Effect of Enrobing and Adding Antioxidants on the Quality of Pork Patties

The effect of applying edible coating and addition of antioxidants (butylated hydroxyanisole (BHA) plus butylated hydroxytoluene (BHT) in a 1:1 ratio) on pork patties were investigated prior to achieve desired physico-chemical, microbiological and sensory qualities. For this, five treatments were conducted as (i) control (neither coated nor antioxidants treated); (ii) coated without antioxidants treated; (iii) coated and antioxidants added in the batter mix only (100 ppm); (iv) coated and antioxidants added in meat mix (100 ppm) only; and (v) coated and antioxidants added both in the batter mix (50 ppm) and the meat mix (50 ppm). Addition of antioxidants both in the batter mix and the meat mix significantly (p<0.05) reduced the microbial loads and thiobarbituric acid (TBA) values. The TBA values significantly (p<0.05) increased up to day 14 and then progressively increased with the advancement of each interval of storage days up to 28 days. Total plate count significantly (p<0.05) increased with the increase in storage days.Coliform and Staphylococcus aureus were absent throughout the storage days in all samples. Staphylococcus aureus however, were present in the control group at day 14 and in enrobed (coated) patties (without antioxidants treated) at 28th day. Addition of antioxidants to batter mix and meat mix did not substantially enhance bacteriostatic activity. Application of coatings and antioxidants retarded the loss of firmness, flavor, changes in appearance and color, and also other sensory attributes. Control patties were better with respect to microbial quality and TBA values but had poorer sensory quality than coated patties. (Asian-Aust. J. Anim Sci 2003. Vol 16, No. 9 :1374-1383)


Effect of Enrobing and Adding Antioxidants on the Quality of Pork Patties
A. K. Biswas*, R. C. Keshri and Sunil Kumar* 1

INTRODUCTION
Numerous post-harvest treatments exist to prolong the shelf life of meat and meat products. Packaging of fresh produce under modified atmospheres and application of anti-microbial chemicals are two means of extending shelf life. Recently, enrobing or edible coatings have been used successfully in preserving freshness of food products. By inhibiting water loss and controlling rate of oxygen and carbon dioxide migration, enrobing can retain quality and extend shelf life. Enrobing can also be used to retard the loss of volatile flavor and aroma compounds and retard changes in textural properties. But, the application of enrobing and edible coatings or films on precooked meat products has not been as extensively studied as their applications on fresh and frozen meat products (Gennadios et al., 1997). Alginate-coated precooked, frozen stored pork patties had improved sensory qualities and were more desirable than uncoated control patties (Wanstedt et al., 1981). Studies have shown that coating with starch-alginate, starch-alginate-tocopherol, and starch-alginate-rosemary reduced warmed-over flavor (WOF) effectively in precooked refrigerated pork chops and beef patties (Hergens-Madsen et al., 1995;Ma-Edmonds et al., 1995;Handley et al., 1996). Herald et al. (196) used corn zein films containing butylated hydroxyanisole to reduce WOF in precooked turkey breast and reported that turkey breast wrapped in corn zein films had less WOF than sample packaged in polyvinylidene chloride (PVDC) plastic wrap. Studies on rosemary extract in meat burgers revealed that it significantly delayed the appearance of rancidity and after 27 days of storage the meat supplemented with the extract (plant antioxidants) did not show any indication of oxidation and maintained low microbial levels (Pszczola, 2002). Formanek et al. (2001) reported that rosemary extract had similar effectiveness to that of BHA/BHT combination of beef patties. Activated lactoferrin, a formulation of lactoferrin, can provide fresh meat with an added level of protection from pathogenic bacteria by preventing microbial attachment to meat surfaces during processing and packaging as well as by inhibiting growth of this bacteria (Pszczola, 2002).
Thus, the present study was conducted with the objective of evaluating the effectiveness of enrobing with bengal gram flour (GF) as a base material and antioxidants on physico-chemical, microbiological and sensory characteristics of pork patties.
The batter mixes were prepared by the following procedure: (1) Flour, carboxymethylcellulose (CMC), skimmed milk powder (SMP), table salt, sugar, spice mix, turmeric and capsicum powder were initially combined and mixed well in the conventional manner on a clean dry glass tray.
(2) Whole egg liquid (WEL) containing test antioxidants (BHA and BHT) was added, and mixed well to avoid clumping. (3) Water was added slowly with intermittent mixing of batter mix, which was then stirred continuously until assured of complete hydration and desired consistency. (4) The batter was covered properly and chilled until used to batter the patties. (6) Three batches of batter mix were prepared for each treatment.

