The Potential Impact of Moringa oleifera for Diminishing the Microbial Contamination and Prolonging the Quality and Shelf-Life of Chilled Meat

This study was implemented to assess the mechanism by which Moringa oleifera leaf extract (MOLE) improves the quality and prolongs shelf-life of the broilers’ breast meat. Ninety Cobb chicks were randomly allocated to 3 groups. A control group received the standard diet, whereas the other two groups received diets containing MOLE at the doses of 250 and 500 mg/kg for 21 days. Inclusion of MOLE in broilers diet significantly reduced the detrimental changes in the overall sensory attribute scores, characteristic color and odor, and the loss of breast muscle elasticity during storage. Furthermore, it significantly reduced concentrations of thiobarbituric acid, total volatile nitrogen, non-esterified fatty acids, and peroxide, during storage compared to the control samples. No effect on the concentrations of heavy metals, such as copper, cadmium, and lead, was observed. Decomposition of samples was delayed as indicated by lower pH values and higher sensory scores at 4 and 6 days of storage in the MOLE groups. Reduced contamination with E. coli and Salmonella species indicated an antibacterial effect of MOLE. Finally, the present study highlights that MOLE supplementation may play a role in improving quality and shelf-life of the chilled breast meat in broilers.

tenderness, low fat, good flavor, good aroma, and juiciness 14 . Another important factor is the safety of meat, which is affected by the extent of microbiological and chemical contamination and controlled by environmental conditions and nutritional supplements. Owing to specific phytochemicals (kaempferitrin, isoquercitrin, rhamnetin, kaempferol and quercetin) and the high content of protein, carotenoids, several minerals, and vitamins; Moringa has been described as a possibly useful feed for animals. It is mainly utilized in chicken nutrition to increase the efficiency of the growth, avoid infections, and enhance appetite by improving meat quality 8,15 . During recent years, considerable interest has been concentrated on utilizing natural antioxidants in food products, due to studies suggesting potential adverse effects that may be related to the consumption of synthetic antioxidants. Natural antioxidants of plant origin are reported to be safe and sound than artificial ones; and did not induce adverse effects on human health 16,17 . Natural compounds are safer and healthier compared to the synthetic antioxidants are increasingly applied in meat systems. They enhance the quality and storage-life of meat and meat-based products when added in animal diets, by decreasing fat and pigment oxidation, which reduces shelf-life and leads to meat quality loss [18][19][20] .
Until now, the use of Moringa oleifera in good quality and safe meat production, with no side effects on meat composition, has been limited. Although it was established that M. oleifera has significance for the quality and safety of meat production, unluckily, the incorporation of Moringa oleifera in production and preservation of meat is still under consideration. Hence, this research was conducted to investigate the possible ways by which Moringa oleifera leaf extract improves the breast meat quality of broilers and prolongs its stability and shelf-life.

MATERIALS AND METHODS Experimental animals
This research was performed in accordance with of the animal ethics rules of the Faculty of Veterinary Medicine, University of Sadat City. Research study was carried out in the animal facility of the previously mentioned faculty. Ninety, one-day old, Cobb chicks of both sexes have been obtained from El-Arabia Poultry Company. Water Journal of Pure and Applied Microbiology and feed were ad libitum provided. Light, naturally, and artificially, was kept 24 hours per day for 2 week, then one hour of darkness was applied daily. Wood shaving was used as bedding material. Strict sanitation practices were applied throughout the experiment. Chicks of all the groups were immunized against infectious bronchitis (IB) strains HIB120 and Newcastle disease (ND) at day 9 of age and against infectious bursal disease (Gumboro) at day 13 of age. There was no vaccination applied during the experimental period (day 14 -35).

Experimental design
Chicks were assigned into three groups, 30 birds each, at day 14 of age. The first group received a standard basal diet (Starter 23% CP 2900 kcal, Grower 20% Crude Protein 3000 kcal, and Finisher 18% CP 31000 kcal) 21 without any treatment and was used as control group (CON). The second group received a diet mixed with methanolic Moringa oleifera leaf extract (MOLE, 250mg/kg feed, MOLE250) from day 14 to day 35 of age. The third group received a diet mixed with MOLE 500mg/kg feed (MOLE500) from day 14 to day 35 of age. All the chicken groups were reared in the same building with identical managing and environmental conditions.

