Investigating the Survival of Microorganisms in Fermented and Dried Meat Products Cured with Various Sources of Nitrite
Open Access
- Author:
- Hunt, Heather
- Graduate Program:
- Animal Science
- Degree:
- Master of Science
- Document Type:
- Master Thesis
- Date of Defense:
- June 26, 2023
- Committee Members:
- Adele Turzillo, Program Head/Chair
Jonathan Alexander Campbell, Thesis Advisor/Co-Advisor
Catherine Nettles Cutter, Committee Member
Edward William Mills, Committee Member - Keywords:
- salami
nitrite
natural nitrite alternatives
cured meat
processed meat
food safety
Salmonella
E. coli
E. coli O157:H7
Listeria monocytogenes - Abstract:
- Salami is a Ready-to-Eat (RTE), cured, fermented, and dried meat product traditionally processed without a thermal lethality step. Previous challenge studies have validated the safety of these products when manufactured with the regulated maximum amount (156 ppm) of in-going sodium nitrite to the raw meat batter. Sodium nitrite is a multifunctional ingredient used in cured, processed meats for its quality (i.e., color, flavor, and antioxidant activity) and food safety attributions to the products. Though most acknowledged as an inhibitor against the germination of Clostridium spp. spores, sodium nitrite has also been documented as an inhibitor against Escherichia coli O157:H7 (EC), Listeria monocytogenes (LM), and Salmonella spp. (S). Despite the vast benefits of nitrite addition to processed meat products, several organizations and agencies have correlated consumption of the ingredient with risk factors for many human cancers. Additionally, consumer demand for clean label products has increased, which has resulted in an increase in the production of meat products manufactured with alternative, natural curing agents. Natural curing agents are sourced from fruits or vegetables that are naturally high and variable in nitrate content. Due to the antimicrobial efficacy of nitrite, concerns have been raised for the safety of meat products manufactured with natural curing alternatives. While there is research that investigates this concern in RTE meat products like deli turkey or boneless ham, there is no published literature that validates these natural alternatives in a RTE, fermented, cured, and dried product manufactured without a thermal lethality step. The primary objective of this study was to determine the fate of EC, LM, and S throughout fermentation, drying, and storage of salami manufactured without a heat treatment and with various sources of nitrite. Preliminary experimentation was conducted prior to beginning two challenge studies. There were two trials in the preliminary experiment. The objective of the first study was to determine the ratio of surrogate organism inocula (mL), E. coli O157:H12 and L. innocua, to pork to achieve at least a 6 log10 CFU/g concentration when organisms were inoculated together and separately. The first trial found that a 6 log10 CFU/g inoculation is achieved when inoculum is added as 1 mL to every 453.6 g of pork. After the completion of trial 1, it was determined necessary to minimize the amount of ingoing liquid from the inoculum while still achieving high levels of pathogen inoculation in the raw meat batter. Therefore, the inoculum was centrifuged to achieve concentrated amounts of the pathogens in the form of a pellet that would be redistributed with a minimum amount of Buffered Peptone Water (BPW) in the raw pork batter. It was unknown, however, what the minimum amount of BPW would be. Therefore, the objective of the second trial was to determine the minimum amount of BPW needed to effectively distribute pellets of mixed cultures of three strains each of EC, LM and S to effectively disperse the pathogen pellets throughout raw, ground pork to achieve at least a 6 log10 CFU/g inoculation of each organism. The results from trial two demonstrated that resuspending the pathogen pellets with 0.1 mL BPW for every 453.6 g of pork achieved at least a 6 log10 log CFU/g inoculation in the meat system. The first challenge study determined the fate of surrogate organisms, E. coli O157:H12 and L. innocua, throughout fermentation and drying of salami manufactured with various sources of commercially available nitrite. Three replications of three treatments were evaluated in this study. Treatments were positive control (purified 6.25% NaNO2; PC), celery powder (CP), and Swiss chard (SC). All treatments were formulated to 156 ppm ingoing nitrite, according to manufacturer recommendations. Surrogate inocula were prepared by individually inoculating single, isolated colonies of the surrogates into 14 mL Tryptic Soy Broth (TSB) which was incubated for 24 h at 36ºC. To prepare salami, pork shoulder butts (IMPS 406) were deboned, cubed (2.54 cm x 2.54 cm), ground (~5mm), vacuum packaged as batches, and stored at ~4ºC overnight until salami manufacturing the following morning. During salami manufacturing, ground