Effect of Moringa oleifera leaf extract on thephysicochemical properties of modifiedatmosphere packaged raw beef

Manzoor Ahmad Shah, Sowriappan John Don Bosco *, Shabir Ahmad Mir

Department of Food Science and Technology, Pondicherry University, Puducherry 605014, India

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 8

a r t i c l e i n f o

Article history:

Received 4 July 2014

Received in revised form

30 September 2014

Accepted 15 October 2014

Available online 29 October 2014

Keywords:

Moringa oleifera

Raw beef

MAP

TBARS

Natural antioxidant

a b s t r a c t

The effect of Moringa oleifera leaf extract (MLE) on the physicochemical properties of raw beef

stored in modified atmosphere packaging (MAP) under 12 days of refrigerated temperature

was investigated. MLE was prepared using water as solvent and its total phenolic content

ranged from 46.13 to 49.45 mg gallic acid equivalent/g of extract. Raw beef chunks were

treated with different concentrations of MLE (0.1, 0.2 and 0.3 g MLE/L solution) and BHT

(0.2 g BHT/L solution) and compared with control (no antioxidant) and packaged in gas

combination of 80% O2 and 20% CO2. MLE had a significant (p < 0.05) effect on pH, TBARS and

color parameters as compared to control but had non-significant effect on water holding

capacity, cooking loss, shear value and microbiological quality of packaged beef. The results

indicate that the MLE can be used as natural antioxidant to preserve raw beef packaged in

high oxygen MAP.

# 2014 Elsevier Ltd. All rights reserved.

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1. Introduction

Fresh meat is one of the most perishable foods in commerce

and its shelf life is influenced by several factors such as pH,

water content, availability of oxygen and composition (Abril

et al., 2001). These factors promote spoilage microbial growth

and oxidative processes which, in turn, lead to deterioration in

flavor, texture and color of meat. Several techniques have

been used to improve fresh meat quality and modified

atmosphere packaging (MAP) is one of the most successful

techniques suitable for meat preservation (Singh, Wani,

Saengerlaub, & Langowski, 2011). In MAP, the gas composition

of package headspace is modified with various gas composi-

tions of different gases like oxygen, carbon dioxide, carbon

monoxide, nitrogen and argon. Typically, MAP containing 80%

* Corresponding author. Tel.: +91 9962149233.E-mail address: sjdbosco@yahoo.com (S.J.D. Bosco).

http://dx.doi.org/10.1016/j.fpsl.2014.10.0012214-2894/# 2014 Elsevier Ltd. All rights reserved.

oxygen and 20% carbon dioxide are used in beef retail markets

as oxygen favors the bright red color of fresh beef which is

appealing to consumers (Kim, Huff-Lonergan, Sebranek, &

Lonergan, 2010), since the consumer purchasing decisions are

influenced more by color than any other quality parameter

(Mancini & Hunt, 2005).

While the high oxygen MAP has a positive effect on fresh

beef color, the elevated O2 levels can also have negative effects

on beef quality. The high levels of oxygen lead to oxidation of

meat lipids resulting in flavor deterioration and off-odors

(Cayuela, Gil, Banon, & Garrido, 2004; Okayama, Muguruma,

Murakami, & Yamada, 1995). Also, high O2 promotes tough-

ness in beef (Kim et al., 2010) as highly oxidative conditions

within the package promote disulphide cross-linking of

proteins (Lund, Hviid, & Skibsted, 2007). Several synthetic

antioxidants have been used to prevent oxidation and to

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 832

extend the shelf life of meat and meat products. But due to

safety concerns about the use of synthetic antioxidants the

search for natural antioxidants has been increased. A huge

number of antioxidants have been prepared from natural

sources mainly of plant origin and applied to meat and meat

products (Shah, Bosco, & Mir, 2014).

The Moringa oleifera commonly known as drumstick, is

native to India, Africa, Arabia, Southeast Asia and South

America and traditionally being used as vegetable. M. oleifera

leaves are of special interest in food preservation because in

addition to contributing taste and aroma to foods, it also

contains a variety of bioactive substances, which are of

considerable use in extending shelf life (Muthukumar,

Naveena, Vaithiyanathan, Sen, & Sureshkumar, 2012). M.

oleifera leaves have been used to extend the shelf life of ghee as

these leaves are rich in several types of natural antioxidant

compounds such as ascorbic acid, carotenoids and phenolic

substances (Siddhuraju & Becker, 2003). M. oleifera leaf extract

(MLE) showed antioxidant properties as revealed by the

following determinations: the Total Antioxidant Activity

(TAA), 2,2-diphenyl-2-picryl hydrazyl (DPPH) radical scaveng-

ing activity and reducing power (Sreelatha & Padma, 2009),

MLE was used as a natural preservative in goat meat patties

(Das, Rajkumar, Verma, & Swarup, 2012) and pork patties

(Muthukumar et al., 2012). The objective of this paper was to

investigate the effect of M. oleifera leaf extract on physico-

chemical properties of modified atmospheric packaged raw

beef stored at refrigerated temperature.

2. Materials and methods

2.1. Materials

Fresh beef was procured from local market of Pondicherry.

Meat was brought to the laboratory of the Department of Food

Science and Technology, under refrigerated conditions. It was

washed with cold water and drained. After removing all the

visible fat and connective tissue it was stored at 4 8C before

use. Butylatedhydroxytoluene (BHT), 2-thiobarbituric acid,

gallic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), peptone and

plate count agar (Himedia, Mumbai, India), trichloroacetic acid

(Merk, Mumbai, India), 1,1,3,3,-tetraethoxypropane (TEP) (Avra

Synthesis, Hyderabad, India) used in the study were of

analytical grade.

