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Safe and shelf-stable natural casing using hurdle technology
Food Control 17 (2006) 127–131 www.elsevier.com/locate/foodcont
Safe and shelf-stable natural casing using hurdle technology S.P. Chawla, Ramesh Chander *, Arun Sharma Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Received 17 February 2004; received in revised form 22 September 2004; accepted 27 September 2004
Abstract Safe and shelf-stable natural casing were prepared using a combination of hurdles viz. reduced water activity, packaging and gamma irradiation. Washed lamb intestines were treated with common salt to reduce water activity to 0.80 ± 0.02, packed in polyethylene bags and subjected to gamma-irradiation (5 and 10 kGy). Control non-irradiated samples had high total viable counts (106 CFU/g), aerobic spores (103 CFU/g), spores of sulphite reducing clostridia (103 CFU/g), potentially pathogenic bacteria such as staphylococci (104 CFU/g) and coliforms (102 CFU/g). Treatment with gamma radiation resulted in a dose dependent reduction in counts of these microbes. A dose of 5 kGy was sufficient to reduce total viable counts by three log cycles; spore counts by two log cycles and completely eliminate staphylococci and coliforms. Samples subjected to a 10 kGy dose were devoid of any viable microbes. The reduced water activity of the product prevented growth of the microbes in natural casings during storage at room temperature. Sausages prepared using hurdle processed natural casing were examined for sensory and textural properties. It was observed that product acceptability and mechanical strength was not affected by radiation processing. Our studies indicated that shelf-stable and safe natural casing could be prepared using a combination of hurdles. Ó 2004 Published by Elsevier Ltd. Keywords: Water activity; Shelf-stable; Hurdle technology; Gamma irradiation; Natural casings; Safety
1. Introduction Natural casing made from intestine of beef, pork, or lamb are used to stuff meat products such as sausages, salami and frankfurters. Although artificial casing made up of collagen, cellulose etc. are available, there is demand for natural casing due to consumer liking. Contamination of natural casing by enteric and exogenous microorganisms is inevitable. Microbial quality of natural casing depends on the hygiene of manufacturing procedure, post processing handling and storage temperature (Trigo & Fraqueza, 1998). Natural casing are contaminated with bacteria of public health significance such as fecal Streptococci, Enterobacteriaceae, coli*
Corresponding author. Tel.: +91 22 25593296/25595374; fax: +91 22 25505151/25519613. E-mail address: [email protected] (R. Chander). 0956-7135/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2004.09.011
forms, sulphite reducing Clostridia (Byun, Lee, Jo, & Yook, 2001; Trigo & Fraqueza, 1998). These could pose health risk to the consumer. Contamination of these products with pathogenic bacteria does not always result in modification of the organoleptic qualities, which results in consumption without suspicion. Currently the natural casing are stored either highly salted or frozen and utilized as early as possible in order to avoid spoilage. Freezing and salting despite being bacteriostatic are not effective in eliminating contaminant and pathogenic microbes. Efficacies of chemical preservatives such as lactic, tartaric, citric acid, hydrogen peroxide and ethanol alone or in combination have been investigated to improve microbiological quality of natural casing. Use of chemical preservatives results in adverse effect on technological properties of casing and problem of toxic residues (Labie, 1987).
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S.P. Chawla et al. / Food Control 17 (2006) 127–131
Radiation processing is a useful technique to improve microbiological quality and to enhance safety of several food commodities by killing pathogens such as Salmonella, Staphylococcus aureus, Campylobacter, Listeria monocytogenes (Farkas, 1998). Previous studies in our laboratories have shown that irradiation treatment can improve shelf life and microbiological quality of buffalo, chicken and lamb meat stored in chilled state (Kanatt, Paul, DÕsouza, & Thomas, 1997; Naik, Paul, Chawla, Sherikar, & Nair, 1994; Paul, Venugopal, & Nair, 1990). A combination of hurdles can ensure stability and microbial safety of preserved food. The most important hurdles applied in food preservation are temperature (high or low), water activity, acidity, redox potential, preservatives and competitive microflora. Each hurdle implies putting microorganisms in a hostile environment, which inhibits their growth or causes their death (Leistner, 1999, 2000). Reduction of water activity is an effective preservation method of perishable material as growth of many of the spoilage bacteria is retarded due to low water activity. There are many reports on development of intermediate moisture (IM) products having reduced water activity (Wang & Leistner, 1993, 1994). We have reported preparation of safe and shelfstable IM ready-to-serve meat products using a combination of hurdles, i.e. reduced water activity, vacuum packaging and gamma-irradiation (Kanatt, Chawla, Chander, & Bongirwar, 2002). Water activity of these products was adjusted to less than 0.85. Vacuum packaging and gamma-irradiation took care of oxidation and microbial contamination, respectively. As for the water activity employed in the study, it was envisaged that Clostridium botulinum cannot grow and Staphylococcus spp. do not produce enterotoxin (Leistner, 1992). These presumptions were confirmed by inoculated pack studies using C. sporogenes and S. aureus (Chawla & Chander, 2004). In the present study we report a process for the preparation of safe and shelf-stable lamb casing using a combination of reduced water activity and gamma-irradiation treatment. Microbiological, textural and sensory qualities of sausages prepared from these radiation-processed casing were investigated.
