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Storage Stability of Pork Emulsion Incorporated with Arjuna (Terminalia arjuna) Tree Bark Extract

Ritika Birla Om Prakash Malav Rajesh V. Wagh Nitin Mehta Manish Kumar Chatli Pavan Kumar
Vol 9(2), 95-109
DOI- http://dx.doi.org/10.5455/ijlr.20180724064413

Arjuna tree bark extract was incorporated at three different levels separately in pork emulsion after replacing the lean meat in the pre-standardized formulation. The control and treatment pork emulsion was packaged in LDPE pouches and stored aerobically under refrigeration (4±1°C) for 9 days and analyzed at regular interval of 1, 3, 5, 7 and 9 days of storage. Water activity decreased significantly (P<0.05) with the advancement of storage period. Redness (a*) value, lightness (L* value) and yellowness (b* values) of control and treatment emulsion, followed a declining trend with the increase in storage time. Oxidative stability parameters followed an increasing (P<0.05) trend throughout the storage period. Microbiological count followed an increasing trend in control and treatment emulsion. Sensory scores showed declining trend with increase in storage time.


Keywords : Antioxidant Colour Profile Extract Storage Sensory

The pig population of India is 10.29 million as the latest livestock census (19th Livestock Census, 2012) it constitutes about 1% of world pig population. In the year 2015-16, pork production was 0.38 million tonnes which constitutes around 6% of total meat produced in India (BAHS, 2016). Pig meat (pork) is a very tasty and popular meat worldwide. But the pork has high fat content with higher proportion of unsaturated fatty acids, which oxidizes more rapidly than chevon and mutton. Therefore pork is more susceptible to lipid oxidation leading to development of warmed-over flavour (WOF) and deteriorative changes in quality thus leading to decreased quality of meat. Lipid peroxidation, the oxidative deterioration of the polyunsaturated lipids of meat and meat products, leads through formation of hydroperoxides to short-chain aldehydes, ketones and other oxidized compounds, which are responsible for causing flavour, texture, colour and nutritional deterioration of meat and meat products. Several adverse health effects, due to the presence of free reactive oxygen species (ROS), such as superoxide radical, hydroxyl radical, peroxyl radical, during lipid oxidation, have been reported (Nissen et al., 2004). Therefore, to avoid or delay the lipid oxidation processes in meat, meat product manufacturers are using several synthetic food additives in the past few decades such as nitrites, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA) etc.

Terminalia arjuna, commonly known as Arjuna, (Family: Combretaceae), contains useful phytoingredients like triterpenoids, β-sitosterol, flavonoids, glycosides etc. Triterpenoids and flavonoids are responsible for its beneficial antioxidant and cardioprotective effect (Dwivedi and Chopra, 2014). Bark of Arjuna has been widely used in Indian system of medicine for a variety of purposes and the bark is particularly effective useful in cardiovascular therapy. Phytochemical screening showed higher concentration, such as phytosterol, lactones, flavonoids, phenolic compounds, tannins and glycosides. The antimicrobial activity of extract showed that greater inhibition zone against gram negative bacteria than gram positive bacteria and the extract showed a promising antioxidant activity (Mandal et al., 2013). Arjuna tree bark contains a very high level of flavonoids compared to other commonly used plant item. Flavonoids detected from its bark are namely arjunolone, flavones, bicalein, quercetin, kempferol and pelorgonidin. The aqueous extract of Arjuna contains 70% polyphenols having a molecular weight greater than 3.5 kDa. Flavonoids are very important because of its antimutagenic and antibacterial property. It is also act as strong anti- proliferative and antioxidant agents due to presence of free radical scavenging action of various phenolic contents (Gupta et al., 2018).

The antioxidant and antimicrobial efficacy of developed Arjuna tree bark extract (ATBE) was valuated in model meat system (In vivo) using pork emulsion in relation to physico-chemical qualities, oxidative stability and instrumental colour attributes microbiological and sensory characteristics during refrigeration (4 ±1C) under aerobic packaging conditions.

