Food Protection Trends - September/October 2016 - 349

methods, including placing product in an acidic environment (reducing the pH), using salt to decrease the water activity (aw), and sealing the storage container to decrease the oxygen level. Leistner and Gorris referred to the use of multiple processing factors to inhibit microorganisms in food as "hurdle technology" (15). The use of multiple, sub-lethal factors to kill and inhibit unwanted microorganisms works by disturbing the homeostasis of microorganisms on multiple fronts simultaneously, resulting in the destruction of microorganisms and allowing for the preservation of the food product (13). Preventing the spoilage of such products is critical to ensure pickled sausages remain of good microbiological quality and are safe for consumption. The preparation of pickled sausage has traditionally used either a cold fill or hot fill process, in which cooked sausages are covered with either a cold or hot brine solution to begin the pickling process. However, hot filling has generally resulted in significant quality defects, including cloudiness, turbidity, or discoloration of the brine, which make the product less appealing to consumers. To avoid these quality defects, many commercial pickled sausage processors may utilize a cold fill process, in which the brine solution is kept at room temperature prior to pickling. The pickling of sausages has been regarded as a safe method of meat preservation by federal regulatory authorities. However, recent incidences of spoilage contamination involving pickled meat products have prompted the United States Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) to reconsider the microbiological stability of pickled sausages subjected to a cold fill pickling process (17). Current U.S. regulations addressing acidified foods (21 CFR parts 114 and 120) (32, 33) are mainly focused on canned food products intended to ensure processors properly address the potential growth of Clostridium botulinum. Title 21 CFR part 114 (32) states that "...the pH must be maintained at or below 4.6..." with no specification to the type or concentration of the acid, while title 21 CFR part 120 (33) states that "...manufacturers of acidified fruit and vegetable products must demonstrate that their process achieves a 5-log reduction in appropriate acid-tolerant pathogenic bacteria, as validation of product safety." It does not appear that either of these policies has been applied to pickled meat products or used to address issues with spoilage. Current USDA-FSIS policies require processors of pickled meat products to demonstrate stability and safety by monitoring and recording several quantitative parameters, including pH, aw, salt concentration, temperature and overall formulations. To date, no specific scientific validation has been performed to demonstrate which intrinsic and extrinsic factors are important to ensure the stability and safety of pickled meat products. The lack of scientific validation has generated additional concerns from Federal regulators about post-processing contamination, since it is unknown if a cold fill process might allow for the survival and growth of spoilage microorganisms. Research on the microflora categorized as spoilage microorganisms associated with acidified meat products is limited. To our knowledge, no scientific literature exists that addresses either: (1) the microbial stability of pickled meat products or (2) the growth or survival of spoilage microorganisms associated with acidified meat and sausage products using a cold fill pickling process. Recent studies have demonstrated the effectiveness of pickling processes using various organic acids to reduce the growth and survival of pathogenic bacteria in fruits and vegetables (1, 13), cucumber purees, vegetable brines and pickles (5, 6, 7) and hard cooked, pickled eggs (22, 26, 29). Research on pickled eggs may be most relevant to pickled meat products, because of their similar protein content and susceptibility to microbial contamination, although these studies did not address spoilage microorganisms such as LAB, yeasts, and molds. Therefore, the purpose of this study was to validate the efficacy of a cold fill pickling process of cooked sausages to prevent the growth and survival of LAB, yeasts, and molds associated with ready-to-eat (RTE) sausage products. MATERIALS AND METHODS In this study, a cooked, smoked, and cured sausage product was placed into an acidified brine (5% acetic acid (wt/vol); 5% salt (NaCl, wt/wt); 22-23ºC; pH ~2.70) solution and held at room temperature for 28 days. Table 1 highlights the spoilage microorganisms chosen for the challenge study, based on their prominence in fresh and RTE meat products, relevant isolation sources, and availability. Antagonism of select lactic acid bacteria and fungi Preliminary studies (data not shown) indicated that the lactic acid bacteria (LAB) cultures (Table 1) identified for use in this challenge study inhibited selected mold cultures. All three LAB cultures inhibited both Penicillium nalgiovense strains; Lactobacillus sakei ATCC 15521 inhibited both Cladosporium cladosporioides strains; and L. curvatus 51436 inhibited C. cladosporioides 11275. No inhibition occurred between the selected yeast and mold cultures. Therefore, the inoculation and spoilage challenge study of sausages by fungi and LAB cultures were performed separately. Preparation of selected lactic acid bacteria cultures and challenges study inoculum LAB cultures were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Lyophilized cultures were transferred into fresh de Man, Rogosa and Sharpe (MRS) broth (Difco, Sparks, MD). Both L. curvatus cultures were incubated aerobically at 30ºC for 48 h, while L. sakei was incubated at 30ºC in a microaerophilic environment (5.0% O2, 10% CO2, 85% N2) in a CO2 incubator (VWR International, West Chester, PA), following ATCC recommendations. Colonies were then re-suspended in fresh MRS broth containing 10% glycerol (VWR, Radnor, PA) and stored at September/October Food Protection Trends 349

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