2.2. Methods

2.2.1. Preparation of M. oleifera leaf extract

Fresh M. oleifera leaves obtained from local market were

washed well with water to remove the adhering dust. They

were dried in a tray drier at 55 8C and ground into powder in

a heavy duty grinder (Compton Greaves, Model CG-DX

Turbo, Mumbai, India) and sieved using a 60 mesh sieve and

packed and stored at room temperature in low density

polyethylene pouches until extraction. MLE was prepared by

mixing about 20 g of dried powder with 100 mL boiled water

and left for 1 h at room temperature, stirring frequently with

a glass rod. The extract was obtained by filtration (Whatman

No. 1) and the residue was again re-extracted with 50 mL

distilled following the same procedure as above. Both the

filtrates were mixed and freeze dried. The resulting extract

was kept in an air tight container and stored for 24 h at 4 8C.

The extract was prepared in duplicates and the analyses was

carried out in triplicates. The extract was analyzed for total

phenolic content, DPPH radical scavenging activity and

reducing power.

2.2.2. Antioxidant properties of M. oleifera leaf extract2.2.2.1. Total phenolics. The total phenolic compounds in the

M. oleifera leaf extracts was determined by the Folin–Ciocalteu

method as described by Singleton and Rossi (1965), with slight

modifications. An aliquot of 0.5 mL of sample (0.1 g MLE

dissolved in 100 mL distilled water) was mixed with 2.5 mL of

Folin–Ciocalteu reagent (diluted 1:10 with distilled water) in

test tubes. After 5 min, 2 mL of a sodium carbonate solution

(7.5%) was added to each tube. The tubes were kept at room

temperature for 2 h, and the absorbance determined spectro-

photometrically (UV-1800; Shimadzu, Japan) against a reagent

blank at 725 nm. The amount of total phenolics was calculated

as gallic acid equivalents in mg/g of plant extract from the

standard curve using different concentrations of gallic acid.

2.2.2.2. DPPH radical scavenging activity. The free radical

scavenging activity of the MLE was determined by using the

stable free radical DPPH (Blois, 2002) with slight modifications.

A aliquot of 2 mL of MLE in water was mixed vigorously with

1 mL of 0.15 mM DPPH solution in ethanol and allowed to

stand at 20 8C for 30 min. The absorbance was read at 517 nm

using a UV spectrophotometer (UV-1800; Shimadzu, Japan).

The DPPH radical scavenging activity was calculated using the

following equation:

DPPH scavenging ð%Þ ¼ ½ðAc � As=AcÞ � 100�

where Ac is the absorbance of the control reaction and As is the

absorbance in the presence of the sample. IC50 value (the

concentration required to scavenge 50% DPPH free radicals)

was calculated.

2.2.2.3. Reducing power. The reducing power of the MLE was

determined by using the method of Yen and Duh (1993) with

slight modifications. 1 mL of MLE of different concentrations

were mixed with 2.5 mL of phosphate buffer (0.2 m, pH 6.6) and

2.5 mL of 1% (w/v) potassium ferricyanide in test tubes. These

tubes were kept at 50 8C for 20 min followed by the addition of

2.5 mL of trichloroacetic acid (10%) and then centrifuged at

9700 � g for 10 min. 2.5 mL supernatant was mixed with

2.5 mL distilled water and 0.5 mL of ferric chloride (0.1%, w/v),

and the absorbance was measured at 700 nm using a UV

spectrophotometer (UV-1800; Shimadzu, Japan). The reducing

power of the sample is indicated by the increase in absorbance

of the reaction mixture. The reducing power of the extract was

compared with that of ascorbic acid (standard).

2.2.3. Sample preparation and MA packagingMeat was cut into small chunks of uniform dimensions of

about 3 � 2 � 2 cm3. These meat chunks were divided into five

equal batches: Control (with no antioxidant), BHT (0.2 g BHT/L

solution), MLE 1 (0.1 g MLE/L solution), MLE 2 (0.2 g MLE/L

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 8 33

solution) and MLE 3 (0.3 g MLE/L solution). BHT was dissolved

in 5 mL of vegetable oil before preparing its solution and equal

quantities of oil was added to other extract solutions and

distilled water for control samples to maintain uniformity.

These chunks were soaked in distilled water, BHT and

different concentrations of MLE solutions in the ratio of 1:2

(w/v) for 15 min at 4 8C, according to the method of Nirmal and

Benjakul (2011). After treatment, the meat pieces were drained

for 5 min at 4 8C.

Then, these samples were placed in laminate pouches (low

density polyethylene and polyamide), of water vapor perme-

ability 4 g/m2/24 h and oxygen permeability 40 mL/m2/24 h at

23 8C. These pouches were gas flushed with a gas composition

of 80% O2 and 20% CO2 and sealed using MAP machine (VAC

Star, S 220 MP, Switzerland). All the treatments were stored for

12 days at 4 8C. The whole experiment was replicated twice

and three measurements were carried out for each parameter

studied in each replica. All the analyses have been carried out

at 1, 3, 6, 9 and 12th day.

2.2.4. pHThe pH of the meat samples was determined by adding 10 g

sample with 50 mL distilled water and homogenizing it for 60 s

in a homogenizer. The pH values were measured using a

digital pH meter (Cyber Scan 5105 pH meter, EUTECH

Instruments, Singapore).

2.2.5. ColorThe instrumental color of raw meat was analyzed using

Hunter Lab Color Flex (model A60-1012-312, Hunter Associates

Laboratory Inc., Reston, VA, USA) with 25 mm aperture set for

illumination D65, 108 standard observer angle. CIE L* (light-

ness), a* (redness) and b* (yellowness) were measured on the

surface of raw meat samples.