Preparation of meat mix
Three different meat mixes containing either no test antioxidant or containing BHA and BHT as antioxidants, each at a level of 25 ppm, or 50 ppm were formulated for preparation of patties. Meat samples required for the formulation of meat mix were obtained from pig slaughtered as per scientific method (Anish, 2002) in the experimental abattoir of Livestock Products Technology Division, IVRI, Izatnagar, Uttar Pradesh, India. According to requirement, hams or portions of ham (mainly biceps femoris, quadriceps femoris, semitendinosus, semimebrinosus and gluteus femoris muscles) was taken pre-rigor within 1 h post mortem, and hot deboning was done in the laboratory, manually. Skin, external fat and fascia, and other separable connective tissue trimmed-off from boneless ham and stored over night at 4±1°C in polyethylene pouches for conditioning. Formulation of meat mix contained 70% lean meat, 15% pork back fat from same pigs, 6% water, 4.5% refined wheat flour (obtained from the local market of Bareilly, India), 1.6% table salt (Brand: Tata Chemicals Pvt. Ltd., Mumbai, India) 0.5% sugar, 0.4% sodium tripolyphosphates (obtained from Central Drug House, New Delhi, India), 0.25% dry ginger powder (obtained from Ashok Griha Udyog Kendra Ltd., Kanpur, India), 150 ppm sodium nitrite (obtained from Polypharm Pvt. Ltd., Mumbai, India), 1.75% spice mix (containing aniseed, bay leaves, black peppers, capsicum, caraway seeds, cardamom, cinnamon, clove, coriander, cumin, nutmeg, mace), and different levels [0, 50 ppm (25 ppm BHA+25 ppm BHT), 100 ppm (50 ppm BHA + 50 ppm BHT)] of antioxidants.
The meat mixes were prepared by the following procedure: (1) Lean meat (6 kg/ batch) samples were cut into 4-5 cm cubes and double minced first with 8 mm plate followed by common grind size of 3 mm plate in a Electrolux mincer, Model-9152, Germany. (2) The minced meat was blended with other ingredients (as mentioned earlier) in a Hobart paddle type mixer (Model No. 50, Germany) for 3 min. (3) The meat mix was taken out and stored at 4±1°C till used. (4) Three trials (batches) were conducted for each treatment.

