EFFECT OF NEUTRAL ELECTROLYZED WATER ON SHELF LIFE OF COLD CHICKEN MEAT

This investigation aimed to assess the impact of 50, 100, and 200 ppm of neutral electrolyzed water (NEW) on the sensory characteristics, chemical composition, and microbiological quality of fresh chicken breast meat kept for six days at 4±1ºC. The results showed that dipping chicken breast meat samples in NEW at three concentrations (50, 100 and 200 ppm) can improve storage stability and reduce microbial populations compared with the control group. The results on the sixth day of storage revealed that the pH values were 6.41±03, 6.34±02c and 6.32±02d, respectively, and the TBARS values were 0.79±0.02, 0.68±0.05 and 0.50±0.02 respectively. While aerobic plate counts, E. coli, and S. aureus counts were 5.58±0.23 , 4.54±0.29, 3.92±0.06; 1.82±0.15, 1.65±0.04, 1.38±0.12 and 1.62±0.07, 1.50±0.01, 1.32±0.02 log CFU/g, respectively, which was lower than the control group in all treatments on day 6. As storage duration extended relative to the control group, dipping chicken breast flesh samples in neutral electrolyzed water, especially at a concentration of NEW200 ppm, obtained the greatest score in sensory characteristics. Over the course of six days of storage, the pH and TBARS values of the meat samples rose in control and all treated groups, but lower values were obtained by treating with 50,100 ppm, while the lowest values were obtained by treating with 200 ppm of NEW. This study found that 200 ppm of NEW is more effective than 50 or 100 ppm. It can be a promising way to increase the shelf life of chicken meat without leaving any hazardous residues and is regarded as a healthy antimicrobial preservative agent for chilled chicken meat kept in a refrigerator.


INTRODUCTION
Chicken meat is one of the main sources of vital polyunsaturated fatty acids, vitamins, minerals, and protein; its consumption has been rising over the past few decades (Alonso-Hernando et al., 2013;OECD-FAO, 2016).In processing plants, contamination of chicken meat can occur throughout processing, packaging and storage until the meat is sufficiently cooked and consumed (Mensah et al., 2002).It may get tainted with harmful and spoiling microbes, turning it into food that is hazardous and unpalatable for customers.The initial count of spoiling microorganisms and the temperature during the production and storage processes determine how long poultry meat will last on the shelf (Yashoda et al., 2001).Chemical, physical, and biological methods are used in certain nations to decrease bacterial contamination and increase shelf life.To reduce or eliminate germs and improve the quality of the goods, numerous chemical treatments based on phosphates, chlorine, and organic acids have been developed for the meat and poultry industries (Loretz et al., 2010).
In food sectors, electrolyzed water (EW) is a safe and effective sanitizer that can lower microbiological hazards (Hricova et al., 2008;Huang et al., 2008).The only substance utilized in the creation of electrolyzed water is sodium chloride.Trihalomethanes, a possible carcinogen, can be produced by aqueous or gaseous chlorine reacting with proteins and carbohydrates in food (WHO, 2000;Huang and Batterman, 2009).In contrast to other sanitizers like hypochlorite, which are not only extremely corrosive but also detrimental to consumer health, NEW is safe for use and has little corrosive effects.Neutral electrolyzed water (NEW) has garnered a lot of attention in recent years as a potential disinfectant.It is safe for the environment, easy to use, and has no negative effects on the user, in addition to being an excellent sanitizer (Shirahata et al., 2012).
Neutral electrolyzed water (NEW), which has been shown in numerous studies to be effective as an antimicrobial agent in the food industry, can eliminate or reduce pathogens, including Salmonella typhi, Escherichia coli, and Campylobacter jejuni, in samples of fresh chicken meat (Saengkrajang et al., 2011) or even reduce them logarithmically (Kim et al., 2000;Han et al., 2018).Additionally, according to Abadias et al. (2008), its broad-spectrum efficacy may eradicate Listeria monocytogenes, Staphylococcus aureus, E. coli, S. typhimurium, and Enterococcus faecalis.In the food industry, this chemical has even been effectively utilized to disinfect and sanitize surfaces (Jiménez et al., 2016).The food's natural physical-chemical conditions, pH, free chlorine concentration, oxidation reduction potential (ORP), type of microorganism present and its microbial load, length of time the electrolyzed water was in contact with the food, and concentration all have impacts on how well the water disinfects (Guerra-Sierra et al., 2019).To achieve the anticipated disinfection of samples without endangering the public's health, it would be convenient to use a lower concentration of neutral electrolyzed water and extend the contact duration of the NEW during the chilling stage (4°C).In the chicken breast flesh samples, the microbial load of S. aureus and E. coli is greatly reduced by neutral electrolyzed water.Its usage is risk-free and is not harmful or corrosive (Guerra Sierra et al., 2022 andPatricia et al., 2023).
The primary cause of the quest for novel alternatives for the preservation of chicken meat is its contamination by harmful bacteria, such as Salmonella or Listeria monocytogenes (CDC, 2023).Chlorine usage in the food business is prohibited in several European nations, including Germany, Denmark, and Belgium (Ramos et al., 2013).By electrolyzing a NaCl solution, Neutral Electrolyzed Water (NEW) is a germicide that produces stable hypochlorous acid under regulated conditions.NEW has previously been used on surfaces and surroundings, as well as to disinfect food items such as pork, strawberry, broccoli, and squid, with positive outcomes (Athyde and Associates, 2017).Its production process is inexpensive, and since it doesn't need handling potentially hazardous chemicals, its use is an environmentally friendly choice.The bacterial wall's amino acid groups and sulfhydryl (-SH) oxidation impact are what cause the drug's antibacterial action, as reported by Kim et al. (2000) and Rivera-Garcia et al. (2019).This impairs the respiration and feeding processes of the bacterium.Therefore, the objective of this study was to assess the effect of different concentrations of neutral electrolyzed water (NEW) at (50, 100, and 200 ppm) on the shelf life, sensory, chemical, and microbiological quality of fresh chicken breast meat stored at 4±1ºC for 6 days.