Preparation of Moringa oleifera leaf extract
Fresh Moringa oleifera (MO) leaves were purchased from a farm at Sadat City, Menoufia governorate, Egypt. For 7 days, fresh MO leaves were collected and shed dried at room temperature (22°C) with relative humidity of 65 %. The dry leaves have been ground, filtered, and drenched in 70 % methanol (50g/L). The extract remained for 24 h with soft shaking at ambient temperature (22°C). Through Whatman filter paper size No.1, extract was then filtered, and the solvent was evaporated by air current for 24 h. Finally, it was entirely dried out in a hot air oven at 40°C for 24 h. The extract was kept in a sealed bottle at 4°C until use 22 .

Sample collection
At the end of the experiment (day 35), chickens were overnightly fasted and euthanized according to the Islamic method of slaughtering. Bleeding of chickens were for 2 min, scalding at 54°C for 2 min, defeathering manually, and evisceration. Six animals per group were randomly selected and sampled. Breast was dissected from the left half of the carcasses for further analyses Journal of Pure and Applied Microbiology by collecting the part from the caudal ending of the sternum headed for a shoulder joint on either side of every carcass and by dragging the breast aside from the back and shattering the link at the shoulder joints. Each breast sample was divided into four equal parts and kept stored in plastic storage bags at 4°C for further analyses at days 0, 2, 4, and 6 post-mortem.

Determination of sensory parameters
Meat samples were evaluated at day 0, 2, 4, and 6 post-mortem. Sensory attributes and organoleptic characterization of raw meat of broiler breast were determined by a rating scale from 1 to 3 presented in Table 1 23 . The examined sensory characteristics were visual appearance (meat and skin color), meat texture, odor, and elasticity. Five qualified judges were trained in the method of tests on raw chicken meat. Also, another five trained judges re-examined the suggested organoleptical method of raw meat assessment. To acquaint the panel with various intensities for the various sensory characteristics of raw chicken meat, and to adapt a scoring scale that should be used, two samplings of 1.5h each have been carried out. The sensory profile utilized was received from the previous two sessions opened. Assessments were carried out in a sensual testing room equipped with the positive air pressure, personal stations, and appropriate illumination 24 .

Meat composition
Crude fiber, protein, fat, water, and ash of broiler breast muscle were determined in accordance with AOAC methods 25 . Moisture has been determined by dry up 1 g of meat in a furnace at 100-105°C till a steady weight has been obtained. Raw protein was determined in accordance with the Kjeldahl method. The raw protein had been obtained as 6.25×N%. Lipid content of the meat was determined in accordance with the Soxhlet extraction method utilizing petroleum ether. Ash content of the meat was determined by burning the sample in a muffle furnace at 550°C for 3h. Crude fiber was determined in accordance with boiling sample with 1.25% diluted H 2 SO 4 , rinsed with water, further boiled with 1.25% diluted sodium hydroxide and the residual deposit after the digestive process had been taken as crude fiber.

Determination of meat quality Determination of pH of the breast muscle
Determination of pH values was performed using a pH Measuring Device (Germany, Testo 205) by penetrating breast muscle.

