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MEAT SCIENCE
Meat Science 79 (2008) 112 www.elsevier.com/locate/meatsci

Development of shelf stable pork sausages using hurdle technology and their quality at ambient temperature (37 1 C) storage
R. Thomas, A.S.R. Anjaneyulu *, N. Kondaiah
1
Division of Livestock Products Technology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP 243 122, India Received 25 May 2007; received in revised form 17 July 2007; accepted 17 July 2007

Abstract Shelf stable pork sausages were developed using hurdle technology and their quality was evaluated during ambient temperature (37 1 C) storage. Hurdles incorporated were low pH, low water activity, vacuum packaging and post package reheating. Dipping in potassium sorbate solution prior to vacuum packaging was also studied. Reheating increased the pH of the sausages by 0.17 units as against 0.11 units in controls. Incorporation of hurdles signicantly decreased emulsion stability, cooking yield, moisture and fat percent, yellowness and hardness, while increasing the protein percent and redness. Hurdle treatment reduced quality deterioration during storage as indicated by pH, TBARS and tyrosine values. About 1 log reduction in total plate count was observed with the dierent hurdles as were reductions in the coliform, anaerobic, lactobacilli and Staphylococcus aureus counts. pH, aw and reheating hurdles inhibited yeast and mold growth up to day 3, while additional dipping in 1% potassium sorbate solution inhibited their growth throughout the 9 days storage. Despite low initial sensory appeal, the hurdle treated sausages had an overall acceptability in the range very good to good up to day 6. 2007 Elsevier Ltd. All rights reserved.
Keywords: Shelf stable; Hurdle technology; Ambient temperature; Quality; Pork sausages

1. Introduction The demand for the processed meat products is increasing in developing countries due to urbanization, improved standards of living and changing life styles. However, the processed meat sector is developing slowly in these countries due to lack of cold chains for eective marketing, since the meat products are perishable. Processing techniques have been standardized for several meat products and most of them have limited shelf life at ambient temperature. Studies have indicated that fermented sausages are less palatable to consumers (Leistner, 1995; Mir Salahuddin & Sharma, 2003). Hence, there is a need to develop safe, nutritious and acceptable shelf stable meat products which can be stored/marketed without refrigeration.
*

Corresponding author. Tel.: +91 9411221554; fax: +91 581 2447284. E-mail address: anneasr@redimail.com (A.S.R. Anjaneyulu). National Research Centre on Meat, Hyderabad, India.

The microbial stability and safety of most foods is due to the combined action of several preservative factors/hurdles. As the main objective is to prevent microbial spoilage and food poisoning, several hurdles are used minimally to obtain the optimum combination to give good sensory qualities, safety and stability as well as savings of energy and money (Das & Radhakrishna, 2001; Grijspaardtvink, 1994; Karthikeyan, Kumar, Anjaneyulu, & Rao, 2000). Shelf stable meat products could be processed by thermal processing in a sealed container or by adjusting hurdles such as pH, water activity (aw) and food preservatives (Kanatt, Chawla, Chander, & Bongirwar, 2002) and/or irradiation (Leistner, 2000; Leistner, Vukovic, & Dresel, 1980) and can be stored without refrigeration. Sausages are versatile and popular products with good acceptability. In view of the limited information on shelf stable meat products, a study was undertaken to develop safe and acceptable pork sausages, stable at ambient temperature for better distribution and marketing using hurdle

0309-1740/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2007.07.022

R. Thomas et al. / Meat Science 79 (2008) 112

technology/combined processes. The successfully developed process can be trialled for dierent shelf stable meat products. 2. Materials and methods 2.1. Lean pork and pork fat Lean pork and fat (back fat) were obtained from crossbred barrows (75% Landrace 25% Desi) (6070 kg live weight) slaughtered as per standard procedure at the experimental abattoir of Livestock Products Technology Division, Indian Veterinary Research Institute, Izatnagar. Meat (about 5 kg in each batch) and fat were obtained from the ham portion of the carcass within 0.5 h of slaughter and debonding was done in the processing plant after conditioning at 4 1 C for about 16 h. Additional back fat was obtained from the loin portion, if required. Skin, fat and meat were separated manually. Meat was cut into cubes of about 3 cm and ground using a 13-mm plate followed by a 8-mm one in a Seydelmann meat grinder (model WD 114, Stutgart, Germany). Fat was ground using a 13mm plate followed by 3-mm plates in the same grinder. Ground meat and fat were packaged in LDPE bags and kept frozen (18 1 C) until subsequent use. Frozen meat and fat were thawed at 4 1 C for 16 h before use. 2.2. Processing of shelf stable pork sausages Hurdles incorporated were low pH ($5.90, using lactic acid (LA-0.5N) and Glucono-delta-lactone (GDL-0.1%)), low water activity ($0.93, using textured soy protein-3%), dipping in 1% potassium sorbate, vacuum packaging and post package reheating (90 C). These hurdles were standardized in the laboratory by conducting a series of preliminary trials (data not shown). In most of the preliminary trials shelf-life was sacriced for sensory attributes, because in developing shelf stable products the aim was to have sausages that are as similar as possible to the control. Nitrite and sodium chloride (NaCl) are normally considered as hurdles, but in this study the term hurdle refers to product hurdles other than nitrite and NaCl. The reason for selecting mild pH and aw hurdles was that sour and dried products are not liked by Indian consumers. Meat emulsions were prepared using a Seydelmann food cutter (Model K20 Ras, Stutgart, Germany) with 20% pork fat. About 7 kg batches were made, namely, 4550 g (65%) lean pork, 1400 g (20%) pork fat, 350 g (5%) condiments mix and rened wheat our, respectively, 140 g (2%) spice mix and rened salt and 35 g (0.5%) cane sugar and sodium tripolyphosphate each. Also, sodium nitrite was added at 0.015%. The spice mix was prepared as per the formulation developed in the laboratory. Onion and garlic were used in the ratio 3:1 as condiments. To the ground lean pork, salt, sugar, sodium nitrite and sodium tripolyphosphate were added and chopped for about 2 min. The condiments mix was then added and chopped again for 2 min (water/ice

