In-Process Microbial Load Dynamics and Production Environment Microbial Hygiene in the Manufacturing of Low-Processed Vacuum-Packed RTE Pork Bar with Dried Plasma

Abstract

Low-processed ready-to-eat (RTE) meat products are highly vulnerable to microbial contamination, yet data on in-process dynamics remain limited. This study investigated microbial dynamics and environmental hygiene during the production of vacuum-packed RTE pork bars containing dried plasma, with a focus on identifying process-inherent contamination risks. Samples were collected at successive processing stages and from food-contact and non-food-contact surfaces. Process hygiene was assessed using indicator organisms (Aerobic Plate Count, Enterobacteriaceae, lactic acid bacteria, yeast and mold, E. coli, S. aureus counts), while food safety relevance was addressed by monitoring Listeria monocytogenes and Salmonella spp. Microbial counts increased by approximately 1.5–2.3 log CFU/g between early processing steps, indicating that these operations are critical contamination-prone steps. Environmental monitoring revealed contamination hotspots on frequently handled surfaces, highlighting the vulnerability of pre- and post-lethality stages. Despite the baking achieving a mean microbial reduction of ~3 log CFU/g, consistent with effective thermal processing, low-level microbial reappearance during packaging and maturation indicated the potential for post-process contamination. The results demonstrate that production-inherent factors largely drive microbial contamination patterns and may persist even in facilities operating under implemented GHP, GMP, and HACCP-based procedures, highlighting step-specific limitations rather than system failure. By providing empirical data on in-process microbial dynamics, this study supports both scientifically based and risk-based approaches within Food Safety Management Systems, offering transferable insights applicable to similar RTE meat production environments. The findings may assist food business operators in optimising targeted control measures and strengthening risk-based decision-making in low-processed RTE meat production.

1. Introduction

Microbiological studies are a primary focus of food science, particularly in terms of ensuring food safety for human health. A special group of products with respect to microbiological profile is ready-to-eat (RTE) products, which, after purchase, do not require any further processing that affects their microbiological quality. Food business operators (FBOs) must test products during their circulation in the market (durability tests) and, very often, characterise the product-specific ability to support or inhibit the growth of microorganisms of concern (challenge tests). Based on challenge test results, food safety criteria (FSC) are determined, along with their limits, as per Regulation EU 2073/2005 [1].

EFSA reports, particularly the annual EFSA–ECDC summary, provide essential data on microbiological hazards for risk assessment, including up-to-date figures on foodborne outbreaks in the EU. The latest report for 2023 noted a 1.2% decrease in reported foodborne outbreaks compared to the previous year, 2022. However, the number of human poisonings (52,127), hospitalisations (2894), and deaths (65) increased by 7.2%, 4%, and 1.6%, respectively [2]. All the top five bacterial zoonoses—campylobacteriosis, salmonellosis, Shiga-toxin-producing Escherichia coli (STEC) infections, yersiniosis, and listeriosis—showed increases of 4.3%, 16.9%, 30%, 13.5%, and 5.8%, respectively. Listeriosis had the highest rates of reported hospitalisations (96.5%) and deaths (mortality rate of 19.7%) in 2023. A close molecular relationship between the current Listeria monocytogenes isolates tested and those recovered previously from similar food and food production sources, as well as from patients with listeriosis, indicates that foods and their production environments may be a potential source of pathogenic strains for humans [3,4].

Globally, pork is one of the most consumed meats due to its high nutritional value, complete amino acid profile, and ease of digestibility [5,6,7]; however, its consumption has also been linked to outbreaks, accounting for an estimated 525,000 infections, 2900 illnesses, and 82 deaths annually in the United States alone [8].

Within the Polish official control and monitoring program, only 0.3% of the analysed food samples (n = 184,439) tested positive for L. monocytogenes. A significant group of products that did not meet the criteria were RTE meat products. Significantly high limit exceedances were found in RTE products, especially those of meat origin, such as fermented pork sausages [9].

Although raw meat is generally intended to be cooked before consumption, inadequate thermal treatment may not ensure the elimination of L. monocytogenes, thereby posing a significant risk to consumers. Moreover, a high incidence of L. monocytogenes in raw materials significantly increases the risk of contamination of processing plant working surfaces and equipment. In turn, L. monocytogenes may contribute to the formation of very difficult-to-remove biofilms on working surfaces, which can lead to contamination of the final product [10].

The safety of a specific product is primarily influenced by its microflora, the design of production processes, and environmental hygiene monitoring during its manufacturing. Certain monitoring is applied to validate production processes, i.e., to assess whether the given technological parameters of the processes (e.g., temperature inside the product during heat treatment, composition of the curing mixture or smoke, duration of the processes, alternative methods of food preservation) specified in the hazard analysis and assessment of critical control points (HACCP) by a team of specialists actually aim to eliminate microorganisms to safe limits.

