The comparative antioxidant and antimicrobial efficacy of grape seed (Vitis vinifera) extract (GSE) at 0.1 % and green tea (Camellia sinensis) extract (GTE) at 0.1 % with synthetic antioxidant i.e. butylated hydroxy anisole (BHA) at 0.01 % was studied in vacuum packaged cooked pork patties during refrigerated storage (4±1°C). The pork patties treated with GSE had significantly (P < 0.05) lower thiobarbituric acid reactive substance (TBARS) values and free fatty acid (FFA) content compared to control (C) and other samples in vacuum environment during refrigerated storage (4±1°C). A significant (P<0.05) lower total plate count and total psychrophilic count were recorded in pork patties added with grape seed extract. Addition of natural antioxidants significantly (P<0.05) reduced the lactobacillus count, coliform count and anaerobic count than control and BHA added vacuum packaged pork patties. GTE added pork patties have significant (P<0.05) lower yeast and mould counts than control and other formulations. The GSE treated pork patties rated significantly (P < 0.05) superior scores of colour, flavor, tenderness, juiciness and overall acceptability than control, BHA and GTE treated samples. Based on the results obtained it can be concluded that GSE had excellent antioxidant and antimicrobial properties compared to control, BHA and GTE treated pork patties during refrigerated storage under vacuum conditions and application of both natural antioxidants and vacuum environment extends shelf life of pork patties up to 30 days in refrigerated storage without any quality deterioration.
Pork is more susceptible to oxidative rancidity than red meat because of its higher content of PUFA. Consumer concern for the composition of foodstuff has increased rapidly in the recent years, the highest and longest sensory quality of food products also being demanded. The meat industry is increasingly searching for natural solutions to minimise oxidative rancidity and extend the shelf-life of meat products rather than synthetic additives. The synthetic antioxidants currently used have been found to exhibit various negative health effects in animals and primates (Saito et al., 2003). Thus, the search for alternative methods to retard oxidative processes in meat has led to the research of alternative natural antioxidants. Green tea leaf extracts are becoming increasingly important as a functional food in the diet because of their high polyphenols contents. Its polyphenols contents can increase up to 36% (dry basis) due to climate, season or variety (Wanasundara and Shahidi, 1998). The tea catechins and other polyphenols are free radical scavengers, metal chelators, inhibitors of transcription factors, and enzymes. Therefore green tea extracts have been used as natural antioxidants, antibacterial and antiviral agents (Higdon and Frei, 2003).
Grape seed extracts (GSE) are industrial derivatives from whole grape seeds that have a great concentration of vitamin E, flavonoids, linoleic acid and OPCs. There is increasing evidence demonstrating the ability of GSE retard lipid oxidation in meat during storage, most likely due to the fact that GSE is a rich source of polyphenolic compounds especially proanthocyanidins (Weber et al., 2007). Therefore, the present study was designed to assess the comparative efficacy of synthetic (BHA) and natural antioxidants i.e.) green tea extract and grape seed extracts on oxidative, microbial and sensory quality of cooked pork patties during refrigerated storage (4±1°C) in vacuum environment.
Materials and Methods
Fresh lean meat (ham and loin) and non-meat ingredients like salt, sugar, corn flour, refined sun flower oil, condiment and spice mix were procured from local market of Tirupati and analytical grade of chemical and food grade additives were procured from standard companies. The natural antioxidants grape seed extract and greed tea extract obtained from Lactonova Pvt. Ltd, Hyderabad, India. The fresh lean meat cut into small chunks and minced in a meat mincer (Sirman, TC 12 E, Italy) through 4 mm plate. The emulsion was prepared by chopping the minced meat along with other non-meat ingredients in a bowl chopper (Scharfen, Model No: TC 11, Germany). The emulsion was prepared by using minced pork is mixed with salt @ 1.5 %, STPP @ 0.4 %, sodium nitrate @ 150 ppm, sodium ascorbate @ 500 ppm, sugar @ 1 % and ice flakes @ 8 % and chopped for one min followed by addition of oil @ 6 % and again chopped for one min and add corn flour @ 3 %, spice mix @ 1.6, condiment mix @ 3 % (onion and garlic: 3:1) chopped for 3 min. The temperature of the emulsion was maintained between 10 and 12ºC. The emulsion was separated into four parts and first part was control (C) (without addition of any antioxidant) and second part was added with synthetic antioxidant i.e.) 0.01% butylated hydroxy anisole (BHA) and third part was added with 0.1 % green tea extract (GTE) and fourth part was added with 0.1% grape seed extract (GSE). In each part, 60 g of pork emulsion was taken for preparation of each raw patty and moulded in round flat shape by using 9.0 cm diameter and 1.2 cm height of bottom glass of petridish and cooked in hot air oven till the patty core temperature reached to 75±3ºC. After cooking of control and treatments separately the pork patties were cooled and vacuum packaged separately in high density polyethylene bags and stored in refrigerated temperature (4±1°C). The patties were evaluated for their quality at five days interval up to 30 days.