Collection and preparation of samples:
The experiment was conducted in the Animal Health Research Institute -Damanhur city.One kilogram of raw, fresh, boneless chicken breast fillet samples was collected from nearby poultry abattoirs in the province of El Behera, sealed in sterile polyethylene bags, and carefully delivered to the laboratory.Within an hour, in separate boxes with cooling packs, and stored at 4±1ºC until they are needed for this inquiry.The samples were split into four groups, each weighing 250 g.The first group served as the control group, and the other three groups of chicken meat samples were used to assess the effects of 50, 100, and 200 ppm concentrations of neutral electrolyzed water (NEW) on the sensory, chemical, and microbiological quality of the samples.

Production of neutral electrolyzed water (NEW) (Rahman et al., 2016; Guerra Sierra et al., 2022):
To prepare electrolyzed water (EW), a membrane exchange device (Lbeg01-17) was utilized.Using Milli-Q water at a pH of 6.7 ± 0.1, which was confirmed with a pH meter (Thermo Scientific Orion 3 Star), the EW was produced from a 5% NaCl solution.The membrane exchange device was run through the salt solution.According to manufacturer recommendations, the obtained electrolyzed water showed an oxide reduction potential (ORP) of 1030 mV (Thermo Scientific Orion Star A221), pH-6.7, and 360 ppm of free chlorine (measured spectrophotometrically using a Merck Pharo-300 and the Spectroquant®Chlorine test-Merck® technique).This electrolyzed water was dissolved into solutions and kept in 220 L drums with threaded lids at concentrations of 50, 100, and 200 ppm (Ref.BR22) for use within 8 h.