According to Regulation 853/2004, FBOs are required to ensure sanitary and technological conditions that minimise the risk of contamination of raw materials and products, as well as minimise the risk of growth and/or survival of pathogenic microorganisms for public health [11]. Large food companies have the resources to establish an effective self-monitoring sampling plan and employ a well-educated quality management board [12,13,14,15,16].

FBOs must plan the technological processes used to eliminate or reduce microbial hazards to acceptable safety levels. Noncompliance with hygiene rules, particularly in food-contact surface washing and disinfection, poses a significant threat to food safety. Monitoring microbiological parameters via swabs from the production environment is crucial for assessing the effectiveness of sanitary procedures, which directly impacts the safety of RTE food production.

The basic division of swabs from production environment surfaces is food-contact surfaces (FCSs) and non-food-contact surfaces (N-FCSs). The FCSs include transport containers, knives, production table surfaces, the interior of cutting, grinding, mixing, and packaging machines, and smoking trolleys primarily. The N-FCSs include the surfaces of floors, walls, and ceilings, ventilation grilles, control and switching panel buttons, and drainage grilles. Environmental hygiene monitoring is typically performed regularly, often weekly and across different production shifts, to capture variability in hygienic conditions. Food safety supervisory authorities in each country should conduct random checks of microbiological parameters in products and on surfaces.

The most used tests to verify the effectiveness and correctness of sanitary and hygiene programs are microbiological tests. Their advantage over audits is the absence of deficiencies, such as missing monitoring documentation or a lack of trained supervisory personnel in the plant. The most frequently used environmental hygiene monitoring included examination of total aerobic count (APC), E. coli, and Enterobacteriaceae. Environmental hygiene monitoring is performed relatively frequently for yeast and mould, as well as for L. monocytogenes and Staphylococcus aureus. The least frequently performed microbiological tests to verify sanitary procedures are for Salmonella spp. and Campylobacter spp. [17].

From a One Health perspective, persistence of foodborne pathogens such as L. monocytogenes in RTE meat processing environments represents a critical interface between food production, environmental contamination, and human health. Even low-level contamination of RTE products may have severe public health consequences, particularly for vulnerable populations. Therefore, understanding microbial dynamics along production lines is essential for risk mitigation beyond regulatory compliance.

Beyond regulatory compliance, increasing attention has been directed toward understanding microbial ecology within RTE meat processing environments, where resident microbiota, process hygiene indicators, and pathogenic species may interact, persist, or re-emerge despite standard sanitation measures. Although official monitoring programs focus on compliance testing, fewer studies have examined stepwise microbial dynamics along low-processed RTE meat production lines, particularly in relation to post-thermal contamination and process hygiene indicators.

The primary objective of this study was to characterize microbial dynamics along a low-processed RTE meat production line under industrial processing conditions. Secondary objectives included (i) the assessment of process and environmental hygiene using selected microbial indicator groups, (ii) the detection and molecular confirmation of L. monocytogenes at successive processing stages, and (iii) the identification of potential points of microbial persistence or recontamination. To achieve these aims, microbiological analyses were performed on product and environmental samples collected at defined processing steps, combined with molecular confirmation of selected isolates [18,19,20].

2. Materials and Methods

2.1. In-Process Sampling Strategy

The production plant supplying the samples operates in the eastern part of the Masovian Voivodeship and processes approximately 10 tons of meat products per week.

The implemented Food Safety Management Systems (FSMS) included Good Hygiene Practice (GHP), Good Manufacturing Practice (GMP), and the HACCP system. Thermal processing was conducted according to the facility’s HACCP plan, with defined time–temperature parameters. The heat treatment processes included baking (external temperature of 130 °C for 30 min in a combi steamer with a smoke generator) and hot smoking (external temperature of 90 °C for 30 min) sequentially.

The final product was an RTE high-protein pork bar with added dried blood plasma. The bar was finely minced, baked, smoked, dried, and vacuum-packed. There was no gluten or casing content. Ingredients included pork (ham), salt, pepper, colour stabiliser at a concentration of 5 g/kg of the meat filling (composed of lactate, sodium acetate, dioxide; SOVIT, Warsaw, Poland), and dried blood plasma (AProPork™, Essentia Protein Solution, Gråsten, Denmark).

The colour stabiliser used in the formulation contained sodium lactate and sodium acetate. While these compounds are recognised for their antimicrobial properties, this study applied them at minimal technological concentrations intended solely for colour stabilisation and pH buffering rather than for primary preservation. This approach aligns with the clean-label objective and multi-hurdle technology system of the RTE pork bar developed.

Nutritional value per 100 g was energy value 726 kJ (173.6 kcal), fat (including saturated fatty acids) 4 g (1.56 g), carbohydrates (including sugars) < 0.5 g (<0.1 g), protein 33.9 g, salt 3.05 g. The manufacturer stored the product at 0–6 °C, required consumption within 24 h after opening, and targeted consumers on weight-reducing, high-protein diets, particularly those engaged in intensive workouts. The production of RTE bars required 18 h and comprised 10 steps, as shown in Figure 1.

Figure 1. Ten-step flow diagram of RTE bar production (ROP: reduced-oxygen packaging) [21].