Determination of Total Volatile Nitrogen (TVN) of the breast muscle
The TVN of the breast muscle was determined according to Conway and Byrne 26 . In a clean dry beaker, 10g of the sample were added to 30 ml of distilled water and thoroughly mixed by a blender for 2 min. Thus, 2 drops of 0.02 M HCl were added to bring the pH value to 5.2. The homogenate was slowly heated to 70°C and then cooled to room temperature and filtered into the inner compartment of Conway dish and then 2ml of 0.01M HC1 were added. The outer ring was filled with 2ml of the sample extract and 1ml of saturated potassium carbonate (KCO3). The Conway unit was rotated as gently as possible and the dish was covered and incubated at 36°C for two hours, HCl in the inner ring was titrated against 0.01M NaOH by using methyl red indicator (T 1 ml). TVN/l00g = 26.88 x (2-T 1 ), Where T 1 = volume of NaOH expended in the titration. 27 TBA test is based on determination of malonaldehyde (MDA) values as a product of lipid peroxidation. The degree of oxidative rancidity is usually stated as TBA number and stated as milligrams of malonaldehyde counterparts per kilogram of samples. A sample of ten grams was mixed with 50ml of distilled water in warring blender for 2 min. The mix was quantitatively transported into Kjeldahl flask by wash with an extra 47.5ml of distilled water. Though, 2.5ml of HCL were added to 125ug/g fat and then a small number of stones was sited to stop pumping. The flask was heated at the maximum heat reachable in the Kjeldahl distillation device. Approximately, 10 min after the boiling were essential to gather 50ml of the concentrate. Therefore, 5ml of the concentrate were mixed to 5ml of TBA reagent in 50ml glass stoppered tube. The contents were immersed and mixed in a boiling water bath for 35 min. Like samples, a distilled water TBA reagent blank was intended and handled. After boiling, cooling under tape water for 10 min was applied. The sample optical density alongside the reference liquid (blank) was measured by using Spectrophotometer (UNICAM969AA Spectronic, USA) at a wavelength of 538 nm. Concentration of malonaldehyde (mg/kg) = Rx7.8, Where R = the absorbance.

Determination of Peroxide Value (PV)
The peroxide value has been determined according to Asakawa et al. 28 . The titre of the sodium thiosulfate solution was determined, where the precise normality of Na 2 S 2 O 3 realizing that in this chemical reaction 1gram-equivalent of Na 2 S 2 O 3 react with 1gram-equivalent of K 2 Cr 2 O 7 (6moles Na2S2O3 react with 1mole K 2 Cr 2 O 7 ) was determined. Peroxide value determination by titration with sodium thiosulfate until blue colour disappeared. The titration procedure was repeated as a minimum 3 times and the single findings should not differ greater than 0.3ml. For all samples, PV was evaluated by the formula: PV = (V 1 -V 0 ) x T x 1000/m (ml equivalent available oxygen/kg). Where, V 1 = volume of thiosulfate solution needed to titrate the sample (ml), V 0 = volume of thiosulfate solution essential to titrate the blank, T= sodium thiosulfate solution titre (normality) and m= sample weight (g).

Determination of Free Fatty Acids (FFA)
Lipid was extracted by using 1 part of the inspected sample to the original extract ratio of 20 parts of 2:1 dichloromethane/methanol 29 . A final ratio of 8:4:3 dichloromethane/ methanol/ water was achieved, by adding a weak salt solution (0.66% NaCl), containing the water included inside the tissue. Titration procedure 30 was modified to the sample nature and quantity. The sample fat extract was vaporized with nitrogen and dissolved in previously neutralized ethanol with cresol purple and 60°C preheated. Three repeats of sample were used titrated with N NaOH to a violet end point Cresol purple has been planned to enhance the sensitivity of the technique and has been used to a titration of lipid fish extracts 31 . In this case, 0.05N NaOH had been used for the titration of an extract to a violet end point cresol purple. Consequently, FFA values were registered as a proportion of oleic acid which equivalent (ml NaOH X NaOH normality X 28.2) / sample weight.

Determination of heavy metals
The standard, blank and digest solutions were aspirated by Atomic Absorption Spectrometer (AAS, VARIAN, Australia, model AA240 FS) and analyzed for content of heavy metal. Investigation was carried out by air/acetylene flow (5.5/1.11/m) flame for Cd, Cu and Pb concentrations (mg/kg wet weight) 25 . Detection limit (ppb) 8-40 for Pb, 0.2-0.8 for Cd and 11-25 for Cu. The three heavy metal levels were verified from the digital scale of AAS and intended rendering to the equation: Metal concentration (ppm) C=Rx(D/W), where C= heavy metal concentration (wet weight), R= digital scale reading of AAS, D= prepared sample dilution and W= sample weight. Each heavy metal concentration in the blank solution was also determined and subtracted from each examined sample 32 .

Bacteriological examination
Samples were prepared for bacteriological examination according to ICMSF 33 . In brief, 225ml of sterile peptone water were added to 25g of the sample, and completely blended utilizing sterile stomacher for 1-1.5 min.