The pH of pork patties were determined by homogenizing 10 g of sample with 50 ml distilled water with the help of tissue homogenizer (Daihan Scientifics, WiseMix, HG-15D, Korea) for 1 min. The pH was recorded using digital pH meter (Thermo Orion, Model 420A+, USA). The TBARS value was determined adopting the procedure of Witte et al. (1970). Free fatty acids per cent was determined according to method described by Koniecko (1979).
All the microbiological parameters of pork patties were determined as per the methods described by APHA (2001).
The pork patties were warmed and served to trained panelists and evaluated for sensory characteristics like appearance, flavour, juiciness, tenderness and overall acceptability using a 8-point hedonic scale (where, 8=extremely desirable, 1= extremely undesirable) as described by Keeton, (1983).
The experiments were repeated four times and the data was analysed using General Linear Model procedure of statistical package for social sciences (SPSS) 15th version and comparison of means tested using Duncan’s multiple range test and significance was considered at P<0.05.
Results and Discussion
Both natural antioxidants ie) GTE and GSE did not significantly (P>0.05) influenced the overall mean pH values of vacuum packaged pork patties during refrigerated storage period (Fig. 1).
Fig. 1: Influence of natural antioxidants and vacuum packaging on pH of pork patties during refrigerated storage (4±1°C)
Bhaskar Reddy et al. (2013) also noted similar findings in restructured mutton slices added with natural antioxidants. The refrigerated storage had significantly (P<0.05) influenced the pH values of vacuum packed pork patties during storage. As the progressing of storage period, there was a significant (P<0.05) increase in pH values between storage days in control and GSE and GTE added antioxidants added pork patties. This increase in pH during storage could be due to protein denturation and liberation of protein metabolites, mainly amines due to bacterial activity. These results are in congruent with Brannan, (2009) in ground chicken breast samples.
Thiobarbituric Acid Reactive Substance (2-TBARS) Value
Addition of GSE and GTE and refrigerated storage significantly (P<0.05) influenced the 2-TBARS values of vacuum packaged cooked pork patties during storage (Fig. 2). Pork patties with GSE had significantly (P<0.05) lower 2-TBARS values compared with control, BHA and GTE added patties. There was significant difference in 2-TBARS values between control, BHA, GTE and GSE samples. GTE showed more anti-oxidative compared to control and BHA added patties. This might be due to anti-oxidative property of GTE. Wanasundara and Shahidi (1998) reported that crude tea catechins have been found to be more effective to reduce the lipid oxidation than a-tocopherol or BHA. Compare to GTE the pork patties added with GSE had lower lipid oxidation. This might be due to the potential antioxidative property of GSE than GTE. The antioxidative property of GSE ismainly due to phenolic compounds, especially polyphenols, such as proanthocyanidins.
Fig. 2: Influence of natural antioxidants and vacuum packaging on 2-TBARS value of pork patties duirng refrigerated storage (4±1°C)
These procyanidins are dimers, trimers and oligomers of themonomeric flavan-3-ols (+)-catechin, (-)-epicatechin and (-)-epicatechin-3-O-gallate (Yilmaz and Toledo, 2004). A significant (P<0.05) increment of 2-TBARS values from 0.175 (0 day) to 0.840 (30 days) was found during 30 days of refrigerated storage, however, these values were not exceeded the threshold value (1 mg malonaldehyde/kg meat). This might be due to combined effect of vacuum environment and antioxidative effect of GSE. These results are in congruent with Babji et al. (2000) in goat meat products.
Free Fatty Acid (FFA) Value
FFA per cent was significantly (P<0.05) influenced by both antioxidants and storage period (Fig. 3).
Fig.3: Influence of natural antioxidants and vacuum packaging on FFA value of pork patties during refrigerated storage (4±1°)
GSE was significantly (P<0.05) lower FFA (0.167) compared to control (0.192) during 30 days of refrigerated storage. The lower FFA is mainly due to delaying of oxidation of oleate fatty esters by GSE polyphenols. Storage period also significantly (P<0.05) affected the FFA per cent and the overall mean of FFA per cent values were increased gradually from 0 day to 30 days of during refrigerated storage under vacuum environment. The increased FFA during storage could be due to microbial lipolytic activities. This is in accordance with Sahoo and Anjaneyulu (1997) who reported that increasing trend of free fatty acid in storage of meat products and suggested that the use of FFA percentage as an analytical indicator to judge the fat quality.
The analysis of variance revealed that a significant (P<0.05) influence of antioxidants, storage period and their interaction on various microbiological profiles viz., total plate, total psychrophilic, coliform, lactobacillus and anaerobic counts of vacuum packaged pork patties during refrigerated storage (Table 1).
