Chemical and Antimicrobial Effects of Pomegranate ...

Vol 23,Vol No.XX 4;Apr , No.2016 XX;

Chemical and Antimicrobial Effects of Pomegranate Peel Aqueous Extract Attia H Atta1, Nermeen H. M. Hassaneen2, Manal I. Abd El Fadeel3, Doaa Sedky4, and Amany M. Mohamed4 1

Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Giza 12211. 2 Food Hygiene Department and 3 Biochemistry Department, Animal Health Research Institute, Dokki. 4Parasitology and Animal Diseases Department, National Research Centre, Dokki, Giza, Egypt. Corresponding author; Attia H Atta1, 1Pharmacology Department, Faculty of Veterinary Medicine, Cairo University, Giza, Tel; +201006540633 E-mail: [email protected]

Abstract This study was designed to investigate the effect of different concentration of pomegranate peel extract on chemical and microbiological quality of frozen bovine minced meat. Sixteen equal parts of minced meat were used as follows: one normal control, three POMP extract- treated parts (0.5, 1.5, and 3 % ), three inoculated parts (S. typhimurium, S. aureus or E. coli) and nine parts (3 POMP concentrations + 3 microorganisms inoculated, i.e. 3 X 3). Physical properties, chemical composition and microbiological count were performed at 0, 7, 15, 30 and 45 days of freezing at -18 ºC. The results showed that POMP aqueous extract maintained protein %, ash%, pH, thiobarbituric acid (TBA) number and total volatile nitrogen (TVN) at levels higher than non-treated non polluted samples and maintained TBA and TVN at levels lower than non-polluted or S. typhimurium or S. aureus but not E. coli - polluted samples. The results revealed that POMP has completely prevented survival of S. typhimurium and S. aureus but not E. coli during storage periods. In conclusion, POMP dried watery extract could be useful as natural preservative for bovine minced meat, since it maintained chemical and microbiological quality of minced meat. Key words: pomegranate peel- additives- natural preservatives -S. typhimurium -S. aureus -E. coli

1. Introduction The epidemiology of food borne diseases is changing and reports from different parts of the world indicate that the resistant strains of food borne pathogens have emerged as public health problem (Siddique et al. 1998). The increased reports on food spoilage

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have led to the wide use of antioxidants such as butylated hydroxytoluene and butylated hydroxyanisole. Synthetic antioxidants are potentially toxic and carcinogenic in living organisms (Tenore et al. 2011). Recently, natural antioxidants are receiving greater attention from consumers due to increasing concerns of human health and/or biology effects. Punica granatum Linn. (Punicaceae), commonly known as pomegranate (POMP) was traditionally recommended for treatment of various disorders such as colitis, headache, diarrhea, dysentery and ulcers (Bachoual et al. 2011). POMP extracts have been reported to possess anti-cancer activity (Brusselmans et al. 2005), antimicrobial activity (McCarrell et al. 2008 and Endo et al. 2010), antidiarrheal activity (Olapour et al. 2009), apoptotic and anti-genotoxic properties (Seeram et al. 2005, Lin et al. 1999), anti-tyrosinase activity (Yoshimura et al. 2005), anti-inflammatory and anti-diabetic activities (Lansky and Newman, 2007). The health benefits of POMP have been attributed to the antioxidant effect of its constituent phytochemicals, which primarily include hydrolysable ellagitannins, anthocyanins and other polyphenols (Patel et al. 2008). Therefore, several POMP containing products have been widely marketed around the world in recent years. POMP fruit peel constitutes about 50% of the total fruit weight (Al-Said et al. 2009), and it is often discarded as waste. However, the fruit peel contains higher amounts of polyphenol compounds than the juice, and it possesses stronger biological activities (Gözlekçi, et al. 201, 1Li, 2006). The purpose of this work is to evaluate the effect of POMP on chemical and microbiological quality of experimentally polluted bovine minced meat to explore its ability to be used as natural food preservative.

2. Materials and methods 2.1. Preparation of POMP aqueous extract The POMP fruits were collected from a well-known market in Giza, Egypt. The fresh peel fruits were cleaned, freeze-dried and grounded into fine powder using an electric blender. The powder was dried in an oven at 40oCfor 24hr. The fine powder (50g) was extracted with 500 ml boiling distilled water for 24hrand then filtered with filter paper. The filtrate was concentrated under vacuum and low temperature (60°C) using rotary evaporator (Gil, et al. 2000). 2.2.