During the 18 h production process, 10 meat samples (raw material, semi-products, or final product)—hereafter referred to as in-process samples—were collected, as shown in Table 1.

Table 1. Sampling points and sample types in the RTE bar production process.

Sampling Point	Sample Type
	In-Process Sample	Environmental Sample
Acceptance of raw material	Pork ham cooled to 6 °C	Swabs from the production table, transport box, and floor
Grinding	Minced ham meat	Swabs from the grinder and floor
Mixing	Minced meat after seasoning	Swabs from the mixer and sewer grate
Shaping	Formed bars from a stuffer	Swabs from the stuffer and production table
Settling	Bars settled on a smoking trolley	Swabs from the smoking trolley and workers’ hands
Baking	Bars after baking	No samples
Smoking	Bars after smoking	No samples
Chilling	Bars after chilling	Swabs from the walls and the sewer grate
ROP (vacuum)	Bars after packaging	Swabs from the table, sewer grate and packaging machine

Immediately after the respective process step, a sample (raw material, semi-finished products, or final product) was aseptically collected and stored at 4 °C [22]. After completing the RTE bar production, all samples were transported to the laboratory for immediate qualitative and quantitative microbiological analyses.

To ensure reproducibility, samples from three independent production series were analysed.

2.2. Production Environment Sampling Strategy

Samples from the production environment, hereafter referred to as environmental samples, were collected after sanitary procedures, before RTE bar production. Sampling sites were selected from environmental surfaces based on risk assessment, considering both the frequency and impact of microbiological hazards.

Food-contact surfaces (FCS) include swabs from transport containers, the production table, the grinder, the mixer, the stuffer, the smoking trolley grates, and the modified atmosphere packaging machine. The presence of pathogens and/or their high count on these surfaces directly impacts product contamination, requiring immediate corrective action. Environmental swabs were not collected during the baking and smoking stages due to high operating temperatures, which prevent reliable recovery of microorganisms and standard swabbing procedures. Monitoring at these points focused solely on the product’s internal temperature to ensure process lethality.

Non-food-contact surfaces (N-FCS) include swabs from floors, walls, drain grates, employee hands, and the rolling elements (wheels) of the smoking trolley. Hand swab samples were collected from food handlers before glove application as part of a routine hygiene assessment. Research indicates that such sites, characterised by constant humidity and limited access to disinfectants, favour biofilm formation, which plays a critical role in secondary contamination of food contact surfaces (FCS) and, subsequently, the food product.

Before the production series used to assess in-process microbial load dynamics, environmental surface swabs were collected with sterile Sponge-Sticks (3M™, Hermes LabSystems s.r.o., Bratislava, Slovakia), either dry or pre-moistened with 10 mL BPW, from food-contact and non-food-contact surfaces at each production step (as presented in Table 1).

Surface swabs were collected according to ISO 18593:2018 using sterile 10 cm × 10 cm templates to define 100 cm² sampling areas [23]. The study performed swabbing in three passes (parallel, perpendicular, and diagonal; 10 strokes each), with additional tracing of template edges and corners to improve recovery from irregular surfaces and biofilms. The study stored swabs at ≤4 °C, transported them to the laboratory within four hours, and analysed them microbiologically on the same day.

2.3. Microbial Analysis of In-Process and Environmental Samples

The study analysed all in-process and environmental samples for six microbiological parameters: aerobic plate count (APC), Enterobacteriaceae (EB), E. coli (EC), yeast and mould (YM), lactic acid bacteria (LAB), and S. aureus (STA). The study conducted the analyses using the TEMPO Most Probable Number (MPN) system (bioMérieux SA, Marcy-l’Étoile, France) [24,25]. According to the TEMPO protocols, 25 g of each in-process sample was diluted with 225 mL of buffered peptone water in a sterile plastic bag and homogenised for 2 min using a paddle homogeniser. Depending on the target microbial group (APC, EB, EC, YM, or LAB), appropriate 1:40 and/or 1:400 dilutions were prepared. Following incubation under specific temperature and time conditions required for each test, bacterial growth was quantified using the TEMPO Reader, which generated results expressed as colony-forming units per gram (log CFU/g).

Environmental samples collected with BPW-soaked sponges were supplemented with 240 mL of buffered peptone water (BPW) and homogenised for 2 min. Microbial counts of APC, Enterobacteriaceae, E. coli, yeast and mould, LAB, and coagulase-positive staphylococci were determined using the TEMPO Most Probable Number (MPN) system with TEMPO AC, EB, EC, YM, and LAB tests.

The limits of detection (LOD) for each TEMPO assay used (APC, Enterobacteriaceae, E. coli, yeast and mould, LAB, and S. aureus) for in-process samples were 1.0 log CFU/g, while for environmental samples were 0.0 log CFU/cm². The limits of quantification (LOQ) were the same as LODs for both sample groups, respectively.

