.3 – 1% of the daily chloroform exposure, whereas water contributes most 99% of the exposure.

The residue levels for both ASC and chlorine were based on using Good Manufacturing Practice (assuming application and post-treatment recommendations are followed).

IN CONCLUSION

This study shown that Salmonella spp. was widespread among the chicken fillet. It may be due to insufficient hygienic condition. This, subjecting the consumer health to great hazards, so the need of antimicrobial agent to minimize and control such organisms is important. Treatment of chicken with ASC and chlorinecan improve the safety of chicken without any physical changes or sensory alterations, besides it minimizing tissue damage and maximizing antimicrobial effects. Additional studies are needed that consider the effect of chemical concentration, spray pressure, contact time, solution recycling, and point of application in commercial processing to ascertain the effectiveness of chemical applications against Salmonella spp. Further studies are needed to improve surveillance strategies to decrease the prevalence of Salmonella spp. in chicken population.

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