2. Materials and methods Fresh intestines of lamb (size 18–20) were procured from a local casing manufacturer (three hanks). They were washed thoroughly with potable water, 10% (w/ w) sodium chloride (food grade table salt) was added and packed in polyethylene bags. The packs were subjected to gamma irradiation at ambient temperature at a dose rate of 5.0 kGy h 1 in a Gamma cell 5000 (Board of Radiation and Isotopes Technology, India) with a 60 Co source. The samples received minimal doses of 5
and 10 kGy. Dosimetry was performed as reported earlier (Kanatt et al., 2002). Three independent experiments were performed. Each hank was used for a separate experiment in order to minimize variation. Water activity of the natural casings before and after the treatment with salts was determined using an Aqua LabCX2T water activity meter (Decagon Devices, USA). 2.1. Microbiological analyses Samples from both the irradiated and the non-irradiated lots were analyzed immediately after irradiation and subsequently at regular intervals during storage at ambient temperature. Samples (10 g) in duplicate were aseptically homogenised for 2 min in a sterile stomacher bag containing 90 ml of sterile saline using stomacher 400 Lab Blender (Seward Medical, UK). Serial dilutions of the homogenate were prepared. To determine spore count vegetative cells were killed by heat treatment at 80 ± 2 °C for 10 min. Colony-forming units were determined using appropriate media. All microbiological media were procured from HiMedia Laboratories, India. The plate count agar was used for the determination of aerobic vegetative bacteria and spores. Baird Parker agar, sodium sulphite polymixin sulphadiazine (SPS) agar, violet red bile agar and potato dextrose agar (PDA) were respectively used for determination of Staphylococcus, spores of sulphite reducing clostridia, coliforms and molds. (Chawla & Chander, 2004; Kanatt et al., 2002). Plates other than PDA were incubated at 37 °C for 48 h. PDA plates were incubated at ambient temperature for 7 days. SPS agar plates were incubated in anaerobic conditions in an Anaerobic Jar (HiMedia, India). 2.2. Preparation of sausages Sausages were prepared by using non-irradiated and irradiated lamb casing on day of irradiation and after 30 days of storage at ambient temperature. 2.3. Sensory analyses A panel of 6–8 staff members analysed the sausages. Panelists were asked to rate the samples as ‘‘acceptable’’ or ‘‘non-acceptable’’ on the basis of appearance, odor, flavour and taste on 10 point scale, where 10 corresponded to a product of highest quality and 0 corresponds to poor quality of product. Scores of 6 and above were considered acceptable. 2.4. Mechanical properties Puncture force required for the sausage prepared using non-irradiated and irradiated casing was deter-
S.P. Chawla et al. / Food Control 17 (2006) 127–131
mined using Instron Universal Testing Machine with cylindrical probe (Bhusan & Thomas, 1998). 2.5. Statistical analysis All results given in figures are mean ± standard deviation. Differences between the variables were tested for significance by one-way ANOVA with TurkeyÕs post test using GraphPad InStat version 3.05 for window 95, GraphPad Software, San Diego California USA www.graphpad.com. Differences at p < 0.05 were considered to be significant.