2.2.6. Thiobarbituric acid reactive substances valueThe lipid oxidation of meat samples was evaluated by

measuring 2-thiobarbituric acid reactive substances (TBARS)

using the method of Siu and Draper (1978) and modified by

Inserra et al. (2014). Aliquots of 2.5 g of meat samples were

homogenized with 12.5 mL of distilled water, in a water/ice

bath. Then, 12.5 mL of trichloroacetic acid (10%, w/v) was

added to precipitate proteins and then the samples were

vortexed. The homogenates were filtered (Whatman No. 1)

and 4 mL of filtrate were added to 1 mL of 0.06 M aqueous

thiobarbituric acid into screw cap tubes. The tubes were kept

in a water bath at 80 8C for 90 min and the absorbance of each

sample was read at 532 nm using a UV spectrophotometer

(UV-1800; Shimadzu, Japan). Results were expressed such as

mg of malonaldehyde (MDA)/kg of meat from the standard

curve using different concentration of TEP (1,1,3,3,-tetraethox-

ypropane).

2.2.7. Water holding capacityWater holding capacity (WHC) was determined by using the

method of Wardlaw, Maccaskill, and Acton (1973) with slight

modifications. Aliquots of 20 g of minced meat was placed in a

centrifuge tube containing 30 mL of NaCl (0.6 M) and was

stirred with a glass rod for 1 min. The tube was then kept at

4 � 1 8C for 15 min, stirred again, and then centrifuged at

3000 � g for 25 min. The supernatant was measured, and the

WHC was expressed as a g/100 g of meat:

WHC ¼ ðinitial weight of NaCl in grams�supernatent weight in gramsÞweight of meat in grams

2.2.8. Cooking lossCooking loss (CL) was calculated as the weight difference

between uncooked and cooked samples relative to the weight

of uncooked samples (Domınguez, Gomez, Fonseca, &

Lorenzo, 2014) and expressed as g/100 g of uncooked sample:

CL ¼ weight of uncooked meat in grams�weight cooked meat in gramsweight of uncooked meat in grams

2.2.9. Shear force valueShear force value was determined by using TA.HD plus-

texture analyzer (Stable Micro Systems, Surrey, UK), equipped

with a Warner–Brazler blade. Six rectangular shaped

(1 cm � 1 cm � 2 cm) samples were prepared and each sample

was sheared once in the center and perpendicular to the

longitudinal orientation of the muscle fibers. The instrument

was set with a 500 kg load cell and a crosshead speed of 2 mm/

s. The maximum shear force (N) was recorded.

2.2.10. Total plate countTotal plate count was determined by using pour plate method

as described in International Commission of Microbiological

Specifications for Foods (ICMSF, 1978). A 10 g of meat sample

was homogenized in 90 mL of sterile peptone water (0.1%).

Appropriate serial dilutions were prepared in 0.1% sterile

peptone water and duplicate plated with plate count agar,

incubated at 37 8C for 48 h. Microbial colonies from the plates

were counted and expressed as log10 cfu/g.

2.2.11. Statistical analysisThe statistical analysis was done by SPPS software package for

windows (SPSS ver. 18; SPSS Inc., Chicago, USA). Data were

analyzed by ANOVA and the means were separated using

Duncan’s multiple range test and statistical significance was

determined at 95% confidence level (p < 0.05). All the data are

presented as the mean with standard deviation.

3. Results and discussion

3.1. Antioxidant properties of M. oleifera leaf extract

Total phenolic content of MLE ranged from 46.13 to

49.45 mg GAE/g of extract. Das et al. (2012) reported the total

phenolics of aqueous extract of MLE were 48.36 mg GAE/g.

while according to Sreelatha and Padma (2009), the total

phenolics of MLE were 45.81 mg GAE/g Since polyphenols are

responsible for the antioxidant activity, the high phenolic

content in the MLE indicates its high antioxidant activity.

The DPPH radical scavenging activity has been widely used

to determine the antioxidant activity of plant extracts and the

antioxidant activity is expressed in terms of IC50 value. MLE

showed an IC50 of 19.31 � 0.87. The scavenging activity of the

MLE progressively increased with the increase in concentration

of the extract and showed a concentration-dependent DPPH

scavenging activity (Muthukumar et al., 2012). Similar results

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 2 4 6 8 10

Abs

orba

nce,

700

nm

Concentration (mg/ml)

Fig. 1 – Reducing power of Moringa oleifera leaf extract

(MLE).

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 834

were obtained by Das et al. (2012) and Sreelatha and Padma

(2009), who reported that increase in concentration of plant

extract, increased the DPPH radical scavenging activity. The

DPPH scavenging ability of the extract may be attributed to its

hydrogen donating ability as antioxidants, on interaction with

DPPH, either transfer an electron or hydrogen atom to DPPH,

thus neutralizing its free radical character (Naik et al., 2003). M.

oleifera leaf extract significantly reduced DPPH radicals. The

degree of discoloration indicates the scavenging potential of the

antioxidant extract, which is due to the radical scavenging

ability (Sreelatha & Padma, 2009).

The reducing power of a compound serves as a significant

indicator of its potential antioxidant activity (Das et al., 2012).

With the increase in MLE concentration, there was an increase

in reducing power of the extract, indicated by the increase in

the absorbance (Fig. 1). MLE showed the highest reducing

power of 0.74 � 0.06 at a concentration of 10 mg/mL of the

Table 1 – Effect of MLE and BHT on pH, WHC and CL of raw be

Parameter Treatment

1 3

pH Control 5.45 � 0.03br 5.47 � 0.04

BHT 5.47 � 0.02abq 5.48 � 0.07

MLE 1 5.48 � 0.01abs 5.53 � 0.02

MLE 2 5.50 � 0.01aq 5.54 � 0.01

MLE 3 5.51 � 0.02as 5.56 � 0.03

WHC (g/100 g) Control 14.27 � 2.34ap 15.63 � 2.44

BHT 14.28 � 1.49ap 16.48 � 2.56

MLE 1 14.52 � 1.87ap 14.65 � 2.21

MLE 2 14.89 � 1.14ap 15.76 � 1.05

MLE 3 15.87 � 2.56ap 15.94 � 2.72

CL (g/100 g) Control 49.53 � 1.42ap 48.56 � 1.11

BHT 48.73 � 1.34ap 48.34 � 2.01

MLE 1 49.82 � 1.09ap 48.77 � 1.74

MLE 2 49.58 � 1.62ap 49.43 � 1.51

MLE 3 49.37 � 1.78ap 48.36 � 1.77

a,b,c superscripts in the same column and p,q,r,s in same rows indicate sig

deviation. MLE = Moringa oleifera leaf extract, BHT = butylated hydr

Control = without antioxidant, BHT = 0.2 g of BHT/L solution, MLE

3 = 0.3 g MLE/L solution.

extract but much lower than that of ascorbic acid. Das et al.

