Applied Microbiology: Open Access
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Study on the Growth, Decline, and Permeability of Dominant Microorganisms in Chilled Pork under Different Temperature Conditions

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Introduction

Chilled meat, also known as cold meat or acid-removing meat, is a nutritious and tasty food that is an important part of meat consumption in China [1]. However, during industrial production, chilled meat is susceptible to microbial contamination due to its high water activity and nutrient-rich composition. This can lead to shortened shelf life and potential food safety risks [2]. In order to address these issues, it is important to analyze the changes in dominant spoilage bacteria in chilled meat under different storage conditions [3]. The growth characteristics of spoilage microorganisms differ depending on environmental conditions, packaging, and storage conditions. Moreover, the ability of different types of spoilage microorganisms to adapt to the surface and inner layers of chilled meat also varies [4].

The purpose of this study is to determine the dominant spoilage bacteria in chilled pork under different storage temperatures (2 ± 2ºC, 10 ± 2ºC, and 20 ± 2ºC) and to investigate the variation of spoilage bacteria in four layers of chilled pork [5]. By understanding the growth patterns and dominance of different spoilage microorganisms in chilled meat, it is possible to identify key control points for targeted preservation of chilled meat and extend its shelf life. Therefore, the aim of this experiment is to measure the number of different types of spoilage microorganisms in chilled meat during storage, analyze the differences between microorganisms in the surface and inner layers of chilled meat [6], and determine the proliferation rate and growth pattern of different spoilage microorganisms in chilled meat to identify the more dominant spoilage microorganisms [7].This paper will provide a theoretical basis for improving precision sterilization and key control point technology in enterprises dealing with chilled meat.

Materials and Methods

Materials, reagents and instruments

The experimental samples were provided by Shuanghui Investment and Development Co. from the slaughterhouse in Luohe City, Henan Province.

The following media were used are plate counting agar medium, nutrient agar medium, Pseudomonas agar medium, VRBGA agar medium, MSA agar medium, STAA agar medium, MRS agar medium, and Bengal red agar medium. These were obtained from Beijing Road and Bridge Technology Co Ltd. Petri dishes were purchased from BRAND (Germany). Sterile homogenization bags and sterile gun tips were purchased from Beijing Road and Bridge Technology Co. Ltd. BUG agar medium, GN/GP-IF inoculum, and GenIII microtiter plates were obtained from Biolog (USA).

The instruments and equipment used are listed in Table 1.

Table 1: Instrumentation used in the experiments.

Sample preparation and storage

The longest back muscle, weighing approximately 200 g, was obtained from the slaughterhouse of Luohe City, Henan Province, Shuanghui Investment and Development Co. The muscle was processed using the normal procedure on the splitting line and then tray packed. The packed samples were stored at three different temperature conditions of 2 ± 2ºC, 10 ± 2ºC and 20 ±b 2ºC. The numbers of total bacteria, Enterobacteriaceae, Pseudomonas, Thermospora, Lactobacillus, Staphylococcus, Micrococcus and Mycobacterium/yeast in the cooled pork were determined every 2 days, 1 day, and 6 hours to determine the pattern of decay of spoilage microorganisms in the cooled pork.

The cooled pork was then divided into four layers under aseptic conditions, with each layer having a thickness of approximately 1 cm. The total number of bacteria, Enterobacteriaceae, Pseudomonas spp., Pseudomonas, Thermospora, Lactobacillus, Staphylococcus/Micrococcus, and Mycobacterium/yeast in each layer was determined to evaluate the penetration of spoilage microorganisms in the cooled pork.

Microbiological analysis

The total bacterial count of the samples was determined according to the national standard GB4789.2-2016 “Microbiological test for food hygiene-Determination of total bacterial count”.

Data analysis

Each experiment was conducted three times, and the data were analyzed using SPSS16.0 software. The results were expressed as Mean (M) ± Standard Deviation (SD). The mean values were compared using Duncan's multiple range test (P<0.05).

Results and Discussion

Analysis of the initial bacterial composition of spoilage microorganisms in cooled pork

The total number of bacteria and the number of colonies of six types of dominant spoilage bacteria were detected in the cooled pork samples using selective media based on the national standard GB4789. The results are presented in Table 2.

