IMPROVING THE QUALITY OF BEEF BURGER BY ADDING THYME ESSENTIAL OIL AND JOJOBA OIL

Essential oils and their components are becoming increasingly popular as naturally occurring antimicrobial and antioxidant agents. Therefore, the present study aimed to investigate the effect of addition of different concentrations (0.02, 0.04, 0.06%) of essential oil of thyme (TEO) and 0.1% oil of jojoba (JO) on the quality and stability of beef burger. The obtained results indicated that the best sensory quality was attained at the highest TEO concentration (0.06%), while slight improvement in sensory quality was noticed in samples treated with JO (1%) as compared with control samples. The storage time significantly affected the TBA (thiobarbituric acid) values, as treated samples with TEO and JO showed lower values of TBA compared with the control samples. Regarding to microbial load, samples treated with 0.04 and 0.06% of TEO revealed significant reduction at 12th day of storage as compared with the control samples. On the other hand, the obtained results illustrated that JO have no significant effect on the microbial load.

SUMMARY Essential oils and their components are becoming increasingly popular as naturally occurring antimicrobial and antioxidant agents. Therefore, the present study aimed to investigate the effect of addition of different concentrations (0.02, 0.04, 0.06%) of essential oil of thyme (TEO) and 0.1% oil of jojoba (JO) on the quality and stability of beef burger. The obtained results indicated that the best sensory quality was attained at the highest TEO concentration (0.06%), while slight improvement in sensory quality was noticed in samples treated with JO (1%) as compared with control samples. The storage time significantly affected the TBA (thiobarbituric acid) values, as treated samples with TEO and JO showed lower values of TBA compared with the control samples. Regarding to microbial load, samples treated with 0.04 and 0.06% of TEO revealed significant reduction at 12 th day of storage as compared with the control samples. On the other hand, the obtained results illustrated that JO have no significant effect on the microbial load.

INTRODUCTION
Modern trends towards production of pre-cooked, refrigerated ready-to-eat meat products have made the control of lipid oxidation increasingly important. Processed meats which are minced and cooked are susceptible to accelerate lipid oxidation, which is one of the main factors responsible for loss of quality of meat products besides microbiological deterioration (Rhee, 1989;and Singh, 1996). Therefore, much attention in recent years has been focused on the use of extracts from herbs and spices to improve sensory characteristics, retard lipid oxidation and extend the shelf life of meat products (Arora and Kaur, 1999;Gulluce et al., 2003 andLagouri andNisteropoulou, 2009).
Thyme is traditionally used as flavouring agents in meat and meat products (Mishra and Dube 1994;Lawless, 1995). Recently, its essential oil is known to include carvacol, borneol, geraniol, but most importantly, thymol. Thyme essential oil also contains a variety of flavonoids, including apigenin, naringenin, luteolin and thymonin (Stahl-Biskup, 1991;Senatore 1996;Peñalver et al., 2005). These flavonoids increase thyme antioxidant capacity (Lacroix et al., 1997). The volatile oil components of thyme have also been known to have antimicrobial activity against different bacteria and fungi species (Dorman and Deans 2000;Nguefack et al., 2009). Staphalococcus aureus, Bacillus subtilis, Escherichia coli and Shigella sonnei are among bacterial species against which thyme has been shown to have antibacterial activity (Kim et al., 1995 andSmith-Palmer et al., 1998). In this regard Soliman and Badeae (2002) and Kalemba and Kunicka (2003) found that thyme essential oil (500 ppm) in vitro possess strong antimicrobial properties towards food born bacteria and fungi, including Aspergillus flavus, A. parasiticus, A. ochracus and Fusarium moniliforme. Although essential oils are well documented in vitro as natural antimicrobial, some food components decrease the antimicrobial effects of the essential oil (Sofos et al., 1998;Lopez-Malo et al., 2000;Nychas and Tassou 2000).
Jojoba oil is the liquid wax produced in the seed of the jojoba (Simmondsia chinensis) plant. The oil makes up approximately 50% of the jojoba seed by weight (Salgm, 2007). It is a straight chain wax ester of 36 to 46 carbon atoms in length. Each molecule consists of a fatty acid and a fatty alcohol joined by an ester bond (Miwa, 1971;Wisniak, 1994;Saguy et al., 1996). Jojoba oil is not affected by prolonged storage or changes in temperature when compared with other vegetable oils, nor does it facilitate microbial growth (Miwa, 1971 andWisniak, 1994). Nowadays, there has been renewed interest in its food potential as a vegetable and salad oil and shortening (Kalscheuer et al., 2006).
The questions concerning the safety of synthetic food additives have encouraged an increase interest in the use of natural substances and request more detailed studies of plant resources and essential oils (Kalemba and Kunicka, 2003). Therefore, the objective of this study was to evaluate the effectiveness of TEO and JO for controlling sensory, physico-chemical and microbiological quality of beef burger during two weeks of refrigeration storage at 5°C.

