Formulation of value added beef meatball using tulsi ( Ocimum sanctum ) leaf extract as a source of natural antioxidant

ARTICLE INFO


Introduction
Lipid peroxidation causes meat spoilage.It occurs during processing and storage when meatballs are exposed to oxygen, heat, and light (Fasseas et al., 2007).Antioxidant have an ability to prevent or reduce the oxidative damage of a tissue indirectly by enhancing natural defense of cell and/or directly by scavenging the free radical species (Verma et al., 2009).Over the years, synthetic antioxidants such as beta hydroxyl anisole, butyrate hydroxyl toluene and tertiary butyl hydroquinone etc. have been widely used to preserve meat and meat products (Fasseas et al., 2007).The use of these antioxidants is questionable since they have been discovered as toxic, mutagenic and carcinogenic effect to human and animal (Hayes et al., 2010).Hence there has been a growing interest in the use of natural antioxidants as alternative remedy for synthetic antioxidants.In addition, consumers have shifted their interest to natural antioxidants since they are considered safer than the synthetic antioxidants (Jung et al., 2010).It has also been reported that these natural antioxidants, especially of plant source, have greater application potential for consumer's acceptability, palatability, stability and shelf-life of meat products (Jung et al., 2010).Tulsi is grown in tropical and sub tropical including Indian Regions (Banerjee et al., 1996).This plant has been evaluated pharmacologically for antimicrobial, immunomodulatory, anti-stress, antiinflammatory, antipyretic, anti-asthmatic, hypoglycemic, hypotensive and analgesic activities.tulsi has been found to be utmost effective in various types of animal models (Chiang et al., 2005).Phenolics and flavonoids are the authentic antioxidants found in tulsi leaf that have been reported to be safe and bioactive (Sreelatha and Padma, 2009).Tulsi is a naturally occurring antioxidants that have many of the same functions as artificial antioxidants like butyrate hydroxyl anisole.It can increase shelf life of stored products without affecting qualities.It contains several compounds having multiple phenolic hydroxyl groups, such as apigenin, luteolin, vitexin, isovitexin, orientin, aesculetin, aesculin, chlorogenic acid and caffeic acid (Koushik and Gopal, 2013).The phenolic compounds, namely, cirsilineol, cirsimaritin, isothymusin, apigenin, rosmarinic acid and eugenol possess good antioxidant activity and have significant ability to scavenge highly reactive free radicals (Pandey and Madhuri, 2010).Tulsi, also known as Ocimum tenuiflorum, is revered as "Queen of herbs" due to its greater medicinal values and often consumed as herbal tea.Marked by its strong aroma and astringent taste, it is regarded as a kind of "elixir of life", as it is believed to promote longevity (Puri and Singh, 2002).Tulsi is known as "Queen of plant", "The mother medicine of nature" (Singh et al., 2010).No investigation on different levels of tulsi leaf extract on beef meatball has been carried out yet.With these view, the present study was undertaken to investigate the possibility of using different levels of tulsi leaf extract in beef meatballs to evaluate shelf-life, nutritional and microbiological status of beef meatball under different storage conditions.

Collection of meat sample
Boneless beef of 2.5kg from freshly slaughtered cattle was collected from "Local market", Bangladesh Agricultural University, Mymensingh at 10.00 a.m.The meat sample was immediately transferred to the "Animal Science Laboratory" and stored at room temperature for 1 hour.

Preparation of jar and other instruments
All necessary instruments and jars or containers were cleaned with hot water and detergent powder and then autoclaved for sterilization and dried before starting the experimental activities.

Sample preparation
About 2.5 kg of fresh beef sample was taken for the preparation of beef meatball.First, the beef was cleaned with fresh water and the fat was trimmed with sharp knife.The beef sample was grinded properly and the spices, garam masala, salt, ice flakes, refined vegetable oil, refined wheat flower, sauce were mixed with grinded beef properly as per experimental design.There were four treatment groups as 0% (T 0 ), 0.1% (T 1 ), 0.2% (T 2 ), and 0.3% (T 3 ) tulsi leaf extract.The beef meatball of proper shape was prepared separately.It was then boiled in hot water for 2-3 minutes, water was removed from the meatball and fried in hot oil until reddish brown color was obtained.After frying the meatballs, they were packaged in polyethylene bags separately and kept into the freezer for further research at different day's of intervals.

Sensory evaluation
Different sensory attributes were examined.The meatball samples were evaluated by a trained 8-member panel.The sensory questionnaires measured intensity on a 5-point balanced semantic scale for the attributes of color, smell, tenderness, juiciness, and overall acceptability.Eight training sessions were held to familiarize the judges with the attributes to be evaluated and the scale to be used (Rubio et al., 2007).Prior to sample evaluation, all panelists participated in the orientation sessions to familiarize with the scale attributes (color, smell, juiciness, tenderness, and overall acceptability) of beef meatball using intensity scale.All samples were served in the Petri dishes.Sensory evaluation was accomplished at 0 day and repeated at 15, 30 and 60 days.