Bacterial strains

The pathogens used are S. typhimurium (NCTC 12023/ ATCC 14028),S. aureus ((NCTC 7447/ ATCC 6538P) and Escherichia coli (NCTC 12241/ ATCC 25922) were obtained from the reference strain bank of food hygiene department Animal Health Research Institute (AHRI). Each strain was deep frozen stored in cryo protective vial containing preservative solution at (– 70 °C). Inoculum of each strain was cultivated in Tryptic Soy Broth over night at 35°C. Then the cells were centrifuged for 10 min at 8000rpm. Supernatant was discarded and cells were washed three times and re-suspended in sterile 0.1% peptone water. The cells were diluted in peptone water adjusted to obtain the desired inoculum level of 1.3×109 CFU/ ml.

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2.3.

Sample treatments

Ten kilograms of bovine minced meat, pursued from butcher shop at Giza were divided into 16 equal parts. The first part was kept without any treatment as normal control. POMP extract was added at concentrations of 0.5,1.5, and 3 %into3 parts. Another three parts were inoculated with S. typhimurium, S. aureus or E. coli only. The 8th – 16th parts were treated with POMP extract at concentration of 0.5, 1.5 or 3.0% before inoculation with S. typhimurium , S. aureus or E. coli . Inoculation of samples was carried out by mixing of 1ml of prepared inoculum into each kilogram minced meat to obtain a level of 106 CFU/gm. The treated and inoculated minced beef meat were kept in refrigerator at 4oC for 1 hour and then placed in polyethylene bags and kept at freezing temperature (– 18oC ) for 0, 7, 15, 30, and 45 days for physical, chemical and bacteriological analysis (EL-Khzaay,2001). Three replicates were performed for each treatment at each of the storage time interval. 2.4.

Physical examinations

Samples were examined for color by naked eye, odor by roasting test using one gram of sample on direct flame and smelling the odor. The texture was also tested by touch and pressure. 2.5.

Chemical examination

Minced meat samples were examined for moisture and protein content as described by (AOAC 1990), pH (ISO, 1974), ash content (Aurand, et al.1987), total volatile basic nitrogen (TVB-N) (ISO, 1974) and thiobarbituric acid value (TBA) as recommended by (Vynck1970). 2.6.

Bacteriological examination

For bacteriological examinations the non-treated non inoculated samples(normal control) and inoculated with or without POMP extract were examined for bacterial counts. Ten grams of meat samples were homogenized with 90 ml of 0.1% sterile peptone water decimal dilutions were prepared for microbiological analysis according to APHA (2001). Then they were enumerated by surface plating on specific media for each examined strain (X.L.D for S. typhymurium according to ISO 6579 (2002), Baird parker for S. aureus according to FDA (2001) and T.B.X for E. coli) according to ISO (2000).

3. Statistical analysis

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Data were expressed as means ± SD. Except for the microbiological analysis, differences between means were tested for significance by the one way analysis of variance (ANOVA) followed by Duncan's test using SPSS. A probability level of p≤0.05 is considered significance

4. Results 4.1.Physical examination The color of the outer surface of minced meat either infected or non-infected (normal control) changed to bright red with flabby texture but of normal odor after four weeks. After six weeks, the color was paler, the odor became rancid and the texture became flabbier. Treatment of samples with different concentration of POMP either polluted or non-polluted showed less changes in meat color, odor and texture as compared with meat samples without POMP extract. 4.2. Chemical examination 4.2.1. Protein percent Protein percent in all samples examined at 0 day was similar, ranged from 19.13 ±0.09 t0 21.97±0.09%). In the normal control samples protein decreased gradually by time and reached to 17.6±0.31% after 6 weeks. POMP aqueous extract maintained protein percent at significantly (P>0.05) higher levels (19.20 ±0.45– 20.33±0.24%) for up to at least 6 weeks compared to normal control (17.60±0.31%). Contaminating samples with S. typhimurium, S. aureus or E. coli decreased protein gradually starting at the 14th day and reached to a level comparable to the non-polluted samples after 45 days. Treatment of S. typhimurium or S. aureus contaminated samples with POMP aqueous extract maintained Protein% at levels higher than normal control or S. typhimurium or S. aureus only spiked samples. However, Protein% in treated E. coli polluted samples was still at lower levels (table 1). 4.2.2. The ash %: The ash % in all samples ranged from 1.12± 0.06 to 1.33±0.05 in fresh samples at zero time with no significant differences. Infection with S. typhimurium, S. aureus or E. coli only as well as treatment with POMP aqueous extract of polluted samples either showed no significant effects on ash % or slightly decreased with no significant difference (table 2).