Materials and Methods

Preparation of Arjuna Tree Bark Extract

Arjuna tree bark was dried and powdered, crushed in mixer and passed through stainless steel sieve. It was powdered into a particle size of 60-80 mesh and successively extracts were prepared, Arjuna tree bark powder was added to a glass thimble, which was placed inside a glass Sohxlet Appratus. This was attached to a condenser and a pre-weighed 500 ml round-bottomed flask (quick-fit, UK) containing 200 ml solvent (distilled water or ethanol) and a few anti-bumping granules. The apparatus was placed on electro mantle, according to Response Surface Methodology (RSM) taken three variants (time, temperature and solvent concentration) were taken. The solvents were distilled off in a rotary vacuum evaporator (RE-300 Yamato Rotary Evaporator, Japan) at different time-temp combination on 210 rpm, leaving the extract in the flask. Re-weighing the flask allowed determination of the weight of the extract. This process was carried out using aqueous and ethanol solvents. The extracts were filtered through Whatman’s filter paper No. 1 after doing all procedure the collected liquid extract was poured in glass petri plates and kept in incubator for 36-48 hrs for drying. Dry extract from glass petri plates was collected and stored in amber coloured bottles at 4±1°C till further use.

Analytical Methods

pH and Water Activity (aw)

The pH of emulsion was determined as per the method described by Trout et al. (1992) with digital pH meter (FE-20-1-KIT, Mettler-Toledo India Pvt. Ltd., Mumbai)  equipped with a combined glass electrode. Ten gram of sample was homogenized with 50 ml of distilled water for 1 min using pestle and mortar. The electrode was dipped into the suspension and the pH value of the sample was recorded. Water activity was determined by using hand held portable digital water activity meter (ROTRONIC HygroPalm AW1, Bassersdorf, Switzerland). Samples were filled up to 80% in moisture free sample cup and water activity was recorded as per specifications.

Peroxide Value (PV) and Free Fatty Acid (FFA)

The peroxide value and free fatty acid were measured as per procedure described by Koniecko (1979).

Thiobarbituric Acid Reactive Substances (TBARS)

The extraction method described by Witte et al. (1970) was used with suitable modifications for the determination of TBARS values of cooked pork nuggets. 10g of sample was triturated with pestle and mortar with 25 ml of pre-cooled 20% trichloroacetic acid (TCA) prepared in 2 M orthophosphoric acid solution for 2 min. The content was then transferred quantitatively to a beaker by rinsing with 25 ml of cold distilled water, well mixed and filtered through ashless filter paper (Whatman filter paper No. 1). Then, 3 ml of TCA extract (filtrate) was mixed with equal amount of 2-thiobarbituric acid (TBA) reagent (0.005 M) in test tubes and placed in dark cabinet for 16 hrs. A blank sample was prepared by mixing 3ml of 10% TCA and 3 ml of 0.005 M TBA reagent. Absorbance (O.D.) was measured at fixed wavelength of 532 nm with a scanning range of 531 to 533 nm using UV-VIS spectrophotometer (SL-159 Elico India Ltd., Mumbai). TBA value was calculated as mg malonaldehyde per Kg of sample by multiplying O.D. value with K factor of 5.2.

Instrumental Colour Profile

Colour profile was measured using CR-400 Konica Chroma meter (Konica Minolta, Japan) set at 2o of cool white light (d65) and known as ‘L’, a, and b values. ‘L’ value denotes (brightness 100) or lightness (0), a (+ redness/- greenness), b (+ yellowness/-blueness) values were recorded on/in a hundreds of pork nugget kept in a plate. The instrument was calibrated using light trap (black hole) and white tile provided with the instrument. Then the above colour parameters were selected. The instrument was directly put on the surface of pork nugget at three different points. Mean and standard error for each parameter were calculated. Delta e (total colour difference) can be calculated by using following formula-

Δe =√ (L*2-L*1)2+ (a*2-a*1) 2+ (b*2-b*1)2

Microbiological Counts

Standard plate counts (SPC), Psychrophilic count, Yeast and Mould counts and Coliforms count in the samples were enumerated following the methods as described by American Public Health Association (APHA 2001) and data were expressed as log10 colony forming units (CFU) per gram sample.

Sensory Evaluation

A seven member experienced panel of judges consisting of teachers and postgraduate students of department of LPT, of College of Veterinary Science, Guru Angad Dev Veterinary and Animal Sciences University evaluated the samples for the sensory attributes viz colour and appearance, flavour, juiciness, texture and overall acceptability using 5-point descriptive scale (Keeton, 1983), where 5=excellent and 1=extremely poor. The test samples were presented to the panelists after assigning the suitable codes. The samples were warmed in a microwave oven for 20 sec before serving to the sensory panelists. The water was served for rinsing the mouth between the samples.