Table 1: Mean ± S.E values of microbial counts of pork patties affected by natural antioxidants during refrigerated storage (4±1°C) under vacuum conditions*
|Samples||Storage Period (days)|
|Total plate count (log10cfu/g)|
|Total psychrophilic count (log10cfu/g)|
|Coliforms count (log10cfu/g)|
|Lactobacillus count (log10cfu/g)|
|Anaerobic count (log10cfu/g)|
Mean values within row and column wise bearing different superscripts are differ significantly (P<0.05); * n=8
Both GSE and GTE and refrigerated storage significantly (P<0.05) influenced the total plate count and total psychrophilic counts of vacuum packaged pork patties during refrigerated storage. Pork patties added with GSE recorded significantly lower total plate count and total psychrophilic counts than control and other treatments. The pork patties added with GTE also showed lower total plate count than control and BHA added samples. Grape polyphenols have a certain antibacterial activity in vitro and the partial hydrophobic nature of the phenolic compounds of GSE is responsible for the antimicrobial activity of GSE (Jayaprakasha et al., 2001). Accumulation and attachments of these phenolics to the bacterial cytoplasmic membrane eventually lead to cell death (Lin et al., 2004). Irrespective of treatments, refrigerated storage period significantly (P<0.05) influenced the total plate counts and total psychrophilic counts of vacuum packaged pork patties. As the storage period progressed, total plate counts and total psychrophilic counts significantly (P<0.05) increased during 30 days of refrigerated storage in control samples, but in treated samples the total plate count and total psychrophilic counts are lower than control. The increase in total plate count and total psychrophilic counts during storage might be due to conducive water activity, change in pH and packaging conditions. Similar results were also noted by Bhaskar Reddy et al. (2013) in restructured mutton slices during refrigerated storage.
Coliforms, considered as indicator of post processing contamination, were detected occasionally during storage. The coliform counts were detected on 5th day in control and BHA and 10thday in GTE and GSE added vacuum packaged pork patties, which might be due to antimicrobial activity of GSE as explained earlier and the mean days coliform counts increased during 30 days of refrigerated storage of vacuum packaged pork patties. Chilling and vacuum environment might have prevented the growth of coliforms at initial days. These results are in congruent with Babji et al. (2000) in goat meat products. Lactic acid bacteria are the major bacterial group associated with spoilage of vacuum packed cooked meat products. Addition of GSE significantly (P<0.05) influenced the lactobacillus counts of vacuum packaged pork patties. As the storage period increased from 0 day to 30 days, the mean day lactobacillus counts increased progressively. However, at this count, lactic acid production was not enough to cause any fermentative repugnant odour, flavour or taste in the pork patties during the entire storage period. Lactobacilli were shown to spoil vacuum packed meat products by causing off-flavour, discolouraton, gas formation and slime production (Labadie, 1999).
Both antioxidants and storage period significantly (P<0.05) influenced the anaerobic counts of vacuum packaged pork patties during refrigerated storage. GSE had significantly (P<0.05) lower anaerobic counts than control, BHA and GTE which might be due to antimicrobial activity of proanthocyanidins and polyphenolics of GSE. As the progressing of storage period, an increase in anaerobic counts were noticed from 1.41 (0 day) to 3.25 log10cfu/g (30 days) in GSE treated samples. It was reported that anaerobes and facultative anaerobes are the most important spoilage bacteria in vacuum packaged refrigerated meat. Similarly a linier increase in anaerobic counts during ambient temperature storage of vacuum packaged hurdle treated chevon keema was reported by Karthikeyan et al. (2000).
Addition of natural and synthetic antioxidants and storage period significantly affected the sensory scores viz. colour, flavour, juiciness, tenderness and overall palatability of pork patties during refrigerated storage (Table 2).
Table 2: Mean ± S.E values of sensory scores of pork patties affected by natural antioxidants during refrigerated storage (4±1°C) under vacuum environment*
|Samples||Storage Period (days)|
Mean values within row and column wise bearing different superscripts are differ significantly (P<0.05); * n=24
In the present study, both natural antioxidants i.e.) GTE and GSE significantly (P<0.05) improved the colour scores than control and BHA during refrigerated storage of aerobic packaged pork patties. The overall mean flavour scores of vacuum packaged pork patties during refrigerated storage were significantly (P<0.05) affected by both antioxidants and storage period. Addition of GSE recorded significantly (P<0.05) higher flavour scores than control BHA and GTE whereas, GTE and BHA was better flavour scores than control. This might be due to antioxidative effect of GSE which reduce the lipid oxidation and also off- flavour development during refrigerated storage (Brannan and Mah, 2007). The addition of GSE showed higher tenderness and juiciness scores than control, BHA and GTE added pork patties during refrigerated storage. The influence of antioxidants and storage days on overall acceptability scores of pork patties during refrigerated storage revealed a significant (P<0.05) difference between treatments. Among all treatments, GSE rated superior overall acceptability scores which might be due to favourable colour, flavour and juiciness scores compared to control, BHA and GTE. As the progressing of storage period, all sensory scores were gradually reduced but all scores are within the acceptable limits during entire storage.
Based on the above results, by comparing the comparative efficacy of both antioxidant and antimicrobial efficacy of BHA, GTE and GSE, it can be concluded that the addition of GSE at 0.1% reduced the lipid oxidation, delays the microbial organism’s multiplication and improves the sensory attributes of pork patties under vacuum environment during refrigerated storage (4±1ºC) up to 30 days without any significant quality deterioration.