HIGHLIGHT ON THE INFLUENCE OF LACTIC ACID AND OZONIZED WATER ON THE SHELF LIFE OF CHICKEN FILLET

The study was supposed to detect the impact of three decontaminators (ozonized water, lactic acid and their blend of them) in improving organoleptic characteristics, chemical and bacteriological quality of poultry meat. Chicken fillet samples were collected from Zagazig city, Egypt. and divided into three groups, Group1: To assess the impact of additives on sensory criteria, chemical quality indices (pH, Thiobarbituric acid, Total Volatile Basic-Nitrogen) and APC of the examined samples with reference to their the shelf life, Group 2: For assessment the additives effectiveness on Enterobacteriacae counts, Group 3: to evaluate the influence of additives on Staphylococcus aureus artificially inoculated in chicken fillet meat samples, each group was divided into 4 subgroups (control, ozonized water 0.38%, lactic acid 1% and blend of both), the data cleared that the immersion of samples in the blend of ozonized water with lactic acid can prolong the shelf life of chicken fillet during chilled storage at 4 o C ±1 with keeping its sensory characteristics and chemical parameters meanwhile a combination of ozonized water and lactic acid can protract the shelf life of chicken fillet by additional 48 hours. Concerning to the antimicrobial efficacy of the different trials, it's interesting to note that the combined uses of ozonized water –lactic acid have greatly a noticeable decrease in aerobic plate count, Enterobacteriacae count and S. aureus count, followed by ozonized water, finally lactic acid. So it’s recommended that using ozonized water-lactic acid blend in poultry meat.


INTRODUCTION
An outstanding nutritional profile is a characteristic of chicken meat as it is vital to include in a diet plan for people of all ages because it is low in fat and nearly entirely composed of unsaturated fatty acids, as well as high biological value protein, vitamins, and minerals (Marangoni et al., 2015) but, as soon as the fresh meat processing process starts, contamination occurs.Usually, the animal is the source of pollution or its external surroundings, as contamination occurs first on the external surface of the meat, and because of the extended production, packing, and transportation chain, meat provides the excellent medium for microbial growth (Capita et al., 2001).The most dangerous contaminant is the bacteria such as Staphylococcus that can be present in most meals for example red meat, chicken and their products which are directly manipulated by humans (Kitai et al., 2005).In addition of producing virulent heat-stable toxins that cause toxic shock syndrome, S. aureus is a particularly dangerous microorganism that can cause hospital infections and food poisoning (Kérouanton et al., 2007).S. aureus is the most familiar reason of food-borne human sickness worldwide (Do Carmo et al., 2004).Enterobacteriaceae are of the most bacteria isolated from chicken meat.The Enterobacteriaceae family is sub grouped into 8 tribes including: Escherichie spp, Edwardsielle spp, Salmonelle spp, Citrobactere spp, Klebsielle spp, Protee spp, Yersine spp, and Erwine spp (Ardrey et al., 1968) Consequently, many consumers have expressed a desire for higher-quality chicken products with a high dietetic value, harmless food additives, prolonged shelf lives, and most importantly, no pathogenic microorganisms.(Sarron et al., 2021).
One of the food improvement techniques able to deactivate bacteria is by ozonation method.Odor control, color removal, organic compound decomposition, and air and water disinfection have all been achieved with ozone.Ozonized water is very hazardous to bacteria due to its great oxidation potential (Qingshi et al., 1989).Furthermore, ozonized water had no adverse effects on the organoleptic criteria of chicken products (Mancini and Hunt, 2005).Ozonized water was accepted by the USDA as an appropriate and safe component for use in preparation of meat and poultry (United States Department of Agriculture, 2002).
Lactic acid is an organic acid with confirmed efficiency as a decontaminant in many categories of food.It can prolong the shelf life of chicken while it is refrigerated by slowing the growth of bacteria that cause spoiling, preventing the production of unwanted compounds, and enhancing sensory qualities (Smaoui et al., 2012).The maximum initial reduction in aerobic mesophilic and psycrophilic bacteria in chicken breast was achieved by lactic acid 3% (Cosansu et al., 2011).
Between the effective treatment practices is the mixing of organic acid solutions and another antibacterial agent that have been tested on the chicken products and showed variable results.Additions of LA to ozonized water enhance its decontamination power (Megahed et al., 2020).So, the current study was aimed to explore the effect of ozonized water 0.38%, lactic acid 1% and their blend on sensory characteristics, biochemical profile, shelf life and microbial load of chicken fillet as a raw material of most chicken products.