Table 2: Initial microflora constitution of chilled pork .

The microbial quantity of cooled pork produced by the normal process of this enterprise was well controlled, and the initial total colony range was generally between 3.33-4.53 × 10⁴ CFU/mL. Among the different categories of microorganisms in cooled pork, S. thermophilus was detected in the highest amount, reaching 2.17 × 10⁴ to 2.64 × 10⁴ CFU/g, accounting for more than 55% of the composition of the bacterial phase. Staphylococcus/Micrococcus had the least amount, ranging from 2.93 × 10² to 1.01 × 10³ CFU/g, accounting for less than 3.00% of the composition of the bacterial phase. Based on Table 2, the initial microbial profile of cooled pork was as follows: the highest amount of S. thermophilus was detected, followed by Enterobacter and Lactobacillus, less Pseudomonas spp. and Staphylococcus/Micrococcus, and the lowest amount of Staphylococcus/Micrococcus was detected.

Pattern of decay of spoilage microorganism growth and comparative analysis in cooled split pork under 2 ± 2ºC Condition

As shown in Figure 1, the total number of bacteria and various spoilage microorganisms in cooled pork stored at 2 ± 2ºC increased to different degrees with the extension of storage time. In the first 3 days of storage, there was almost no growth, and then it entered a period of rapid growth. Among them, S. thermophilus had the highest initial number, and it also maintained the highest number during storage. The initial number of Enterobacteriaceae was significantly smaller than that of S. thermophilus, but it grew rapidly during storage and was second only to the number of S. thermophilus. The number of colonies of Pseudomonas spp. was low at the beginning of storage, but it proliferated faster during storage and became one of the main dominant bacteria under this temperature condition. Although the initial number of Lactobacillus was higher, it gradually lost its numerical advantage during storage. Mold/Yeast grew more slowly during storage and gradually stabilized after 7 days. Staphylococcus/Micrococcus had the lowest number and growth rate during storage, as shown in Figure 1. Based on the analysis of Figure 1, the main dominant spoilage bacteria of cooled pork at 2 ± 2ºC were S. thermophilus, Enterobacteriaceae, and Pseudomonas spp (Table 3).

Table 3: The change of microflora constitution of chilled pork storage at 2 ± 2ºC.

Figure 1: The growth of spoilage microorganism in chilled pork stored at 2 ± 2ºC. Note:

The results from Table 3 and Figure 1 suggest that during the storage of cooled pork at 2 ± 2ºC, the proportion of Enterobacteriaceae and Pseudomonas spp. increased significantly, indicating that these two microorganisms have a high growth rate at this temperature condition and are important factors contributing to pork spoilage. On the other hand, the proportion of Lactobacillus, Staphylococcus/Micrococcus, and Mycobacterium/yeast decreased during storage, indicating that these microorganisms were not dominant in the spoilage of cooled pork at this temperature condition. The results also suggest that S. thermophilus, which had the highest initial number, remained the dominant microorganism throughout the storage period. Overall, the results indicate that controlling the growth of Enterobacteriaceae and Pseudomonas spp. during the storage of cooled pork at 2 ± 2ºC is crucial for extending the shelf life of the product.

The study analyzed the microbial changes in different layers of chilled pork during storage at 2 ± 2ºC. The results showed that the first layer of cooled pork had the fastest growth rate and the largest number of microorganisms, with the total number of colonies exceeding 10⁸ CFU/mL on the 9^th day of storage. The second layer had a slower growth rate, with the total number of colonies of Thermokill Soxhlet exceeding 10⁶ CFU/mL and the number of colonies of Enterobacteriaceae increasing rapidly and approaching 10⁶ CFU/mL. The third layer had the slowest growth rate, with the total number of colonies of Thermokill Soxhlet close to 10⁴ CFU/mL and the number of colonies of Enterobacteriaceae still higher than that of Pseudomonas. The fourth layer had relatively lower numbers of microorganisms, with the total number of Thermokill Soxhlet colonies close to 10³ CFU/mL and the number of Enterobacteriaceae colonies being the second highest after Thermokill Soxhlet (Figure 2).

Figure 2: The spoilage microorganism quantity in 4-layer chilled meat at 2 ± 2°C.