akes were not added to reduce the water activity in the sausages). Ground pork fat was slowly incorporated while chopping which was continued until the fat was completely dispersed in the batter (34 min). Spice mix and rened wheat our were added and chopping was continued for another 1 min to get a ne viscous emulsion. About 2 kg of emulsion was separated out for processing the control sausages (without any hurdles). To the remaining emulsion, textured soy protein (3%) was added as humectant (over and above 100%) and chopped again for 0.5 min. pH of the treated emulsion was then adjusted to about 5.90 using lactic acid (0.5N) and GDL (0.1%). Meat emulsions, control and treated ones, were stued into 25 mm diameter cellulose casings (Viskase Nojax, Viskase Co. Inc., Chicago, USA) using a hydraulic sausage ller (Mainca, Model EP-25, Spain) and linked manually at about 12 cm intervals. Cooking was done in a steam oven without pressure to an internal temperature of 75 C, as recorded by a digital probe thermometer (Model CT-809, Century Instruments (P) Ltd, Chandigarh). The sausages were cooled to room temperature and the casings removed. Sausages made from the treated group (with hurdles) were divided into two treatments (Treatment I and Treatment II). Sausages from treatment II were dipped in 1% warm (6070 C) potassium sorbate solution in ltered water and allowed to dry at room temperature (about 15 min) in a closed chamber in the processing plant (potassium sorbate dipping was tried as a separate hurdle to reduce the possible growth of yeast and mold during storage). Sausages from all the three groups (control, treatment I and treatment II) were then vacuum packaged in laminates of Nylon/LDPE bags using a Rochermatic packaging machine (Model VM 195, Osnabruck, Germany) in such a way that each packet contained 7 or 8 sausages. Vacuum packaged sausages from treatments I and II were reheated to an internal temperature of 90 C and were then allowed to cool to room temperature. The internal temperature of the sausages was monitored continuously using a digital probe thermometer (Model CT-809, Century Instruments (P) Ltd, Chandigarh). One packet from each group was used for evaluation of physico-chemical, microbiological and sensory attributes on the day of processing. The remaining packets in each of the three groups were divided into two subgroups (six packets in rst subgroup and four packets in second subgroup). Sausages from the rst sub group were stored in an incubator pre-set at 37 1 C, while sausages from the second subgroup were stored at 4 1 C. Sampling was at 3 and 6 days for both ambient temperature and refrigerated stored samples. The experiment was repeated three times. 2.3. Analytical procedures pH was determined using a digital pH meter (Elico, Model LI 127, India). The weight of sausages was recorded before and after initial cooking and the yield was calculated (cooking yield = weight of cooked sausages/weight of raw

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sausages 100) and expressed as percentage. The procedure of Kondaiah, Anjaneyulu, Rao, Sharma, and Joshi (1985) was followed to measure emulsion stability. About 25 g of meat emulsion was taken in a LDPE bag and heated in a thermostatically controlled water bath at 80 C for 20 min. The stability was calculated from weight loss during cooking and expressed as a percentage of the initial weight. Moisture, fat and protein contents of the sausages were determined as per standard procedures(AOAC, 1995). The procedure of Tarladgis, Watts, Younathan, and Dugan (1960) was followed to estimate the TBARS (thiobarbituric acid reacting substances) number as mg of malonaldehyde per kg of sample. The water activity (aw) of the sausages was measured by Paw kit water activity meter (Decagon, Devices, USA). The procedure of Strange, Bendedict, Smith, and Swift (1977) was followed to determine the tyrosine value. The method described by Koniecko (1979, chapter 6) was followed for measuring titratable acidity. Titratable acidity was expressed as ml of 0.01N NaOH/g sample required to neutralize the ltrate as suggested by Wang (2000). Accelerated TBARS, which is a measure of potential protection against lipid oxidation, was measured by the procedure described by Juncher et al. (2000). In this method, lipid oxidation was accelerated by temperature (50 C) and the addition of FeSO4. 2.4. Lovibond Tintometer colour units The colour of the cooked pork sausages were measured using a Lovibond Tintometer (Model F, Greenwich, UK). Samples from three dierent sites of the sausages were placed in the sample holder and secured against the viewing aperture. The sample colour was matched by adjusting red (a) and yellow (b) units, while keeping the blue units xed at 2.0. The corresponding colour units were recorded. The hue and chroma values were determined using the formula, (tan1)b/a (Little, 1975) and (a2 + b2)1/2 (Froehlich, Gullet, & Usborne, 1983), respectively, where a = red unit, b = yellow unit. 2.5. Texture prole analysis Texture prole analysis (TPA) was conducted as per the procedure described by Bourne (1978) using a Stable Microsystems Texturometer (Stable Microsystems Ltd. Surrey, UK) model TA-XT2 texture analyzer attached to a software, texture expert. Uniform sized pieces (1.5 cm3) were used as the test samples. They were placed on the platform in a xture and compressed to 50% of their original height at a cross head speed of 50 mm/s through a two cycle sequence, using a 25 kg load cell. The texturometer was also used to measure shear force and work of shearing using a WarnerBratzler blade. Uniform sized samples (1 cm3) were radially sheared with a V-shaped blade attached to plunger at 50 mm/min crosshead speed.

