Published: September 12, 2011 r2011 American Chemical Society 10869 dx.doi.org/10.1021/jf201124v | J. Agric. Food Chem. 2011, 59, 10869–10878 ARTICLE pubs.acs.org/JAFC Immobilization of Subtilisin on Polycaprolactam for Antimicrobial Food Packaging Applications Prabhawathi Veluchamy, † Ponnurengam Malliappan Sivakumar, † and Mukesh Doble* Department of Biotechnology, Indian Institute of Technology Madras, Adyar, Chennai 600 036, India ABSTRACT: Subtilisin was immobilized on polycaprolactam and used for food packaging applications to reduce the transference of microorganisms from the packaging material to the packaged food material. The optimized conditions for subtilisin immo- bilization was as follows: pH, 8; temperature, 4 °C; glutaraldehyde, 0.5%; incubation time, 25 h; and subtilisin concentration, 600 μL. The formation of ÀCHdNÀ at 1576 cm À1 in the Fourier transform infrared (FTIR) spectrum confirmed the immobilization. Subtilisin-immobilized polycaprolactam (SIP) exhibited the highest residual activity of 106.67 ( 4.41% and 104.67 ( 0.88% at 40 °C and pH 8 and retained residual activity of 94% at the end of 56 days when compared to 21.33 ( 4.10% in the case of free subtilisin. SIP significantly (p < 0.05) lowered the colony forming units (CFU), dry weight, and protein and carbohydrate contents in bacterial and fungal biofilm. Practical application of the SIP on ham steaks at 4 and 20 °C showed a 2À3 times reduction of Staphylococcus aureus as well as Escherichia coli cells in the range of p < 0.05. KEYWORDS: Polycaprolactam, adhesion, subtilisin, immobilization ’ INTRODUCTION There is an increased demand on easily prepared, minimally processed fresh produce without any foodborne microbial con- tamination. 1 This has intensified the research on antimicrobial packaging technologies. 2 Microbial contamination of food occurs primarily at the sur- face, because of post-processing handling, especially during food packing. The microbes that are adhered to these packages be- come transferred to the packed food. At first, these microbes are bound to the substratum using flagella. 3 After a while, they secrete sticky extracellular polymeric substances (EPS), forming a biofilm matrix embedded by cells. 4 EPS are predominantly composed of polysaccharides, proteins, 5,6 nucleic acids, uronic acids, and humic substances. 7 Polysaccharides are partly responsible for bacterial adhesion and the formation of the biofilm on the surface. 8 EPS serve some of the important functions, including forming and maintaining the microcolony, 9 enabling the bacteria to capture nutrients, 10 facilitating cellÀcell communication, 11 and function- ing as a stabilizer of the biofilm structure and as a barrier against hostile environments. 12,13 Therefore, any substance that disturbs the integrity of EPS will be ideal for preventing biofilm. Effects of coating various surfaces, including cloth and polymer, with antibacterial agents that disturb the biofilm and slime have been reported. 14,15 In the food industry, antibacterial sprays or dips have been reported. However, direct surface application of antibacterial substances onto foods have limited benefits because the active substances are neutralized on contact or diffuse rapidly from the surface into the food mass. 16 Antimicrobial food packaging materials have to extend the lag phase and reduce the growth rate of microorgan- isms, which will extend the shelf life and maintain product quality and safety. 17 This solution appears to lead to the lowest risk to the consumer. 18 Currently, there is a strong interest in the use of renewable and nontoxic supports for immobilization to make the process more ecofriendly. 19 Protease exhibits both antibacterial and antifungal properties. Protease from Bacillus proteolyticus CFR3001 exhibits anti- bacterial activity against Escherichia coli, Listeria monocytogenes, Bacillus cereus, and Yersinia enterocolytica by lysing the cell walls. 20 Protease produced by Pseudomonas aeruginosa M-1001 exhibited antifungal activity against pathogenic fungi, Fusarium solani. 21 The protease subtilisin is widely used in commercial products, for example, in laundry and dishwashing detergents, skin-care ointments, contact-lens cleansers, cosmetics, food processing, and pharmaceuticals. 22 In the food industry, they are used for various applications, including meat tenderization, cheese ripen- ing as a digestive and an animal feed supplement, flavor devel- opment, and milk coagulation. 23 These applications are because of its peptidase and esterase activities. It also exhibits antimicro- bial, antifungal, and antibiofilm characteristics. Polycaprolactam belongs to a class of polymers called poly- amine, which contains amine and carbonyl groups and comes in the trade name, Nylon 6. It is better suited than polyethylene and polypropylene when immobilized with protease. 24 In this study, subtilisin has been immobilized on polycaprolactam and used for antimicrobial food packaging applications. The main drawback of the enzyme in such applications is the loss of its activity and stability during prolonged use. Therefore, an attempt has been made to improve its long-term stability and then test its suitability as an ideal active food wrapper. ’ MATERIALS AND METHODS Materials. Subtilisin, a protease from Bacillus subtilis (EC 3.4.21.62), was purchased from Sigma (St. Louis, MO). Polycaprolactam was Received: June 3, 2011 Revised: August 12, 2011 Accepted: September 8, 2011