Bacterial screening E. coli screening
Immediately after slaughter, samples were screened for E. coli according to ISO 34 . Concisely, 1 milliliter from the initial dilution, was injected into MacConkey broth tubes supplemented by inverted Durham's tubes. The injected tubes remained at 37°C for 24 hours. From positive MacConkey tube, 1 milliliter was injected into one more MacConkey broth tube and developed at 44°C for 24 hours. Loopfuls from the positive MacConkey broth tubes, were individually streaked onto Eosin Methylene Blue agar medium (EMB), which was then developed at 37°C for 24 hours. The presumed colonies were seemed like metallic green in color. Into slope nutrient agar tubes, the presumed colonies were then inoculated and purified for further recognition.

Salmonella screening
Salmonella screening was carried out according to the method described by Harvey and Price 35 . In brief, one milliliter of the initial dilution was injected into nine milliliter Rappaport Vassilidis broth tube, and then the tube was stored at 43°C for 24 hours. On Xylose lysine desoxychoclate (XLD) agar medium, loopfuls from the inoculated tubes were individually streaked and developed at 37°C for 24 hours. The presumed colonies were looked as red with or without black centers. On nutrient Journal of Pure and Applied Microbiology results are presented as LSmean ± standard error (SE). Differences were deemed to be significant if Padj≤0.05.

Composition of broiler breast muscle
Feeding of broilers on diets containing MOLE at concentrations of 250 or 500mg/kg of diet (groups MOLE250 and MOLE500, respectively) did not have any significant impact (P>0.05) on composition of meat as shown in Table 2. Similar values were obtained in all three groups for ash, protein, fat, crude fiber, and water concentrations of the breast muscle.

Sensory characteristics during storage
The sensory quality of broiler breast muscle declined during storage in all three groups as indicated in Table 3, as expected. Meat of the CON group became unacceptable for human consumption already after day 2 of storage. This was obvious in all individual sensory characteristics and overall scores. Addition of MOLE in broiler diet delayed the sensory changes of the breast muscles over the storage period until unacceptability as in Table 3. External aspect differed between CON and MOLE500 samples already at day 0. External aspect, muscular elasticity, and overall score were different between CON and MOLE500 at day 2 of storage. Color and odor were not different between CON and both MOLE groups. However, the overall score was higher (P<0.05) in both MOLE group samples at days 2 and 4 of storage and was highest (P<0.05) in the MOLE500 group samples at day 6. The positive effect of MOLE on sensory quality of broiler meat was slightly stronger with higher MOLE concentration in the diet.

Objective quality parameters
Storage of the breast muscles at 4°C increased (P<0.001) meat pH and the concentrations of TVN, TBA, peroxide, and NEFA as shown in Table 4. Inclusion of MOLE in the diet of broiler chicks at concentrations 250 and 500mg/kg of diet significantly delayed detrimental changes. Compared to CON, the pH was decreased (P<0.05) from fourth day of storage through the MOLE group samples. The concentrations of TVN, TBA, peroxide, and NEFA were lower (P<0.05) in MOLE500 samples and in most cases also in MOLE250 samples from day 2 of storage. Differences between both MOLE groups were agar plates, the presumed colonies were then reared and sub-cultivated at 37°C for 24 hours. On slope nutrient agar, the purified suspected colonies were selected and streaked for further recognition.

Staphylococcus aureus screening
Staphylococcus aureus screening was performed according to ICMSF 33 . On duplicated plates of Baired Parker agar (BPA) and using a sterile glass spreader, 0.1ml from formerly ready dilution was spread. At 37°C for 48 hours, the injected and control plates were incubated. On nutrient agar slopes, the black colonies surrounded with clear zone have been chosen up and purified for further proof of identity.

Morphological, biochemical, and serological identification
According to Cruickshank et al. 36 , morphological examination has been conducted, while biochemical identification of E. coli was conducted according to the Krieg and Holt 37 . According to Kok et al. 38 , isolates were serologically recognized by using rapid diagnostic E. coli antisera sets (DENKA SEIKEN Co., Japan). The presumptive colonies of Salmonella were further subjected to biochemical tests; indole, methyl red, Voges-Proskauer and Citrate test (IMViC) as per the standard test protocol described in Bacteriological Analytical Manual FDA 39 . Salmonellae were serologically identified according to Kauffman-White scheme 40 for the determination of flagellar (H) and Somatic (O) antigens using Salmonella antiserum (DENKA SEIKEN Co., Japan). Staphylococcus aureus was tested for production of catalase, free coagulase, yellow pigment, and thermonuclease (TNase) according to Lancette et al. 41 .