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Table 1: Effect of POMP aqueous extract on protein % of bovine minced meat Groups Normal control

0 20.4±0.32cd

POMP treated 0.5% 20.0±0.12bc samples 1.5% 21.23±0.15abc 3.0% 20.50±0.17ab Infected Salm. 19.27±0.15f samples Staph. 19.13±0.09 f E coli 19.47±0.26 f

Storage period (days) 7 15 30 20.03±0.89bc 19.5±0.81b 19.1±0.21bc

45 17.60±0.31ef

20.13±0.46bc 20.0±0.46bc 19.97±0.49ab 19.20±0.45abc 20.97±0.89ab 20.1±0.69ab 20.4±0.23a 19.90±0.21ab 21.47±0.15a 20.9±0.35a 20.6±0.31a 20.33±0.24a 18.23±0.15de 18.0±0.25de 17.5±0.29de 16.70±0.35fg 18.57±0.23d 18.3±0.33d 18.1±0.49cde 17.17±0.44fg 18.10±0.1de 18.0±0.2de 17.97±0.55cde 17.0±0.58fg

Infected POMP Salm. 20.8±0.15bc 20.40±0.31ab 20.0±0.36bc 19.1±0.31bc 18.67±0.38cde - Treated Staph. 21.0±0.12bc 20.10±0.38bc 20.0±0.33ab 19.90±0.21ab 19.03±0.55bcd (0.5%) E.coli 19.83±0.44def 18.53±0.32d 18.3±0.22d 18.0±0.29cde 17.53±0.32ef Infected POMP Salm. 20.70±0.46bcd 19.10±0.31cd 18.9±0.33b 18.77±0.39b 18.53±0.42cde - Treated Staph. 21.43±0.23ab 18.87±0.41d 18.6±0.22cd 18.50±0.29cd 17.90±0.21def (1.5%) E.coli 19.50±0.29ef 18.20±0.38de 18.0±0.33de 17.83±0.44cde 16.83±0.44fg Infected POMP Salm. 21.97±0.09a 18.27±0.54de 18.0±0.19de 17.50±0.29de 17.27±0.15fg - Treated Staph. 21.10±0.56abc 18.87±0.73d 18.4±0.71d 18.13±0.55cde 17.53±0.26fg (3.0%) E coli 19.83±0.44def 17.37±0.32e 17.2±0.33e 17.17±0.44e 16.23±0.43g Values are means ± SE; Means in the same column with different superscripts are significantly different at p≤ 0.05, Salm.; S. typhimurium , Staph.; S. aureus.

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Table 2: Effect of POMP aqueous extract on Ash % of bovine minced meat. Groups Normal control

0 1.12±0.07b

Storage Period (days) 7 15 30 b b 1.12±0.06 1.09±0.03 1.06±0.03ab

45 0.99±0.02ab

POMP treated 0.5% samples 1.5% 3.0%

1.26±0.06ab 1.24±0.06ab 1.33±0.05ab

1.177±0.08 b 1.14±0.05ab 1.17±0.07 b 1.16±0.05b 1.29±0.05 b 1.22±0.05ab

1.12±0.06ab 1.15±0.03ab 1.20±0.06ab

1.10±0.05 a 1.12±0.03 a 1.12±0.06 a

Salm. 1.12±0.06 b Staph. 1.18±0.03ab E. coli 1.12±0.06 b

1.06±0.04 b 1.0±0.03 b 1.38±0.31 a 1.22±0.05ab 1.05±0.03 b 1.02±0.06ab

0.98±0.02ab 1.0±0.06ab 1.00±0.06ab

0.85±0.03ab 0.8±0.06ab 1.03±0.07 a

Infected Salm. 1.31±0.01ab POMP Staph. 1.25±0.01ab Treated (0.5%) E. coli 1.17±0.03ab