2.4. Detection of Salmonella spp. and L. monocytogenes

Following the use of 1 mL of the homogenate from the in-process and environmental sample for TEMPO enumeration, the residual homogenate was subjected to Salmonella spp. detection using the VIDAS SPT assay (bioMérieux SA, Marcy-l’Étoile, France). The method employs the Enzyme-Linked Fluorescent Assay (ELFA) principle, enabling rapid, automated screening for Salmonella spp. [26].

For L. monocytogenes detection, 25 g of in-process sample was mixed with 225 mL of Half-Fraser broth in a sterile bag and homogenised for 2 min. The homogenate was incubated at 37 °C for 24 h to detect L. monocytogenes in the sample using the VIDAS LMX test (bioMérieux SA, Marcy-l’Étoile, France) [27].

In contrast to the in-process samples, environmental sponge samples required the addition of 250 mL of Half-Fraser liquid medium, with subsequent procedures identical to those used for L. monocytogenes detection in in-process samples.

In the case of a positive VIDAS LMX result, meat samples or swabs were streaked onto ALOA chromogenic agar (Ottaviani and Agosti), and characteristic growth and colour changes in the medium identified typical colonies. For growth, further microbiological identification was performed according to the VIDAS LMX manufacturer’s instructions and ISO 11290-1 [28], which included Gram staining and biochemical tests.

2.5. Biochemical Profiling and PCR Confirmation of L. monocytogenes

For the biochemical identification of Gram-positive bacilli with a positive VIDAS LMX result, the Oxoid Microbact Listeria 12 L Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used, including tests for esculin hydrolysis, mannitol fermentation, xylose, arabitol, ribose, rhamnose, trehalose, tagatose, glucose-1-phosphate, methyl-D-mannose, methyl-D-glucose, and hemolysis.

The study suspended microscopically confirmed pure colonies of Gram-positive bacilli in sterile diluent, vortexed the suspension for 10 s, dispensed 100 μL into the reagent wells of the test strips, and assessed haemolytic activity by adding a drop of heparinised sheep blood equilibrated to room temperature. The study then incubated the tests at 37 °C for 22 h before reading.

Isolates showing positive results for rhamnose fermentation and hemolysis, and negative results for xylose fermentation, were subjected to the CAMP test with Rhodococcus equi and S. aureus. In addition, PCR using the L. monocytogenes-specific primer lmo0733, which encodes a protein similar to a transcriptional regulator, was performed, followed by electrophoresis. Positive PCR results definitively identified the isolates as L. monocytogenes.

After two passages of the isolates on nutrient agar, the study performed the CAMP test on Columbia agar supplemented with 5% sheep blood and assessed haemolysis zone enlargement after 24 h of incubation at 35 °C.

2.6. PCR Amplification of the lmo0733 Gene

For DNA isolation, a thick layer of monoculture was suspended in 300 µL of DNA-free water, boiled at 99 °C for 10 min, cooled, and centrifuged at 1500 rpm for 6 min. Then, 200 µL of the supernatant containing Listeria spp. was collected for further analysis.

PCR was performed using primers lmo0733F (5′-CGCAAGAAGAAATTGCCATC-3′) and lmo0733R (5′-TCCGCGTTAGAAAAATTCCA-3′). The 25 µL reaction mixture contained 12.5 µL DreamTaq DNA Polymerase (5 U/µL; Thermo Fisher Scientific, Waltham, MA, USA), 1 µL of each primer, 9.5 µL of DNA-free water, and 1 µL of template DNA.

PCR cycling conditions included an initial denaturation at 95 °C for 2 min, followed by 25 cycles of denaturation at 95 °C for 30 s, annealing at 49 °C for 30 s, and extension at 72 °C for 45 s, with a final extension at 72 °C for 2 min. Amplicons were visualised by electrophoresis on a 2% agarose gel stained with ethidium bromide for 70 min.

2.7. Data Processing and Statistical Analysis

Statistical analysis was conducted in Microsoft Excel 2019 (Windows, Redmond, WA, USA) using a one-way analysis of variance (ANOVA) at α = 0.05. The study fitted polynomial trend equations to in-process pathogen counts to characterise changes across production steps.

Although samples were collected sequentially along the production process, the statistical analysis did not constitute true repeated measures, as independent samples were collected at each processing step and no individual unit was measured repeatedly over time. Therefore, one-way ANOVA was applied to compare mean microbial counts between processing steps. The analysis aimed to identify stage-specific differences rather than to model within-sample temporal dependence. To further characterise process-related dynamics, polynomial trend analyses were additionally performed.

One-way ANOVA was used to assess differences in mean microbial counts among processing steps. When significant overall effects were detected (p < 0.05), differences were interpreted at the group level rather than through pairwise causal attribution. Results are presented as mean ± standard deviation. Polynomial trendlines were applied solely to describe overall process-related patterns and were not used as evidence of biological validation or predictive reliability.

3. Results

3.1. Microbial Dynamics and Pathogen Detection in In-Process Samples of RTE Pork Bars

Figure 2 presents the dynamics of trends in mean counts of individual microorganisms obtained from quantitative analyses after each production step of low-processed RTE meat bars with dried plasma.

