Nonstarter Bacterial Communities in Aged Cheddar Cheese: Patterns on Two Timescales.

The aim of this study was to investigate the temporal stability of microbial contamination during cheddar cheese production by examining patterns of nonstarter bacteria in 60-day aged cheddar collected from the start and end of 30 consecutive production days. Further, we explored the source of these temporal microbial variations by comparing microbial communities in the aged cheese to those on food contact surfaces from a piece of cheesemaking equipment previously identified as a major source of nonstarter bacteria in the same processing environment. 16S rRNA metabarcoding and culture-based sequencing methods identified two Streptococcus sequence variants significantly associated with the end of the production day in both the aged cheese and the cheese processing environment. Closer inspection of these sequence variants in the aged cheese over the 40-day sampling period revealed sinusoidal-like fluctuations in their relative ratios, which appeared to coincide with the Lactococcus starter rotation schedule. These results demonstrate that the microbial composition of finished cheese can vary according to the timing of processing within a production day. Further, our results demonstrate that time-of-day microbial differences in cheese can result from bacterial growth on food contact surfaces and that the composition of these microbial differences is subject to change day-to-day and may be linked to routine changes in the Lactococcus starter culture. IMPORTANCE Long production schedules used in modern cheese manufacturing can create circumstances that support the growth of microorganisms in the cheese processing environment. This work demonstrates that this growth can lead to significant changes in the microbial quality of aged cheese produced later in the production day. Further, we demonstrate that the dominant bacteria associated with these microbial changes throughout production are subject to change between days and might be influenced by specific cheese manufacturing practices. These findings improve understanding of microbial contamination patterns in modern food manufacturing facilities, thereby improving our ability to develop strategies to minimize quality losses due to microbial spoilage.

Population dynamics of coliforms in a commercial Cheddar cheese production facility.

The detection of coliforms in young cheese is a potential indication of undesirable microbial growth within the processing environment. The aim of this study was to investigate sources and conditions that lead to the intermittent detection of coliforms (1-3 log cfu/g) in young Cheddar cheese at a single commercial facility. Analysis of historical production data, in combination with iterative investigative sampling events, was performed to determine coliform levels in milk, whey, curd, and surfaces at the beginning, middle, and end of the production day. After sanitation, conveyor belt pieces from the draining and matting conveyor (DMC) were collected and evaluated for bacterial survivors using culture-based methods and scanning electron microscopy. Production data analysis indicated that cheese produced later in the production day (≥16 h) was significantly more likely to test positive for coliforms than cheese made earlier in the production day (<12 h). Enumeration of coliforms in raw and heat-treated milk demonstrated that the subpasteurization thermal treatment (67-70°C, 26-28 s) was effective at reducing, but not eliminating, coliforms. Repeated sampling identified the DMC, particularly the drain belt and belt 1, as a critical area that supported coliform growth during the production day. Coliform levels in whey entering the weir maintained a level of <1 cfu/mL throughout production; however, coliform levels in whey below the drain belt increased from <1 cfu/mL at midday (8 h) to 5.04 log cfu/mL by the end of the production day (~18 h). Routine sanitation inside the DMC resulted in undetectable coliform levels on easily accessible surfaces. However, enrichment and scanning electron microscopy of belt sections revealed pockets of viable coliforms and other bacteria in cracks and defects in conveyor belts, indicating that sanitation did not eliminate all viable bacteria. Low levels of coliforms are present in heat-treated milk and survive sanitation in the DMC and could serve as the initial seed for high levels of coliforms at the end of the production day.

Growth potential and biofilm development of nonstarter bacteria on surfaces exposed to a continuous whey stream.

Commercial Cheddar cheese production uses an automated, continuous production system that provides favorable conditions for specific undesirable bacterial subpopulations in certain sections of the processing system. The draining and matting conveyor (DMC) is a large, fully enclosed series of conveyor belts that separates curd and whey on the first drain belt and supports the cheddaring process in subsequent sections. In a previous study, we demonstrated that coliforms increase in the draining section of the DMC (pH 6.0-6.3, 36°C) over a typical 18-h production shift and can lead to detectable coliforms in finished cheese. Sampling at the commercial plant indicated 2 sources of very low levels of coliforms: (1) subpasteurized whey and curd entering the DMC and (2) surfaces in the DMC after sanitation. Mitigation of these sources would require different approaches. The aim of this study was to investigate whether naturally low levels of coliforms in whey could increase in the bulk liquid and attach to different surface materials within 18 h. A laboratory-scale system was created to mimic the conditions of the initial draining section of the DMC and consisted of single-pass, naturally contaminated whey (pH 6.3, 35°C) flowing through a bioreactor (1.11 L/h) containing coupons of surface types found in the DMC (stainless steel and polypropylene). Whey inside the bioreactor chamber and surface coupons were enumerated for bacterial subpopulations on selective media for planktonic and attached bacteria, respectively, at 0, 12, 15, and 18 h. Bacterial isolates were identified by 16S rDNA sequencing. Nonstarter bacteria present in the whey at 0 h included coliforms (Enterobacter), Pseudomonas, and Acinetobacter (0.80, 2.55, and 2.32 log cfu/mL, respectively), with each increasing significantly in whey (6.18, 7.00, and 5.89 log cfu/mL) and on coupons (5.20, 6.85, and 5.29 log cfu/cm2, respectively) after 18 h in the continuous flowing system. Scanning electron microscopy confirmed bacterial attachment on both surfaces, with early biofilm development evident on polypropylene coupons by 18 h. Results from this laboratory-scale study demonstrated that naturally low levels of coliforms entering the DMC in the whey could replicate within the conditions of the draining section of the DMC to the levels found in the commercial production environment.