Proximate composition
Proximate composition such as Dry Matter (DM), Ether Extract (EE), Crude Protein (CP) and Ash were carried out according to the methods of AOAC (1995).

Physicochemical properties measurement
All pH value of raw, cooked and cooking loss meatball was measured using pH meter (Hanna HI99163) from raw meatball homogenate.It was prepared by blending 5 g of meat with 10 ml distilled water.

Biochemical analysis and microbial assessment
Free Fatty Acid (FFA), Per Oxide Value (POV) and Thiobarbituric Acid Reactive Substances (TBARS) value were determined according to Sharma et al, (2012).Total Viable Count (TVC), Total Coliform Count (TCC) and Total Yeast Mold Count (TYMC) were determined according to Ikhlas et al., (2011).All determination was done in triplicate and mean value was reported.

Statistical analysis
Data were analyzed using 4x4 factorial experiment in completely randomized design replicated three times per cell using SAS 9.1.3version Statistical Discovery software, NC, USA.Duncan's Multiple Range Test (DMRT) test was used to determine the significance of differences among treatments means.

Sensory evaluation
It was found that sensory quality after fortification with tulsi leaf extracts was deteriorated with increased storage period.The range of overall observed color score at different treatment was 3.75 to 4.33, flavor score was 3.75 to 4.17, tenderness score was 3.58 to 4.17, juiciness score was 3.83 to 4.00 and overall acceptability score was 3.83 to 4.08 (Table 1).The range of different day's intervals of overall observation of overall acceptability score was 3.08 to 4.58.Among four treatments most preferable color and juiciness was observed from 0.2 % (T 2 ), tenderness and overall acceptability was observed from 0.1% (T 1 ) and flavor was observed from 0.3% (T 3 ) tulsi leaf extract.Texture attributes viz, hardness, springiness, color, odor, gumminess and flavor as well as the nutritional quality of the product were significantly higher for clove and cardamom burfi.Sensory evaluation revealed that among different herbs, cardamon is highly preferred in burfi followed by ginger, turmeric , clove, curry leaves and tulsi (Prasad et al., 2017).It is in agreement with the present findings where tulsi leaf extract significantly affect different sensory attributes.

Proximate components
Overall DM content at different treatment was 53.99 to 59.29%.The highest value was observed in 0 day and decreased gradually up to 60 th day (Table 2).The highest amount of DM content indicates this product is more preferable.DM content was increased with increased storage period because moisture loss was decreased with storage period.Similar results were reported for Indonesian traditional meatballs with a moisture content ranged from 69.52 to 71.17% (Purnomo and Rahardiyan 2008).The CP content at different treatments was 41.87 to 51.93.Synthetic antioxidant group contain more amount of CP than control group.The most preferable CP content was observed at 0.3 % (T 4 ) tulsi leaf extracts group.The CP content at different days of interval was 23.32 to 22.24%.The most preferable CP content was observed at 0 day and less preferable at 60 th day.The EE content at different treatments was 11.38 to 12.06 %.The most preferable EE content was observed at 0.2 (T 2 ) and 0.3% (T 3 ) tulsi leaf extracts.The range of EE content at different days of interval was 11.82 to 11.89%.The most preferable EE content was observed at 0 day and less preferable EE content was at 60 th day (Table 2).Serdaroglu et al. (2005) reported a similar fat content ranged from 7.9 to 8.8% in low-fat traditional Turkey koefte beef meatballs.Overall ash content at different treatments was 3.49 to 3.67%.Control group contain higher amount of ash than treated with tulsi leaf extracts group except T 3 .

Physicochemical properties
The range of overall observed raw pH at different treatments was 5.69 to 5.81%.Among four treatments most preferable raw pH was observed at 0.3% (T 3 ) tulsi leaf extract (Table 3).The highest amount of raw pH indicated the higher preferability of the products for consumers' health.The raw pH among the treatments was decreased with increased storage period.The raw p H at different days of interval was 5.70 to 5.79%.The most preferable raw p H was observed at 15 th day and less preferable was observed at 60 th day.Overall cooked p H at different treatments was 5.93 to 6.10%.The most preferable cooked p H was observed at 0.3% (T 3 ) tulsi leaf extract.The most preferable cooked p H was observed at 30 th day and less preferable cooked p H at 60 th day.These results are similar to those of Sallam et al. (2004), who reported that storage time had a significant (p< 0.05) effect on pH values, which tended to increase with storage time (up to 15 days with present finding).The overall cooking loss at different treatments was 23.97 to 24.95%.The highest cooking loss was observed at 0.3% (T 3 ) tulsi leaf extract.The overall cooking loss at different days of interval was 22.35 to 26.32%.The lowest cooking loss was observed at 60 th day and the highest cooking loss was observed at 0 day of observation.Cooking loss refers to the reduction of weight of meatballs during cooking process (Jama et al., 2008).