3. Results and discussion Fresh untreated natural casing had a water activity of 0.95 ± 0.02. Water activity of casing was reduced to 0.80 ± 0.02 by addition of 10% salt. The results of microbiological analyses are compiled in Table 1. The fresh natural casing had total viable counts (TVC) of more than 106 CFU/g. The counts of potentially pathogenic bacteria such as staphylococci and coliforms were more than 104 CFU/g and 102 CFU/g respectively. Both the aerobic spore and anaerobic spore (sulphite reducing clostridia) counts were found to be 103 CFU/g. The untreated casing spoiled within few hours at ambient temperature. As can be seen, the salt treatment resulted in some reduction in counts of vegetative cells but had no effect on spore count. Radiation treatment resulted in a dose dependent reduction in counts of these mi-
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crobes. A dose of 5 kGy was sufficient to eliminate staphylococci, coliforms and molds but not the bacterial spores. TVC reduced by about three log cycles in samples irradiated at 5 kGy. All vegetative bacterial cells, mold and bacterial spores were eliminated at 10 kGy (Table 1). These results suggested that non-irradiated natural casing were heavily contaminated with various bacteria including, potentially pathogenic bacteria such as staphylococci and coliforms, spores and molds and radiation processing could significantly improve their microbiological quality. Our results are in agreement with Byun et al. (2001) who reported that washed natural casing had total microbial load, entrococci and coliforms in the range of 3–5 log CFU/g. Similarly Trigo and Fraqueza (1998) also reported high level of aerobic counts, coliforms, enterocci and spores in both fresh and dried natural casing. Improvement of microbial quality of fresh natural casing by radiation treatment has also been reported earlier (Byun et al., 2001; Trigo & Fraqueza, 1998). In the present study a 5 kGy dose was required to reduce bacterial counts by three log cycles. Radiation sensitivity of bacteria is affected by number of factors such as water activity, composition, irradiation temperature, presence of oxygen (Mendonca, 2002). Higher resistance of bacterial cells in salted natural casing could be attributed to lower water activity. In the presence of water cell injury during irradiation is due to both direct damage to cell DNA and indirect damage due to the reactivity of the radiolytic products with cell components (Grecz, Rowley, & Matsuyama, 1983). Also, in complex systems
Table 1 Microbiological analyses of salted natural casings from sheep during storage at ambient temperature Treatment
Counts log CFU/gm for storage period Analysis
Initial
15 days
30 days
60 days
90 days
Non-irradiated
TVC SPC SRCC SC CC FC
5.5–5.9 3.5–3.7 3.0–3.9 2.6–3.7 1.9–2.3 +++
4.4–5.6 3.4–3.7 2.9–3.5 2.6–3.3 NVC +++
4.7–5.3 3.2–4.0 3.2–4.0 2.3–2.6 NVC ++
4.7–5.1 3.4–3.7 2.3–2.7 2.0–2.6 NVC ++
3.79–4.04 3.26–3.58 2.90–3.08 2.48–2.90 NVC ++
Irradiated (5 kGy)
TVC SPC SRCC SC CC FC
2.3–3.5 2.4–2.8 2.4–2.6 NVC NVC NVC
2.6–3.8 2.0–2.8 2.0–2.5 NVC NVC NVC
2.7–3.9 2.3–2.7 2.0–2.3 NVC NVC NVC
2.7–4.0 2.5–3.0 2.0–2.7 NVC NVC NVC
2.14–2.57 2.84–3.23 2.57–2.70 NVC NVC NVC
Irradiated (10 kGy)
TVC SPC SRCC SC CC FC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
TVC—total viable count; SPC—spore count; SRCSC—sulphite reducing clostridia spore count; SC—staphylococcus count; CC—coliform count, FC—fungal count, (+) growth of mold on PDA, NVC—no viable count detected. Fresh washed natural casing had TVC of 6.3, SPC of 3.1, SRCSC of 2.9, SC of 4.5, and CC of 2.4 log CFU/g. Results shown are of three independent experiments involving intestines of different animals.
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some of the constituents such as proteins are believed to compete with the bacterial cell for interaction with free radicals thereby reducing the net effect of radiation damage and making the organism more resistant (Urbain, 1986). The viable counts of vegetative cells, Staphylococcus and coliforms decreased by about 1–2 log in the case of non-irradiated samples during the storage period of three months. This could be attributed to the fact that the presence of salt disturbs homeostasis (internal equilibrium) of bacterium and it utilizes every possible repair mechanism in order to overcome the hostile environment. In the process of overcoming the hostile environment, the bacterium uses all its energy and dies; this eventually leads to reduction in cell viability (Leistner, 2000). In this case the main stress is reduced water activity. The most of bacteria fail to grow at water activity below 0.85 (Ledward, 1981). However, each bacterial species has different capability to overcome a particular stress. Staphylococcus is one of the most resistant bacterial species capable of tolerating low water activity. Maximum water activity that inhibits growth of S. aureus in IM pork during storage is reported to be 0.88 (Plitman, Park, & Sinskey, 1973). S. aureus was able to grow at water activity higher than 0.90 in mutton kababs (Kanatt et al., 2002) as well as in traditional Korean semi-dried seafood Kwamegi (Chawla et al., 2003). However, it failed to multiply when water activity was less than 