(2012) and Muthukumar et al. (2012), also reported a

concentration dependent increase in reducing power of

MLE. The reducing properties are generally associated with

the presence of reductones and the antioxidative action of

reductones is based on the breaking of free radical chains by

the donation of hydrogen atom (Gordon, 1990; Muthukumar

et al., 2012).

3.2. pH

The pH of raw beef packaged in modified atmosphere and

stored under refrigerated temperature is given in Table 1. The

mean pH was slightly different among the control and treated

samples. The mean pH increased gradually in all treatments

during the storage period. On day 1, the pH of control samples

was lowest (5.45) while that of MLE 3 was highest (5.51).

According to Virgilia, Saccania, Gabbaa, Tanzia, and Soresi

Bordini (2007) changes in pH may be affected by low-molecular

weight compounds formed from endogenous and exogenous

activities in the product. Accumulation of microbial metab-

olites may cause an increase in pH. According to Gill (1983),

bacteria on exhaustion of stored glucose, utilize amino acids

released during protein breakdown, leading to the formation

and accumulation of ammonia which increases the pH.

According to Biswas, Keshri, and Bisht (2004) the pH values

of precooked pork patties increased significantly during

storage. Similar trend was reported by Muthukumar et al.

(2012), for raw pork patties incorporated with MLE.

3.3. Water holding capacity

Water holding capacity affects both the economic and sensory

attributes of meat (Oeckel, Warnants, & Boucque, 1999). The

water holding capacity percent of raw beef packaged in

ef in MAP at refrigerated temperature.

Storage period (days)

6 9 12br 5.53 � 0.03bcq 5.60 � 0.01ap 5.63 � 0.01abp

bq 5.50 � 0.01cq 5.54 � 0.06apq 5.59 � 0.02bp

abr 5.56 � 0.05abqr 5.59 � 0.01apq 5.63 � 0.01abp

abq 5.56 � 0.02abq 5.57 � 0.08apq 5.67 � 0.06ap

ar 5.61 � 0.02aq 5.64 � 0.03apq 5.68 � 0.03ap

ap 16.22 � 2.37ap 16.75 � 2.80ap 17.01 � 1.96ap

ap 17.51 � 2.03ap 17.94 � 2.51ap 18.21 � 1.39ap

ap 15.53 � 1.41ap 16.26 � 2.34ap 17.18 � 2.16ap

ap 16.81 � 2.67ap 16.94 � 1.61ap 16.37 � 1.20ap

ap 17.13 � 2.08ap 18.21 � 1.13ap 18.87 � 2.65ap

ap 48.17 � 1.31ap 47.94 � 1.17ap 47.34 � 1.21ap

ap 48.07 � 1.20ap 47.74 � 1.23p 46.25 � 1.89p

ap 48.57 � 1.67ap 47.76 � 1.84ap 47.46 � 2.03ap

ap 48.23 � 1.39ap 47.54 � 1.57ap 47.23 � 1.13ap

ap 48.20 � 1.47ap 47.21 � 1.54ap 46.01 � 1.32ap

nificant differences ( p < 0.05). Values are given as mean � standard

oxyl toluene, WHC = water holding capacity, CL = cooking loss,

1 = 0.1 g MLE/L solution, MLE 2 = 0.2 g MLE/L solution, and MLE

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 8 35

modified atmosphere and stored under refrigerated temper-

ature is shown in Table 1. Water holding capacity showed

non-significance variation (p < 0.05) among the MLE treated

samples when compared with control. Control samples

showing the lowest average water holding capacity

(14.27 g/100 g), while MLE 3 treated showed the highest value

(15.87 g/100 g) on day 1. Also, with the increase in storage

period WHC showed a non-significant difference among

the control and treated samples but showed an increased

trend. The higher WHC values in antioxidant (BHT and

MLE) treated samples as compared to control may be due to

the increased pH (Bernthal, Booren, & Gray, 1991). As pH value

is increased above the isoelectric pH of proteins, there is

an increase in WHC. Similar results were reported by

Muthukumar et al. (2012), and Das et al. (2012) for raw pork

patties and in goat meat patties incorporated with MLE. The

relatively lower WHC value of the control sample in our study

may be due to slight denaturation of sarcoplasmic proteins,

which play an important role in determining WHC (Joo,

Kauffman, Kim, & Park, 1999). The above results thus indicate

that the treatment with MLE improved the functional

properties of muscle proteins. According to Hayes et al.

(2010) an increase in water holding capacity was observed in

raw beef patties treated with ellagic acid and olive leaf

extract. When a polyphenol–protein complex is formed,

charge distribution alteration occurs which may led to an

in increased water holding capacity (Hayes et al., 2010).

3.4. Cooking loss

The cooking loss of meat packaged in modified atmosphere

and stored under refrigerated temperature is shown in Table 1.