Statistical Analysis

Data was analyzed statistically on ‘SPSS-16.0’ (SPSS Inc., Chicago, II USA) software package as per standard methods (Snedecor and Cochran, 1994). Duplicate samples were drawn for each parameter and the whole set of experiment was repeated three times to have total number of observations. Sensory evaluation was performed by a panel of seven member judges, Total observations of all parameters were six (n=6) except sensory parameters where n=21. The average values were reported along with standard error. The statistical significance was estimated at 5% level (P<0.05) and evaluated with Duncan’s Multiple Range Test (DMRT).

Results and Discussion

Three different levels of selected phyto-extracts of Arjuna (Terminalia arjuna) tree bark (ATB) viz: (T-1=0.2%, T-2=0.5%, T-3=1.0%) was incorporated separately in pork emulsion after replacing the lean meat in the pre-standardized formulation. The control and treatment pork emulsion was packaged in LDPE pouches and stored aerobically under refrigeration (4±1°C) for 9 days and analysed at regular interval of 1, 3, 5, 7 and 9 days of storage.

The results for physico-chemical properties i.e. (emulsion pH, water activity (aw), instrumental colour profile, oxidative parameters {(Thiobarbituric acid reactive substances (TBARS)}, free fatty acid contents (FFA) and peroxide value), microbiological quality (SPC, coliforms, psychrophilic, yeast and molds count) and sensory evaluation (5-point descriptive scale), (APPEARANCE, odour and overall acceptability) of pork emulsion incorporated with the three different levels of  i.e. 0.20%, 0.50% and 1.0% ethanolic Arjuna tree bark extract (ATBE) are presented in Tables 1 to 5. The control and treatment emulsion was stored aerobically under refrigeration  (4±1°C) for 9 days and analyzed at regular interval of 1, 3, 5, 7 and 9 days of storage.

Physico-chemical Properties

The pH of pork emulsion varied significantly (P<0.05) amongst treatments and days of storage with the incorporation of 0.20, 0.50 and 1.0 % Arjuna (Terminalia arjuna) tree bark extract (ATBE). In the present study the pH values were comparable in all treated samples on 1st day of storage, thereafter followed an increasing trend during storage, irrespective of the added amount of phyto-extract. The pH was significantly (P<0.05) lower for T-3 and T-2 than T-1 on the (9th) day of storage and significantly (P<0.05) higher in control than all the treatments throughout the storage period. The increase in the pH during storage might be due to the low carbohydrate content, formation of N non-protein compounds and basic ammonium ions coupled with buffering action of protein (Rathour et al., 2017 and Kumar et al., 2015). Similarly, Villalobos-Delgado et al., (2017) reported that an increase in pH is associated with the accumulation of ammonia caused by the degradation of proteins and amino acids. It has been widely recognized that the concept of water activity (aw) is of great importance in meat preservation, because measured values generally correlated well with the potential for growth and metabolic activity of microorganisms (Chirife and Buera, 1995).

Water activity decreased significantly (P<0.05) with the advancement of storage period (Table 1). The variation in aW of treatment pork emulsion, might be due to the addition of 0.20, 0.50 and 1.0 % ATBE and microbial growth during storage. Similar findings were reported by Verma et al. (2018) in pork emulsion incorporated with blood hydrolysate. The aW was highest in T-2 and lowest in control on last (9th) day of storage. The values in the present study were in conformity with the earlier findings on ground raw pork by Wagh and Chatli (2017).

 

 

Table 1: Effect of different levels of Arjuna tree bark extract (ATBE) on the physico-chemical parameters of pork emulsion during refrigerated (4±1°C) aerobic storage (Mean±S.E.)*

Treatments/Days                                      Refrigerated Storage period (Days)
Day 1 Day 3 Day 5 Day 7 Day 9
pH
C (Control) 5.77±0.06A 5.78±0.06AB 5.85±0.01AB 5.90±0.05B 5.92±0.05Aa
T1 (0.20%) 5.80±0.05A 5.83±0.02A 5.87±0.09A 5.93±0.03AB 6.06±0.06Bb
T2 (0.50%) 5.78±0.05A 5.81±0.06A 5.84±0.06A 5.89±0.04AB 6.02±0.01Bb
T3 (1.0%) 5.78±0.045A 5.81±0.06A 5.84±0.06A 5.89±0.04AB 6.02±0.01Bb
Water Activity (aW)
C (Control) 0.92±0.006C 0.90±0.005B 0.89±0.005ABa 0.88±0.007Aa 0.87±0.006Aa
T1 (0.20%) 0.93±0.006B 0.92±0.010AB 0.91±0.006Ab 0.90±0.005Ab 0.90±0.006Ab
T2 (0.50%) 0.93±0.007A 0.92±0.006A 0.92±0.005Ab 0.91±0.005Ab 0.92±0.006Ab
T3 (1.0%) 0.93±0.005B 0.91±0.011A 0.90±0.005Aab 0.90±0.006Ab 0.91±0.006Ab