0.85 (Chawla & Chander, 2004). The spore counts of the samples remained constant during storage. In irradiated samples no viable cells of Staphylococcus or coliforms were detected during the study period. However, spores were detected in the samples irradiated at 5 kGy. Molds were observed in non-irradiated samples during storage and irradiation treatment was required to inactivate molds. Growth of mold in IM foods having water activity above 0.75 has been reported (Labuza, Cassil, & Sinskey, 1972). We have also reported mold growth in IM mutton kababs (having water activity 0.85 ± 0.02) when stored at ambient temperature (Chawla & Chander, 2004). Counts of spores of aerobic bacteria and anaerobic sulphite reducing clostridia remained constant during storage at ambient temperature. Results confirm the fact that spores are much more resistant in their ability to survive in adverse conditions, i.e. reduced water activity. Spores however failed to germinate due to low water activity of the casing. In the irradiated samples there was no significant change in bacterial counts during storage, suggesting failure of radiation damaged cells to recover during storage. Bacterial spores were detected in samples irradiated at 5 kGy, whereas, no viable spores were observed in samples irradiated at 10 kGy. The results of these storage studies indicated that the natural casing with reduced water activity were not
favourable for multiplication of the organisms. Results suggest bacteriostatic effect of the hurdle employed. However, Staphylococcus and bacterial spores survived during storage suggesting that additional hurdles have to be employed to eliminate these microbes. In inoculum packed studies with meat products with reduced water activity, S. aureus cells and C. sporogenus spores survived during storage at ambient temperature (Chawla & Chander, 2004). Sensory analyses of sausages prepared from natural casing on day of irradiation and after 30 days of storage at ambient temperature were carried out. Appearance, odor, flavour, and overall acceptance scores of sausages prepared using irradiated casings were comparable with that prepared from non-irradiated casings at both the analyses points. Results of sensory analyses immediately after treatment are presented in Fig. 1. Similar observations were made with irradiated IM meat products, where acceptability of products was not affected by irradiation treatment (Kanatt et al., 2002). Textural analyses of sausages prepared from natural casing on day of irradiation and after 30 days of storage at ambient temperature were conducted. There was no significant difference in the puncture strength of sausages prepared using irradiated casing were comparable with that prepared from non-irradiated casing on day of irradiation and after 30 days of storage at ambient temperature. Results of puncture strength of sausages prepared from natural casing after 30 days of irradiation treatment are presented in Fig. 2. These results are in contrast to that of Byun et al. (2001), who reported reduction in shear force in sausages prepared using irradiated casing. These differences could be attributed to differences the type of casings used and irradiation conditions in both the studies. Currently the natural casings are stored either highly salted or frozen and utilized as early as possible in order to avoid spoilage. Our results have shown that microbiological quality of natural casing used by meat
10
Non-irradiated 5 kGy 10 kGy
8
Sensory scores
130
6
4
2
0 Color
Odour
Texture
Overall acceptability
Fig. 1. Sensory qualities of sausages prepared from variously treated natural casings. Results are mean ± SD of eight penal members.
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Puncture strength (kg)
2.0
Hank 1 Hank 2 Hank 3
1.5
1.0
0.5
0.0
Non-irradiated
5 kGy
10 kGy
Fig. 2. Puncture strength of sausages prepared from variously treated natural casings. Results are mean ± SD of eight sausages prepared from same treatment.
processing industry is very poor. Reduced water activity in combination with radiation processing can improve the safety of natural casings without affecting their functional properties. Thus there is scope to develop safe and shelf-stable natural casing using a combination of hurdles including radiation. References Bhusan, B., & Thomas, P. (1998). Quality of apples following gammairradiation and cold storage. International Journal of Food Science and Technology, 49, 485–492. Byun, M. W., Lee, J. W., Jo, C., & Yook, H. S. (2001). Quality properties of sausage made with gamma irradiated natural pork and lamb casing. Meat Science, 59, 223–228. Chawla, S. P., & Chander, R. (2004). Microbiological safety of shelfstable meat products prepared by employing hurdle technology. Food Control, 15, 559–563. Chawla, S. P., Kim, D. H., Jo, C., Lee, J. W., Song, H. P., & Byun, M. W. (2003). Effect of gamma irradiation on the survival of pathogens in Kwamegi a traditional Korean semidried seafood. Journal of Food Protection, 66, 2093–2096. Farkas, J. (1998). Irradiation as a method for decontaminating food. International Journal of Food Microbiology, 44, 189–204. Grecz, N., Rowley, R. B., & Matsuyama, A. (1983). The action of radiation on bacteria and viruses. In E. S. Josephson & M. S.