Figure 2. Polynomial trends in mean counts of APC (blue), Enterobacteriaceae (violet), E. coli (green), yeast and mould (yellow), and LAB (red) in-process samples across successive production steps.

Before heat treatment, APCs ranged from 5.0 to 5.5 log CFU/g, with significant increases observed after cooling and seasoning (p < 0.05). Baking reduced the counts by approximately 1 log unit, and smoking caused a further reduction of 1.5 log units. Subsequent maturation and packaging steps were associated with increases of 0.42 and 0.72 log CFU/g, respectively.

Enterobacteriaceae counts (~4 log CFU/g) decreased slightly during early processing, peaked after grinding, and became undetectable after heat treatment and maturation; however, they reappeared after packaging (0.5 ± 0.71 log CFU/g). Notably, Enterobacteriaceae reappeared specifically after the vacuum-packaging step, despite their prior elimination during thermal processing (p < 0.05).

E. coli counts increased after the cooling, grinding, and forming, reaching a maximum of 2.0 ± 0.82 log CFU/g, then decreased after the seasoning, portioning, and baking. The study detected no E. coli in any post-heating processing steps.

LAB counts decreased after cooling but increased sharply by ~2 log units during grinding. Slight increases during seasoning and portioning resulted in counts reaching a maximum of 4.48 ± 0.29 log CFU/g. Subsequent forming, baking, and smoking caused reductions of 0.19, 2.14, and 1.38 log units, respectively. Post-processing maturation resulted in a minor increase (0.89 ± 1.25 log CFU/g), with no significant differences observed between maturation and packaging (p > 0.05).

Yeast and mould counts declined gradually from 4.35 log CFU/g at the cutting step to 3.95 log CFU/g at the forming step. After baking and throughout all subsequent post-heating steps, the study detected no yeast or mould.

Polynomial trendlines were fitted to describe changes in mean microbial counts across processing steps (Table 2).

Table 2. Polynomial trend equations and R² values for each tested microbial group.

Microbiological Parameter	Polynomial Trend Equation	R2 Value
APC	y = 0.0093x4 − 0.1889x3 + 1.2165x2 − 2.8588x + 7.2842	0.886
Enterobacteriaceae	y = 0.0105x4 − 0.2067x3 + 1.2262x2 − 2.6009x + 5.5092	0.957
E. coli	y = 0.0177x3 − 0.3163x2 + 1.4002x − 0.0533	0.916
Yeast and mould	y = 0.0099x4 − 0.1886x3 + 1.0712x2 − 2.2539x + 5.6967	0.902
LAB	y = 0.0127x4 − 0.2542x3 + 1.5163x2 − 2.7369x + 4.1592	0.935

Table Footnote: APC, aerobic plate count; LAB, lactic acid bacteria. Polynomial equations describe microbial trends; R² indicates the goodness-of-fit of the model.

Molecular confirmation via PCR targeting the lmo0733 gene was performed on purified isolates obtained from VIDAS LMX–positive samples following selective enrichment, thereby confirming the presence of viable L. monocytogenes. PCR-confirmed isolates accounted for 27.3% of all in-process samples. The highest prevalence was observed at the raw material acceptance stage (4 samples), and, notably, viable isolates were also recovered from two samples collected after the baking stage.

No evidence of Salmonella spp., nor detectable counts of E. coli or S. aureus, was observed in any samples at any production step.

Overall, 66.7% (n = 33) of product samples tested positive in the VIDAS LMX assay across production steps. All samples (100%, n = 3) collected from raw material acceptance to settling were positive, while 67% remained positive after baking. From hot smoking to the end of production, all in-process samples tested negative.

Only 77.3% (n = 22) of isolates from LMX VIDAS-positive product samples exhibited biochemical and morphological features consistent with L. monocytogenes, as confirmed by PCR using the specific primers lmo0733F and lmo0733R, followed by electrophoresis.

PCR confirmed L. monocytogenes isolates in 58.8% (n = 17) of in-process samples previously positive in microscopic and biochemical tests, representing 27.3% (n = 33) of all in-process samples. PCR-positive isolates originated only from series 1 (33.3%) and 3 (66.7%), most frequently after raw material acceptance (4 samples), grinding (2 samples), and baking (2 samples). The study obtained one positive electrophoresis result from environmental swabs collected after mixing and shaping. The study detected no genetically confirmed L. monocytogenes in environmental samples collected after settling, smoking, chilling, or vacuum packaging (ROP).

3.2. Microbial Assessment of the Production Environment

No evidence of Salmonella spp., nor detectable counts of E. coli and S. aureus (>1 CFU/cm²), were found in environmental surface swabs at the end of any production step. Table 3 presents mean values ± standard deviations of APC, Enterobacteriaceae, and yeast and mould counts collected at each production step.

Table 3. Mean values ± standard deviation of APC, EB, and YM counts in environmental samples (log CFU/cm²) from FCS, non-FCS, and overall, collected before each production step.