1.24±0.02 b 1.20±0.03 b 1.177±0.03b 1.15±0.04ab 1.08±0.04 b 1.06±0.03ab

1.14±0.04ab 1.12±0.06ab 1.04±0.04ab

1.07±0.03 a 0.88±0.04 a 1.04±0.04bc

Infected Salm. 1.34±0.04ab POMP Staph. 1.32±0.01ab Treated (1.5%) E. coli 1.24±0.03ab

1.277±0.03 b 1.22±0.03 a 1.19±0.04 a 1.04±0.04 a 1.20±0.06 b 1.17±0.050ab 1.11±0.060ab 0.88±0.02 a 1.12±0.06 b 1.0±0.03 b 0.98±0.02ab 1.07±0.03bc

Infected Salm. 1.36±0.03 a POMP Staph. 1.35±0.03 a Treated (3.0%) E. coli 1.21±0.06ab

1.28±0.03 b 1.20±0.04 b 1.29±0.04 b 1.20±0.03 b 1.09±0.05 b 1.0±0.03 b

Infected Samples

1.14±0.06ab 1.19±0.05 a 0.92±0.04 b

1.07±0.04 a 0.78±0.02 a 0.99±0.02 a

Values are means ± SE; Means in the same column with different superscripts are significantly different at p ≤ 0.05. Salm.; S. typhimurium , Staph. S. aureus.

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4.2.3. The moisture content The moisture decreased in non-treated control significantly after 30 days. POMP aqueous extract alone maintained moisture% at higher levels up to 45 days compared to non-polluted non-treated control at respective times. S. Typhimurium, S. aureus and E. coli decreased moisture gradually by time in a manner similar and comparable to normal control at respective times. Treatment with POMP aqueous extract of S. typhimurium or S. aureus , but not E. coli - polluted samples maintained moisture at levels higher than POMP aqueous extract alone or S. typhimurium or S. aureus alone (table 3) 4.2.4. Effect on pH The pH of non-treated non polluted samples was increased by time from 5.75±0.03 at zero time to 6.22±0.04 after 6 weeks of refrigeration. Moreover, the pH of all samples was increased by time of refrigeration. However, the pH of POMP alone – treated samples was lower than non-treated non polluted samples with a significant (P >0.05) difference at 6th week. However, samples polluted with S. typhimurium, S. aureus or E. coli only showed no significant effect on the pH for 6 weeks of preservation. POMPtreated of S. typhimurium or S. aureus but not E. coli - Polluted samples showed a decreased pH at 6th week compared to polluted only samples (table 4). 4.2.5. Thiobarbituric acid (TBA) value TBA increased in the normal control from 0.25±0.01 at zero time to 1.33±0.2 after 6 weeks storage period at 4 °C. POMP aqueous extract alone increased TBA from 0.24±0.02– 0.26± 0.02 at zero time to 0.48±0.03– 0.49±0.02at 6 weeks storage time. Pollution of minced meat samples with S. typhimurium, S. aureus or E. coli increased TBA from 0.24±0.01– 0.28± 0.01 at zero time to 0. 86 ±0.08 – 0.89±0.08 after 45 days storage at -18°C. Spiking with POMP aqueous extract of S. typhimurium, S. aureus inoculated samples maintained TBA at values at levels significantly (P>0.05) lower than that of the normal control but nearly similar to those treated with POMP alone. However samples with E. coli, TBA values, although lower than the normal control but it was significantly higher than that treated with POMP alone (table 5).

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Table3: Effect of POMP aqueous extract on moisture % of bovine minced meat. Groups Normal control

Storage period (days) 0 7 15 30 45 a ab ab b 72.43±0.23 71.27±0.15 70.0±0.25 67.57±0.35 65.80±0.36 cd

POMP treated 0.5% samples 1.5% 3.0%

72.97±0.15 a 72.1±0.23 a 72.57±0.24 a 71.7±0.38 a 72.8±0.25 a 72.1±0.40 a

Infected samples

71.8±0.17 a 71.0±0.33 a 71.9±0.33 a

71.03±0.15 a 69.97±0.24 a 70.70±0.21 a 69.93±0.12 a 71.13±0.35 a 69.97±0.38 a

Salm. 72.33±0.24 a 70.07±0.033 b 69.0±0.27b Staph. 72.30±0.17 a 70.6±0.38ab 70.0±0.33b E. coli 72.17±0.23 a 71.23±0.79ab 70.0±0.35b