2.6. Microbiological evaluation All the microbiological parameters of shelf stable pork sausages were determined as per the methods described by ICMSF (1996). Ready made media from Hi-Media Laboratories (P) Ltd, Mumbai, were used for the enumeration of dierent microbes. Duplicate plates were prepared and the counts were expressed as colony forming units (cfu) per gram. Preparation of samples and serial dilution of pork sausages were done near the ame in a horizontal laminar ow apparatus (Model YSI-188, Yarco Sales (P) Ltd., New Delhi) which was pre-sterilized by ultraviolet irradiation, observing all possible aseptic precautions. About 10 g of sample was aseptically weighed and transferred to a sterile mortar and homogenized for 2 min using a sterile pestle, while adding 90 ml of 0.1% sterile peptone water to make a 101 dilution. Sterile peptone water (0.1%) was used as diluent for making further dilutions. The plates (M 091) for mesophilic counts were incubated at 37 1 C for 48 h and plates showing 30300 colonies were counted. For psychotrophic counts, the plates were prepared similarly but incubated at 8 1 C for 10 14 days. Coliform count was by using Violet Red Bile Agar (M 049A) and the plates were incubated at 37 1 C for 48 h. The number of redpurple colonies with about 0.5 mm diameter surrounded by a zone of precipitated bile was counted. Anaerobic (anaerobic agar, M228), lactobacilli (MRS Agar, M6411) and Staphyloccus aureus (Baired Parker agar, M 1140) counts were measured by incubating the plates at 37 1 C for 48 h. Potato Dextrose agar (M 096) was used to enumerate yeast and mold counts. The plates were incubated at 25 1 C for 5 days. Black, white, red or greenish black coloured colonies appearing on the plates were counted. Colonies judged to be border line cases were also counted. 2.7. Sensory evaluation Standard sensory evaluation method using 8-point descriptive scale (Keeton, 1983) was followed with modications where 8 = excellent; 1 = extremely poor. The experienced panel (seven members) consisted of scientists and post-graduate students of Division of Livestock Products Technology, IVRI, Izatnagar. The panelists were trained and well acquainted with dierent sensory attributes during their post graduate/doctoral programmes. They were briey given the nature of the experiment without disclosing the identity of samples. Pork sausages, vacuum packaged in laminates of Nylon/LDPE bags and held at ambient temperature (37 1 C) were drawn at 3 days interval. Samples were warmed (4045 C) using a microwave oven (LG Electronics India (P) Ltd., Mumbai) for 1 min and served to the panelists. The panelists evaluated the samples for appearance, avour, juiciness, texture, binding and overall acceptability using a standard score sheet. Sensory evaluations were conducted between 3.30

R. Thomas et al. / Meat Science 79 (2008) 112

and 4 pm and ltered tap water was provided to the panelists for rinsing their mouth in between samples. 2.8. Statistical analysis The experiments were replicated a minimum of three times and the data generated for dierent quality characteristics were compiled and analyzed using a randomized block design at the institutes computer centre. The data were subjected to analysis of variance (one way ANOVA for quality parameters and 2-way ANOVA for storage data), least signicant dierence, paired t-test (Snedecor & Cochran, 1995) and Duncans multiple range test (Steel & Torris, 1981, chapter 8) for comparing the means to nd the eects between treatments, storage periods and their interaction for various parameters in the dierent experiments. The smallest dierence (D5%) for two means to be signicantly dierent is (P < 0.05). 3. Results and discussion 3.1. Quality evaluation of hurdle treated shelf stable pork sausages (SSPS) 3.1.1. Physico-chemical characteristics of SSPS The emulsion pH, which was adjusted to 5.95, was signicantly lower than that of control and was obviously reected in the product pH. Emulsion stability and cooking

yield of the treatment group was signicantly lower (P < 0.01) and could be attributed to its lower pH compared to control (5.95 vs. 6.27) (Table 1). Reheating increased the pH of the sausages by 0.17 units as against 0.11 units in the control. Dipping of sausages in 1% K-sorbate solution further decreased their pH by 0.04 units (treatment II) compared to treatment I. The increase in product pH with reheating might be attributed to the higher degree of protein denaturation and the release of free SH groups (Lawrie, 1998). A higher product pH for bologna sausages, with increase of processing temperature was reported (Mangalassary, Dawson, Reick, & Han, 2004). A signicant decrease (P < 0.01) in moisture content was found with reheating and this could be attributed to increased moisture loss as the core temperature of the products increased. Similarly, (Puolanne & Kukkonen, 1983) reported a decrease in moisture content of sausages with increase in product core temperature. Sausages from treatment groups had signicantly higher (P < 0.01) protein contents than the control and this could be due to loss of moisture and fat from the sausages on reheating and incorporation of TSP in the formulation. As mentioned above, reheating reduced the fat content of the sausages by 3.3%. This may be attributed to the low pH and the subsequent denaturation of muscle proteins resulting in lower fat binding (Lawrie, 1998) and expansion and liquefaction of fat with reheating and thereby loss from the product (Whiting, 1988). Dipping of the sausages in 1% K-sorbate

Table 1 Eect of potassium sorbate dipping and post package reheating on the physico-chemical parameters of SSPS Parameter Control Treatment I Physico-chemical characteristics Emulsion pH Product pH Emulsion stability (%) Cooking yield (%)# Moisture (%) Protein (%) Fat (%) Water activity Shear force (N) Work of shearing (N s) Instrumental colour scores Redness (a) Yellowness (b) Hue Chroma Texture proles Hardness (N/cm2) Adhesiveness (N s) Springiness (cm) Cohesiveness (ratio) Gumminess (N/cm2) Chewiness (N/cm) Fracturability (N) 6.27 0.01a 6.38 0.03a 94.58 0.05a 96.34 0.07a 55.95 0.05a 18.10 0.03b 21.93 0.02a 0.96 0.00a 12.73 0.05a 45.93 0.58a 3.47 0.02b 4.96 0.02a 55.03 0.01a 6.05 0.02a 51.42 0.23a 0.014 0.05 0.907 0.02a 0.512 0.01a 26.47 0.16a 23.99 0.21a 0.392 0.08a 5.95 0.01b 6.12 0.02b 92.47 0.08b 94.13 0.08b 52.83 0.07b 20.47 0.05a 18.63 0.01b 0.93 0.00b 11.49 0.08b 40.21 0.31b 3.80 0.01a 4.29 0.02b 48.49 0.08b 5.74 0.01b 37.4 0.19b 0.013 0.06 0.883 0.07b 0.413 0.01b 15.45 0.04b 13.84 0.04b 0.342 0.01b II 5.95 0.01b 6.08 0.03b 92.47 0.08b 94.13 0.08b 52.76 0.06b 20.54 0.03a 18.61 0.02b 0.93 0.00b 11.44 0.08b 40.32 0.23b 3.87 0.01a 4.37 0.02b 48.46 0.21b 5.84 0.01b 36.68 0.33b 0.0130 0.05 0.881 0.02b 0.410 0.06b 14.86 0.15b 13.12 0.17b 0.345 0.08b

n = 9; # n = 3. Control = no hurdles; Treatment I = hurdles + reheating to 90 C; Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping. Means with dierent superscripts in the same row indicate signicant dierence (P < 0.05).