Bile Salt-Stimulated Lipase Activity in Donor Breast Milk Influenced by Pasteurization Techniques.

Breast milk contains bile salt-stimulated lipase (BSSL), which significantly increases the fat digestion capacity of newborns who have limited pancreatic lipase secretion in the first few months after birth. Problematically, Holder pasteurization used in non-profit milk banks to ensure the microbiological safety of donor milk for infants, particularly preterm infants (<37 weeks gestation age), destroys milk BSSL, thus limiting infant fat absorption capacity. Alternative strategies are needed to ensure the safety of donor milk while preserving BSSL activity. Three alternative pasteurization techniques-high-pressure processing (HPP, 550 MPa, 5 min), gamma cell irradiation (IR, 2.5 Mrads) and UV-C (254 nm, 0-33,000 J/L)-were compared with Holder pasteurization (low-temperature long-time, LTLT, 62.5°C, 30 min) for retention of BSSL activity in donor breast milk. As the time required for donor milk pasteurization by UV-C in published methods was not clear, donor breast milk was spiked with seven common bacterial strains and treated by UV-C for variable time periods and the minimum UV-C dosage required to achieve a 5-log10 reduction of CFU/mL was determined. Eight thousand two hundred fifty J/L of UV-C exposure was sufficient to achieve 5-log10 reduction of each of bacterial targets, including Bacillus and Paenibacillus spores. The retention of BSSL activity was highest after HPP (retaining 62% of the untreated milk BSSL activity), followed by UV-C (16,500 J/L), IR and LTLT (35, 29, and 0.3% retention, respectively). HPP was an effective alternative to pasteurize milk with improved retention of BSSL activity compared to Holder pasteurization. Future work should investigate the effect of alternative pasteurization techniques on the entire array of bioactive components in donor breast milk and how these changes affect preterm infant health outcomes. Implementation of HPP technique at milk banks could improve donor milk-fed infant fat absorption and growth.

Application of Sample6 DETECT™ HT/L Kit for the Detection of Listeria in Mixed Leafy Greens.

BACKGROUND: Early and accurate detection of Listeria in foods is vital. Current methods require 24 h enrichment for detection. OBJECTIVE: This study aimed to demonstrate enhanced early detection of Listeria in mixed leafy greens using Sample6 DETECT™ HT/L, a phage-based detection system. METHODS: A method comparison between the reference method (U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10) for the detection of Listeria spp. and the Sample6 DETECT HT/L using a new proprietary R2 Medium was performed in mixed leafy greens. RESULTS: Using the R2 Medium enrichment with Sample6 DETECT HT/L, detection of L. innocua was possible at 12 h (with a centrifugation step), and L. monocytogenes was detected by 18 h, with equivalent performance as the 24 h enrichments using the reference method detection. The Sample6 DETECT HT/L gave an equivalent performance for L. innocua at 15 h as the reference method at 24 h. Detection was accomplished by the addition of reagents in the kit, following the package insert, and reading results in a detection chamber using a 96-well plate reader that detects a fluorescent signal. CONCLUSIONS: Results indicate the new R2 Medium and Sample6 DETECT HT/L allowed for earlier detection of Listeria spp. in mixed leafy greens. HIGHLIGHTS: Sample6 DETECT HT/L with the new R2 Medium allowed the early detection of Listeria spp. and may be applied in other food matrices and environmental samples.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Rapid Detection of Listeria in Ice Cream in 13 Hours Using the Roka Listeria Detection Assay.

Background: Listeria contamination is a major concern in the ice cream industry; therefore, early and accurate detection is vital. Current detection methods require about a 24 h enrichment period for detection. Objective: Enhance the early detection of Listeria in ice cream using the highly sensitive isothermal ribosomal RNA-based Roka/Atlas Listeria Detection Assay. Methods: The R2 Medium was developed for Listeria enrichment by Molecular Epidemiology, Inc. (Seattle, WA). Comparative growth curve studies were performed on the new R2 Medium for Listeria and the currently validated media for the Roka Listeria Detection Assay. Subsequently, a method comparison between the Roka Listeria Detection Assay and the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 reference method on ice cream was carried out. Results: The R2 Medium supports the growth of L. monocytogenes better than Buffered Listeria Enrichment Broth, Demi-Fraser broth, and Modified University of Vermont Broth, as indicated by the faster growth rate of the organism. When used as an enrichment medium in a method comparison study of ice cream, the results showed that R2 Medium-enriched samples tested with the Roka Listeria Detection Assay gave an equivalent performance compared with the 24 h FDA-BAM reference method at 10 and 18 h post-enrichment for Listeria. Conclusions: The results from this study indicate that the new R2 Medium and the highly sensitive Roka Listeria Detection Assay allowed for the rapid detection of Listeria species in ice cream in 13 h. Highlights: The Roka Listeria Detection Assay, in conjunction with a new media formulation (R2 Medium), allowed for the early detection of Listeria in ice cream and may be applied in other food matrixes and environmental samples.