Biochemical properties
The biochemical effects of tulsi leaf extract on beef meatball is shown in Table 4.The overall FFA, POV and TBARS value at different treatment was 0.36 to 0.44%., 4.47 to 4.20 and 0.49 to 0.59, respectively.The overall FFA, POV and TBARS at different days of interval were 0.35 to 0.44%., 4.03 to 4.59% and 0.40 to 0.69%, respectively.The most preferable FFA value was observed at 0 day and less preferable FFA value was observed at 60 th day.The most preferable value was observed from 0.3% (T 3 ) tulsi leaf extract.The FFA value (0.44) in the control group was significantly (P < 0.01) higher than the values of the samples treated with 0.1, 0.2, and 0.3% tulsi leaf extracts.Lee and Kunz (2005) found that fermented sausages showed an increasing FFA content over time.It has been reported that these natural antioxidants, especially of plant source, have greater application potential for consumer's acceptability, palatability, stability and shelf-life of meat products (Jung et al., 2010).Throughout the storage time, POV were generally higher in control group compared to treatment groups (Table 4).The most preferable POV was observed at 0.3% (T 3 ) tulsi leaf extracts.The lowest amount of POV indicates that this product is most preferable for consumers health.The control sample showed a higher level of TBARS than samples treated with 0.1, and 0.3% except 0.2% tulsi leaf extracts.This difference was especially significant (p < 0.01) after 60th days of storage time.Natural antioxidants, in particular polyphones, are the major plant compounds which have the ability to attenuate the oxidative damage of a tissue indirectly by enhancing natural defenses of cell and/or directly by scavenging the free radical species combat pathological disorders generated by physicochemical Reactive Oxygen Species (ROS) (Du et al., 2010).Antioxidants have an ability to prevent the oxidative damage of tissue indirectly by enhancing natural defenses of cell and directly by scavenging the free radical species (Verma et al., 2009).It has also been reported that these natural antioxidants, especially of plant source, have greater application potential for consumer's acceptability, palatability, stability and shelf-life of meat products (Jung et al., 2010).Tulsi leaf extracts can be a potential source of natural antioxidant which can be used in meat products.

Microbiological assessment
Effect of tulsi leaf extracts on beef meatball on microbial population is shown in Table 5. TVC value of fresh beef was 5.12 logs CFU/g beef, indicated good quality beef.The overall total viable count, TCC value and TYMC of beef meatball was 4.74 to 4.86, 1.07 to 1.13, and 1.47 to 1.69 (log CFU/g), respectively at different treatment levels.The range of TVC value, TCC value and TYMC values at different days of interval was 4.50 to 5.13, 1.01 to 1.18 and1.11 to 1.89, respectively.The TCC value of fresh beef was 1.13 logs CFU/g beef.Among these four treatments, the TCC in the control sample (1.13 logs CFU/g) was significantly higher than in the samples treated with 0.1, 0.2, and 0.3% of tulsi leaf extracts.The different superscript was observed from different treatment indicated that there were significant differences of TYMC values among these four treatment groups.Among four treatments, the total yeast-mold count in the control sample (1.69 log CFU/g) were significantly (p<0.01)higher than in the samples treated with, 0.1, 0.2, and 0.3% of tulsi leaf extracts.Some bacteria may be present in the product, but their growth is controlled under storage conditions (Fernandez-Lopez et al., 2005).Sensory scores were 5 for excellent, 4 for very good, 3 for good, 2 for fair, and 1 for poor.Mean in each row having different superscript varies significantly at values *P < 0.05.Again, mean values having same superscript in each row did not differ significantly at P > 0.05.T 0 = 0% tulsi leaves extract, T 1 = 0.1% tulsi leaves extract, T 2 = 0.2% tulsi leaves extract, T 3 = 0.3% tulsi leaves extract, DI=Days of Intervals, Treat= Treatment, T*DI=Interaction of Treatment and Day Intervals.

Conclusion
From the study it may be concluded that 0.3% of tulsi leaf extracts as natural antioxidant may be used in beef meatballs preparation.On the basis of sensory evaluation, physicochemical properties, biochemical analysis and microbial assessment indicated that 0.3% tulsi leaf extracts showed better results in the preparation of beef meatball compare to control and other two treatments.