MATERIALS AND METHODS:
1-Preparation of chicken fillet samples: Chicken fillets were collected from the city market in Zagazig, handled under aseptic conditions and transported an ice box container without delay to microbiology laboratory at Animal Health Research Institute, Zagazig Lab., for more examination.
Aerobic Plate Count, Enterobacteriacae and S. aureus count was were detected according to APHA (2001) and ISO 21528-2, 2004and FDA (2001) in a preliminary, unreported trial to obtain the samples of choice for further treatment trials with different decontaminators, i.e. samples with a known total bacterial and enterobacteriacae count and S.aureus free samples.broth until the turbidity was adjusted to match a 0.5 McFarland standard tube (1.5 x 10 8 CFU) and incubated at 37 o C for 24h, Then centrifugation for 15 minutes at 3000 rpm to obtain a pellet of bacterial cells, then washing twice in phosphate buffered saline (PBS) and diluted to 1.0 x 10 6 CFU/ ml in PBS for inoculating of the samples Saad et al. (2015).

Design of the experiment:
Three groups of chicken fillet samples were subjected to the following treatment Group 1: For evaluation of the effectiveness of additives on sensory criteria, chemical quality indices (pH, TBA and TVB-N) and APC of the examined samples, the analysis were done initially after 1 hr.(1 st day) then day after day until decomposition take occur.
Group 2: For evaluation of the effectiveness of additives on Enterobacteriacae count in treated and control samples Group 3: For evaluation of the effectiveness on S. aureus count artificially inoculated in chicken fillet samples after 30, 60 and 120 minutes of dipping in decontaminators.
The control and treated groups were chilled at 4 o C ±1 with interval period (1-9 days) were subjected to further sensory, biochemical and bacteriological analysis

4-Sensory evaluation:
The coded samples were organoleptically examined in the period of refrigerated storage for color, odor, texture and overall acceptability by panel consisting of 10 members from the Animal Health Research Institute.The panelists used a 9-rating scale for their scores as described by Pearson and Tauber (1984).

5-Chemical quality parameters: 5.1-pH (Pearson, 2006):
Approximately, 10 grams of the sample and 10 ml of neutralized distilled water were mixed in a blender.After 10 minutes of continuous shaking at room temperature, the homogenate was allowed to settle.Then use an electrical pH meter and record results.

6-Bacteriological evaluation:
Preparation of samples: ISO 6887-3 (2017): From control and treated groups, 10 g test portion was taken under aseptically, transferred into a sterile 90 ml peptone water 0.1% (Merck) to obtain a dilution 10 -1 , a millimeter from this suspension (10 -1 ) was transferred with a sterile pipette to a tube of sterile peptone water 0.1 % (9 ml) to obtain a dilution of (10 -2 ), repeat this step for more serial dilutions.

Enumeration and isolation procedure:
1-Aerobic plate count (APC): APHA (2001): Aseptically, 0.1 ml of each dilution was spread onto the surfaces of two sets of plate count agar solid media in petri dishes that had already been labeled.After letting the medium absorb the inoculum, invert the plates and incubate them for 48 hours at 35 ± 2 °C.The duplicate plate average count was counted, and the APC/g was calculated.

2-Enumeration of S. aureus (BAM, FDA,
Chapter 12): Pouring of 1 ml of the prepared dilution into plates containing the specific media (Baird Parker -Egg yolk-Tellurite emulsion) incubated at 35 -37 ºC for 48 hours.To detect the count of S. aureus per gram, distinctive black colonies with a zone of clearing and a narrow white margin surrounding them were counted.

3-Enumeration of Enterobacteriacae (ISO
21528-2, 2004) Using poured-plate technique 1 ml of the initial suspension is inoculated in two Petri dishes then pouring of Violet red bile glucose agar (44 o C to 47 o C).After solidification a layer of the same medium is added and allows solidifying.The dishes are incubated at 37 °C for 24 h ± 2 h.Confirmation Subculture of colonies of presumptive Enterobacteriaceae on nonselective medium, and confirmation by means of tests for fermentation of glucose and presence of oxidase.

7-Statistical analysis
The recorded results were statistically analyzed using analysis of variance (ANOVA) test and comparative of means were performed according to Duncan, using SPSS ver. 14 (2006).All bacterial counts were changed to log10 (log CFU/g).Results were recorded as mean ± standard errors (SE).The value of P<0.05 was used to indicate statistical significance.

RESULTS
Table 1: Sensory evaluation of treated and untreated chicken fillet samples.

