2304 JOURNAL OF FOOD SCIENCE—Vol. 67, Nr. 6, 2002 © 2002 Institute of Food Technologists Food Microbiology and Safety JFS: Food Microbiology and Safety Pasteurization of Milk Using Pulsed Electrical Field and Antimicrobials K. SMITH, G.S. MITTAL, AND M.W. GRIFFITHS ABSTRACT: The inactivation of naturally occurring microorganisms in raw skim milk by pulsed electric field (PEF) treatment alone and combined with the antimicrobial agents nisin and lysozyme, added both singly and together, was investigated. A 7.0-log reduction of microorganisms found in raw skim milk was achieved through a combina- tion of PEF treatment (80 kV/cm, 50 pulses), mild heat (52 °C), and the addition of both the natural antimicrobials nisin (38 IU/mL) and lysozyme (1638 IU/mL). The combination of PEF, mild heat, and antimicrobials resulted in a much higher microbial inactivation than the sum of the individual reductions achieved from each treatment alone, indicating synergy. Varying the pH from 6.7 to 5.0 had no effect on microbial inactivation. Keywords: pulsed electrical field (PEF), antimicrobial, milk, pasteurization, nonthermal processing Introduction S EVERAL COUNTRIES REQUIRE MILK TO undergo a mandatory pasteurization at about 72 °C for 16 s using a High Tempera- ture Short Time (HTST) pasteurizer. Al- though thermal processing is effective at eliminating pathogens (Adams and Moss 1995), there is some concern that the sen- sory and nutritional properties of milk may decline because of protein denatur- ation and the loss of vitamins and volatile flavors. In addition, the HTST process is energy-intensive. The desire to produce milk with a fresh-like taste has generated interest in nonthermal processes that of- fer the advantages of low processing tem- peratures, low energy use, and the reten- tion of nutrients and organoleptic qualities, while still inactivating patho- genic microorganisms to levels that do not pose a public health risk. The innovative technology of using a high-voltage pulsed electric field (PEF) for food preservation appears promising, especially when com- bined with other preservation methods. The popularity of minimally processed foods has brought increased attention to preservation through the use of antimicro- bials, which inhibit or destroy the growth of microorganisms. Antimicrobials are pro- duced by animals, plants, and microor- ganisms. Among the most widely investi- gated antimicrobials are nisin and lysozyme. Lactic acid bacteria produce a wide range of antimicrobial proteins known as bacteriocins. Nisin, produced by Lactococcus lactis subsp. lactis is a mem- ber of the class of bacteriocins known as lantibiotics, which contain the amino acid lanthionine. Nisin exhibits inhibitory ac- tivity against gram-positive bacteria, in- cluding L. lactis subsp. lactis, Lactococcus lactis subsp. cremoris, L. bulgaricus, Sta- phylococcus aureus, and Listeria monocyto- genes, and prevents the outgrowth of spores of many Clostridium and Bacillus spp. (Harris and others 1992; Hurst and Hoover 1993; Abee and others 1995). Al- though gram-negative bacteria are nor- mally resistant to nisin, they are sensitized by sublethal injury due to heat treatment (Kalchayanand and others 1992), hydro- static pressure, and electroporation (Kal- chayanand and others 1994). Nisin has also been shown to destabilize the outer membrane vesicles of gram-negative bac- teria (Gao and others 1991). Nisin is heat- stable (Hurst 1981); however, it becomes increasingly ineffective in solutions that approach a neutral pH (Daeschel 1993). The antimicrobial action of nisin is most powerful at pH 6.5 to 6.8 (Luck and Jager 1995), although its stability in this pH range is very poor. Jung and others (1992) investigated the activity of nisin against L. monocytogenes in milk and observed that the efficacy of nisin decreases with in- creasing fat content. They hypothesized that nisin absorbed to milk fat globules, reducing its availability to inhibit cells. Lysozyme, which is an enzyme found in foods of animal origin, occurs naturally in milk at a concentration of approximately 0.13 g/mL (Reiter 1978). Gram-positive bacteria are more susceptible to the action of lysozyme because of the relatively sim- ple structure of their cell wall, which con- tains up to 90% peptidoglycan. Gram-neg- ative bacteria are more resistant to lysozyme because of the smaller amount of peptidoglycan contained in the cell wall. Lysozyme is effective against several gram-positive bacteria, including Micro- coccus, Sarcina, Lactobacillus, and Bacillus, as well as several gram-negative bacteria, such as Salmonella, Pseudomonas, Aero- monas, and Escherichia coli (Proctor and Cunningham 1988). Hughey and Johnson (1987) found lysozyme was able to effec- tively inhibit Clostridium botulinum and L. monocytogenes, while Ibrahim and others (1996) showed that heat denaturation of lysozyme resulted in an increase in activi- ty against gram-negative bacteria such as E. coli. The activity of lysozyme is highest in the pH range of 3 to 7 (Proctor and Cun- ningham 1988). Nisin and lysozyme have both been granted GRAS (generally re- garded as safe) status by the U.S. Food and Drug Administration (Luck and Jager 1995). According to Martin and others (1996), the inactivation of E. coli using PEF is more limited in skim milk than in a buffer solution when exposed to simi- lar treatment conditions of field strength and number of pulses because of the complex composition of skim milk and the presence of proteins. The influence of milk fat on the PEF inactivation of mi- croorganisms is unclear. Reina and oth- ers (1998) observed no significant differ- ences in the inactivation of L. monocytogenes in whole milk, 2% milk, and skim milk. Less than a 1-log differ- ence between the inactivation of E. coli in milk and in phosphate buffer was re- ported by Dutreux and others (2000). However, experiments conducted by Grahl and Markl (1996) indicated that