Processing and treatment of samples (Moghassem Hamidi et al., 2021):
Three of the four groups were treated with neutral electrolyzed water (NEW) at concentrations of 50, 100, and 200 ppm for 15 minutes at room temperature, followed by storage at 4±1ºC for six days.The control group was submerged in sterile distilled water.Fresh chicken breast meat samples from all groups were tested right away for sensory evaluation, chemical (pH and TBARS value), and initial microbial load of aerobic plate count (APC), E. coli and S. aureus, sp counts, at zero-day and on days 2, 4, and 6 of storage at 4 o C. The proportion of treatment solutions to chicken breasts was 1:1 (w/v).Subsequently, each was packaged separately into sterile zippered polyethylene bags and kept at 4±1ºC.

Microbiological analysis:
4. 1. Preparation of serial dilutions (APHA, 1992): 25 grams of meat samples were weighed and put into a sterile homogenizer flask that contained 225 milliliters of peptone water (0.1%) under aseptic circumstances.The contents of each flask were homogenized at 14000 rpm for 2.5 minutes in order to yield a 10 -1 dilution.After that, a sterile pipette was used to transfer 1 ml to a sterile test tube containing 9 ml of (0.1%) peptone water.Subsequently, a decimal serial dilution was made in increments of 10 -10 to accommodate the entire range of anticipated sample contamination.The following formula was used to count and record the number of colonies in colony forming units per gram (cfu/g) of meat samples for microbial counting: cfu/g = level of dilution plated x number of colonies counted/volume plated.These were further expressed in mean colony forming units per gram (mean cfu/g) and converted to log10 base values (log10cfu/g).

Total aerobic plate count (APC) (Jay, 2002):
After being weighed and placed into a sterile stomacher bag containing 0.1% sterile buffered peptone water (1:9 W/V), the treated chicken breast meats were gently washed for two minutes.Then, 0.1% sterile buffered peptone water was used to serially dilute the rinse solution 10 times. 1 ml of the suitable diluent was plated in triplicate using the pour-plate technique on the plate count agar (Merck, Germany) for the enumeration of (APC).The plates were then incubated at 32 o C for 48 hours and 7 o C for 10 days, respectively.

E. coli count
Using a sterile bent glass spreader, 100 μl of each previously prepared serial dilution was evenly distributed over duplicate plates of Eosin methylene blue (EMB) agar (OXOID, CM0 069).For twenty-four hours, the control and inoculation plates were incubated at 37 °C (FDA, 2001).The greenish metallic colonies suspected to be E. coli had a dark purple center.The number of colonies and their expression as log CFU/g of material were recorded.

Staphylococcus aureus count:
According to (FDA, 2001) the serial dilution was distributed on Baird-Parker Agar medium (CM 215) supplemented with egg yolk tellurite emulsion (SR54) plates at 35 o C for 48 hours.Colonies that looked suspiciously black and glossy, with a halo zone surrounding them, were selected for morphological analysis and biochemical identification.

Chemical analysis:
On days 0, 2, 4, and 6 of the storage, the following chemical analyses of every treatment were performed:

Measurement of pH (ES 63-11/2006):
We confirmed with a Digital Jenco 609 pH meter.By mixing a 10 g sample with 90 ml deionized water for two minutes, the pH was determined.A digital pH meter was used to determine pH.

Measurement of Thiobarbituric acid reactive substance (TBARS) (ES 63-9/2006):
A ten-gram sample and forty-eight milliliters of distilled water were combined.Add two milliliters of 4% ammonium chloride (to raise the pH to 1.5) to the previously mentioned components, blend for two minutes, and let the mixture sit at room temperature for ten minutes.After being put into Kjeldal flasks, the mixture was rinsed with a further 50 mL of distilled water, an antifoaming solution, and a few glass beads.After the flask was heated to 50 °C, the Kjeldal distillation apparatus was put together.Ten minutes after the boiling started, distillates were collected.A stopped glass tube was filled with the distillates (50 ml), which had been mixed.After adding 5 milliliters of TBA reagent (0.2883/100 milliliters of glacial acetic acid), the tube was sealed, shaken, and left in a bath of boiling water for 35 minutes.Similar to the sample, a blank was made by mixing 5 mL of TBA reagent with 5 mL of distilled water, and it was handled the same way.The tube was heated and then allowed to cool for ten minutes under tap water.A section was moved to a curette, and then a spectrophotometer (Perkin Elmer, 2380, USA) was set to read the sample's optical density (D) against the blank at a wavelength of 538 nm.When comparing the sample to the blank, the TBA value (mg malondialdehyde/Kg of the sample) is equal to Dx7.8 D.