Preparation of test samples
Three batches of five different patties (i) control (neither enrobed nor antioxidant treated) T1; (ii) enrobed without antioxidants treated T2; (iii) enrobed and antioxidants added in batter mix (50 ppm BHA+50 ppm BHT) only T3; (iv) enrobed and antioxidants added in meat mix (50 ppm BHA +50 ppm BHT) only T4; and (v) enrobed and antioxidants added in batter mix (25 ppm BHA+25 ppm BHT) and meat mix (25 ppm BHA+25 ppm BHT) T5 were prepared by using the previously processed batter mixes and meat mixes by following way (1) 75 g of meat was portioned and moulded into a round flat shape by using a 90 mm diameter x12 mm height glass petri dish. (2) Patties were deep fat fried (DFF) at 125°C for 7 min in refined sunflower oil (Brand: Sundrop Agro Tech. Food (P) Ltd., Jaipur, India) using a Batjiboi Gannet thermo-controlled deep fat fryer to a final internal temperature of 82±2°C as measured with a probe thermometer. (3) Patties were turned upside down and again DFF at the same temperature for uniform colour development. (4) Excess oil was drained off and patties were predusted by rolling over the heap of flour for a uniform coating. (5) Patties were battered with the appropriate batter mix having temperature of 25°C by dipping into it, and then allowing excess batter drain off over a few sec for getting average batter pick up 28% on the basis of raw patties (75 g). (6) Patties were DFF a second time at 150°C for 6 min with turning at 3 min. (7) Patties were drained off excess oil and cooled at room temperature (25°C) before being packed in polypropylene film and stored at refrigeration temperature (4±1°C) for from 0 to 28 days for subsequent measurement of physico-chemical properties, microbiological quality, and sensory characteristics.
The determined parameters included cooking yield, fat content, moisture content, pH, 2-thiobarbituric acid (TBA) value, shear value, microbial count and sensory panel test. All analysis were performed thrice with duplicate for each treatment.
Cooking yield : Yields were determined in batches for each treatment by dividing the cooked weight by raw weight and multiplied by hundred.
Fat content : The fat content in cooked patties was estimated by solvent extraction method as given in AOAC (1995).
Measurement of moisture content: The moisture content (n=6) was determined by oven drying the sample to a constant weight at 100°C for 16 h (AOAC, 1995).
Determination of pH : The pH of the sample was determined (Trout et al., 1992) by using a digital (Century, Model: CP-901) pH meter equipped with a combined glass electrode. For this, 10 g of the sample was homogenized with 50 ml distilled water for 1 min using an Ultra Turrex T25 tissue homogenizer (Janke and Kunkel, IKA, Labortecnik, Germany).

Determination of 2thiobarbituric acid (TBA) value
2-thiobarbituric acid value was determined by the method of Witte et al. (1970) with slight modifications. The samples (20 g) were homogenized by using Ultra Turrex T25 tissue homogenizer (Janke and Kunkel, IKA, Labortecnik, Germany) at high speed for 1 min in the solution containing 20% trichloroacetic acid (50 ml) and distilled water (50 ml). The homogenate was filtered through Schleicher and Schuell ashless filter paper (equivalent to Whatman # 42), Ref. 300210, supplied by E. Merck (India) Limited, Mumbai. 3 ml of TCA extract was mixed with 3 ml of TBA reagent (0.1 g of 2-thiobarbituric acid dissolved in l00 ml of distilled water) in a graduated test tube and placed in a boiling water bath for 35 min after covering the tube with an inverted small glass beaker. The contents in the tubes were cooled under running tap water and absorbance measured using a spectrophotometer (Du-640, Beckman Instrument, San Ramon, CA, USA.). TBA was calculated as mg malonaldehyde/kg of sample by referring to a standard graph.

Measurement of shear force
The shear force value of the sample was measured following the method of Berry and Stiffler (1981) with slight modification. The pork patties were cut into 1 cm diameterx 1 cm thickness at different locations with a sharp blade. The cut pieces were then sheared in a Warner-Bratzler shear press (Model: 81031307, G.R. Elec, Mfg., Co., USA.). Ten observations were recorded for each sample to obtain the value of shear force in kg/cm2.

Microbiological analysis
Samples from the refrigerated stored pork patties were used to determine the total plate count (TPC), psychrotrophic count (PC), coliform count (CC), Staphyloccus aureus count (SAC) and yeast and mold count (YAM) according to the standard methods of APHA (1984). Analysis were performed at each week interval to monitor microbial quality prior to the sensory tests. The pour plate method in duplicate was used for analyzing the samples. Standard plate count agar (Hi-Media Laboratories Pvt. Ltd., Mumbai, India, Code No M091) was used to determine TPC and PC after incubation for 48 h at 35±2°C and 10 d at 4±1°C, respectively. Enumeration of coliforms was done by Violet red bile agar (Hi-Media Laboratories Pvt. Ltd., Mumbai, India, Code No M49), staphylococcus by Baird Parkar Agar (Hi-Media Laboratories Pvt. Ltd., Mumbai, India,Code No 43) and in both the cases, plates were incubated at 35±2°C for 48 h. Yeast and mold were enumerated by Potato dextrose agar (Hi-Media Laboratories Pvt. Ltd., Mumbai, India, Code No M096) after incubation for 5 d at 25°C.