n=6; C= Control (without phyto-extract); T-1= 0.2% Arjuna tree bark extract, T-2= 0.50% Arjuna tree bark extract; T-3= 1.0 % Arjuna tree bark extract. *Mean±S.E. with different superscripts row wise (capital alphabets) and column wise (small alphabets) differ significantly (P<0.05).

Instrumental Colour Profile

The results of instrumental colour profile of pork emulsion incorporated with the three different levels of i.e. 0.20%, 0.50% and 1.0% ethanolic Arjuna tree bark extract (ATBE) are presented in Table 2.

Table 2: Effect of different levels of Arjuna tree bark extract (ATBE) on the instrumental colour profile of pork emulsion during refrigerated (4±1°C) aerobic storage (Mean±S.E.)*

Treatments/ Days Refrigerated Storage Period (Days)
Day 1 Day 3 Day 5 Day 7 Day 9
L* (Lightness)
C (Control) 55.76±0.64B 54.16±0.58AB 54.08±0.53AB 53.89±0.58AB 52.78±0.64A
T1 (0.20%) 54.81±0.60A 54.59±0.52A 54.15±0.60A 54.10±0.35B 53.03±0.24A
T2 (0.50%) 54.38±0.18B 54.14±0.19AB 53.99±0.25AB 53.59±0.33A 53.74±0.30AB
T3 (1.0%) 54.55±0.41AB 54.76±0.25B 54.69 ± 0.59B 53.73±0.38AB 53.35±0.32A
a* (Redness)
C (Control) 12.83±0.56C 12.19±0.50C 11.93±0.53BC 10.34±0.23AB 9.38±0.21Aa
T1 (0.20%) 12.11±0.56A 12.03±0.50A 12.06±0.53A 11.79±0.23A 11.48±0.21Ab
T2 (0.50%) 11.89±0.61A 11.85±0.56A 11.67±0.59A 10.88±0.61A 10.91±0.53Ab
T3 (1.0%) 11.94±0.63A 11.78±0.55A 11.66±0.66A 10.98±0.33A 10.80±0.63Ab
b* (Yellowness)
C (Control) 15.83±0.59C 15.19±0.53C 14.93±0.56BC 13.34±0.26AB 12.39±0.24Aa
T1 (0.20%) 15.11±0.59A 15.03±0.53A 15.06±0.58A 14.79±0.26A 14.48±0.24Ab
T2 (0.50%) 14.89±0.64A 14.85±0.59A 14.67±0.62A 13.88±0.64A 13.91±0.56Ab
T3 (1.0%) 14.94±0.60A 14.78±0.58A 14.66±0.69A 13.98±0.35A 13.80±0.66Ab

n=6; C= Control (without phyto-extract); T-1= 0.2% Arjuna tree bark extract, T-2= 0.50% Arjuna tree bark extract; T-3= 1.0% Arjuna tree bark extract. *Mean±S.E. with different superscripts row wise (capital alphabets) and column wise (small alphabets) differ significantly (P<0.05).

Lightness (L* value) of control and treatment emulsion, followed a declining trend with the increase in storage time however colour scores decreased significantly (P<0.05) for treatments on the 9th day of the storage which is attributed to the fact that salt greatly enhanced the process of meat discoloration due to the pro-oxidative activity as well as its ability to release iron from heme pigments (Devatkal et al., 2014). The rate of decrease in treatments was lower than control, which might be due to the ability of the ATBE to maintain the colour of the product by retarding the oxidation reaction (Kumar et al., 2015). Calvo et al. (2008) also reported a decrease in L* value in tomato peel incorporated beef and beef products. Similar observations were recorded in raw beef hamburgers incorporated with dried tomato peel (Garcia et al., 2009), in pork patties incorporated with tomato powder (Kim et al., 2009), in beef patties containing commercial lycopene preparation (Escalante et al., 2003) and Rathour et al. (2017) in chevon rolls incorporated with aloe vera and cinnamon bark extracts stored under aerobic packaging at 4 ± 1ºC.