Production Step	APC			Enterobacteriaceae			Yeast and Mould
	FCS	Non-FCS	OVERALL	FCS	Non-FCS	OVERALL	FCS	Non-FCS	OVERALL
Acceptance	6.22 ± 0.62	6.36 ± 0.58	6.27 ± 0.58	3.62 ± 1.26	3.78 ± 1.27	3.67 ± 1.27	3.31 ± 2.21	3.13 ± 2.21	3.13 ± 2.21
Cooling	5.91 ± 1.52	6.69 ± 0.29	6.17 ± 1.30	3.42 ± 1.08	4.30 ± 0.81	3.71 ± 1.60	2.96 ± 2.10	4.80 ± 0.16	3.57 ± 1.93
Grinding	4.98 ± 0.31	6.44 ± 0.39	5.71 ± 0.81	2.83 ± 0.31	4.62 ± 0.10	3.73 ± 0.92	4.24 ± 0.11	4.47 ± 0.31	4.36 ± 0.26
Mixing	4.90 ± 1.58	6.28 ± 0.43	5.82 ± 1.17	1.59 ± 0.07	2.53 ± 1.79	2.26 ± 1.58	2.59 ± 0.00	1.07 ± 0.97	1.50 ± 1.07
Shaping	5.54 ± 0.94	5.35 ± 0.53	5.44 ± 0.72	3.14 ± 0.96	2.48 ± 0.88	2.78 ± 0.93	3.53 ± 0.78	2.50 ± 1.25	2.97 ± 1.15
Settling	3.09 ± 0.25	4.82 ± 0.74	3.88 ± 1.01	0.34 ± 0.76	1.31 ± 1.15	0.78 ± 1.07	0.64 ± 0.91	2.55 ± 1.06	1.51 ± 1.36
Chilling	n/s	2.07 ± 0.28	2.07 ± 0.28	n/s	0.00 ± 0.00	0.00 ± 0.00	n/s	1.54 ± 1.54	1.54 ± 1.54
ROP	3.20 ± 1.49	4.70 ± 0.74	4.02 ± 1.36	0.86 ± 1.73	0.78 ± 1.75	0.82 ± 1.74	1.69 ± 1.06	3.62 ± 0.27	2.74 ± 1.22

Table Footnote: APC: aerobic plate count; FCS: food-contact surfaces; non-FCS: non-food-contact surfaces; n/s, no sample available in this group.

The highest APCs in environmental swabs were recorded at the time of raw material acceptance, while the lowest occurred during chilling. Grinding yielded the highest mean counts of Enterobacteriaceae and yeast and mould, whereas chilling showed virtually no detectable Enterobacteriaceae. Among post-heat-treatment steps, ROP samples showed significantly higher yeast and mould counts, and APCs than those chilled (p < 0.05).

Overall, 44.0% (n = 84) of environmental samples tested positive in the LMX VIDAS assay. The highest proportion was observed in series 1 (46.4%), followed by equal proportions in series 2 and 3 (42.9%, n = 28). The study detected no positive results during either the chilling or ROP step.

Only 40.5% (n = 37) of isolates obtained from LMX VIDAS-positive environmental samples exhibited characteristics suggestive of L. monocytogenes in microscopic and biochemical tests and were therefore subjected to PCR using primers lmo0733F and lmo0733R, followed by electrophoresis.

The analysis confirmed the genetic identity of L. monocytogenes in 46.7% (n = 15) of these isolates. Confirmed isolates originated more frequently from series 2 (57.1%) than from series 1 (42.9%, n = 7). Within the production process, the study most frequently detected L. monocytogenes at the raw material acceptance and shaping steps (three samples each), less frequently at the mixing step (one sample), and at none of the remaining stages (p < 0.05).

4. Discussion

Growing consumer demand for ‘clean label’ products has prompted manufacturers to eliminate traditional chemical preservatives, such as nitrites and sorbates. However, as demonstrated in this study, the absence of these barriers significantly increases reliance on thermal processing and stringent environmental hygiene [29,30]. Our findings show that even minor fluctuations in the microbial quality of raw materials or environmental recontamination can compromise the safety of low-processed RTE pork bars.

The microbial dynamics observed throughout the production of low-processed RTE pork bars provide insight not only into the effectiveness of specific technological steps implemented in the investigated facility, but also into contamination patterns that are inherent to minimally processed RTE meat production systems operating without chemical preservatives. The observed changes in microbial counts across processing stages are consistent with patterns previously reported for thermally processed meat products and reflect general process-related risks rather than facility-specific anomalies [31].

Enterobacteriaceae and E. coli counts exhibited characteristic fluctuations typical of raw meat processing. The increase observed after grinding and forming (p < 0.05) supports earlier findings demonstrating that mechanical disruption, increased surface area, and homogenisation promote microbial redistribution and contamination, particularly from food-contact equipment [32,33,34]. These observations confirm that grinding represents a universally critical step in raw meat processing and should be systematically classified as a high-risk operation in HACCP-based hazard analyses for RTE meat products, regardless of plant size or production scale.