Non-significant difference (p < 0.05) was observed in cooking

loss among the different treatments compared with control

samples and within the storage period. But cooking

loss decreased slightly with the storage period in all the

Table 2 – Effect of MLE and BHT on color (L*, a* and b*) propert

Parameter Treatment

1 3

L* Control 45.54 � 0.94ar 46.59 � 1.30a

BHT 45.80 � 1.90aq 45.56 � 0.80a

MLE 1 46.30 � 1.89ap 46.53 � 2.05a

MLE 2 45.85 � 0.61ar 46.49 � 1.33a

MLE 3 45.51 � 1.18ar 45.75 � 1.15a

a* Control 14.66 � 0.23ep 11.95 � 0.37d

BHT 19.34 � 0.38ap 17.65 � 1.03a

MLE 1 15.29 � 0.32dp 13.13 � 0.42c

MLE 2 16.41 � 0.18cp 14.07 � 0.43b

MLE 3 17.24 � 0.35bp 14.88 � 0.64b

b* Control 20.10 � 0.66abp 19.14 � 0.83a

BHT 21.81 � 0.21ap 18.23 � 1.71a

MLE 1 17.45 � 0.76cp 19.49 � 2.67a

MLE 2 18.49 � 1.92bcp 17.52 � 1.03a

MLE 3 19.15 � 0.83bcpq 19.89 � 1.86a

a,b,c,d,e superscripts in the same column and p,q,r,s,t in same rows

mean � standard deviation. MLE = Moringa oleifera leaf extract, BHT = bu

of BHT/L solution, MLE 1 = 0.1 g MLE/L solution, MLE 2 = 0.2 g MLE/L solut

treatments. According to Kannan, Kouakou, and Gelaye (2001),

the cooking loss was higher at 0 day than at 4, 8 or 12 days of

storage for goat steaks. Cooking loss is a combination of liquid

and soluble matter lost during cooking and with increasing

temperature, water content decreases while fat and protein

contents increase indicating that the main part of cooking loss

is water (Brugiapaglia & Destefanis, 2012). During cooking, the

various meat proteins denature and cause structural changes,

such as the destruction of cell membranes, shrinkage of meat

fibers, and gel formation of myofibrillar and sarcoplasmic

proteins (Jung et al., 2012; Tornberg, 2005). MLE can actively

scavenge free radicals and thus prevent cellular damage

(Sreelatha & Padma, 2009). The increased meat pH by the

extract can also account for the observed decrease in cooking

loss (Hazra, Biswas, Bhattacharyya, Das, & Khan, 2012;

Thomsen & Zeuthen, 1988).

3.5. Color

Meat color is another important parameter affecting consum-

er acceptance as color is regarded as an indicator of perceived

quality and freshness of meat and is the first limiting factor in

the shelf-life of meat (Smith, Belk, Sofos, Tatum, & Williams,

2000). The effects of MLE on Hunter Lab color values on raw

beef stored under MAP during the refrigerated storage are

shown in Table 2. Lightness, L* values did not vary significantly

(p < 0.05) for the all the treatments on day 1. However, with the

increase in storage time, L* values increase for all the

treatments, but the highest increase was in control samples

(45.54–50.54). Franco et al. (2012) reported the similar increase

in L* values for beef steaks stored in MAP at 4 8C.

Redness, a* values of all the samples vary significantly

(p < 0.05) on day 1, with highest for BHT (19.34) treated sample

and lowest for control (14.66) sample. However with the

increase in MLE concentration the redness values also

increased. The a* values decreased gradually with the increase

ies of raw beef in MAP at refrigerated temperature.

Storage period (days)

6 9 12qr 48.38 � 1.74apq 49.81 � 0.75ap 50.54 � 0.83ap

q 46.57 � 1.22apq 47.61 � 0.84bpq 48.35 � 0.98bp

p 47.48 � 1.91ap 48.63 � 0.91abp 49.28 � 0.32abp

r 47.15 � 0.42aqr 48.63 � 1.20abpq 49.38 � 0.75abp

qr 46.54 � 0.87apqr 47.56 � 0.90bpq 48.13 � 0.80bp

q 11.25 � 0.25br 10.16 � 0.16cs 9.33 � 0.42at

q 16.10 � 1.18ar 12.99 � 0.53as 10.43 � 0.63at

q 11.36 � 1.14bqr 10.12 � 0.31cr 9.21 � 1.84ar

cq 11.54 � 1.24br 10.46 � 0.49bcrs 9.76 � 0.56as

q 12.86 � 1.13br 10.88 � 0.24bs 9.99 � 0.40as

pq 18.49 � 1.73apqr 16.88 � 1.22aqr 16.32 � 2.07ar

q 17.53 � 2.03aq 16.98 � 0.90aqr 15.03 � 0.79abr

p 18.04 � 1.21ap 17.36 � 2.45ap 11.18 � 0.73cp

pq 18.01 � 0.57apq 16.10 � 1.31aq 12.79 � 0.49bcr

p 18.66 � 0.22apq 17.17 � 0.26aq 15.07 � 1.44abr

indicate significant differences ( p < 0.05). Values are given as

tylated hydroxyl toluene, Control = without antioxidant, BHT = 0.2 g

ion, and MLE 3 = 0.3 g MLE/L solution.

0

10

20

30

40

50

60

70

80

90

1 3 6 9 12

Shea

r fo

rce,

N

Storage period (Days)

Control BHT MLE 1 MLE 2 MLE 3

Fig. 3 – Effect of MLE and BHT on shear force (Newtons, N)

values of raw beef in MAP at refrigerated temperature.

MLE = Moringa oleifera leaf extract, BHT = butylated

hydroxyl toluene, Control = without antioxidant,

BHT = 0.2 g of BHT/L solution, MLE 1 = 0.1 g MLE/L solution,

MLE 2 = 0.2 g MLE/L solution, and MLE 3 = 0.3 g MLE/L

solution.

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 836

in storage time for all the samples. Similar trend was reported

by Maqsood and Benjakul (2010) for ground beef treated with

tannic acid stored under MAP and refrigerated temperature.