Redness (a* value) is an indicator of freshness of the meat and criteria for quality evaluation by the consumers. Redness (a*) value showed the pattern T-3>T-2>T-1 amongst treatments and followed a decreasing trend in all the treatments throughout the storage. This might be due to gradual oxidation of myoglobin and accumulation of metmyoglobin with storage time (Mancini and Hunt, 2005). However, a* value was higher in ATBE treated samples than control throughout the storage period and rate of decrease in a* value was significantly (P<0.05) lower in treated products than control which might be due to antioxidant properties of ABTE. Several authors have studied the colour of meat and meat products and reported that meat oxidation causes a decrease in a* value (Lavieri and Williams, 2014; Kumar et al., 2015).

Yellowness (b* values) also followed a decreasing trend for all the treated emulsion as well as control throughout the storage period irrespective of the level of incorporation of ATBE. A decrease in b* values for meat is an indicative of the development or presence of the metmyoglobin brown pigment (Lin et al., 2015). Realini et al. (2015) also reported decrease in b* values of beef patties containing Acerola fruit extract. Selgas et al. (2009) reported same trend for a* and b* values in hamburgers containing dried tomato powder. Similar observations were recorded in oxidative stability of pork emulsion containing tomato products and pink guava pulp during refrigerated (4±1ºC) aerobic storage by Joseph et al. (2014).

Oxidative Quality Parameters

The oxidative stability of pork emulsion during storage was evaluated on the basis of TBARS, peroxide value and free fatty acid content (Table 3). The deteriorative changes produced from the oxidation of lipids are responsible for colour deterioration, rancid flavour and aroma as well as formation of toxic compounds. TBARS values followed an increasing (P<0.05) trend throughout the storage period irrespective of added level of ATBE in the pork emulsion (Fig. 1). TBARS value was measured lowest in T-3 and highest in T-1 among treatments and similar trend continued throughout the storage. However, TBARS was significantly (P<0.05) higher in control than ATBE treated products throughout the storage period. TBARS values were well below the permissible level of 1.0, indicator of acceptability of meat products (Witte et al., 1970).

Table 3: Effect of different levels of Arjuna tree bark extract (ATBE) on the oxidative parameters of pork emulsion during refrigerated (4±1°C) aerobic storage (Mean±S.E.)*

Treatments/ Days Refrigerated Storage Period (Days)
Day 1 Day 3 Day 5 Day 7 Day 9
TBARS Value (mg malonaldehyde/Kg)
C (Control) 0.38±0.009Ab 0.56±0.005Bd 0.85±0.013Cc 1.24±0.052Dc 1.75±0.013Eb
T1 (0.20%) 0.38±0.007Ab 0.48±0.007Bc 0.65±0.008Cb 0.79±0.007Db 0.87±0.013Ea
T2 (0.50%) 0.35±0.011Aa 0.44±0.004Bb 0.63±0.006Cb 0.73±0.004Db 0.86±0.003Ea
T3 (1.0%) 0.33±0.006Aa 0.41±0.010Ba 0.52±0.008Ca 0.65±0.010Da 0.79±0.017Ea
Free Fatty Acid (%)
C (Control) 0.19±0.008Ac 0.29±0.016Bc 0.342±0.008Cd 0.46±0.004Dd 0.48±0.008Dc
T1 (0.20%) 0.17±0.003Ab 0.23±0.005Bb 0.267±0.003Cc 0.29±0.004Dc 0.30±0.008Db
T2 (0.50%) 0.11±0.004Aa 0.14±0.003Ba 0.189±0.003Cb 0.24±0.011Db 0.29±0.006Eb
T3 (1.0%) 0.10±0.004Aa 0.14±0.004Ba 0.174±0.003Ca 0.19±0.007Da 0.21±0.002Ea
Peroxide Value (meq/Kg)
C (Control) 5.93±0.15Ab 6.24±0.13Bc 6.757±0.059Cc 7.59±0.04Dd 7.96±0.09Ed
T1 (0.20%) 5.49±0.09Aa 5.99±0.14Bc 6.45±0.065Cb 6.88±0.09Dc 7.09±0.07Dc
T2 (0.50%) 5.29±0.03Aa 5.67±0.06Bb 6.25±0.067Cb 6.64±0.04Db 6.73±0.02Db
T3 (1.0%) 5.22±0.16Aa 5.32±0.03Aa 5.45±0.079Aa 5.73±0.07Ba 5.81±0.04Ba

n=6; C= Control (without phyto-extract); T-1= 0.2% Arjuna tree bark extract, T-2= 0.50% Arjuna tree bark extract; T-3= 1.0% Arjuna tree bark extract. *Mean±S.E. with different superscripts row wise (capital alphabets) and column wise (small alphabets) differ significantly (P<0.05).