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Peterson (Eds.), Preservation of food by ionizing radiation (pp. 167–218). Boca Raton, FL: CRC Press. Kanatt, S. R., Chawla, S. P., Chander, R., & Bongirwar, D. R. (2002). Shelf-stable and safe intermediate moisture (IM) meat products using hurdle technology. Journal of Food Protection, 65, 1628–1631. Kanatt, S. R., Paul, P., DÕsouza, S. F., & Thomas, P. (1997). Effect of gamma-irradiation on the lipid peroxidation in chicken, lamb and buffalo meat during chilled storage. Journal of Food Safety, 17, 283–294. Labie, C. (1987). Les contaminants des boyaux naturals. Viande et Produitis Carnes, 8, 73–79. Labuza, T. P., Cassil, S., & Sinskey, A. J. (1972). Stability of intermediate moisture foods. 2 Microbiology. Journal of Food Science, 37, 160–162. Ledward, D. A. (1981). Intermediate moisture meats. In R. A. Lawrie (Ed.). Developments in meat science (vol. 2, pp. 159–194). England: Applied Science Publisher. Leistner, L. (1992). Food preservation by combined method. Food Research International, 25, 151–158. Leistner, L. (1999). Combined methods of food preservation. In M. Shafiur Rahman (Ed.), Handbook of food preservation (pp. 457–485). New York: Marcel Dekker. Leistner, L. (2000). Basic aspects of food preservation by hurdle technology. International Journal of Food Microbiology, 55, 181–186. Mendonca, A. F. (2002). Inactivation by irradiation. In V. K. Juneja & J. N. Sofos (Eds.), Control of foodborne pathogens (pp. 75–104). New York: Marcel Dekker. Naik, G. N., Paul, P., Chawla, S. P., Sherikar, A. T., & Nair, P. M. (1994). Influence of low dose irradiation on the quality of fresh buffalo meat stored at 0–3 °C. Meat Science, 38, 307–313. Paul, P., Venugopal, V., & Nair, P. M. (1990). Shelf-life enhancement of lamb meat under refrigeration by gamma irradiation. Journal of Food Science, 55, 865–866. Plitman, M., Park, Y., & Sinskey, A. J. (1973). Viability of Staphylococcus aureus in intermediate moisture meats. Journal of Food Science, 38, 1004–1008. Trigo, M. J., & Fraqueza, M. J. (1998). Effect of gamma radiation on microbial population of natural casings. Radiation Physics and Chemistry, 52, 125–128. Urbain, W. M. (1986). Biological effects of ionizing radiation. In Food irradiation. Food science and technology series (pp. 83–117). London: Academic Press. Wang, W., & Leistner, L. (1993). Shafu: a novel dried meat product of China based on hurdle technology. Fleischwirtschaft, 73, 854–856. Wang, W., & Leistner, L. (1994). Traditional Chinese meat products and their optimization by hurdle technology. Fleischwirtschaft, 74, 1135–1145.
Safe and shelf-stable natural casing using hurdle technology S.P. Chawla, Ramesh Chander *, Arun Sharma Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India Received 17 February 2004; received in revised form 22 September 2004; accepted 27 September 2004
Abstract Safe and shelf-stable natural casing were prepared using a combination of hurdles viz. reduced water activity, packaging and gamma irradiation. Washed lamb intestines were treated with common salt to reduce water activity to 0.80 ± 0.02, packed in polyethylene bags and subjected to gamma-irradiation (5 and 10 kGy). Control non-irradiated samples had high total viable counts (106 CFU/g), aerobic spores (103 CFU/g), spores of sulphite reducing clostridia (103 CFU/g), potentially pathogenic bacteria such as staphylococci (104 CFU/g) and coliforms (102 CFU/g). Treatment with gamma radiation resulted in a dose dependent reduction in counts of these microbes. A dose of 5 kGy was sufficient to reduce total viable counts by three log cycles; spore counts by two log cycles and completely eliminate staphylococci and coliforms. Samples subjected to a 10 kGy dose were devoid of any viable microbes. The reduced water activity of the product prevented growth of the microbes in natural casings during storage at room temperature. Sausages prepared using hurdle processed natural casing were examined for sensory and textural properties. It was observed that product acceptability and mechanical strength was not affected by radiation processing. Our studies indicated that shelf-stable and safe natural casing could be prepared using a combination of hurdles. Ó 2004 Published by Elsevier Ltd. Keywords: Water activity; Shelf-stable; Hurdle technology; Gamma irradiation; Natural casings; Safety
1. Introduction Natural casing made from intestine of beef, pork, or lamb are used to stuff meat products such as sausages, salami and frankfurters. Although artificial casing made up of collagen, cellulose etc. are available, there is demand for natural casing due to consumer liking. Contamination of natural casing by enteric and exogenous microorganisms is inevitable. Microbial quality of natural casing depends on the hygiene of manufacturing procedure, post processing handling and storage temperature (Trigo & Fraqueza, 1998). Natural casing are contaminated with bacteria of public health significance such as fecal Streptococci, Enterobacteriaceae, coli*
Corresponding author. Tel.: +91 22 25593296/25595374; fax: +91 22 25505151/25519613. E-mail address: [email protected] (R. Chander). 0956-7135/$ - see front matter Ó 2004 Published by Elsevier Ltd. doi:10.1016/j.foodcont.2004.09.011
forms, sulphite reducing Clostridia (Byun, Lee, Jo, & Yook, 2001; Trigo & Fraqueza, 1998). These could pose health risk to the consumer. Contamination of these products with pathogenic bacteria does not always result in modification of the organoleptic qualities, which results in consumption without suspicion. Currently the natural casing are stored either highly salted or frozen and utilized as early as possible in order to avoid spoilage. Freezing and salting despite being bacteriostatic are not effective in eliminating contaminant and pathogenic microbes. Efficacies of chemical preservatives such as lactic, tartaric, citric acid, hydrogen peroxide and ethanol alone or in combination have been investigated to improve microbiological quality of natural casing. Use of chemical preservatives results in adverse effect on technological properties of casing and problem of toxic residues (Labie, 1987).