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solution before reheating had no signicant inuence on the composition. As expected, the aw of the hurdle treated groups were signicantly lower (P < 0.01), due to the addition of TSP as humectant. Accelerated TBARS, which is a measure of potential protection against lipid oxidation by the addition of hurdles, indicated that addition of the hurdles signicantly reduced the chance of lipid oxidation during storage (Fig. 1). K-sorbate dipping reduced the accelerated TBARS values slightly. It was reported that addition of 2% lactic acid and 0.25% GDL signicantly reduced accelerated TBARS values in cooked cured emulsion type meat products (Juncher et al., 2000). WB shear force and work of shearing were decreased signicantly (P < 0.01) in the hurdle treated sausages (Table 1). This could be attributed mainly to the pH and reheating hurdles, where the lower pH and increased core temperature might have increased protein denaturation leading to decreased binding properties. Incorporation of dierent hurdles signicantly increased (P < 0.01) the lovibond-tintometer red units (a-values) of pork sausages, while yellowness (b-values), hue angle and chroma had a decreasing trend (Table 1). Moreover, K-sorbate dipping slightly improved the dierent colour units of the hurdle treated sausages. The increase in redness and decrease in yellowness in the treated samples could mostly be attributed to pH and reheating hurdles, as they have been reported to increase the conversion of myoglobin to metmyoglobin in cooked meat products (Maca, Miller, Bigner, Lucia, & Acu, 1999) and increased concentration of haem pigments due to increased moisture and fat loss. Similar ndings were reported with decreasing in pH in cooked meats (Papadopoulos, Miller, Ringer, & Cross, 1991) and beef top rounds (Maca, Miller, Maca, & Acu, 1997). A signicant decrease (P < 0.01) in all the textural attributes, except adhesiveness, was observed by the application of the dierent hurdles (Table 1). Hurdle treatment decreased the hardness by 27%, while further dipping in K-sorbate decreased it by 28.6% compared to the control sausages. The better fat and water retention due to higher pH and absence of reheating probably contributed to the signicantly higher hardness and cohesiveness in the con0.09

trol sausages. It might be due to the formation of good quality gel matrix as a result of reduced muscle protein denaturation. Feng and Xiong (2002) observed that the quality of the gel matrix had an important role in determining the textural properties of cooked frankfurters. The slightly lower hardness and cohesiveness of sausages from treatment II compared to those from treatment I might be due to the relatively lower pH, from K-sorbate dipping. The signicantly lower springiness observed in hurdle treated sausages could be attributed to their lower elastic properties resulting from decreased fat binding. Gumminess, chewiness and fracturability showed the same trend as observed for springiness. A decrease in chewiness, gumminess and hardness in fermented sausages (Gou, Guerrero, Gelabert, & Arnau, 1996) and decrease in springiness in salami type products (Barbut, 2005) with decreasing pH has been reported. Moreover, Voller, Dawson, and Han (1996) observed a decrease in various textural characteristics of cooked fowl meat gels at high processing temperatures. 3.2. Quality changes in SSPS during ambient temperature storage (37 1 C) The sausages were not subjected to sensory evaluation on the day they were spoiled, but physico-chemical and microbiological parameters were determined. The acceptable/unacceptable distinction was made mostly on the basis of avour changes detected at the time of opening of the packets. The colour changes and sliminess developed were also considered. 3.2.1. Physico-chemical characteristics The pH of the control sausages increased by about 0.4 units on day 3 of storage, while that of treatment I and II showed only a slight increase of 0.09 and 0.10 units, respectively (Table 2). This could be attributed to the higher microbial load in the control sausages (Table 3). Increases in spoilage organisms results in more protein degradation in meat products (Lawrie, 1998), which in turn increases their pH, attributed to the accumulation of more metabolites (Bell & Shelef, 1978). However, the inhibitory action of the hurdles, especially lower pH and reheating, on microbial growth, will contribute to the lower increase in pH on day 3 of the treated groups compared to control. A gradual increase in pH of hurdle treated caprine keema with ambient temperature storage has been reported (Karthikeyan et al., 2000). On day 6, the pH of the sausages from the treated groups showed a decreasing trend compared to day 3 which was signicant in treatment I, but not in treatment II. The addition of sugar and TSP in their formulation, combined with vacuum packaging, could have favoured the growth of lactic acid producing bacteria in the products. It was reported that organic acids, mainly lactic acid, are formed in vacuum packaged sausages during storage as a result of carbohydrate fermentation, which decreased pH (Incze,

Absorbance (532 nm- 600 nm)

0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 Control Treatment I Treatment II

Fig. 1. Eect of hurdle treatments on the accelerated TBARS values of shelf stable pork sausages.