Sensory panel test
Five different types of patties at a temperature of 35 40°C were assessed per sitting under incandescent light for their appearance and colour, coating adhesion, flavour, juiciness, texture and overall acceptability by a seven member panel of judges consisting of scientists and post graduate students of the Livestock Products Technology Division at the Indian Veterinary Research Institute using an 8 point descriptive hedonic scale. A length of 2-3 cm pieces of patties were offered to the tasters conducting 3 sittings (n=21) at each storage interval of 0, 7, 14, 21 and 28 days (Baker and Scott-Kline, 1988;Keeton, 1983), where 8=extremely desirable and 1=extremely undesirable.

Statistical analysis
The statistical design of this study was 5 (treatment) x 3 (replication) randomized block design and data were analyzed on a window 98 computer using statistical software packages developed by following the procedures of Snedecor and Cochran (1989). All the analysis were conducted in duplicates. There were seven sensory judges for each treatmentxreplication combination. Data were subjected to one way analysis of variance and means were compared by critical difference test. The storage data were analyzed on the basis of 5 (treatments)x5 (storage days)x3 (replications) with two way analysis of variance. Duncan's Multiple Range test and critical difference were determined at 5% significant level.

Cooking yi이d
The results in Table 1 show the cooking yield of pork patties varied from 85.18±0.68 to 87.42±0.31.Enrobed patties exhibited significantly (p<0.05) higher cooking yield might be due to moisture barrier and fat intake properties during DFF. Higher cooking yield also related to adhesion of batter mix on patties as good adhesion improved the cooking yield (Hanson and Fletcher, 1963). Furthermore, starch granules present in flours has moisture absorption properties as they swell by taking moisture from batter mix at the time of heating and thereby increased cooking yield (Miller et al., 1973). However, in this study, treated patties (T2, T3, T4 and T5) did not vary in cooking yield.

Fat content
Results in this study showed that treated patties had significantly (p<0.05) higher fat content (Table 1), which might be due to absorption of oil by enrobing materials as incorporation of egg yolk content in batter mix formulation increase oil absorption. Mohammad et al. (1998) found that incorporation of liquid egg yolk in batter mix make it lipophilic in nature and thereby increased fat contents in enrobed patties.

Moisture content
The results of this study showed that treatment means for the overall moisture content of pork patties ranged from 56.75±0.62 to 52.62±0.75 (Table 2)  .81) after 28 days of storage at 4°C. The rate of moisture loss in enrobed patties over the 28 days was less because of coating which released comparatively less moisture than uncoated control. The moisture barrier properties of the coating might be due to incorporation of skimmed milk powder (Cunningham, 1989) and carboxymethylcellulose (Luck, 1968) in the batter mix. This result of moisture content in the present study is in agreement with the work of Wu et al. (2000), who reported that uncoated samples exhibited higher relative moisture loss than coated products of 3 days of storage at 4°C. Chinnan (1997) and Chinnan et al. (1997) also reported that incorporation of corn zein (CZ), methylcellulose (MC), carboxymethylcellulose (CMC) and hydroxypropyl methylcellulose (HPMC) in batter mixes reduced moisture loss. However, overall moisture content in enrobed without antioxidant treated (T2) and enrobedantioxidants treated (T3, T4, T5) samples were similar (Table  2).

pH
Results in Table 2 show that pH was significantly (p<0.05) higher in T1 (control) group compared to T4 and T5  group and non-significantly higher than T3 and T2 group. However, no change in overall pH (Table 2) was observed with the advancement of storage days in all treatments. Significant decreased in pH values were noticed only over 28 days of chilled storage. The decrease in pH values might be due to significant (p<0.05) increases in psychrotrophic and lactobacillus count at the above storage days producing lactic acid by break down of carbohydrates. The result observed in this investigation was supported by the result reported by Incze (1992). Papadima and Bloukas (1999) also reported decrease in pH of traditional Greek sausages during storage condition. The non-significant variation in pH of cooked patties with added BHA/BHT antioxidants has also been observed by McCarthy et al. (2000).