Sensory evaluation (Moghassem Hamidi et al., 2021)
Six trained sensory panelists performed the sensory evaluation of the chicken breast flesh on days 0 and 6 of storage.Every sample was assessed three times.A straightforward four-point scoring system was used to assess color, odor, and texture following the guideline table.The following formula was used to determine the sensory index.

SI =
(2 X C) + (2 X O) + T 5 Where C stands for colour, O for odour, T for texture, and SI for sensory index.Assessing the colour, texture, and odour of chicken breast flesh to determine its sensory quality score Qualities 4 (Highest quality) 3 (Good quality) 2 (Fair quality) 1 (Poor quality) Terms used to describe breast chicken flesh used to assess its sensory qualities  , 1955).Data presented as three replicates' mean ± SD.A column's means that are separated by distinct letters differ significantly from one another (P < 0.05).

RESULTS
Table 1): The mean scores for the sensory attributes of samples of chicken breast flesh stored at 4±1ºC for six days while being treated with varying amounts of neutral electrolyzed water (NEW).The panelists discovered that the newly prepared chicken breast samples (day 0) in both the treated and untreated samples with neutral electrolyzed water (NEW) were well in all sensory qualities, as can be seen from the findings shown in Table (1) which displays the results of the sensory analysis performed on the treated chicken breast meat samples from days 0 to 6 of storage.

Parameter
Throughout storage, all of the treatments' sensory indices for odour, colour, and texture declined.In contrast to the control group, which had the lowest score at the end of storage (day 6), the chicken breast meat samples treated with neutral electrolyzed water (NEW) at concentrations (200 ppm) had the highest sensory index score on day 0, when all treatments were observed to have higher scores than the control group.According to Rasooli, (2007), sensory profiles enable us to assess meat quality and occasionally spot undesired impurities.The sensory assessment findings showed that the chicken breast samples treated with neutral electrolyzed water (NEW) scored higher than the control samples in every sensory category.Neutral electrolyzed water (NEW) is added at concentrations of 50, 100, and 200 parts per million.Up to the end of the sixth storage day, there was a noticeable improvement in the chicken breast meat's appearance, softness, and flavour, notably at the concentration (200 ppm).
After four days of storage, the sensory quality of the chicken breast samples, particularly the control sample, markedly declined and was no longer fit for cooking.Sensory attribute alterations were less noticeable in the samples of chicken breasts treated with NEW (200 ppm), compared to control samples and other treatment groups.
These findings concur with those of (Moghassem Hamidi et al., 2021) who found that chicken meat stored in a refrigerator had improved sensory qualities due to the application of neutral electrolyzed water (NEW).Also, (Guerra-Sierra et al., 2022) reported that neutral electrolyzed water enhances the favorable sensory qualities of chicken meat, such as taste, colour, odour, texture, and the overall acceptability in chillers.Furthermore, other foods treated with neutral electrolyzed water (NEW) during refrigeration have been shown to retain their sensory qualities.This means that NEW can extend the shelf-life of food items, in addition to providing them with the proper colour and flavor (Patricia et al., 2023).Because of the physicochemical stability of chickens, the obtained findings indicated that NEW may be employed throughout the preparation of chicken meat.