128
S.P. Chawla et al. / Food Control 17 (2006) 127–131
Radiation processing is a useful technique to improve microbiological quality and to enhance safety of several food commodities by killing pathogens such as Salmonella, Staphylococcus aureus, Campylobacter, Listeria monocytogenes (Farkas, 1998). Previous studies in our laboratories have shown that irradiation treatment can improve shelf life and microbiological quality of buffalo, chicken and lamb meat stored in chilled state (Kanatt, Paul, DÕsouza, & Thomas, 1997; Naik, Paul, Chawla, Sherikar, & Nair, 1994; Paul, Venugopal, & Nair, 1990). A combination of hurdles can ensure stability and microbial safety of preserved food. The most important hurdles applied in food preservation are temperature (high or low), water activity, acidity, redox potential, preservatives and competitive microflora. Each hurdle implies putting microorganisms in a hostile environment, which inhibits their growth or causes their death (Leistner, 1999, 2000). Reduction of water activity is an effective preservation method of perishable material as growth of many of the spoilage bacteria is retarded due to low water activity. There are many reports on development of intermediate moisture (IM) products having reduced water activity (Wang & Leistner, 1993, 1994). We have reported preparation of safe and shelfstable IM ready-to-serve meat products using a combination of hurdles, i.e. reduced water activity, vacuum packaging and gamma-irradiation (Kanatt, Chawla, Chander, & Bongirwar, 2002). Water activity of these products was adjusted to less than 0.85. Vacuum packaging and gamma-irradiation took care of oxidation and microbial contamination, respectively. As for the water activity employed in the study, it was envisaged that Clostridium botulinum cannot grow and Staphylococcus spp. do not produce enterotoxin (Leistner, 1992). These presumptions were confirmed by inoculated pack studies using C. sporogenes and S. aureus (Chawla & Chander, 2004). In the present study we report a process for the preparation of safe and shelf-stable lamb casing using a combination of reduced water activity and gamma-irradiation treatment. Microbiological, textural and sensory qualities of sausages prepared from these radiation-processed casing were investigated.
2. Materials and methods Fresh intestines of lamb (size 18–20) were procured from a local casing manufacturer (three hanks). They were washed thoroughly with potable water, 10% (w/ w) sodium chloride (food grade table salt) was added and packed in polyethylene bags. The packs were subjected to gamma irradiation at ambient temperature at a dose rate of 5.0 kGy h 1 in a Gamma cell 5000 (Board of Radiation and Isotopes Technology, India) with a 60 Co source. The samples received minimal doses of 5
and 10 kGy. Dosimetry was performed as reported earlier (Kanatt et al., 2002). Three independent experiments were performed. Each hank was used for a separate experiment in order to minimize variation. Water activity of the natural casings before and after the treatment with salts was determined using an Aqua LabCX2T water activity meter (Decagon Devices, USA). 2.1. Microbiological analyses Samples from both the irradiated and the non-irradiated lots were analyzed immediately after irradiation and subsequently at regular intervals during storage at ambient temperature. Samples (10 g) in duplicate were aseptically homogenised for 2 min in a sterile stomacher bag containing 90 ml of sterile saline using stomacher 400 Lab Blender (Seward Medical, UK). Serial dilutions of the homogenate were prepared. To determine spore count vegetative cells were killed by heat treatment at 80 ± 2 °C for 10 min. Colony-forming units were determined using appropriate media. All microbiological media were procured from HiMedia Laboratories, India. The plate count agar was used for the determination of aerobic vegetative bacteria and spores. Baird Parker agar, sodium sulphite polymixin sulphadiazine (SPS) agar, violet red bile agar and potato dextrose agar (PDA) were respectively used for determination of Staphylococcus, spores of sulphite reducing clostridia, coliforms and molds. (Chawla & Chander, 2004; Kanatt et al., 2002). Plates other than PDA were incubated at 37 °C for 48 h. PDA plates were incubated at ambient temperature for 7 days. SPS agar plates were incubated in anaerobic conditions in an Anaerobic Jar (HiMedia, India). 2.2. Preparation of sausages Sausages were prepared by using non-irradiated and irradiated lamb casing on day of irradiation and after 30 days of storage at ambient temperature. 2.3. Sensory analyses A panel of 6–8 staff members analysed the sausages. Panelists were asked to rate the samples as ‘‘acceptable’’ or ‘‘non-acceptable’’ on the basis of appearance, odor, flavour and taste on 10 point scale, where 10 corresponded to a product of highest quality and 0 corresponds to poor quality of product. Scores of 6 and above were considered acceptable. 2.4. Mechanical properties Puncture force required for the sausage prepared using non-irradiated and irradiated casing was deter-
S.P. Chawla et al. / Food Control 17 (2006) 127–131
mined using Instron Universal Testing Machine with cylindrical probe (Bhusan & Thomas, 1998). 2.5. Statistical analysis All results given in figures are mean ± standard deviation. Differences between the variables were tested for significance by one-way ANOVA with TurkeyÕs post test using GraphPad InStat version 3.05 for window 95, GraphPad Software, San Diego California USA www.graphpad.com. Differences at p < 0.05 were considered to be significant.