R. Thomas et al. / Meat Science 79 (2008) 112

Table 2 Eect of potassium sorbate dipping and post package reheating on the physico-chemical parameters of SSPS during ambient temperature storage (37 1 C) Treatment/parameter Storage period (days) 0 pH Control I II TBARS value (mg malonaldehyde/kg) Control I II Tyrosine value (mg/g) Control I II Titratable acidity (ml 0.01N NaOH/g) Control I II 6.38 0.08b1 6.12 0.05c2 6.08 0.08c2 0.075 0.01b2 0.093 0.01d1 0.090 0.05d1 0.306 0.05b3 0.327 0.02d2 0.337 0.02d1 0.917 0.02a2 1.27 0.02a1 1.30 0.02a1 3 6.79 0.01a1 6.21 0.01a2 6.18 0.02b3 0.582 0.03a1 0.154 0.08c2 0.148 0.01c2 0.800 0.02a1 0.443 0.05c2 0.427 0.02c3 0.717 0.02b2 1.02 0.02c1 1.07 0.02c1 6 SP 6.16 0.01b 6.15 0.01b SP 0.231 0.05b 0.209 0.08b SP 0.503 0.02b 0.487 0.05b SP 1.13 0.03b 1.13 0.02b 9A 6.25 0.01a 6.23 0.01a 0.284 0.08a 0.275 0.01a 0.617 0.010a 0.613 0.01a 0.90 0.01d 1.00 0.01d

n = 9; SP = Spoiled. Control = no hurdles; Treatment I = hurdles + reheating to 90 C. Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping. Means with dierent superscripts (letters in the same row and numbers in the same column) indicate signicant dierence (P < 0.05). A Data in the same column on day 9 was analyzed by paired t-test. Table 3 Eect of potassium sorbate dipping and post package reheating on the microbiological characteristics of SSPS during ambient temperature storage (37 1 C) Treatment/parameter Storage period (days) 0 Total plate count (log cfu/g) Control I II Coliform count (log cfu/g) Control I II Total anaerobic count (log cfu/g) Control I II Lactobacillus count (log cfu/g) Control I II 4.27 0.08b1 3.28 0.03d2 3.21 0.05d2 1.83 0.05 ND ND 2.56 0.04b1 1.31 0.01d2 1.30 0.08d2 1.53 0.13a1 1.13 0.17d2 1.14 0.12d2 3A 6.74 0.16a1 3.99 0.10c2 3.89 0.04c3 1.77 0.06 1.79 0.03 ND 3.45 0.05a1 1.63 0.12c2 1.63 0.12c2 1.95 0.14b1 1.22 0.13c2 1.23 0.17c2 2.49 0.15 ND ND 1.95 0.37 1.39 0.17 ND 6 SP 4.76 0.05b1 4.65 0.09b2 SP ND ND SP 2.47 0.09b1 2.31 0.09b2 SP 1.37 0.12b1 1.32 0.04b2 SP 1.69 0.17 1.61 0.13 SP 1.95 0.15 ND 9A 6.29 0.10a 6.23 0.03a ND 1.81 0.06 3.68 0.05a1 3.40 0.08a2 1.68 0.14a 1.62 0.02a 1.96 0.16 1.99 0.12 2.19 0.12 ND

Staphylococcus aureus count (log cfu/g) Control 2.11 0.13 I ND II ND Yeast and mold count (log cfu/g) Control I II 1.34 0.07 ND ND

n = 9; SP, spoiled; ND, not detected. Control = no hurdles; Treatment I = hurdles + reheating to 90 C. Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping. Means with dierent superscripts (letters in the same row and numbers in the same column) indicate signicant dierence (P < 0.05). A Data in the same column on day 9 was analyzed by paired t-test.

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1992). Similar ndings were observed in vacuum packaged frankfurters (Blickstad & Molin, 1983) and pasteurized sliced ham (Andersen, Bertelsen, Ohlen, & Skibsted, 1990). However, pH was increased signicantly (P < 0.01) on day 9 in sausages from both treatment I and II groups, compared to day 6, attributed to the substantial increase in their microbial load, especially TPC, which might have nullied the eect of increased lactobacillus count. The slightly lower pH values observed throughout storage in treatment II compared to the treatment I could be due to their lower initial pH as a result of K-sorbate dipping. Estimation of TBARS value, which indicates the oxidative stability of products, showed that hurdle treated sausages had signicantly higher (P < 0.01) TBARS values on the day of processing compared to the control (Table 2). This may be attributed to the post package reheating of the sausages resulting in release of more non-heme iron (Verma, Paranjape, & Ledward, 1985), which catalytically increased lipid oxidation in the products. Higher TBARS values with increase in processing temperature for cooked bualo meat has been reported (Kesava Rao, Kowale, Babu, & Bisht, 1996). However, the increase in TBARS values was very signicant (P < 0.01) in control sausages compared to hurdle treated samples on day 3 (0.45 units vs. 0.06 units). TBARS values showed an increasing trend during subsequent storage, but no signicant dierence was found (P > 0.05) among treatments I and II at any storage time. The signicantly lower TBARS values of sausages from the treatment groups compared to controls on day 3 could be attributed to the presence of the hurdles. These hurdles might have led to the decrease in microbial load of treated sausages, resulting in lower TBARS values, as a positive correlation between microbial load and TBARS value was reported in ground bualo meat (Sahoo & Anjaneyulu, 1997). Although, the tyrosine value cannot be considered specic for proteolysis, it has been regarded as a good general index of meat protein breakdown (Pearson, 1968) and could prove useful for the assessment of spoilage in meat and meat products. The tyrosine value was signicantly higher for the sausages containing hurdles on the day of processing (Table 2). This could be due to denaturation and subsequent proteolysis of both meat and soy proteins during heat processing which might be accentuated by lactic acid and GDL (Syed, Rao, & Amla, 1995). A similar result was reported for hurdle treated goat meat keema compared to controls (Karthikeyan et al., 2000). On day 3, tyrosine values of the control sausages increased from 0.30 to 0.80 mg/g, the increase was markedly lower in hurdle treated samples, probably due to increased proteolysis in the control sausages due to their increased microbial load (Table 3). With storage, a signicant linear increase in tyrosine value was observed for all the samples. Moreover, the sausages subjected to K-sorbate dipping exhibited a slightly lower tyrosine values throughout storage compared to undipped samples (treatment I). An increase in tyrosine value for hurdle treated goat meat keema with