The lower TBARS value in treated products can be attributed to oxidative stability provided by ATBE which contain high levels of antioxidant components, such as polyphenol, flavonoid, tannins. The results of the present study were in consonance with the findings reported by Biswas et al. (2012) and Verma et al. (2018) who incorporated curry (Murraya koenigii L.) and mint (Mentha spicata) leaf extracts and porcine blood protein hydrolysate in pork emulsion stored under aerobic packaging condition at 4±1ºC respectively.

Fig. 1: Effect of different levels of ATBE on TBARS values of pork emulsion during refrigerated storage. (Control: Without any phytoextracts; T-1: 0.2% ATBE; T-2: 0.5% ATBE; T-3: 1.0% ATBE. Bar represents the standard error (n = 6))

Peroxide value of pork emulsion followed an increasing trend in control and treatments (Fig. 2). The Peroxide value (PV) was significantly (P<0.05) higher in control than all the other treatments throughout storage period of 9 days. It might be due to the formation of hydroperoxides during storage than their degradation into secondary oxidation products and antioxidant properties of added amount of phytoextracts. T-3 measured lowest PV followed by T-2 and T-1. The lower peroxide value in treatment emulsion is an indication of antioxidant potential of ATBE, attributed to the phenolic groups i.e. polyphenols (mainly flavonoids) which provides a labile hydrogen atom by free radicals like alkoxyl or peroxyl radicals and get transformed into phenoxyl radical. Similar observations were recorded in pork patties incorporated with sea buckthorn extract (Wagh et al., 2017) and  in pork emulsion containing tomato products and pink guava pulp (Joseph et al., 2014) stored under aerobic packaging condition at 4±1ºC respectively.

Fig. 2: Effect of different levels of ATBE on peroxide value of pork emulsion during refrigerated storage. (Control: Without any phytoextracts; T-1: 0.2% ATBE; T-2: 0.5% ATBE; T-3: 1.0% ATBE. Bar represents the standard error (n = 6))

Microbiological Quality

The results of microbiological quality of pork emulsion incorporated with the three different levels of i.e. 0.20%, 0.50% and 1.0% ethanolic Arjuna tree bark extract (ATBE) are presented in Table 4. The standard plate count (SPC) of pork emulsion followed an increasing trend in control and treatment emulsion. The standard plate count (SPC) was significantly (P<0.05) higher in control than all the treatments throughout storage period of 9 days. It might be due to the antimicrobial properties of phytoextract incorporated in the treatment emulsion. ATBE contains phenolic compounds, flavonoids, tannins, steroids, alkaloids, and triterpenoids which exhibited a strong antimicrobial effect on fresh pork (Zhang et al., 2009). Similarly, Reddy et al. (2017) reported that rosemary and green tea extract in chicken meat sausages and Wagh and Chatli (2017). Sea buckthorn extract in raw ground pork, were found to be significantly (P<0.05) effective in decreasing the standard plate count in treatment products.

Table 4: Effect of different levels of Arjuna tree bark extract (ATBE) on the microbiological parameters of pork emulsion during refrigerated (4±1°C) aerobic storage (Mean±S.E.)*