3. Results and discussion Fresh untreated natural casing had a water activity of 0.95 ± 0.02. Water activity of casing was reduced to 0.80 ± 0.02 by addition of 10% salt. The results of microbiological analyses are compiled in Table 1. The fresh natural casing had total viable counts (TVC) of more than 106 CFU/g. The counts of potentially pathogenic bacteria such as staphylococci and coliforms were more than 104 CFU/g and 102 CFU/g respectively. Both the aerobic spore and anaerobic spore (sulphite reducing clostridia) counts were found to be 103 CFU/g. The untreated casing spoiled within few hours at ambient temperature. As can be seen, the salt treatment resulted in some reduction in counts of vegetative cells but had no effect on spore count. Radiation treatment resulted in a dose dependent reduction in counts of these mi-
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crobes. A dose of 5 kGy was sufficient to eliminate staphylococci, coliforms and molds but not the bacterial spores. TVC reduced by about three log cycles in samples irradiated at 5 kGy. All vegetative bacterial cells, mold and bacterial spores were eliminated at 10 kGy (Table 1). These results suggested that non-irradiated natural casing were heavily contaminated with various bacteria including, potentially pathogenic bacteria such as staphylococci and coliforms, spores and molds and radiation processing could significantly improve their microbiological quality. Our results are in agreement with Byun et al. (2001) who reported that washed natural casing had total microbial load, entrococci and coliforms in the range of 3–5 log CFU/g. Similarly Trigo and Fraqueza (1998) also reported high level of aerobic counts, coliforms, enterocci and spores in both fresh and dried natural casing. Improvement of microbial quality of fresh natural casing by radiation treatment has also been reported earlier (Byun et al., 2001; Trigo & Fraqueza, 1998). In the present study a 5 kGy dose was required to reduce bacterial counts by three log cycles. Radiation sensitivity of bacteria is affected by number of factors such as water activity, composition, irradiation temperature, presence of oxygen (Mendonca, 2002). Higher resistance of bacterial cells in salted natural casing could be attributed to lower water activity. In the presence of water cell injury during irradiation is due to both direct damage to cell DNA and indirect damage due to the reactivity of the radiolytic products with cell components (Grecz, Rowley, & Matsuyama, 1983). Also, in complex systems
Table 1 Microbiological analyses of salted natural casings from sheep during storage at ambient temperature Treatment
Counts log CFU/gm for storage period Analysis
Initial
15 days
30 days
60 days
90 days
Non-irradiated
TVC SPC SRCC SC CC FC
5.5–5.9 3.5–3.7 3.0–3.9 2.6–3.7 1.9–2.3 +++
4.4–5.6 3.4–3.7 2.9–3.5 2.6–3.3 NVC +++
4.7–5.3 3.2–4.0 3.2–4.0 2.3–2.6 NVC ++
4.7–5.1 3.4–3.7 2.3–2.7 2.0–2.6 NVC ++
3.79–4.04 3.26–3.58 2.90–3.08 2.48–2.90 NVC ++
Irradiated (5 kGy)
TVC SPC SRCC SC CC FC
2.3–3.5 2.4–2.8 2.4–2.6 NVC NVC NVC
2.6–3.8 2.0–2.8 2.0–2.5 NVC NVC NVC
2.7–3.9 2.3–2.7 2.0–2.3 NVC NVC NVC
2.7–4.0 2.5–3.0 2.0–2.7 NVC NVC NVC
2.14–2.57 2.84–3.23 2.57–2.70 NVC NVC NVC
Irradiated (10 kGy)
TVC SPC SRCC SC CC FC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
NVC NVC NVC NVC NVC NVC
TVC—total viable count; SPC—spore count; SRCSC—sulphite reducing clostridia spore count; SC—staphylococcus count; CC—coliform count, FC—fungal count, (+) growth of mold on PDA, NVC—no viable count detected. Fresh washed natural casing had TVC of 6.3, SPC of 3.1, SRCSC of 2.9, SC of 4.5, and CC of 2.4 log CFU/g. Results shown are of three independent experiments involving intestines of different animals.