The decrease in a* value is due to the oxidation of myoglobin

and formation of metmyoglobin (Mancini & Hunt, 2005).

Higher a* values of BHT and MLE 3 compared to control implies

the fact that the added antioxidants were responsible for the

color stabilization.

Yellowness, b* values vary significantly among the treat-

ments and also with the storage period. At day 1, BHT treated

samples showed the highest b* (21.81) values while MLE 1

treated samples showed the lowest (11.18) value. Muthukumar

et al. (2012), also reported a decrease in b* values for raw pork

patties incorporated with MLE, during storage.

3.6. Thiobarbituric acid reactive substances value

Lipid oxidation, a major cause of chemical spoilage in meat,

results in the production of free radicals which may lead to the

oxidation of meat pigments and generation of rancid odors

and flavors (Faustman & Cassens, 1990) and the production of

potentially toxic compounds. The effect of BHT and MLE on

TBARS values of raw beef stored under MAP during refrigerat-

ed storage is shown in Fig. 2. In comparison with control

samples, TBARS values for all the treated samples vary

significantly (p < 0.05) and with the increase in the concentra-

tion of the MLE, there was a decrease in TBARS values among

the treatments. Samples treated with different concentrations

of MLE showed lower TBARS values than control, although the

BHT treated samples showed the lowest lipid oxidation

throughout the storage in comparison with control and the

MLE treated samples. Also, with the increase in storage time,

there was significantly (p < 0.05) increase in TBARS values in

the samples but lower values for BHT and MLE treated samples

as compared with the control samples. Das et al. (2012)

reported that MLE (0.1%) retarded lipid oxidation of cooked

goat meat patties stored at 4 8C for 15 days. According to

Muthukumar et al. (2012) MLE can be used as a natural

antioxidant to prevent lipid oxidation in ground pork patties.

0

0.2

0.4

0.6

0.8

1

1.2

1 3 6 9 12

TB

AR

S va

lue

(mg

mal

onal

dehy

de p

er k

g m

eat

Stora ge peri od (Day s)

Control BHT MLE 1 MLE 2 MLE 3

Fig. 2 – TBARS values (mg malonaldehyde per kg of sample)

of MLE and BHT treated beef in MAP at refrigerated

temperature during 12 days. MLE = Moringa oleifera leaf

extract, BHT = butylated hydroxyl toluene,

Control = without antioxidant, BHT = 0.2 g of BHT/L

solution, MLE 1 = 0.1 g MLE/L solution, MLE 2 = 0.2 g MLE/L

solution, and MLE 3 = 0.3 g MLE/L solution.

Phenolic compounds present in the plant extracts inhibit the

formation of rancid off-flavors in meat products by serving as

radical scavengers by hydrogen atom or electron donators

(Jadhav, Nimbalkar, Kulkarni, & Madhavi, 1995). Mustafa,

Abdul Hamid, Mohamed, and Abu Bakar (2009) also demon-

strated a strong correlation between the TPC and DPPH

antioxidant assay (r = 0.86) in 21 tropical plant extracts. Hayes

et al. (2010) observed that olive leaf extract treated beef patties

showed lower level of lipid oxidation as compared to control in

MAP conditions at concentrations of 100 and 200 mg/g muscle,

which reduced the lipid oxidation by an average of about 76%

and 86%, respectively.

3.7. Shear force value

Shear force values as measured by Warner–Bratzler shear

force device are shown in Fig. 3. Shear force values were not

significantly different for control and the treated samples

throughout the storage period, but shear force values

decreased with the increase in storage time up to day 9, and

again increased slightly at day 12. Tenderness as measured in

terms of shear force values depends on factors such as the

treatment of the animal prior to slaughter, postmortem

methodologies, the method of sample preparation (Webb,

Casey, & Simela, 2005) and muscle type (Jongberg, Wen,

Torngren, & Lund, 2014). The decrease in shear force values up

to day 9 in our study, may be due to the postmortem

proteolysis by endogenous proteases causes a weakening of

myofibril structures and associated proteins leading to the

tenderization (Kemp & Parr, 2012). But high oxygen MAP has

been shown to decrease the tenderness value of beef (Kim

et al., 2010), which may be the reason of increase in the shear

force values on day 12. According to Lund et al. (2007) the high

oxygen MAP affects the meat tenderness negatively through

protein disulphide cross-linking. Lower shear values for

antioxidant treated samples in the present study may be

due to inhibition of protein cross-linking and aggregation by

the MLE and BHT treatment.

3

4

5

6

7

8

9

1 3 6 9 12

Tot

al p

late

cou

nt (l

og C

FU

/g)

Storag e period (D ays)

Contr ol BHT MLE 1

MLE 2 MLE 3

Fig. 4 – Total plate count of MLE and BHT treated beef in MAP

at refrigerated temperature during 12 days. MLE = Moringa

oleifera leaf extract, BHT = butylated hydroxyl toluene,

Control = without antioxidant, BHT = 0.2 g of BHT/L

solution, MLE 1 = 0.1 g MLE/L solution, MLE 2 = 0.2 g MLE/L

solution, and MLE 3 = 0.3 g MLE/L solution.

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 8 37

3.8. Total plate count

The effects of antioxidants and storage time on microbial

quality of raw beef are shown in Fig. 4. Total plate count

increased in both the control and the treated samples,

throughout the storage period but the control samples showed

slightly higher microbial load than the treated samples.

Similar results were reported by Muthukumar et al. (2012),

for raw and cooked pork patties incorporated with MLE and

this may be due to the lower doses applied which impart less

antimicrobial effect. According to Mitsumoto, O’grady, Kerry,

and Buckley (2005), the shelf life of raw meat is limited by

microbial spoilage and usually has a shelf life of about 7 days

under refrigerated conditions, depending on hygiene and

preservation conditions.