Decomposed Border line
Fit for human utilization Keeping quality 7 th day 5 th -6 th day 0-5 th day Control samples (Untreated) 8 th day 6 th -7 th day 0-6 th day Samples treated ozonized water 11 th day 9 th -10 th day 0-9 th day Samples treated with lactic acid 11 th day 9 -10 th day 0 -9 th day Treated samples with ozonized waterlactic acid blend

DISCUSSION
Shelf life, which includes appearance, texture, flavor, color, and nutritional value, is the amount of time that passes between a product's packaging and consumption during which the product properties stay acceptable to the user of product (Singh and Singh 2005).The meat and meat products shelf life ' s depends on the level of its microbial contamination.
Thus, food microbiologists and technologists had critical objectives, including raising the keeping quality of meat and reducing or getting rid of food-borne infections and Sensory evaluation of chicken fillet through the period of refrigerated storage was cleared in table 1 and showed that sensory characteristics were affected by different methods of treatments like lactic acid and a blend of ozonized water and lactic acid, these were proven to be highly effective in delaying sensory changes in refrigerated chicken fillet and its shelf life extending and decomposed to 9 th and 11 th day respectively, meanwhile ozonized water alone extend the shelf life one day more than control samples.The sensory changes were attributed to proteolysis and lipid oxidation in untreated samples (control) that were more obvious in shorter time than those in treated samples due to progressive growth of microbial load, this specific spoilage fault is a result of an accumulation of extremely high numbers of microbial cells instead of being produced by any specific metabolic activity of the microorganisms.These results similar with Moghassem Hamidi et al. (2021) who found that chicken meat stored in a refrigerator had improved sensory properties due to the using of neutral electrolyzed water.
Regarding to the results cleared in the table 2 the initial pH values were almost identical in each group.The values of pH diminished immediately after treatment with lactic acid and lactic acid -ozonized water mixture, in comparison to control samples, similar to our findings Aktas et al., 2003) declared that the lactic acid addition to the meat systems resulted in diminish the pH initial values.The pH mean values On the 3 rd day increased gradually within cold storage at 4 ± 1°C; however it remain acceptable, at the 5 th day the untreated control samples had pH value (6.61±0.024)higher than treated groups (6.18±0.015,6.02±0.01,6.07±0.015,6.14±0.014)ozonized water, Lactic acid and ozonized water -lactic acid blend respectively, at the seventh and ninth day the pH value exceed the permissible limit, this is due to the breakdown of protein /lipid as a result of chemicals, physical and microbiological damage with formation of alkyl group and accumulation of ammonia.The initial spoilage occurs at pH 6.2 (Pearson, 2006).High pH value recorded at the 5 th day of storage at control group was similar to that reported by Hernandez-Pimentel et al. (2020), declared that an increase in pH values in the chicken fillet samples (treated or control groups) at the sixth day throughout the refrigerator storage period.Slight reduction of the pH caused by organic acids is fulfilling to avoid the growth of several bacteria (Stratford and Anslow, 1998).Therefore, treatment with lactic acid and its mixture with Ozonized water stabilized the pH of the injected chicken breast.
The measurement of total volatile Basic nitrogen (TVB-N) is a traditional chemical mean it common used to assessment the degree of meat spoilage and it shouldn't over than 20 mg TVN/ 100 g according to (ESO, 2005(ESO, /1651)).The content of TVN in chicken point for evaluating the freshness of chickens as an important reference index, (Ozogul and Özogul, 2000).Results tabulated in table 3 measured normal values of (TVB-N mg %) in the untreated and treated samples until the day 5, we note that the average value of TVB-N in the untreated control samples (21.30±0.36) is slightly higher than the permissible limits.
and the treated samples continued to be within the normal range, on day 7 the TVB-N values was 22.26±0.49for ozonized water treated samples and the others still within normal values going to the ninth day it reach to 20.41 ±0.33 and 21.63±0.31for lactic acid and ozonized water -lactic acid blend treated groups respectively.This is almost in line with findings from Rukchon et al. (2011) that TVB-N was significantly found in fresh chicken and that its level increased with storage period, as well as findings from Khalafalla et al. (2016) that showed TVB-N at the third and sixth days of storage was significantly higher in the Control group than all treated groups.It's possible that microbial activity at low temperatures is the cause of this TVB-N rise (Ibrahim and Desouky, 2009).
However, Shenouda (1980) described that the rise in TVB-N is not associated with microbiological activity, but rather is typically brought on by autolytic enzymes and deamination.
Trimethylamine (TMA), Ammonia and Dimethylamine (DMA) make up the majority of TVB-N compounds found in chicken, and their concentrations rise as the chicken spoils due to enzymatic or bacterial degradation (Khalafalla et al. 2016).According to Ndaw et al. (2008), TBA is a useful diagnostic for determining the quality of meat and the extent of oxidative rancidity and lipid oxidation.Malondialdehyde (MDA) is the main byproduct of oxidative rancidity and is responsible for the bad flavor of oxidized fat.