storage at ambient temperature has been reported (Karthikeyan et al., 2000). Titratable acidity (TA) was signicantly higher (P < 0.01) for hurdle treated sausages (Table 2) due to the addition of LA and GDL in their formulation. Titratable acidity of products depends upon the initial concentration of acid used (Acton & Johnson, 1973). All the samples showed a reduction in TA on day 3, while it increased in hurdle treated sausages on day 6, but further decreased on day 9. The decrease in TA of sausages on day 3 could be attributed to their increased pH. The lactic acid produced by increased lactobacillus organisms might have resulted in an increase in TA on day 6. The further reduction in TA on day 9 could be due to increased accumulation of bacterial metabolites as a result of a substantial increase in total plate count, masking the eect of increased lactobacillus counts. The present ndings conrm an inverse relationship between TA and pH. A reduction in TA in chicken emulsions stored under refrigeration, with storage has been reported (Rajani, Kondaiah, Anjaneyulu, & Thomas, 2007). 3.2.2. Microbiological characteristics Microbiological changes in SSPS during ambient temperature storage (37 1 C) are presented in Table 3. About 1 log reduction in total plate count (TPC) was observed in the sausages on incorporation of the dierent hurdles compared to controls. Dipping in 1% K-sorbate solution resulted in a further reduction of TPC, but not signicantly compared with treatment I. The lower TPC counts observed initially in the hurdle treated sausages is probably due to the destruction of microbes by post package reheating and reduced water activity. The slightly lower pH resulting from K-sorbate dipping might explain the lower TPC observed in treatment II compared to treatment I. Negbenebor, Balami, and Igene (1995) also observed a lower TPC in patties made from pre rigor beef dipped in 4% K-sorbate solution. TPC increased signicantly in all samples throughout storage and the increase was more pronounced in control sausages. It reached about 7 log cfu/g in the control sausages on day 3, while it was only about 6 log cfu/g in hurdle treated sausages on day 9, due to the direct destruction of vegetative cells due to reheating (Lawrie, 1998) and metabolic injury to micro-organisms as a result of the pH and aw hurdles (Leistner, 2000). Sliminess was detected in hurdle treated sausages on day 9. Several researchers reported signicant increase in TPC in meat products during room temperature storage, Torres et al. (1994) in charqui, Karthikeyan et al. (2000) in hurdle treated goat meat keema and Chawla and Chander (2004) in mutton kababs. It has been reported that spoilage of meat products is not noticed until bacterial counts are more than 108 cells/cm2 (Jay, 1996), however, in this study, spoilage of SSPS was detected at bacterial counts below 7 log cfu/g. Also, the phenomenon of auto-sterilization that is reported to take place in hurdle treated foods during storage at ambient temperature (Leistner, 2000) was not

R. Thomas et al. / Meat Science 79 (2008) 112

observed, which could be due to the higher aw and pH level used compared to values used by other researchers. Coliforms, considered an indicator of post processing contamination, were detected occasionally during storage. They were detected only in control sausages on the day of processing which clearly indicates the benet of post package reheating. Moreover, coliforms were reported to occur in signicantly higher numbers in vacuum packaged meat and meat products (Goktan, Tuncel, & Unluturk, 1988), however, in the present study the incorporation of dierent hurdles, especially reheating, reduced their occurrence. Incorporation of hurdles signicantly decreased (P < 0.01) the anaerobic counts and this could be attributed to the destruction of large numbers of vegetative cells during reheating (Leistner & Gorris, 1995). Anaerobic counts reached approximately 3.5 log cfu/g on day 3 in the control sausages, but it took about 9 days for hurdle treated samples to reach this level. Similarly, a linear increase in anaerobic bacterial count during ambient temperature storage of vacuum packaged hurdle treated chevon keema was reported by Karthikeyan et al. (2000). Enumeration of lactobacillus counts showed a similar trend as observed for anaerobes. Lactic acid bacteria were reported to be the major bacterial group associated with spoilage of vacuum packaged cooked meat products (Samelis & Georgiadou, 2000) as well as slime production (Pexara, Metaxopoulos, & Drosinos, 2002). The species of lactic acid bacteria responsible for spoilage was reported to depend on the product composition (Korkeala & Makela, 1989). Lactobacillus were shown to spoil vacuum packaged meat products by causing o-avour, discolouration, gas formation and slime production (Pexara et al., 2002) and in the present study o-avour and sliminess were detected when lactobacillus counts reached approximately 1.68 log cfu/g. Application of dierent hurdles was found to inhibit Staph. aureus up to day 3, but their growth appeared on day 6 of storage. Although, Staph. aureus was reported to survive at aw 0.86 (Jay, 1996), they were not detected in the pork sausages at aw of 0.93. This could be due to the presence of the other hurdles, especially low pH and post package reheating. Hurdles such as low pH, low aw and reheating were sufcient to inhibit yeast and mold growth up to day 3, but additional dipping in 1% K-sorbate solution inhibited their growth throughout the 9 days. Although, yeasts and molds were reported to survive the pH and aw levels used in the present study, their absence in treatment I on the day of processing could be attributed to post package reheating. K-sorbate is well known for its inhibitory eect on yeast and molds (Ledward, 1990). However, the available literature shows that their inhibitory level varies with the method of application. For example, incorporation of 0.2% K-sorbate in the formulation was found to be eective in inhibiting the growth of yeast and molds in meat balls (Hsu & Sun, 2006) and fresh pork sausages (Lee,