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some of the constituents such as proteins are believed to compete with the bacterial cell for interaction with free radicals thereby reducing the net effect of radiation damage and making the organism more resistant (Urbain, 1986). The viable counts of vegetative cells, Staphylococcus and coliforms decreased by about 1–2 log in the case of non-irradiated samples during the storage period of three months. This could be attributed to the fact that the presence of salt disturbs homeostasis (internal equilibrium) of bacterium and it utilizes every possible repair mechanism in order to overcome the hostile environment. In the process of overcoming the hostile environment, the bacterium uses all its energy and dies; this eventually leads to reduction in cell viability (Leistner, 2000). In this case the main stress is reduced water activity. The most of bacteria fail to grow at water activity below 0.85 (Ledward, 1981). However, each bacterial species has different capability to overcome a particular stress. Staphylococcus is one of the most resistant bacterial species capable of tolerating low water activity. Maximum water activity that inhibits growth of S. aureus in IM pork during storage is reported to be 0.88 (Plitman, Park, & Sinskey, 1973). S. aureus was able to grow at water activity higher than 0.90 in mutton kababs (Kanatt et al., 2002) as well as in traditional Korean semi-dried seafood Kwamegi (Chawla et al., 2003). However, it failed to multiply when water activity was less than 0.85 (Chawla & Chander, 2004). The spore counts of the samples remained constant during storage. In irradiated samples no viable cells of Staphylococcus or coliforms were detected during the study period. However, spores were detected in the samples irradiated at 5 kGy. Molds were observed in non-irradiated samples during storage and irradiation treatment was required to inactivate molds. Growth of mold in IM foods having water activity above 0.75 has been reported (Labuza, Cassil, & Sinskey, 1972). We have also reported mold growth in IM mutton kababs (having water activity 0.85 ± 0.02) when stored at ambient temperature (Chawla & Chander, 2004). Counts of spores of aerobic bacteria and anaerobic sulphite reducing clostridia remained constant during storage at ambient temperature. Results confirm the fact that spores are much more resistant in their ability to survive in adverse conditions, i.e. reduced water activity. Spores however failed to germinate due to low water activity of the casing. In the irradiated samples there was no significant change in bacterial counts during storage, suggesting failure of radiation damaged cells to recover during storage. Bacterial spores were detected in samples irradiated at 5 kGy, whereas, no viable spores were observed in samples irradiated at 10 kGy. The results of these storage studies indicated that the natural casing with reduced water activity were not
favourable for multiplication of the organisms. Results suggest bacteriostatic effect of the hurdle employed. However, Staphylococcus and bacterial spores survived during storage suggesting that additional hurdles have to be employed to eliminate these microbes. In inoculum packed studies with meat products with reduced water activity, S. aureus cells and C. sporogenus spores survived during storage at ambient temperature (Chawla & Chander, 2004). Sensory analyses of sausages prepared from natural casing on day of irradiation and after 30 days of storage at ambient temperature were carried out. Appearance, odor, flavour, and overall acceptance scores of sausages prepared using irradiated casings were comparable with that prepared from non-irradiated casings at both the analyses points. Results of sensory analyses immediately after treatment are presented in Fig. 1. Similar observations were made with irradiated IM meat products, where acceptability of products was not affected by irradiation treatment (Kanatt et al., 2002). Textural analyses of sausages prepared from natural casing on day of irradiation and after 30 days of storage at ambient temperature were conducted. There was no significant difference in the puncture strength of sausages prepared using irradiated casing were comparable with that prepared from non-irradiated casing on day of irradiation and after 30 days of storage at ambient temperature. Results of puncture strength of sausages prepared from natural casing after 30 days of irradiation treatment are presented in Fig. 2. These results are in contrast to that of Byun et al. (2001), who reported reduction in shear force in sausages prepared using irradiated casing. These differences could be attributed to differences the type of casings used and irradiation conditions in both the studies. Currently the natural casings are stored either highly salted or frozen and utilized as early as possible in order to avoid spoilage. Our results have shown that microbiological quality of natural casing used by meat
10
Non-irradiated 5 kGy 10 kGy
8
Sensory scores
130
6
4
2
0 Color
Odour
Texture
Overall acceptability
Fig. 1. Sensory qualities of sausages prepared from variously treated natural casings. Results are mean ± SD of eight penal members.
S.P. Chawla et al. / Food Control 17 (2006) 127–131
Puncture strength (kg)
2.0
Hank 1 Hank 2 Hank 3
1.5
1.0
0.5
0.0
Non-irradiated
5 kGy
10 kGy
Fig. 2. Puncture strength of sausages prepared from variously treated natural casings. Results are mean ± SD of eight sausages prepared from same treatment.
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