It has been suggested that 0.9 mg/kg is the highest TBA value that denotes high-quality chicken meat (ESO, 2005(ESO, /1651)).As cleared in table 4, TBA mean values for all treated and untreated groups nearly the same and significant increase with the advanced storage till the 5 th day.At the 7 th day control untreated sample show undesirable increase which indicate spoilage, followed by ozonized water treated samples, meanwhile samples treated with lactic acid and the ozonized water -lactic acid blend still within the acceptable limit until the day ninth.According to obtained result, lactic acid alone or in combination with ozonized water effectively prevented lipid oxidation in samples of chicken meat.These data support the findings of Lee et al. (2023)  and sensory evaluation were seen in all control and treated groups telling that using of ozonized water-lactic acid blend improve the quality of chicken fillet to an extent.However, the study of their effect on bacteriological quality is still required.
Table 5 indicates a comparison of effectiveness of ozonized water, Lactic acid and Ozonized water -lactic acid blend used separately on microbial stability of the chicken fillet samples which stored at 4 °C.Regarding to treated samples, APC significantly reduced from the first day of treatment, Ozonized water -lactic acid blend was the variable that most significantly reduce the microbial growth followed by Lactic acid>ozonized water,.By the third and fifth day all treatment trials also showed a significant reduction in APC.Meanwhile at the 7 th day signs of deterioration appear on control samples and no further bacteriological evaluation could be done.At the same time the three treatment groups declared a delayed growth in APC till the day 7th by ozonized water and to the day 9 by Lactic acid and ozonized water -lactic acid blend.But the highest effect recorded by Ozonized water -lactic acid blend.According to Casas et al. (2021) and Rangel et al. (2021), one of the main causes of ozone's disinfecting power is its oxidation-reduction potential (2.08 eV) and rise in intracellular reactive oxygen species (ROS), which cause bacterial cell lysis and have a negative impact on nucleic acid.Similarly, According to Jindal et al. (1995), immersion of chicken drumsticks in ozonized water at 0.44 to 0.54 ppm for 45 minutes resulted in 1.11-log CFU/cm 2 reductions in aerobic plate counts.This in agreement with Karamah and Wajdi (2018) findings, which showed that when chicken fillets are contacted with ozonized water containing 0.38 mg/l of ozone for 120 minutes at 3˚C, the amount of aerobic mesophilic bacteria is reduced by 1 unit log cfu/g.This is in line with the findings of Alahakoon et al. (2014) found that there were a significant reductions in the total aerobic counts of treated samples by calcium chloride that were combined with 0.002% or 0.01% lactic acid and untreated control samples.
Concerning to the impact of lactic acid, ozonized water and their mixture on Enterobacteriacae group it was clear in Table ( 6) that they have a significant effect with a log reduction of 0.73, 0.87 and 0.78 after half hour, 0.91, 1.13 and 1.2 after one hour and 1.01, 1.19 and 1.22 after two hours of immersion in ozonized water, lactic acid and their combination respectively.Similarly Smaoui et al. (2012) (2008) who discussed that at 25 ppm ozone reduced the number of 15 bacterial species both Gram negative and Gram positive bacteria by greater than 3 log cfu/ml.furthermore, Yang and Chen (2007) found that throughout the whole refrigerated period, the broiler parts treated with ozone had microbial counts that were lower than the control parts.According to the findings of EL Dahshan et al. (2013), after 9 days of storage, the treated groups that received 40, 60, and 70 ppm of ozone for 20 minutes remained within the permissible limits authorized (EOS-1090/2005); these indicated that the ozone treatment extended the shelf-life of the chicken breasts.
Table 7 illustrated that the three groups of treatments were significantly reduce the count of S. aureus artificially inoculated in the examined chicken fillet samples and arranged as follow Ozonized water -lactic acid blend > lactic acid > ozonized water with a log reduction of 2.64, 2.19 and 2.15 respectively after two hours and it was approved that ozonized water-Lactic acid blend for 2 hours is the trial of choice, this come in agreement with (Kazemi Taskooh et al., 2016)

Table 2 :
The mean pH values in examined treated and untreated chicken fillet samples.

Table 3 :
The mean TVB-N values in examined treated and untreated chicken fillet samples.

Table 4 :
The mean TBA values in examined treated and untreated chicken fillet samples.

Table 5 :
The mean APC±S.E (Log10 CFU/g) of untreated and treated groups of chicken fillet samples.Means inside the same column with different superscripts are different significantly at (p< 0.05) according to Duncanʼs multiple comparisons.R: Rejected samples.

Table 6 :
The mean counts ±S.E.(log10 CFU/g) of Enterobacteriacae in control untreated and treated chicken fillet samples (n = 3 for each group).

Table 7 :
The mean counts ±S.E.(log10 CFU/g) of the inoculated S. aureus in control untreated and treated chicken fillet samples (n = 3 for each group).