4. Conclusion

The present study showed that the MLE is a good source of

phenolic compounds with significant radical scavenging

activity and reducing power. The raw beef treated with MLE 3

had a significant (p < 0.05) effect on the reduction of TBARS

values compared with control. But the BHT showed the highest

inhibitory effect followed by MLE 3 on lipid oxidation compared

to other treatments. Also, MLE had a significant effect on color

parameters as compared to control but had non-significant

effect on water holding capacity, cooking loss, shear value and

microbiological quality of packaged beef. These results indicate

that MLE can be used as a natural antioxidant to prevent lipid

oxidation in raw meat, packaged in high oxygen MAP.

Acknowledgements

The authors are grateful to the Department of Food Science

and Technology, Pondicherry University for providing the

laboratory facilities. The first author (M. A. Shah) is thankful to

UGC for providing Moulana Azad National Fellowship.

r e f e r e n c e s

Abril, M., Campo, M. M., Onenc, A., Sanudo, C., Alberti, P., &Negueruela, A. I. (2001). Beef color evolution as a function ofultimate pH. Meat Science, 58, 69–78.

Bernthal, P. H., Booren, A. M., & Gray, J. I. (1991). Effect ofreduced sodium chloride concentration and tetrasodiumpyrophosphate on pH, water holding capacity andextractible protein of pre rigor and post rigor ground beef.Meat Science, 29, 69–82.

Biswas, A. K., Keshri, R. C., & Bisht, G. S. (2004). Effect ofenrobing and antioxidants on quality characteristics ofprecooked pork patties under chilled and frozen storageconditions. Meat Science, 66, 733–741.

Blois, M. S. (2002). Antioxidant determination by the use of astable free radical. Nature, 26, 1199–1200.

Brugiapaglia, A., & Destefanis, G. (2012). Effect of cookingmethod on the nutritional value of Piemontese beef. InProceedings of the 58th international congress of meat science andtechnology.

Cayuela, J. M., Gil, M. D., Banon, S., & Garrido, M. D. (2004). Effectof vacuum and modified atmosphere packaging on thequality of pork loin. European Food Research and Technology,219, 316–320.

Das, A. K., Rajkumar, V., Verma, A. K., & Swarup, D. (2012).Moringa oleifera leaves extract: A natural antioxidant forretarding lipid peroxidation in cooked goat meat patties.International Journal of Food Science and Technology, 47, 585–591.

Domınguez, R., Gomez, M., Fonseca, S., & Lorenzo, J. M. (2014).Effect of different cooking methods on lipid oxidation andformation of volatile compounds in foal meat. Meat Science,97, 223–230.

Faustman, C., & Cassens, R. G. (1990). The biochemical basis fordiscoloration in fresh meat: A review. Journal of Muscle Foods,1, 217–243.

Franco, D., Gonzalez, L., Bispo, E., Latorre, A., Moreno, T.,Sineiro, J., et al. (2012). Effects of calf diet, antioxidants,packaging type and storage time on beef steak storage. MeatScience, 90, 871–880.

Gill, C. O. (1983). Meat spoilage and evaluation of the potentialstorage life of fresh meat. Journal of Food Protection, 46, 444–452.

Gordon, M. F. (1990). The mechanism of antioxidant action invitro. In B. J. F. Hudson (Ed.), Food antioxidants (pp. 1–18).London: Elsevier Applied Science.

Hayes, J. E., Stepanyan, V., Allen, P., O’Grady, M. N., O’Brien, N. M.,& Kerry, J. P. (2010). Effect of lutein, sesamol, ellagic acid andolive leaf extract on the quality and shelf-life stability ofpackaged raw minced beef patties. Meat Science, 84, 613–620.

Hazra, S., Biswas, S., Bhattacharyya, D., Das, S. K., & Khan, A.(2012). Quality of cooked ground buffalo meat treated withthe crude extracts of Moringa oleifera (Lam.) leaves. Journal ofFood Science and Technology, 49, 240–245.

International Commission on Microbiological Specifications forFoods (ICMSF). (1978). Microorganisms in foods. 1. Theirsignificance and methods of enumeration. Canada: University ofToronto Press.

Inserra, L., Priolo, A., Biondi, L., Lanza, M., Bognanno, M.,Gravador, R., et al. (2014). Dietary citrus pulp reduces lipidoxidation in lamb meat. Meat Science, 96, 1489–1493.

Jadhav, S. J., Nimbalkar, S. S., Kulkarni, A. D., & Madhavi, D. L.(1995). Lipid oxidation in biological and food systems. In D.L. Madhavi, S. S. Deshpande, & D. K. Salunkhe (Eds.), Foodantioxidants: Technological, toxicological, and health perspectives(pp. 5–64). New York: Marcel Dekker, Inc.

Jongberg, S., Wen, J., Tørngren, M. A., & Lund, M. N. (2014). Effectof high-oxygen atmosphere packaging on oxidative stability

f o o d p a c k a g i n g a n d s h e l f l i f e 3 ( 2 0 1 5 ) 3 1 – 3 838

and sensory quality of two chicken muscles during chillstorage. Food Packaging and Shelf Life, 1, 38–48.

Joo, S. T., Kauffman, R. G., Kim, B. C., & Park, G. B. (1999). Therelationship of sarcoplasmic and myofibrillar proteinsolubility to colour and water holding capacity in porcinelongissimus muscle. Meat Science, 52, 291–297.

Jung, E. Y., Yun, I. R., Go, G. W., Kim, G. D., Seo, H. W., Joo, S. T.,et al. (2012). Effects of Radix puerariae extracts onphysicochemical and sensory quality of precooked porksausage during cold storage. LWT-Food Science and Technology,46, 556–562.

Kannan, G., Kouakou, B., & Gelaye, S. (2001). Color changesreflecting myoglobin and lipid oxidation in chevon cutsduring refrigerated display. Small Ruminant Research, 42, 67–75.