Treatments/ Days Refrigerated Storage Period (Days)
Day 1 Day 3 Day 5 Day 7 Day 9
Standard Plate Count (log10 cfu/g)
C (Control) 2.56±0.06Ad 4.42±0.06Bc 5.83±0.05Cc 5.20±0.05Dc 6.88±0.13Ec
T1 (0.20%) 2.04±0.07Ac 3.46±0.07Bb 4.19±0.07Cb 5.55±0.05Db 5.80±0.06Eb
T2 (0.50%) 1.35±0.04Aa 3.51±0.06Ba 3.92±0.05Ca 4.41±0.07Da 4.74±0.06Ea
T3 (1.0%) 1.68±0.05Ab 3.56±0.06Ba 3.93±0.04Ca 4.54±0.06Da 4.72±0.05Ea
Coliforms Count (log10 cfu/g)
C (Control) 1.99±0.009Ab 2.05±0.01Ac 2.20±0.02Bb 2.26±0.03Bb 3.02±0.05Cc
T1 (0.20%) 1.90±0.02Ab 1.98±0.03Bb 2.05±0.02Ca 2.00±0.02BCa 2.33±0.02Db
T2 (0.50%) 1.80±0.06Aa 1.88±0.03ABa 2.00±0.02BCa 2.05±0.03Ca 2.14±0.07Ca
T3 (1.0%) 1.89±0.02Ab 1.96±0.01ABb 2.04±0.03BCa 2.08±0.03Ca 2.29±0.02Db
Psychrophilic Count (log10 cfu/g)
C (Control) 2.44±0.03Ad 2.90±0.02Bd 3.24±0.02Cd 4.20±0.02Dd 4.66±0.10Ec
T1 (0.20%) 2.11±0.01Ac 2.84±0.03Bc 2.93±0.02Cc 3.28±0.02Dc 3.75±0.02Eb
T2 (0.50%) 1.15±0.02Aa 1.91±0.03Ba 2.29±0.03Ca 3.14±0.04Da 3.19±0.03Ea
T3 (1.0%) 1.31±0.03Ab 2.05±0.07Bb 2.88±0.04Cb 3.25±0.02Db 3.33±0.02Ea
Yeast and Molds Count (log10 cfu/g)
C (Control) ND ND 1.36±0.06Bb 2.76±0.05Cb 2.93±0.05Dc
T1 (0.20%) ND ND ND ND 1.05±0.06Bb
T2 (0.50%) ND ND ND ND ND
T3 (1.0%) ND ND ND ND ND

n=6; C= Control (without phyto-extract); T-1= 0.2% Arjuna tree bark extract T-2= 0.50% Arjuna tree bark extract; T-3= 1.0% Arjuna tree bark extract. *Mean±S.E. with different superscripts row wise (capital alphabets) and column wise (small alphabets) differ significantly (P<0.05).

Psychrophilic count (PC) followed similar increasing trend as that of SPC in pork emulsion throughout the storage period. Psychrophilic count was found comparable in T-2 and T-3 on 3rd day of storage and onward throughout storage period of 9 days. These findings are in consonance with Dhanze et al. (2013) who found that there was a considerable reduction in the numbers of pathogens on raw chicken meat incorporated with sea buck thorn leave extract during refrigeration storage of 7 days. Coliforms count followed an increasing trend with the progress of storage period. Coliform count were significantly (P<0.05) higher in control than treatment emulsion which might be due to the antimicrobial properties of ATBE. T-2 showed significantly lower (P<0.05) coliforms count throughout storage period of 9 days.

Yeast and mold count were detected on 5th day of storage in control and 9th day in T-1 whereas, in all other treatments it was not detected even on 9th day of storage. Similarly, Verma et al. (2018) reported the slower growth rate of yeast and mould counts in pork emulsion incorporated with porcine blood hydrolysate than control which might be due to the anti-fungal properties of the protein hydrolysate.

Sensory Quality Parameters

The results of sensory quality parameters of pork emulsion incorporated with the three different levels of of i.e. 0.20%, 0.50% and 1.0% ethanolic Arjuna tree bark extract (ATBE) are presented in Table 5 and Fig. 3.

Table 5: Effect of different levels of Arjuna tree bark extract (ATBE) on the sensory parameters of pork emulsion during refrigerated aerobic storage (Mean±S.E.)