The absence of E. coli on food-contact surface (FCS) swabs after washing indicates that the observed changes in E. coli counts in the product were primarily attributable to processing steps rather than ineffective sanitation. The grinding step resulted in a marked increase in E. coli counts (p < 0.05), whereas the addition of food ingredients and subsequent thermal treatment significantly reduced contamination levels in the final product [35]. The absence of E. coli after smoking confirms the effectiveness of thermal inactivation. From a broader food safety management perspective, these findings reinforce existing recommendations that operators should conduct grinding under strict temperature control (≤4 °C) and use hygienically designed stainless steel equipment with regularly sharpened cutting elements to minimise microbial growth and cross-contamination [36,37].

Despite effective thermal treatment, the reappearance of Enterobacteriaceae at low levels after packaging (mean: 0.5 log CFU/g) suggests potential post-lethality contamination or environmental exposure during handling [38]. Importantly, this phenomenon reflects a well-documented vulnerability of post-thermal steps in RTE meat production rather than a failure specific to the investigated facility. Studies on minced and emulsified meat products have reported similar increases in microbial counts during packaging, where homogenisation and manual handling contribute to microbial redistribution [39]. These results underscore the importance of treating packaging and maturation as critical, hygiene-sensitive stages within FSMS.

Regarding Salmonella spp., the findings indicate that raw materials are the primary source of contamination, originating from upstream slaughter or meat-cutting operations where slaughter techniques and sanitary conditions may not fully meet regulatory requirements [40]. The absence of Salmonella spp. in cooled raw material, environmental swabs, semi-finished products, and the final RTE product confirms that in-plant washing and subsequent processing steps were effective. More broadly, this highlights the importance of supply chain control and demonstrates that pathogen risk management in RTE meat production cannot rely solely on in-plant hygiene measures; instead, it must encompass upstream slaughter and primary processing stages.

The detection of L. monocytogenes by the LMX VIDAS assay at multiple processing steps, particularly during raw material acceptance, grinding, and baking, indicates that early production stages remain susceptible to contamination, despite effective thermal inactivation later in the process. PCR confirmation in 27.3% of in-process samples is notable, given the pathogen’s ability to persist under cold, low-oxygen conditions [41,42,43,44]. The detection of L. monocytogenes DNA via PCR after the baking stage, despite the core temperature reaching safety limits, may be attributed to two factors. First, it may represent the presence of non-viable (dead) cells whose genetic material remained detectable. Second, it could indicate the presence of cells in a ‘Viable but Non-Culturable’ (VBNC) state, induced by the thermal stress [45]. In products lacking chemical preservatives, the potential for recovery of these cells during shelf life poses a critical food safety risk that requires further investigation through challenge testing.

Previous research has shown that L. monocytogenes occurs almost three times more frequently in N-FCS than in FCS [12,46,47,48]; however, our results do not support this pattern, as the LMX VIDAS test identified positive environmental samples more often in FCS (59.4%) than in N-FCS (40.6%, n = 37). L. monocytogenes contamination in environmental swabs is much more frequent in plants where animal slaughter and subsequent meat processing are in the same building (more than twice). The processing plant from which the samples came was a small facility that carried out animal slaughter and further processing in the same building, with separate entrances and different staff [41].

Previous studies have also demonstrated that cross-contamination of the environment and equipment constitutes one of the primary routes through which L. monocytogenes enters RTE products [44,49,50,51]. Therefore, the presence of genetically confirmed L. monocytogenes in samples underscores the need to strengthen not only process controls but also the hygienic design and cleaning validation of food contact surfaces, particularly during the grinding, forming, and packaging stages.

The significant reduction in APC after thermal processes such as baking (approx. 1 log CFU/g) and smoking (a further 1.5 log CFU/g) confirms the bactericidal efficiency of the two-step heat treatment (p < 0.05). These reductions are consistent with prior studies, which showed that heat treatments above 70 °C significantly reduce the total microbial load, including spoilage organisms and vegetative mesophilic pathogenic bacteria [52,53,54,55]. However, the slight increase in APC observed during maturation and packaging suggests possible post-lethality contamination or favourable conditions for microbial regrowth during vacuum-packed storage [56,57].

We obtained similar APC and Enterobacteriaceae contamination in raw pork at the acceptance step as in the previous results [58].

Our results demonstrate a direct link between the microbial load on Food Contact Surfaces (FCS) and final product quality. Although the sanitation procedure protocols are generally performed effectively, the persistent low-level presence of APC on surfaces such as the meat stuffer and the ROP machine (Table 3) correlates with the microbial shifts observed in the product. These findings confirm that, in meat bars containing dried plasma and no preservatives, environmental monitoring must extend beyond standard pathogens to include indicator organisms that predict shelf life stability.

However, slight increases in APC during maturation and packaging suggest the potential for post-process recontamination or microbial growth under favourable conditions, such as vacuum-packaging and extended shelf life. While the final APC remained within acceptable limits, these findings underscore the importance of maintaining hygienic conditions during post-thermal steps and the potential benefit of introducing hurdle technologies to extend shelf life.