EXTRACTION OF TEO
Extraction had been done in the Department of Biochemistry, Faculty of Agriculture, Cairo University, by hydrodistillation of dried plant leaves followed by evaporation under vacuum according to Cosentino et al. (1999). While JO was obtained by direct extraction of jojoba seeds without refining.

BEEF BURGER PRODUCTION
Was carried out in agreement with Egyptian standard specification for burger (ESS 1688/1991) as follows: Twenty kilo-grams of freshly beef chuck 24 hours postmortem was purchased from local butcher shop at Giza market-Egypt and directly transported to the laboratory in an ice box to be minced in electrical mincer (4 mm). Minced meat 65%, fat 20%, soybean 5%, black pepper 0.3%, salt 1.8% and water 10% were thoroughly mixed for five minutes and divided into five portions. First portion was used as control, while the other portions were either mixed with TEO (0.02, 0.04, 0.06%), or jojoba oil (0.1%), respectively. The obtained pastes were formed into 50 g beef burger using cardboard meat box, packed in foam plates and stored at refrigerator shelf at 5ºC. Three samples for each treatment were examined every three days for two weeks as follows:

SENSORY QUALITY
Odour, colour and overall acceptability of raw beef burger samples were assessed by 5-7 members of Food Hygiene and Control Department (with past experience in burger processing and evaluation) to evaluate their sensory characteristics. Sensory hedonic scheme, ranged from 0 (very bad) to 8 (very good) following the procedures of AMSA (1995), was applied.

MICROBIOLOGICAL QUALITY
Samples homogenate and serial decimal dilutions were prepared following the recommendation of Spencer and De Spencer (2001). The serial dilutions of each sample were investigated for count of Enterobacteriacae on violet red bile dextrose agar, Staphylococci on Barid Parker agar, total mould on Saboureaud dextrose agar, proteolytic bacteria using 10% skim milk agar and lipolytic bacteria using tributyrine agar applying the techniques described by APHA (1992).

STATISTICAL ANALYSIS
The results are presented as the mean of three replicates with standard deviation. The data generated were analyzed by statistical software package using standard procedures for analysis of variance and Duncan multiple range test (Duncan, 1955) to compare the means and determine the effect of treatments (SAS, 1995). The probability value of p≤0.05 was used as the criteria for significant differences. Figure 1 represents odour, colour and overall acceptability values for all examined samples for the first three days of storage. Decline of sensory attributes begin after the third day of storage with marked reduction of odour, colour and overall acceptability values in the control samples at the 6 th day with rejectionable characteristics at the 9 th . While TEO treated samples revealed acceptability for 12 and 15 days for 0.02% and 0.04, 0.06% respectively.

SENSORY EVALUATION
It is worth to mention that significant improvement of odour and overall acceptability of investigated samples were observed at 0 day due to addition of thyme essential oil, this could be referred to its aromatic effect (Mishra and Dube, 1994;Lawless, 1995). Furthermore, highest concentration (0.06%) TEO treated samples showed the best sensory quality. On the other hand, a noticed improvement in sensory characteristics could be noticed in samples treated with jojoba oil as compared with control samples. Addition of TEO and TEO (0.04, 0.06%) treated samples with highest odour, and overall acceptability value for 0.06% TEO treated samples.