68.30±0.17b 65.80±0.25cd 68.43±0.34b 65.57±0.29 d 68.37±0.32b 65.4±0.29 d

Infected Salm. 72.93±0.12 a 71.97±0.09 a 71.0±0.15 a 70.67±0.24b 69.63±0.27ab POMP Staph. 72.60±0.15 a 71.43±0.29ab 71.1±0.27ab 70.77±0.39 a 69.4±0.29ab Treated (0.5%) E. coli 72.13±0.09 a 70.37±0.32ab 69.0±0.33b 68.37±0.23b 66.70±0.35c Infected Salm. 73.03±0.2 a 71.9±0.15 a 70.0±0.25 a 70.57±0.26 a 69.03±0.15ab POMP Staph. 73.17±0.49 a 71.6±0.30ab 70.1±0.31b 70.73±0.32 a 68.6±0.31ab Treated (1.5%) E. coli 73.03±0.2 a 70.50±0.31ab 69.0±0.33ab 68.20±0.15b 65.47±0.33 d Infected Salm. 73.17±0.49 a 71.7±0.32 a POMP Staph. 72.23±0.19 a 71.8±0.29 a Treated (3.0%) E. coli 73.0±0.17 a 70.4±0.29ab

70.8±0.22 a 70.3±0.25 a 69.0±0.17b

70.07±0.29 a 69.03±0.15ab 69.87±0.29 a 68.80±0.25ab 68.0±0.15b 65.30±0.36 d

Values are means ± SE; Means in the same column with different superscripts are significantly different at P ≤0.05. Salm.: S. typhimurium , Staph.: S. aureus.

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Table 4: Effect of POMP aqueous extract on pH of bovine minced meat. Groups Storage period (days) 0 7 15 30 c c c Normal control 5.75±0.03 5.88±0.03 5.90±0.03 6.07±0.04a

45 6.22±0.04ab

POMP - treated 0.5% samples 1.5% 3.0%

5.77±0.03c 5.79±0.02bc 5.78±0.03bc

5.87±0.01 a 5.89±0.03a 5.89±0.01 a 5.93±0.02 a 5.91±0.02 a 5.89±0.02a

5.93±0.02a 5.96±0.01a 6.01±0.01a

6.06±0.04 c 6.05±0.04 c 6.06±0.02 c

Infected samples

Salm. Staph. E. coli

5.71±0.01c 5.73±0.02bc 5.78±0.02bc

5.87±0.01 a 5.89±0.05 a 5.91±0.02 a 6.0±0.04a 5.92±0.03 a 6.1±0.03 a

5.91±0.23a 6.10±0.06a 6.18±0.04a

6.28±0.05 a 6.28±0.04 a 6.31±0.04 a

Infected POMP - Salm. Treated (0.5%) Staph. E. coli

5.76±0.04bc 5.78±0.03bc 5.80±0.02 a

5.89±0.01 a 5.9±0.02 a 5.90±0.02 a 5.93±0.03a 6.04±0.07 a 6.1±0.02 a

5.97±0.02a 5.99±0.02a 6.22±0.01a

6.09±0.01 c 6.09±0.02 c 6.29±0.03 a


5.79±0.03bc 5.80±0.02bc 5.82±0.02ab

5.89±0.01 a 5.92±0.03a 5.89±0.01 a 5.99±0.01 a 5.99±0.07 a 6.1±0.03 a

5.98±0.03a 6.01±0.03a 6.21±0.03a

6.09±0.01 c 6.11±0.01bc 6.32±0.03 a


5.81±0.02bc 5.79±0.03bc 5.78±0.05a

5.89±0.02 a 5.96±0.02bc 5.90±0.01 a 5.97±0.01bc 6.04±0.07 a 6.1±0.05a

6.0±0.04 a 6.01±0.04a 6.24±0.02a

6.11±0.01bc 6.11±0.01bc 6.34±0.03 a

Values are means ± SE; Means in the same column with different superscripts are significantly different at P ≤0.05. Salm.: S. typhimurium , Staph.: S. aureus