Statistical analysis
Data obtained in this research were analyzed by using SAS statistical software (Version 9.3, SAS Inst., Cary, USA), data obtained in this research were investigated. Heavy metal concentration and meat composition data were analyzed with simple ANOVA and Tukey's posthoc test. Quality traits and Sensory data were analyzed by ANOVA using the MIXED procedure with fixed factor group and repeated factor storage day (with autoregressive covariance structure) and group x storage day interaction. The Tukey-Kramer correction was used to manage the Family-wise Error Rate (referred to as Padj). The  observed at day 6 of storage in TBA and peroxide concentrations indicating dose dependency of the beneficial effects.

Concentrations of heavy metals
Cadmium and lead were detected in about 50% of the investigated samples of all groups, whereas copper was measurable in all specimens. The concentration of heavy metals into individual specimens was highly variable.
The analysis of variance revealed no significant influence (P>0.05) of supplementation with MOLE, irrespective of the amount applied, on the concentrations of heavy metals in broiler breast muscle as in Table 5.

Bacterial contamination
Four out of 6 samples of the CON group were contaminated with potentially pathogenic bacteria. One sample was tested positive for Data are expressed as LSmeans ± SE; CON -control group, MOLE250 -group supplemented with 250 mg MOLE / kg diet, MOLE500 -group supplemented with 500 mg MOLE / kg diet. a-d different lowercase superscript letters indicate significant differences between storage days within a group (P<0.05) A,B different capital superscript letters indicate significant differences between groups within a storage day (P<0.05) pH (acidity), TVN (total volatile nitrogen, mg/100g), TBA (thiobarbituric acid, mg/kg), FFA (free fatty acids, mg KOH/g), Peroxide (meq)