TBA-VALUE
TBA-value is a valuable test in determination of lipid oxidation (Pikul et al., 1983). Table I indicates gradual increase of TBAvalues of all examined samples during storage time. No significant difference (p≤0.05) were observed among treated samples and control at 0 d, which indicates that oxidative deterioration of beef burger lipid occurred during storage time.
At the third day of storage significant reduction (p≤0.05) of TBA-values of TEO and JO treated beefburger samples was obvious. Moreover, an inverse trend was observed with addition of TEO, where, the lowest TBA-values were recorded in samples containing 0.06% followed by 0.04 and respectively. Such findings may be attributed to the high antioxidant effect of TEO, which is related to the scavenger nature of its flavonoids and phenolic content as apigenin, naringenin, luteolin, thymonin, carvacrol and thymol (Stahl-Biskup, 1991;Senatore, 1996;Lacroix et al., 1997;Peñalver et al., 2005;Skerget et al., 2005;Wojdylo et al., 2007 andAmarowicz et al., 2009).
Regarding JO treated beef burger, TBAvalues were significantly lower those than from control samples.These values were however significantly higher than samples treated with 0.04 and 0.06% TEO.
In contrast the JO proved weak antioxidant effect which is referred to its molecular stability (Miwa, 1971 andWisniak, 1994).
It's worth to mention that at the 12 th day of storage both 0.02% TEO and Jojoba oil treated samples showed TBA-values over 0.5 mg mal/kg which rendered these samples unacceptable.

PH-VALUE
pH-value of examined samples reveals that no significant difference (p≤0.05) could be established among treated beef burger  during all storage time. Where pH of control, 0.02% TEO, 0.04% TEO 0.06% TEO and JO were 6.53 ± 0.23, 6.38 ± 0.23, 6 .27 ± 0.22, 6.26 ± 0.22 and 6.33 ± 0.22 respectively at 12 th day of storage. Such finding could be referred to the low quantity of oil added in the burger formulation and due to buffering effect of meat (Djenane et al., 2004).

MICROBIOLOGICAL QUALITY
Microbial quality of beef burger was assessed through estimation of Enterobacteriaceae, Staphylococcus, proteolytic, lipolytic bacterial and total mould counts (tables II-VI) whereas microbial load could have been implicated for public hazard. Enterobacteriaceae, Staphylococcus, proteolytic, lipolytic bacteria and total mould counts of control samples were significantly higher (p≤0.05) than all treated samples and continued for the end of the experiment.
Addition of TEO and JO to beef burger formulation significantly minimized (p≤0.05) microbial load of examined samples than control one. At 0, and 3 d no significant difference could be established between treated samples for each examined bacterial group with expect ion of Staphylococcus count, where 0.06% TEO treated samples (2.3±0.083) were significantly lower than JO  (2.78±0.1) treated samples (Mohsenzadeh,-M 2007). After six days of storage, 0.06% TEO treat beef burger revealed the significant lowest count for Enterobacteriaceae, Staphylococcus, proteolytic, lipolytic bacteria and total mould counts followed by 0.04% TEO treated one and continued for two weeks of refrigerated storage. Meanwhile, microbial load of samples treated with JO was significantly higher than that of samples treated with different concentration of TEO. These results may emphasize the antimicrobial activity of phenolic compounds in TEO mainly thymol and carvacrol in (Cosentino et al., 1999;Peñalver et al., 2005;Šegviae et al., 2006 andViuda et al., 2008).
Considering the antimicrobial effects of TEO added in burger formulation the data given in tables (II-VI) indicated that 0.04 and 0.06% concentrations significantly minimized microbial load than control, 0.02% thyme and JO treated samples. As 0.04 and 0.06% thyme treated samples revealed 4. 6, 4.11, 3.09, 2.22, 2.3 and 5.9, 4.47, 3.39, 3.4 and 3.3 log cfu/g respectively reduction in counts of Enterobacteriaceae, Staphylococcus, proteolytic, lipolytic bacterial and  total mould counts than control samples respectively at 12 th day of storage. These obtained result confirmed the possibility of using thyme essential oils in meat products to prevent the growth of either spoilage and pathogenic bacteria and extend their shelf-life.

CONCLUSION
From the obtained results, it could be concluded that addition of TEO to beef burger formulation at concentration of 0.04 and 0.06% not only minimize lipid oxidation but also improved its sensory characteristics and enhanced the wholesomeness of the product during two weeks of refrigerated storage. JO incorporation in beef burger formulation also showed lower TBA-value and microbial load than control samples. Furthermore, during refrigerated storage shelf life of 0.02% TEO and jojba O treated samples was extended for three days more than control. While beef burger treated with 0.04 and 0.06% TEO showed five days more than Control burger. With superior quality for TEO 0.06% treated burger at the end of storage time.