Chemical analysis of treated chicken meat: 2.1. Hydrogen ion concentration (pH)
According to Hernandez-Pimentel et al.
(2020), lipid/protein degradation brought on by chemicals, microbes, and physical damage resulted in a rise in pH values in the chicken breast meat samples (control or treated groups) throughout the refrigerator storage period until the end of the sixth day.
The obtained results in Table (2) showed the changes in the pH value of the chicken breast meat samples, the initial pH on zeroday was 6.26±0.02 in group I (control).
While at the end of storage (day 6), the control group had the highest pH value of 6.57±02 while the last treated group with 200 ppm had the lowest value of 6.32±.02 and treated groups with 50 and 100 values were 6.41±03 and 6.34±02 respectively.
The action of microbial or endogenous enzymes like lipase and protease, which raise the concentration of volatile bases over extended storage, may be the source of rising pH readings (Hernández Pimentel et al., 2020).Furthermore, a statistically significant difference was seen on day six between the groups who received treatment and the control group.This is supported by the findings of Rahman et al. (2012) and Sheng et al. (2018).The rise in pH during storage might be attributed to the microbial production of ammonia and the degradation of protein components (Gill, 1983).The control group experienced a higher pH value than the other treatment groups, and on day six of storage at 4 o C, when the pH value reached >6.5, the first signs of spoiling were noticed.

Thiobarbituric acid reactive substances (TBARs):
The thiobarbituric acid reactive substances (TBARS) test is a widely used method for the identification of secondary oxidation products.Malondialdehyde (MDA) is the primary product of oxidative rancidity and is responsible for the off flavour of oxidized fat.Products made from chicken breast flesh have low quantities of antioxidants and high levels of polyunsaturated fatty acids, which make them vulnerable to oxidative degradation (Dawson and Gartner, 1983).It was shown by the data in Table (3) that the TBA of control samples' mean values were increased from 0.43±0.05at day zero to 1.13±0.03at day 6 of storage.While TBA values of the chicken breast treated with neutral electrolyzed water (NEW) at concentrations (50, 100 and 200 ppm) increased to 0.79±0.02,0.68±0.05and 0.50±0.02mg MDA/kg respectively at day 6 of storage.

Regardless
of treatment, TBARS progressively rose as storage time increased, but TBA levels in treated samples dramatically lowered malondialdehyde (MDA) levels in comparison to the control sample.The control and treated chicken samples exhibited modest levels of lipid oxidation, with lipid oxidation levels below 0.5 mg MDA/kg, indicating no oxidative rancidity throughout the storage period.TBA in chicken meat shouldn't exceed 0.9 mg/kg of chicken meat, according to (ES 1651(ES /2005)).Samples treated with (NEW) at (50 ppm and 100 ppm) are good for consumption until day 4; samples in the control group are valid until day 2 only.On the other hand, the samples treated with (NEW) at concentration (200 ppm) were safe to eat up to day 6 of storage.According to the obtained data, the optimum group for minimizing TBA level was that treated with neutral electrolyzed water (NEW) at concentration (200 ppm).These samples had the lowest TBA value compared to other treated groups.When compared to the control group, the groups treated with neutral electrolyzed water (NEW) exhibited considerably (P<0.05) less lipid oxidation.NEW may have a higher antioxidant capacity because of the high concentration of phenolics it contains.These findings concur with the findings of Patricia et al. (2023).