Faustman, Djordjevic, Faraji, & Decker, 2006), while Negbenebor et al. (1995) reported that dipping of pre-rigor beef strips in 4% K-sorbate solution inhibited the growth of yeasts and molds in patties processed from them. 3.2.3. Sensory attributes Changes in sensory attributes of pork sausages during ambient temperature storage are shown in Table 4. It is evident that incorporation of dierent hurdles resulted in an initial reduction in all the sensory attributes, however, they maintained these attributes for a longer time compared to control. Sensory evaluation of the control sausages was done only on the day of processing due to the development of o-odour and sliminess on the 3rd day. The signicant decrease (P < 0.01) observed in appearance due to incorporation of hurdles could be attributed to the higher rate of conversion of myoglobin to brownish metmyoglobin, partly due to low pH and high temperature resulting from reheating and to the concentration of meat pigments due to increased moisture and fat loss. Dipping
Table 4 Eect of potassium sorbate dipping and post package reheating on the sensory attributesB of SSPS during ambient temperature storage (37 1 C) Treatment/parameter Storage period (days) 0 Appearance Control I II Flavour Control I II Juiciness Control I II Texture Control I II Binding Control I II Overall acceptability Control I II 7.00 0.131 6.83 0.24a2 6.83 0.24a2 7.16 0.241 6.83 0.24a2 6.83 0.24a2 7.16 0.151 6.67 0.11a2 6.67 0.15a2 7.00 0.151 6.83 0.15a2 6.83 0.15a2 7.00 0.24 6.50 0.11a 6.50 0.10a 7.00 0.241 6.83 0.15a2 6.83 0.15a2 3A ND 6.56 0.24b 6.67 0.24b ND 6.68 0.24b 6.72 0.11b ND 6.50 0.15b 6.50 0.15b ND 6.61 0.11b 6.57 0.10b ND 6.50 0.15a 6.50 0.15a ND 6.60 0.20b 6.66 0.11b 6A 6.45 0.11c 6.42 0.15c 6.47 0.24c2 6.56 0.10c1 6.50 0.09b 6.50 0.10b 6.37 0.24c 6.32 0.15c 6.00 0.15b 6.00 0.15b 6.47 0.11c 6.51 0.20c

n=21; ND = not detected. Control = no hurdles; Treatment I = hurdles + reheating to 90 C. Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping. Means with dierent superscripts (letters in the same row and numbers in the same column) indicate signicant dierence (P < 0.05). A Data in the same column on day 3 and 6 were analyzed by paired t-test. B Based on 8-point descriptive scale.

R. Thomas et al. / Meat Science 79 (2008) 112

of sausages in 1% K-sorbate solution prior to packaging had no signicant eect on their appearance. The decrease in the colour scores observed with storage could be due to non-enzymatic browning due to reaction between lipid oxidation products and amino acids as reported by Che Che Man, Bakar, and Mokri (1995). Incorporation of hurdles signicantly reduced (P < 0.01) the avour of pork sausages on the day of processing. This may be due to the higher fat loss, since the fat content of the meat products was reported to have signicant eects on avour (Pearson & Gillett, 1997). The signicant reduction in avour with storage could be due to increased fat oxidation (Santamaria, Lizarraga, Astiasaran, & Bello, 1992) as well as higher microbial load (Sahoo & Anjaneyulu, 1997). The comparatively better avour scores observed in sausages subjected to K-sorbate dipping could result from a lower rate of lipid oxidation due to their lower microbial load. Juiciness of hurdle treated pork sausages was signicantly lower on the day of processing (P < 0.01) due to the low aw and increased fat and moisture loss. The decrease in juiciness observed with storage might be due to further drying occurring during ambient temperature storage. Dipping of sausages in 1% K-sorbate solution prior to reheating had no eect on juiciness. Addition of hurdles resulted in a signicant decrease (P < 0.01) in texture of the sausages, probably due to increased denaturation of proteins and subsequent decrease in fat and water binding properties. The products subjected to K-sorbate dipping exhibited lower textural characteristics compared to the undipped samples during storage, probably due to their comparatively lower pH. The decrease in texture of the sausages from both treatments during ambient temperature storage may be attributed to the changes in disulphide bonds due to increased protein denaturation (Santamaria et al., 1992) as a result of increased microbial activity (Jay, 1996). Binding of the sausages decreased significantly with hurdle treatment and the reasons discussed for the textural changes are probably applicable. Overall acceptability of the hurdle treated sausages followed the same pattern observed for the other sensory attributes. The slightly higher overall acceptability scores for sausages subjected to K-sorbate dipping could be attributed to their better avour scores. The overall acceptability was in the range very good to good up to day 6 for sausages from both treatment groups. Despite good quality characteristics on the 9th day, due to the development of slight o-avours and sliminess, it was concluded that the hurdle treated sausages had a shelf life of 6 days at 37 1 C. 3.3. Comparison of keeping quality of hurdle treated pork sausages stored at ambient (37 1 C) and refrigerated (4 1 C) temperatures Quality changes of sausages stored under ambient and refrigerated temperatures were compared on the days

where sampling coincided. Control sausages spoiled before day 3 of storage at ambient temperature. Therefore, the comparison was between treatment I and II. As the sausages for both ambient and refrigerated storage were processed from same batch of emulsion, there was no dierence among treatments on day zero. Thus, there was only one day where sampling coincided i.e. day 6, the end of the shelf life of the treated sausages. 3.3.1. Physico-chemical characteristics Physico-chemical characteristics of pork sausages stored at ambient and refrigeration temperatures on day 6 are shown in Table 5. Storage at ambient temperature signicantly increased (P < 0.05) their pH, presumably to their higher microbial load, especially TPC (Table 5). TBARS values increased signicantly (P < 0.01) in sausages stored at 37 1 C compared to their refrigerated counterparts during the 6 days storage, due to their increased rate of lipid oxidation (Pearson & Gillett, 1997). Tyrosine values of sausages signicantly increased (P < 0.01) during 6 days of storage at ambient temperature, compared to the refrigerated samples, presumably due to higher rates of proteolysis Ambient temperature storage for 6 days signicantly decreased (P < 0.01) the titratable acidity of pork sausages compared to the refrigerated ones, due to the higher pH. 3.4. Microbiological characteristics Comparison of microbiological changes in hurdle treated pork sausages during ambient and refrigerated storage is presented in Table 5. As expected storage of hurdle treated pork sausages at ambient temperature for 6 days increased their TPC by about 1 log cfu/g compared to refrigeration temperature. During enumeration of microbial proles of minced sheep meat, Narasimha Rao and Ramesh (1988) observed a 5 log cfu/g increase in TPC during 8 h storage at 30 1 C. Coliforms were not detected in the hurdle treated samples during storage, both at 37 and 4 C. Anaerobic counts increased signicantly (P < 0.01) in both treatments, by about 1 log cfu/g during storage at 37 1 C compared to those kept at 4 1 C. Similarly, lactobacillus counts increased signicantly (P < 0.05) in the samples kept at ambient temperature, but the rate of increase was markedly lower (i.e. about 0.2 log cfu/g). Pexara et al. (2002) reported a signicant increase in lactobacillus counts for cooked pork sausages stored at 10 C compared to those stored at 4 C. Staph. aureus organisms were detected in hurdle treated sausages at less than 2 log cfu/g on day 6 at 37 1 C, while they were absent in the refrigerated samples. During ambient temperature storage for 6 days, yeast and mold were appeared only in sausages of treatment I. Their absence in treatment II could be attributed to the K-sor-