Kemp, C. M., & Parr, T. (2012). Advances in apoptotic mediatedproteolysis in meat tenderisation. Meat Science, 92, 252–259.

Kim, Y. H., Huff-Lonergan, E., Sebranek, J. G., & Lonergan, S. M.(2010). High-oxygen modified atmosphere packagingsystem induces lipid and myoglobin oxidation and proteinpolymerization. Meat Science, 85, 759–767.

Lund, M. N., Hviid, M. S., & Skibsted, L. H. (2007). The combinedeffect of antioxidants and modified atmosphere packagingon protein and lipid oxidation in beef patties during chillstorage. Meat Science, 76, 226–233.

Mancini, R. A., & Hunt, M. C. (2005). Current research in meatcolour. Meat Science, 71, 100–121.

Maqsood, S., & Benjakul, S. (2010). Preventive effect of tannicacid in combination with modified atmospheric packagingon the quality losses of the refrigerated ground beef. FoodControl, 21, 1282–1290.

Mitsumoto, M., O’grady, M. N., Kerry, J. P., & Buckley, D. J. (2005).Addition of tea catechins and vitamin C on sensoryevaluation, colour and lipid stability during chilled storagein cooked or raw beef and chicken patties. Meat Science, 69,773–779.

Mustafa, R. A., Abdul Hamid, A., Mohamed, S., & Abu Bakar, F.(2009). Total phenolic compounds, flavonoids and radicalscavenging activity of 21 selected tropical plants. Journal ofFood Science, 75, C28–C35.

Muthukumar, M., Naveena, B. M., Vaithiyanathan, S., Sen, A. R.,& Sureshkumar, K. (2012). Effect of incorporation of Moringaoleifera leaves extract on quality of ground pork patties.Journal of Food Science and Technology http://dx.doi.org/10.1007/s13197-012-0831-8.

Naik, G. H., Priyadarsini, K. I., Satav, J. G., Banavalikar, M. M.,Sohoni, P. P., Biyani, M. K., et al. (2003). Comparativeantioxidant activity of individual herbal components usedin Ayurvedic medicine. Phytochemistry, 63, 97–104.

Nirmal, N. P., & Benjakul, S. (2011). Retardation of qualitychanges of Pacific white shrimp by green tea extracttreatment and modified atmosphere packaging duringrefrigerated storage. International Journal of Food Microbiology,149, 247–253.

Oeckel, M. J., Warnants, N., & Boucque, Ch. V. (1999).Comparison of different methods for measuring water

holding capacity and juiciness of pork versus onlinescreening methods. Meat Science, 51, 313–320.

Okayama, T., Muguruma, M., Murakami, S., & Yamada, H.(1995). Studies on modified atmosphere packaging of thinsliced beef. 1. Effect of 2 modified atmosphere packagingsystems on pH value, microbial growth, metmyoglobinformation and lipid oxidation of thin sliced beef. Journal ofthe Japanese Society for Food Science and Technology, NipponShokuhin Kagaku Kogaku Kaishi42, 498–504.

Shah, M. A., Bosco, S. J. D., & Mir, S. A. (2014). Plant extracts asnatural antioxidants in meat and meat products. MeatScience, 98, 21–33.

Siddhuraju, P., & Becker, K. (2003). Antioxidant properties ofvarious solvent extracts of total phenolic constituents fromthree different agroclimatic origins of drumstick tree(Moringa oleifera Lam.) leaves. Journal of Agriculture and FoodChemistry, 51, 2144–2155.

Singh, P., Wani, A. A., Saengerlaub, S., & Langowski, H.-C. (2011).Understanding critical factors for the quality and shelf-lifeof MAP fresh meat: A review. Critical Reviews in Food Scienceand Nutrition, 51, 146–177.

Singleton, V. L., & Rossi, J. A. (1965). Colorimetry of totalphenolics with phophomolybdicphosphotungtic acidreagents. American Journal of Enology and Viticulture, 16,144–158.

Siu, G. M., & Draper, H. H. (1978). A survey of the malonaldehydecontent of retail meats and fish. Journal of Food Science, 43,1147–1149.

Smith, G. C., Belk, K. E., Sofos, J. N., Tatum, J. D., & Williams, S.N. (2000). Economic implications of improved colourstability in beef. In E. A. Decker, C. Faustman, & C. J. Lopez-Bote (Eds.), Antioxidant in muscle foods – Nutritional strategies toimprove quality (pp. 297–426). New York: Wiley.

Sreelatha, S., & Padma, P. R. (2009). Antioxidant activity andtotal phenolic content of Moringa oleifera leaves in twostages of maturity. Plant Foods for Human Nutrition, 64, 303–311.

Thomsen, H. H., & Zeuthen, P. (1988). The influence ofmechanically deboned meat and pH on the water holdingcapacity and texture of emulsion type meat products. MeatScience, 22, 189–201.

Tornberg, E. (2005). Effects of heat on meat proteins.Implications on structure and quality of meat products.Meat Science, 70, 493–508.

Virgilia, R., Saccania, R., Gabbaa, L., Tanzia, E., & Soresi Bordini,C. (2007). Changes of free amino acids and biogenic aminesduring extended ageing of Italian dry-cured ham. LWT-FoodScience and Technology, 40, 871–878.

Wardlaw, F. B., Maccaskill, L. H., & Acton, J. C. (1973). Effect ofpostmortem muscle changes in poultry meat loafproperties. Journal of Food Science, 38, 421–424.

Webb, E. C., Casey, N. J., & Simela, L. (2005). Goat meat quality.Small Ruminant Research, 60, 153–166.

Yen, G. C., & Duh, P. D. (1993). Antioxidative properties ofmethanolic extracts from peanut hulls. Journal of theAmerican Oil Chemistry Society, 70, 383–386.