pork was combined with surrogate inocula, dry ingredients according to treatment, and a starter culture (Safepro® B-LC 007 starter culture; CHR Hansen; Hoersholm, Denmark) that was suspended in DI water. After distribution of inocula and additional ingredients, raw batter was stuffed into 55 mm fibrous casings, fermented (pH < 5.0) at 24-26ºC and then dried to a target water activity (aw) of ≤ 0.88. Individual salami (n=9) were sampled on days (D) 0, 1, 2, 3, 7, 14, and 28 for surrogate survival, pH, and aw (N=216). Unique comparisons between sampling days within treatment were analyzed using a General Linear Model procedure (SAS OnDemand Version 9.4). Comparisons across treatments within organism on the same day were analyzed using a mixed model procedure in SAS. Treatment, pH, aw, and treatment by sampling day interaction were included in the model as fixed effects. A significance level of P < 0.05 was used to determine significant differences in all analyses. Using the procedures described above, salami achieved a pH of 4.73 ± 0.17 by D3 of fermentation. Neither E. coli O157:H12 nor L. innocua, were significantly impacted by pH (p > 0.05). Salami never achieved the target aw. Ultimate aw of all salami treatments was 0.90 ± 0.4 on D28. L. innocua was found to be significantly impacted by aw (p = 0.0319), whereas E. coli O157:H12 was not (p = 0.0678). Treatment group had a significant impact on both organisms (p < 0.0001). Total reductions from D0 to D28 of E. coli O157:H12 in PC, CP, and SC were 0.52, 1.76, and 0.93 log10 CFU/g respectively. Total reductions from D0 to D28 of L. innocua in PC, CP, and SC were 0.52, 1.94, and 1.51 log10 CFU/g, respectively. The second challenge study determined the fate of three strains each of EC, LM, and S in RTE salami manufactured with various sources of nitrite and without a heat treatment. EC isolates EDL933 (ATCC 43895; ground beef outbreak) Sakai, and PA-2 (Hartzell, et al., 2011), LM serotypes Scott A, 1/2a isolate FSL R2-603 (deli meats outbreak) and 4b isolate H3396 (hot dog outbreak), and S serovars Typhimurium (ATCC 14028; chicken organs), Montevideo isolate SMvo13, and Derby (ATCC 7378; human isolate) were identified for use in this study. Twenty-four hour cultures of each organism were prepared by individually inoculating single, isolated colonies of each pathogen strain into 25 mL of TSB in duplicate which were incubated for 24h at 36ºC. After incubation, cultures were centrifuged (~20ºC for 5 minutes, at 11,000 x g). The pathogen pellets were resuspended within strain with 2.5 mL BPW to be distributed in the raw pork. Three replications of four treatment groups were evaluated in this study: negative control (no nitrite source; NC), PC, SC, and Prosur® Natpre T-10 (dried fruit extract; T-10). The ingoing salt content of NC was adjusted to meet that of PC. PC and SC were formulated to 156 ppm ingoing nitrite. SC and T-10 were utilized according to manufacturer recommendations. Salami were prepared as previously described in the first challenge study. Upon achieving the target water activity, salami were vacuum packaged and stored at ambient temperatures (20 ± 0.003ºC). Salami were evaluated in triplicated for pathogen survival, pH, and aw on days 1, 2, 3, 7, 14, 21, 28, 35, 42, 49, and 118 (n=9; N=432). Results were analyzed using the same procedures as described for the first challenge study. The fixed effects of pH and aw did not have a significant impact on any of the resulting pathogen population differences between treatments(p > 0.05). All salami treatments achieved a pH < 5.0 after the first 24h of fermentation. Additionally, all salami treatments achieved a aw of ≤ 0.88 by the third week of manufacturing. Treatment had a significant impact on all pathogen populations during the study (p < 0.0001). Reductions of EC between D0 (raw batter) and D21 (when salami met the target aw) were 1.33, 2.61, 0.78, and 2.14 log10 CFU/g for NC, PC, SC, and T-10, respectively. LM reductions between D0 and D21 were 1.06, 2.35, 2.57, and 1.19 log10 CFU/g for NC, PC, SC, and T-10, respectively. S reductions between D0 and D21 were 0.58, 2.17, 2.3, and 0.73 log10 CFU/g for NC, PC, SC, and T-10, respectively. All treatments achieved at least a 5 log10 reduction of EC and S by D118. NC was the only treatment group to not achieve a 5 log10 reduction of LM (4.55 log10 CFU/g) by D118. This research was the first to evaluate pathogen survival in non-heat treated salami manufactured with various sources of nitrite and during extended, reduced oxygen, ambient storage. If processors use purified sodium nitrite or Swiss chard at 156 ppm nitrite, they may use this research as scientific validation of a 2 log10 CFU/g reduction of LM and S in pork salami, in combination with a raw material sampling plan, Hazard Analysis and Critical Control Point plan, and Good Manufacturing Practices according to the Blue Ribbon Task Force Option #5.