Food Protection Trends - September/October 2016 - 350

Table 1. Spoilage microorganisms inoculated onto sausages prior to cold-fill pickling Spoilage Microorganism Lactic Acid Bacteria (LAB) Yeast Mold Genus/Species ATCC1/ID Isolation Source Lactobacillus curvatus Lactobacillus curvatus Lactobacillus sakei Debaryomyces hansenii Debaryomyces hansenii Candida zeylanoides Candida zeylanoides Cladosporium cladosporioides Cladosporium cladosporioides Penicillium nalgiovense Penicillium nalgiovense 51436 PTA5150 15521 90624 10619 26318 24745 60549 11275 96457 96460 fermented sausage Kimchi (fermented Korean dish) moto (sake starter culture) salty pickle horse meat sausage Spanish sausage grape must frozen lamb carcass wheat bread mold-ripened sausage mold-ripened salami American Type Culture Collection (ATCC); Manassas, VA. 1 -80ºC until future use. Working stocks were maintained on MRS agar (Difco) and stored at 4ºC. To prepare the separate LAB inoculum, frozen stocks were transferred twice into fresh MRS broth and streaked onto MRS agar, under the same incubation conditions already described. Single colonies of each LAB were isolated and transferred into 10 ml of fresh MRS broth. Following incubation, 10 ml of each culture were transferred to 990 ml of fresh MRS broth and incubated for an additional 24 h at the same temperature and atmospheric conditions as previously described to obtain a cell concentration of ~8 log10 CFU/ml. Each single LAB inoculum was prepared in duplicate, providing a total inoculum volume of 6 liters. Two liters of each LAB culture were mixed in a sterilized metal container under a biological safety hood to produce a homogenous LAB cocktail (6 liters total) for the sausage inoculation immersion bath as described previously (22, 26). This procedure was repeated in the same manner for each of the 3 replications of the challenge study. Yeast cultures Yeast cultures were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Upon receipt, yeast cultures were transferred into fresh yeast mold (YM) broth (Difco, Sparks, MD) and incubated aerobically at 25ºC for 72 h, following ATCC recommendations. Yeast cultures were then re-suspended in YM broth containing 10% glycerol (VWR, Radnor, PA), and stored at -80ºC. To create working stocks, the yeast freezer stock cultures were streaked onto Malt Extract Agar (MEA; Difco, Santa Maria, CA) slants in sterile, screw top test tubes, incubated at 25ºC for 72 h, and stored at 4ºC until use. 350 Food Protection Trends September/October To prepare the yeast inoculum, single colonies of each yeast strain were individually transferred with a sterile 1 µl loop from MEA slants to 10 ml of fresh YM broth and allowed to grow aerobically at 25ºC for 24 h. Incubated yeast cultures (10 ml) were transferred to 490 ml of fresh YM broth and incubated aerobically at 25ºC for an additional 72 h to obtain a cell concentration of ~6 log10 CFU/ml for each of the yeast cultures. Five hundred ml of each of the four yeast cultures were mixed in a sterilized metal container under a biological safety hood to produce a homogenous yeast cocktail (2 liters total) for the sausage inoculation immersion bath. This procedure was repeated in the same manner for each of the 3 replications of the challenge study. Mold cultures Mold cultures were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Each mold culture was transferred to fresh YM broth and incubated aerobically at 25ºC for 96 h, following ATCC recommendations. Molds were then re-suspended in YM broth containing 10% glycerol (VWR, Radnor, PA), and stored at -80ºC for future use. Cultures were transferred from freezer stocks, using a sterile 10 µl loop, to fresh MEA slants prepared in 125-ml glass bottles, by spreading the inoculated loop across the agar slant surface. Agar slants were then incubated aerobically at 25ºC for 96 h and stored at 4ºC until use. To prepare the mold inoculum, 50 ml of sterile 2% (wt/ vol) buffered peptone water (BPW; HiMedia, Mumbai, India) with Tween 80 (Acros Organics, Geel, Belgium) at 0.03% (wt/vol) was poured into each MEA bottle slant, and a sterile 10 µl loop was used to scrape off and collect the mold spores for transfer into the BPW. Tween 80 was added to the

Table of Contents for the Digital Edition of Food Protection Trends - September/October 2016