TBA value
Enrobed (T2) and enrobed and antioxidants treated (T3, T4 and T5) patties had significantly (p<0.05) higher TBA values (Table 3) which might be due to additional oil absorption by enrobing materials than control. The overall TBA values significantly (p<0.05) increased up to 14th day and non-significantly increased from 21st to 28th day. These findings are in the agreement with Jo et al. (1999), who observed an increase in TBA value with an increase in fat content in pork sausage during storage. It could be due to increased lipid oxidation and production of volatile metabolites in the presence of oxygen during aerobic packaging. Among all enrobed and enrobed and antioxidants treated samples, T5 had comparatively lower TBA values on the day 21 and 28. This might be due to synergistic effect of combination of antioxidants (BHA+BHT) added both in batter mix and meat mix. McCarthy et al. (2001) reported that BHA/BHT had most beneficial effect in raw meat oxidative stability during refrigeration storage. T3 and T4 group of patties also exhibited lower (p<0.05) TBA values on the day 7 and 14, respectively, but were differed non-significantly from control patties.

Shear value
Shear values from the Warner Bratzler shear device can be used to identify if meat products contain a high amount of variability in total shear value. Differences in shear values can be used to determine if differences exist in total shear force between meat samples. Results in Table 3 show that enrobed patties had significantly (p<0.05) lower shear force values which might be due to incorporation of flours and absorption of fat and possibly due to reduction in proportion of muscle fibre and unseparable connective tissues present in the formulations. But no interaction was observed between treatment groups and storage days. Significant (p<0.05) decrease in shear force value at 28th day might be due to myofibrillar fragmentations by muscle or microbial enzyme during storage at 4°C.

Total plate count (TPC)
Significantly (p<0.05) increasing trends of TPC were observed with the advancement of each interval of storage days compared to control (Table 4). This might be due to increase in handling and addition of various ingredients in batter mix. This statement was supported by Reddy et al. (1990), who reported higher aerobic count after breading and battering of fish finger. But from the day 14 to 28 a significant (p<0.05) decrease in TPC in T5 sample was due to antimicrobial effect of BHA/BHT antioxidants added both in batter mix and meat mix. Similar finding has been   reported by Gailani and Fung (1984) in ground pork patties. This effect might be due alteration of cell permeability by phenolic antioxidants and also by interaction with cell membrane protein to cause disruption of membrane structure. The overall TPC showed a linear increasing trend at each interval of storage days up to 28th day. Chicken patties stored at refrigeration temperature also showed a linear increase in total plate count (Nath and Mahapatra, 1995). Table 4 shows that overall PC had a significantly (p<0.05) increasing trends from 14th day to the end of the experiment. No PC was detected in T1, T4 and T5 groups from '0' to 14th day and T2 and T3 groups up to 7 th day due to sufficient heat treatment during cooking which drastically injured and killed the psychrotrophic population in patties (Jay, 1996). PC was more in enrobed patties which might be due to additional handling and incorporation of different ingredients in batter mix. However, T3, T4 and T5 group of samples had lower overall PC compared to T2 sample at the day 28, which might be due to inhibitory action of BHA/BHT antioxidants in all the three types of treated patties. This is in agreement with Gailiani and Fung (1984), who reported an inhibitory effect of BHA antioxidants of PC in ground pork after 4 weeks of refrigeration storage.