Microbiological analysis 3.1 Total aerobic plate count (APC):
High APC may be attributed to the contamination of the chicken meat from different sources or unsatisfactory processing, as well as unsuitable conditions during storage (Zahran, 2004).NEW has an antibacterial impact on a variety of microorganisms, including pathogens, according to the findings of many researches.The product and kind of microbe determine the antibacterial action.In addition to being very caustic, some sanitizers, including hypochlorite, are harmful to consumers' health.In contrast, NEW is safe for consumers and has low corrosive effects.Data presented in Table (4) showed that aerobic plate count mean values were increased from day zero to day 6 of storage.The control APC mean values ranged from 3.17±0.06at day zero to 6.93±0.02log10 cfu/g at day 6 of storage.
While APC mean values of the chicken breast treated with neutral electrolyzed water (NEW) at concentrations (50, 100 and 200 ppm) reached 5.58±0.23,4.54±0.29 and 3.92±0.06at day 6 of storage.Compared to the control group, samples treated with NEW exhibited a substantial reduction in the count of aerobic bacteria, particularly at concentrations of 200 ppm.The overall bacterial count should not be more than 10 5 /g (ES 1651/2005).On day 2, the APC count for the control samples was 4.95±0.02,which was almost at the maximum recommended limit, while on day 4, the APC count for control samples was 5.64±0.05which was over the maximum recommended limit and indicated that the untreated control chicken breast samples had a shelf-life of less than six days.The treated samples exhibited a delayed growth for APC till day 6, and a larger decreasing impact on total bacterial count was noted in (NEW 200 ppm) when (NEW) concentrations were raised to 100 ppm and 200 ppm.This indicates that under chilled storage, the samples' shelf life was extended to six days.The APC values for the samples treated with (NEW 50 ppm) were still valid for consumption until day 4 of storage.
Moghassem Hamidi et al. (2021) found that (NEW 200 ppm) significantly reduces the total viable count of chicken breast flesh during refrigerated storage, which lends credence to this conclusion.
Treated chicken samples with (NEW) at concentrations 100 ppm and 200 ppm do not exceed the permissible limit 10 5 cfu/g even after storage for 6 days.The bacterial wall's amino acid groups and sulfhydryl (-SH) oxidation impact, which influences bacterial respiration and feeding is the source of NEW's antibacterial activity (Rivera-Garcia et al., 2019 andKim et al., 2000).The bactericidal activity of NEW is also produced by the controlled electrolysis of NaCl solution, resulting in the regulated generation of stable hypochlorous acid (Rahman et al., 2011;Athayde et al., 2017;Hernández-Pimentel et al., 2020).(Rahman et al., 2016).

S.aureus count:
The emergence of multi-resistant zoonotic strains of pathogenic foodborne bacteria, such as S. aureus, from foods originating from animals, has led to a significant problem of food poisoning in underdeveloped countries, causing tremendous economic losses and public health issues (Guerra Sierra et al., 2022) Poult. Res. 2020, 29, 622-635. Hricova, D.;Stephan, R. and Zweifel, C. (2008): Electrolyzed water and its application in the food industry.Journal of Food Protection 71: 1934-1947.

Table 2 :
Pattern of pH of chicken breast meat samples treated with different concentrations of neutral electrolyzed water (NEW) during refrigerated storage at 4±1ºC for 6 days.

Table 3 :
The TBARS values (mg/kg) of chicken breast flesh samples treated with varying concentrations of neutral electrolyzed water (NEW) were analyzed after six days of refrigeration at 4±1ºC.

Table 4 :
Chicken breast flesh samples treated with varying concentrations of neutral electrolyzed water (NEW) during six days of refrigeration at 4±1ºC were examined for patterns of aerobic plate (bacterial) count APC (log10cfu/g).

Table 5 :
Pattern of E.coli count (log10cfu/g) in chicken breast meat samples treated with different concentrations of neutral electrolyzed water (NEW) during refrigerated storage at 4±1ºC for 6 days.

Table 6 :
Pattern of S.aureus count (log10cfu/g) in chicken breast meat samples treated with different concentrations of neutral electrolyzed water (NEW) during refrigerated storage at 4±1ºC for 6 days.
Egyptian Standards for Chilled Poultry and Rabbits: Egyptian Organization for Standardization and Quality Control.Ministry of Industry, Arab Republic of Egypt, Cairo, Egypt.ES (63/11-2006): Egyptian Organization for Standardization and quality control.Egyptian Standards for poultry meat products treated with heat.Methods of analysis and testing for meat and meat products Part: 11 Measurement of pH.ES (63/9-2006): Egyptian Organization for Standardization and quality control.Egyptian Standards for poultry meat products treated with heat.Methods of analysis and testing for meat and meat products part: 9, determination of thiobarbituric acid FDA "Food and Drug Administration" (2001): Center for Food safety and applied nutrition.www.FDA.org.Guerra-Sierra, B.E.; Sandoval-Meza, A.X. and García-Sánchez, L.T. (2019):