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Table 5: Effect of POMP aqueous extract on TBA of bovine minced meat \

Storage period (days) 0 7 15 30 45 0.25±0.01ab 0.38±0.01ab 0.43±0.01ab 0.74±0.02a 1.33±0.2 a

Normal control

0.24±0.02ab 0.31±0.02 b 0.35±0.02c 0.38±0.02c 0.48±0.03c 0.25±0.01ab 0.31±0.02 b 0.34±0.03c 0.39±0.02c 0.49±0.03c 0.26±0.03c 0.32±0.01 b 0.39±0.03c 0.41±0.02c 0.49±0.02c

POMP treated samples

0.5% 1.5% 3.0%

Infected samples

Salm. 0.24±0.01ab 0.40±0.02ab 0.50±0.03b 0.58±0.03b 0.86±0.08b Staph. 0.25±0.01ab 0.43±0.03ab 0.55±0.04b 0.61±0.04b 0.87±0.09b E. coli 0.28±0.02a 0.46±0.05 a 0.59±0.02a 0.64±0.03b 0.89±0.08b

Infected POMP - Salm. 0.24±0.01ab 0.29±0.01 b 0.33±0.01c 0.38±0.01c 0.43±0.01c Treated (0.5%) Staph. 0.26±0.01ab 0.31±0.01 b 0.34±0.01 b 0.37±0.02c 0.46±0.02c E. coli 0.25±0.01ab 0.39±0.05ab 0.54±0.07b 0.63±0.04b 0.85±0.07b Infected POMP - Salm. 0.23±0.01 b 0.29±0.01 b 0.30±0.01 b 0.39±0.01c 0.47±0.02c Treated (1.5%) Staph. 0.24±0.01ab 0.31±0.01b 0.33±±0.01b 0.39±0.01c 0.47±0.02c E. coli 0.25±0.02ab 0.40±0.05ab 0.52±0.02ab 0.61±0.06b 0.80±0.05b Infected POMP - Salm. 0.24±0.01ab 0.38±0.05ab 0.40±0.01ab 0.44±0.01c 0.5±0.01c Treated (3.0%) Staph. 0.23±0.01ab 0.33±0.05ab 0.39±0.03c 0.42±0.04c 0.49±0.05c E. coli 0.23±0.01 b 0.36±0.05ab 0.44±0.02b 0.53±0.04b 0.84±0.02b Values are means ± SE; Means in the same column with different superscripts are significantly different at P ≤0.05. Salm.: S. typhimurium , Staph: S. aureus .

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4.2.6. Total volatile nitrogen (TVN) TVN was increased in the normal control from 12.53±0.15 at zero time to 20.57±0.75 after 45 days storage at 4 °C.POMP alone increased TVN from 12.27±0.51– 12.40± 0.51 at zero time to15.4±0.26 – 16.00±0.12after 6 weeks of storage. Pollution of meat samples with S. typhimurium, S. aureus or E. coli increased TVN from 12.87±0.43 – 13.03± 0.24 at zero time to 21.90 ±1.0– 22.76±0.66 after 6 weeks of storage. Spiking with POMP of S. typhimurium (14.7±0.12 to 14.9±0.06), S. aureus (14. 4 ±0.11 to 15. 03 ±0.09) polluted samples maintained TVN at values which were significantly lower than that of the normal control (20.57 ± 0.75) but nearly similar to those reported in samples treated with POMP alone (15.4±0.26 – 16.00±0.12). However in the samples polluted with E. coli, TVN values (21.07±1.09 – 21.93±0.84) were nearly similar to that of the normal control (20.57 ± 0.75) (table 6).

4.2.7. Bacteriological examination No bacterial growth in the non-inoculated samples indicating that there was no environmental contamination of samples. The total bacterial count in polluted minced non-treated meat remained nearly at the initial counts up to 6 weeks of freezing preservation (6.5– 6.7 × 106 for S. typhimurium, 6.2– 6.6 × 106 for S. aureus and 6.8– 6.9 × 106 for E. coli ).Treatment of samples with POMP extract at concentration of 0.5%, 1.5% and 3.0% decreased S. typhimurium counts in minced meat from 6.5 × 106 to 3.7 x 104,1.2 × 104 and 9.103 respectively, after one week. The count was gradually decreased to