Coliforms, Staphylococcus aureus and yeast and mold count
Coliforms (CC), Staphylococcus aureus (SAC) and yeast and mold (YAM) were not detected at any point of time in the present investigation, except that the SAC were detected in control patties at 14th day and in enrobed (T2) patties at 28th day only (Table 5). YAM were also detected only in control (T1) and enrobed (T2) patties at 14th day. It might be due to destruction of bacteria during DFF of patties (above 82±2°C central core temperature) which have the thermal death points of 57°C. Good hygienic condition during preparation of patties could also be one of the reasons for absence of these bacteria. Moreover, use of antioxidants in patty preparation might also have had important role in inhibition of growth of these bacteria. But SAC and YAM infrequently appeared and might be due to post processing contamination. Table 6 shows significantly (p<0.05) higher appearance and colour scores in enrobed patties (T2, T3, T4 and T5) compared to control, because of golden brown colour imparted by enrobing and frying. But overall appearance and colour scores significantly (p<0.05) decreased at 7th day onwards. Progressive decrease in appearance and colour scores during storage might be due to pigment and lipid oxidation's resulting in non-enzymatic browning. It could also be due to relative moisture loss (RML) from the surface of coating. Wu et al. (2000) reported similar observation in precooked beef patties stored in natural antioxidants treated coatings and films. However, in this experiment enrobed-antioxidants treated patties (T3, T4 and T5) did not show any significant different in overall appearance and colour scores over 28 days of storage period. Similar report was mentioned by Resurreccion and Reynolds (1990) as no effective colour stability was observed by BHA/BHT antioxidants up to 18 days of storage in frankfurter containing chicken and pork. But treatment T2 had lower (p<0.05) appearance and colour value than treatment T3, T4 and T5 on the day 21 and this supported the result obtained by McCarthy (2001).
Higher juiciness scores at high fat level have been reported by several workers (Cross et al., 1980;Keeton, 1983;Berry and Leddy, 1984;Kregel et al., 1986 andReitmeier andPrusa, 1987). Significantly (p<0.05) lower juiciness scores were observed at 7th day and 28th day due to loss of moisture from patties. A linear decrease in juiciness scores of chicken patties was reported by Rao and Reddy (1997). The result observed in the present study is also in agreement with Wu et al. (2000) in showing that enrobed pork chop and beef patties were more juicy than uncoated control products during refrigeration (4±1°C) storage. Control (T1) had significantly lower overall textural scores than both enrobed (T2) and enrobed-antioxidants treated (T3, T4 and T5) patties. The overall textural scores of T2, T3, T4 and T5 samples were nearly similar. But these scores significantly (p<0.05) decreased with increase in the storage days (Table 7). Higher textural scores in enrobed patties were due to the fact that coated patties lost moisture more slowly than uncoated patties. However, significant decreased in textural scores might also be due to changes in functional properties during storage at 4°C. Table 7 shows that enrobed (T2) enrobed-antioxidants treated (T3, T4 and T5) patties had significantly (p<0.05) higher overall acceptability scores than control (T1). Among all enrobed products, sample T4 and T5 had significantly (p<0.05) higher acceptability scores than sample T2 and T3 as these were vary on day 21. However, no significant differences were observed in between T4 and T5 samples.
The overall acceptability scores significantly (p<0.05) decreased at 7th day onward which might be due to decrease in other sensory attributes namely appearance and colour, coating adhesion, flavor, juiciness and texture. The enrobed and antioxidants treated products had higher overall acceptability scores which might be due to enrobing with antioxidants having some beneficial effect on sensory parameters.

CONCLUSIONS
For precooked pork patties the addition of antioxidants simultaneously in both batter mix and the meat mix (T5) have some beneficial effect on TBA values, microbial qualities and few sensory parameters relative to adding into batter mix (T3) or pork meat mix (T4) alone. Similarly, treatments T3 and T4 have some beneficial effect than other pork patties though no way is better than treatment T5. In light of these observations, it can be concluded that enrobing with a batter and antioxidant treatments (antioxidants added in batter mix or meat mix or both in batter mix and meat mix) significantly increased product quality and shelf life, where T5 product is superior to others.