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Table 5 Comparison of physico-chemical and microbiological characteristics of hurdle treated pork sausages stored at ambient (37 1 C) and refrigerated (4 1 C) temperatures on 6th day Parameter Physico-chemical characteristics pH TBARS value (mg malonaldehyde/kg) Tyrosine value (mg/g) Titratable acidity (ml 0.01N NaOH/g) Microbiological characteristics Total plate count (log cfu/g) Total anaerobic count (log cfu/g) Lactobacillus count (log cfu/g) Staph. aureus count (log cfu/g) Yeast and mold count (log cfu/g) Treatment I II I II I II I II I II I II I II I II I II 4 1 C 6.12 0.08 6.08 0.01 0.095 0.05 0.091 0.08 0.330 0.02 0.325 0.05 1.27 0.03 1.30 0.02 3.33 0.05 3.28 0.09 1.36 0.09 1.40 0.09 1.16 0.12 1.15 0.04 ND ND ND ND 37 1 C 6.16 0.01 6.15 0.05 0.231 0.08 0.209 0.08 0.503 0.01 0.487 0.08 1.13 0.08 1.13 0.08 4.76 0.02 4.65 0.01 2.47 0.08 2.31 0.17 1.37 0.13 1.32 0.09 1.69 0.17 1.61 0.13 1.95 0.15 ND t-Value 2.965* 3.78* 88.00** 89.13** 26.00** 37.79** 7.63** 19.08** 177.82** 758.95** 87.65** 82.15** 2.98* 2.88*

n = 9; * P < 0.05; ** P < 0.01. Treatment I = hurdles + reheating to 90 C; Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping.

bate dipping prior to packaging. Refrigerated storage inhibited the growth of yeast and mold in sausages of both treatments up to 6th day. 3.4.1. Sensory attributes Sensory evaluation of hurdle treated pork sausages revealed that storage at ambient temperature caused a marked decrease in all the sensory attributes compared to refrigerated storage (Table 6). This reduction was more signicant (P < 0.01) for appearance, avour, texture and binding compared to juiciness and overall acceptability (P < 0.05). The signicant decrease in appearance of sausages kept at 37 1 C could be attributed partly to the concentration of meat pigments due to increased moisture loss. Increased lipid oxidation in samples kept at ambient temperature might contribute to their signicantly lower avour scores. Das and Radhakrishna (2001) reported that avour changes were more marked in room temperature stored ready-to-eat mutton curry developed using hurdle technology, compared to refrigerated storage. As mentioned above, the decrease in moisture content of sausages during ambient temperature storage could have led to their lower juiciness scores and the decrease in texture and binding prop-

erties. Despite these changes, the hurdle treated pork sausages had an overall acceptability in the range of good to very good on day 6 of storage.
Table 6 Comparison of sensory attributesA of hurdle treated pork sausages stored at ambient (37 1 C) and refrigerated (4 1 C) temperatures on 6th day Parameter Appearance Flavour Juiciness Texture Binding Overall acceptability Treatment I II I II I II I II I II I II 4 1 C 6.83 0.11 6.83 0.15 6.83 0.24 6.83 0.10 6.67 0.09 6.67 0.10 6.83 0.24 6.83 0.15 6.50 0.15 6.50 0.15 6.67 0.11 6.67 0.20 37 1 C 6.45 0.15 6.42 0.12 6.47 0.11 6.54 0.11 6.50 0.16 6.50 0.13 6.37 0.16 6.32 0.10 6.00 0.15 6.00 0.15 6.47 0.15 6.52 0.12 t-Value 15.81** 15.81** 11.06** 11.06** 2.65* 2.65* 12.64** 12.64** 13.16** 13.16** 3.28* 2.93*

n = 21; * P < 0.05; * P < 0.01; Treatment I = hurdles + reheating to 90 C; Treatment II = hurdles + reheating to 90 C + 1% potassium sorbate dipping. A Based on 8-point descriptive scale.

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11

4. Conclusions Even though incorporation of hurdles decreased the various quality parameters of pork sausages compared to controls on the day of processing, they maintained good quality characteristics during storage. Hurdle treatment signicantly retarded quality deterioration during ambient temperature storage, especially lipid oxidation and microbiological characteristics. Hurdle treatment extended the shelf life of SSPS to 6 days. Further, dipping of pork sausages in 1% K-sorbate solution prior to packaging was found to improve their characteristics, especially yeast and mold counts and sensory attributes, particularly avour. Storage of hurdle treated pork sausages at ambient temperature (37 1 C) resulted in signicant deterioration in most of the quality parameters on day 6, compared to their refrigerated counterparts. However, dierent physico-chemical and microbiological parameters were within the acceptable limit recommended for meat products. It is concluded that hurdle treated pork sausages retained good acceptability and could be stored safely for 6 days at ambient temperature (37 1 C). Acknowledgement The authors acknowledge the nancial assistance in the form of Senior Research Fellowship by Director, Indian Veterinary Research Institute. References
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