Treatments/Days                              Refrigerated Storage Period (Days)
Day 1 Day 3 Day 5 Day 7 Day 9
Appearance
C (Control) 4.25±0.06C 4.01±0.05BCa 3.74±0.08Ba 3.03±0.04Aa 2.97±0.07Aa
T1 (0.20%) 4.28±0.11B 4.25±0.06Bb 4.19±0.04Bb 3.12±0.04Aa 3.08±0.07Aa
T2 (0.50%) 4.29±0.06C 4.22±0.05Cb 4.18±0.04BCb 4.02±0.06ABb 3.86±0.06Ab
T3 (1.0%) 4.36±0.06C 4.29±0.05Cb 4.25±0.04BCb 4.09±0.06ABb 3.93±0.056Ab
Odour
C (Control) 4.05±0.02Da 3.81±0.05Ca 3.75±0.02Ca 2.47±0.03Ba 2.06±0.03Aa
T1 (0.20%) 4.11±0.07Da 4.07±0.04Db 3.84±0.04Ca 3.25±0.05Bb 2.98±0.05Ab
T2 (0.50%) 4.73±0.01Cb 4.68±0.02Cc 4.59±0.02Cb 4.21±0.05Bc 3.91±0.03Ac
T3 (1.0%) 4.80±0.01Cb 4.75±0.02Cc 4.66±0.02Cb 4.28±0.05Bc 3.98±0.03Ac
Overall Acceptability
C (Control) 4.16±0.05Ca 4.05± 0.05Ca 3.75±0.06Ba 3.07±0.05Aa 2.95±0.05Aa
T1 (0.20%) 4.235±0.06Ba 4.16± 0.05Bab 4.00±0.09Bb 3.46±0.04Ab 3.33±0.10Ab
T2 (0.50%) 4.44±0.06Bb 4.33± 0.06Bbc 4.27±0.08Bc 3.98±0.05Ac 3.87±0.05Ac
T3 (1.0%) 4.51±0.06Bb 4.40± 0.06Bc 4.34±0.08Bc 4.05±0.05Ac 3.94±0.05Ac

n=21; C= Control (without phyto-extract); T-1= 0.2% Arjuna tree bark extract, T-2= 0.50% Arjuna tree bark extract; T-3= 1.0% Arjuna tree bark extract. *Mean±S.E. with different superscripts row wise (capital alphabets) and column wise (small alphabets) differ significantly (P<0.05).

Sensory scores for the entire parameters showed declining trend with increase in storage time. The rate of decrease of sensory score was significantly (P<0.05) higher in control than treatment emulsion during storage. T-3 showed highest sensory scores for appearance than other treatments and control emulsion during the storage period. The slower rate of decrease in appearance might be due to the antioxidant properties of incorporated phyto-extract, these results are also supported with the findings of instrumental colour profile. Similar findings were reported by Pogorzelsk et al. (2018) during refrigerated storage of raw ground pork meat incorporated with Haematococcus pluvialis extract rich in astaxanthin. Joseph et al. (2014) also reported that the colour scores decreased (P<0.05) throughout the storage period in control and treatment pork emulsion incorporated with tomato products and guava pulp.

On day one of storage study sensory scores of treatment emulsions were higher than control emulsion. Similarly, Selgas et al. (2009) reported increase in visual colour scores with the incorporation of lycopene in raw hamburgers. Colour and appearance are among the most important sensorial properties by which consumers judge the quality of meat and influence them when they decide to purchase (Villalobos-Delgado et al., 2017). Odour score followed similar decreasing trend as that of the appearance score during storage. The results revealed significantly higher (P<0.05) odour score of T-3 followed by T-2 and T-1 pork emulsion throughout the storage period. Joseph et al. (2014) also reported that odour scores decreased significantly (P<0.05) throughout storage period. Villalobos-Delgado et al. (2017) reported that odour was most affected during the storage period, probably because of lipid oxidation and ammonia production from protein breakdown.

The score of overall-acceptability of pork emulsion incorporated with 0.20, 0.50 and 1.0 % ATBE (Fig. 3) was very important for the selection of level of incorporation of phytoextract in the present study. The critical analysis of sensory quality attributes revealed that the sensory panelists awarded highest overall acceptability scores to T-3 that was significantly (P<0.05) higher than T-2 and T-1 and graded it as best among all the treatments and control emulsion. Liang et al. (2018) reported that the score of overall-acceptability of dried minced pork slices with mulberry polyphenols was higher than control. The values in the present study were in conformity with the earlier findings on ground raw pork by Wagh and Chatli (2017).

Fig. 3: Effect of different levels of ATBE on over acceptability of pork emulsion during refrigerated storage. (Control: Without any phytoextracts; T-1: 0.2% ATBE; T-2: 0.5%ATBE; T-3: 1.0% ATBE. Bar represents the standard error (n = 21)).

Conclusion

Hence, it was concluded from above results and discussions that the incorporation of 1.0 % level of Arjuna tree bark extract (T-3) in the pork emulsion lead to the improvement in the various physico-chemical qualities, antioxidant potential, microbiological and sensory quality during 9 days refrigerated storage. Development of shelf-stable pork nuggets incorporated with Arjuna tree bark extract at 1.0 % level might be novel approach to provide a product which will provide all the appropriate nutrients in a convenient form to the consumers.

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