disCussiON
With the intention to produce valueadded meat and to determine its acceptance, consumers and processor consider several meat quality traits, including sensory characteristics, quality parameters, nutritious value, and health effects 42,43 . Firstly, the visual aspect quality traits (external aspect, color, and odor) and the muscular elasticity are regarded as the key characteristics that determine consumers' initial preference for meat 43 . Feed additives are used to improve performance of broilers, and at the same time meat quality and shelf-life. Moringa oleifera leaves are used as feed additive and due to their high nutritive value improve feed intake and digestibility and lead to enhanced performance in monogastric animals 15 . Moringa oleifera has been described to enhance the storage-life as well as the quality of broiler meat 8,44 . We used two concentrations of MOLE in the current study as feed additive and investigated the effects on meat quality and shelflife of broiler breast muscle. Meat composition was not influenced by MOLE supplementation. However, the findings of our research indicated that the breast muscle sensory attributes of broilers fed a standard diet without MOLE (control group) became earlier unacceptable upon storage under chilling condition. The inclusion of MOLE in broilers diet delayed the unacceptable changes in the sensory quality of the breast muscles over the period of storage (6 days). Thus, it can be concluded that MOLE improves the shelf-life of broiler meat, as reflected in part of the sensory parameters (overall scores, external aspect, and muscle elasticity), under chilling condition. These findings were consistent with that of Cui et al 45 , who concluded that Moringa oleifera can be used as feed ingredient for broilers to improve meat quality and consistent with Karthivashan et al. 46 , who stated that feeding of broilers on diet supplemented with MOLE, 0.5% w/w, improved its meat quality. In another study 8 , MOLE as feed additive affected the color of broiler breast meat as well as the color stability over time until day 6. The effects were pronounced with the highest level of MOLE (1000g/ton diet). On the other hand, Qwele et al. 21 observed no effect of dietary supplementation of Moringa (5:20% of the diet) formulated diets on broilers meat color.
The improvement of meat quality in broilers fed diets containing MOLE may be attributed to the anti-oxidative potential of MOLE 47 . Meat quality attributes as ultimate pH, TVN, TBA, peroxide and NEFA and shelf-life are key parameters taken into consideration in the evaluation of meat by the consumer as well as the processor. Meat quality has been affected, to a great extent, by the percentage of ultimate pH, and its drop in the muscles after slaughter 42,43 . The pH was not different among the groups in our study until day 2 of storage. However, the pH increased thereafter much faster in CON than MOLE samples indicating decay initiation. Storage of breast muscles of the CON group under chilling condition deteriorated values of quality parameters (pH and TVN, TBA, peroxide and NEFA) induced by conversion and dismounting of lipids and proteins of the meat. Inclusion of MOLE in broiler diets improved the quality showing a reduced increase in values of pH, TVN, TBA, peroxide and NEFA and increasing shelf-life compared to meat of the CON group. These results were in accordance with that of Wapi et al. 8 who mentioned that MOLE as an additive affects the pH of broiler breast meat as well as its stability over time until day 6. The effects were pronounced with the highest level of MOLE (1000g/ton MOLE). In contrast, dietary supplementation of broilers with Moringa formulated diets had no effect on the meat pH in the study of Qwele et al. 21 . Type of extract and used concentrations may be responsible for the observed discrepancies.
The results of our study support conclusions that intake of feed supplemented with MOLE could be useful to improve the meat quality for human consumption. It was reported that MOLE is abundant in antioxidant compounds, such as terpenoids, coumarin, flavonoids, triterpenoids, sterols, saponins, alkaloids, and phenolics [48][49][50] . Antioxidant function of MOLE reduces the intensity of malondialdehyde (MDA) and fat peroxidative products 51,52 . Moreover, it increases the intensity of glutathione (GSH) as well as the activities of antioxidant enzymes superoxide dismutase and catalase [52][53][54][55] , because it is also rich in further antioxidant compounds such as vitamin c, selenium, oleic acid, phenolic compounds, and carotenoids. M. oleifera might also become a perfect candidate as a food preservative by inhibiting proteolysis via its protease inhibitor, in food industry 56 .
The other possible cause for improving the meat quality and prolonging its shelf-life is the reducing potential of MOLE through its low content of heavy metals, such as copper (Cu), cadmium (Cd), and lead (Pb). Heavy metals induce reactive oxygen species (ROS) generation and cause lipid peroxidation, membrane dismantling and damage to DNA, protein, and carbohydrates 57 .
Our study revealed no difference in the heavy metal's concentration of Cd, Cu, and Pb in broilers breast muscle compared to the control. Heavy metals such as cadmium which are possibly toxic are missing from Moringa oleifera leaves, thereby making their inclusion safe in poultry diets 58 . Thus, a daily intake of considerable amounts of toxic heavy metals Pb, Cd, and Cu, resulting in poor quality of poultry meat 59 , is prevented. Cd, Cu, and Pb, are problematic for humans through ingestion by food in our daily diet 59 . Monitoring of heavy metals in poultry meat and meat products is therefore important for providing good quality poultry meat products for human consumption. Poultry meat which exceeds the maximum permissible limits of heavy metals may be declared as unsuitable for human consumption 59 .
Another positive aspect of feeding broiler with diets containing MOLE, especially 500 mg/ kg diet, is the reduced bacterial contamination of their meat. The anti-bacterial activity of MOLE against different species of bacteria such as: E. coli, S. aureus and S. typhi has been highlighted by many authors. It was attributed to flavonoids, tannins, steroid, alkaloid, saponins contained in MOLE which have anti-bacterial properties 60 . Moringa oleifera leaf meal could serve as alternative to antibiotics in broiler production as this is in attempt to reduce antimicrobial resistance 61 .

CONClusiON
Compared to CON samples, sensory parameters (overall scores, external aspect, and muscle elasticity) were improved, values of ultimate pH, TVN, TBA, peroxide, and NEFA were decreased, and bacterial contamination was reduced in muscle samples of MOLE supplemented broilers over storage time compared to CON. Based on previous findings, M. oleifera as natural feed supplement improved the quality and prolonged the stability and shelf-life of the breast meat from broilers stored under chilling condition.