The fluctuations in LAB counts are also significant between production steps (p < 0.05). The increase in LAB after seasoning and portioning aligns with their adaptation to meat matrices and potential early dominance due to salt and spice tolerance [59,60,61]. Although thermal steps substantially reduced their counts, their post-maturation resurgence indicates the survival of sublethally injured cells or recontamination. While LAB are generally associated with fermentation, considered unharmful, and may have a protective role against pathogens, uncontrolled growth may lead to spoilage and undesired acidification of sensory properties in low-processed meat formulations [62,63,64,65].

The rapid and complete elimination of yeast and mould after baking suggests their high thermal inactivation, as expected given their lower heat resistance compared to bacterial spores [66]. Their presence in pre-heat steps further underscores the importance of controlling raw material quality and air hygiene, as these fungi often originate from environmental sources; their elimination ensures better fungal safety and a lower risk of spoilage during storage.

High coefficients of determination (R²) indicate good mathematical fit of the trendlines to the observed data; however, these models were used for descriptive purposes only and do not imply biological validation or causal inference [67]. Other studies on RTE meat products have successfully applied predictive models to describe microbial behaviour during processing [68,69].

Monitoring practices among small- and medium-sized food business operators (FBOs) remain heterogeneous with respect to sampling frequency, sampling locations, and detection methods. Limited resources often constrain the implementation of extensive environmental monitoring programs, particularly in facilities undergoing expansion or reconstruction [46,70]. Taken together, the presented data reinforce the concept that even a minimal FSMS, based on GMP, GHP, and HACCP procedures, fundamentally relies on two complementary pillars—a scientifically based approach and a risk-based approach—and that empirical evidence on microbial dynamics, such as provided in this study, is essential to inform other facilities on how contamination patterns may evolve despite the implementation of prerequisite programmes. In this context, the present study demonstrates that targeted, process-oriented sampling strategies may provide a feasible and scientifically sound alternative to extensive monitoring programmes, enabling improved risk control in resource-limited production environments.

Together, these FSMS reinforced the reliability and safety of the production process by ensuring consistent personnel hygiene, equipment sanitation, and environmental hygiene, while systematically identifying and controlling critical points where microbial contamination could occur. Their application, among others, was to minimise cross-contamination and fluctuations in microbial load throughout production, thereby contributing to the effective inactivation of pathogens such as L. monocytogenes during thermal processing and supporting the overall microbiological safety of low-processed RTE pork bars.

The incorporation of dried plasma as a functional protein source did not appear to introduce specific microbial hazards if operators controlled the initial quality of raw materials. However, the high initial APC load observed at raw material acceptance suggests that the synergistic effect of meat trimmings and functional additives requires a robust heat treatment to eliminate background microflora, which would otherwise compromise the safety margins of the final product.

Overall, although heat treatment was sufficient to eliminate L. monocytogenes in the finished RTE product, the observed reappearance of APC and LAB during packaging and maturation highlights the importance of post-lethality control measures. These may include post-process interventions or the application of natural antimicrobials to mitigate microbial regrowth [71,72]. Taken together, the findings suggest that the most critical contamination window lies between raw material handling and post-baking operations, reinforcing the need for integrated, risk-based FSMS across all processing stages.

5. Conclusions

In summary, this study demonstrates that a two-step heat treatment comprising baking and smoking effectively reduces the microbial load of low-processed RTE pork bars, including the inactivation of major foodborne pathogens. Nevertheless, the detection of L. monocytogenes and elevated counts of indicator microorganisms during early processing stages, together with low-level recontamination observed after packaging, highlight critical vulnerabilities that cannot be addressed solely by thermal processing.

The findings confirm that process-inherent factors primarily drive microbial risks in low-processed RTE meat products, particularly during raw material handling, grinding, forming, and post-lethality operations. Even when facilities operate under acceptable sanitation conditions and implement GMP-, GHP-, and HACCP-based programmes internally, these stages remain susceptible to contamination and microbial redistribution. These results underscore the need to adopt a holistic FSMS that integrates upstream raw material control, hygienic process design, and robust post-process safeguards.

The empirical data on microbial dynamics generated in this work offer transferable knowledge that other food business operators can use to understand how contamination patterns can evolve despite the implementation of prerequisite programmes, thereby supporting more informed, evidence-based risk management decisions.

The application of predictive modelling, supported by the high reliability of observed microbial trends across processing steps, represents an additional tool for improving risk assessment, identifying critical contamination windows, and guiding process optimisation. Such approaches may enhance decision-making within FSMS and support a more effective allocation of control measures, particularly in small- and medium-sized production environments [73].

Overall, the results confirm that ensuring the microbiological safety of low-processed RTE meat products requires an integrated strategy extending beyond lethality treatments. Effective control depends on the continuous management of contamination risks throughout the entire production chain, from raw material sourcing to post-packaging handling, reinforcing the importance of system-based, risk-oriented food safety management applicable across comparable RTE meat processing environments.