Overall, the results suggest that the growth rate and number of microorganisms in chilled pork are influenced by the storage conditions and location in the meat. Therefore, proper storage conditions and handling practices are essential to ensure the safety and quality of chilled pork. It appears that under the storage conditions of 2 ± 2ºC, the growth rate and number of colonies of Thermokill Soxhlet and Enterobacter were relatively high in all four layers of the chilled pork. In contrast, Pseudomonas and lactic acid bacteria showed weaker permeability under these conditions, possibly due to changes in oxygen availability. It is worth noting that the permeability of microorganisms can vary depending on the storage conditions, as well as the specific strain of microorganism in question. Therefore, it is important to monitor the microbial changes and adjust the storage conditions accordingly to ensure the safety and quality of the chilled pork.

Growth and decline law and comparative analysis of spoilage microorganisms in cooled and segmented pork at 10 ± 2ºC

When cooled pork is stored at 10 ± 2ºC, the total number of bacteria and the number of spoilage microorganisms increase to varying degrees with the extension of storage time, as shown in Figure 3. The indicators remain stable for the first 2 days of storage before entering a period of rapid growth. The growth tends to stabilize after 5 days. Among the spoilage microorganisms, Thermokill Soxhlet had the highest initial number and maintained the highest number throughout storage. The initial number of Enterobacteriaceae was significantly smaller than that of Thermokill Soxhlet, but it grew rapidly in storage and became the second highest number after Thermokill Soxhlet. Pseudomonas had a high proliferation rate in the middle and late stages of storage, exceeding the number of lactic acid bacteria on the 3rd day. Although the initial number of lactic acid bacteria was high, it lost its numerical advantage at the 4th day. Mold/yeast growth was slower in storage, and Staphylococcus/Micrococcus had the lowest number and growth rate in storage, both significantly lower than those of Thermokill Soxhlet, Enterobacteriaceae and Pseudomonas, as shown in Figure 3. Thus, under these storage conditions, Thermokill Soxhlet, Enterobacteriaceae and Pseudomonas are the dominant spoilage bacteria for cooled pork (Figures 3 and 4, Table 4).

Table 4: The change of microflora constitution of chilled pork storage at 10 ± 2ºC.

Figure 3: The growth of spoilage microorganism in chilled pork stored at 10 ± 2ºC.

Figure 4: The spoilage microorganism quantity in 4-layer chilled meat at 10 ± 2°C.

When cooled pork was stored at 10 ± 2ºC, the proportion of Enterobacteriaceae and Pseudomonas in the bacterial community increased significantly, with Enterobacteriaceae increasing from 12.12% to more than 24.48% and Pseudomonas increasing from 3.91% to 13.44%. In contrast, the proportion of Thermokill Soxhlet remained stable, accounting for about 60% of the community. However, the proportions of lactic acid bacteria, Staphylococcus/Micrococcus and mold/yeast decreased over time, with lactic acid bacteria decreasing from 21.73% to less than 2.00%, Staphylococcus/Micrococcus decreasing from 1.17% to less than 0.01%, and mold/yeast decreasing from 3.06% to less than 0.05%. This decline was mostly replaced by Enterobacteriaceae and Pseudomonas. Combining Figure 3 and Table 4, it can be concluded that Thermokill Soxhlet, Enterobacteriaceae, and Pseudomonas are the dominant spoilage microorganisms in cooled pork when stored at 10 ± 2ºC.

The number of spoilage microorganisms present in different layers of cooled pork stored at 10 ± 2ºC is depicted in Figure 4. The microbial changes tend to stabilize after 5 days of storage as compared to 2 ± 2ºC. The results revealed that, on the 5th day of storage, the total number of Thermokill Soxhlet colonies in the first layer of cooled pork was close to 10⁹ CFU/mL, while the total number of colonies of Lactic Acid bacteria, Enterobacteriaceae and Pseudomonas exceeded 10⁷ CFU/mL, and the total number of colonies of Staphylococcus/Micrococcus exceeded 10⁴ CFU/mL. In the second layer, the total number of Thermokill Soxhlet and Enterobacteriaceae colonies was close to 10⁶ CFU/mL, while the number of Lactic Acid bacteria and Pseudomonas was close to 10⁵ CFU/mL, and the growth of Staphylococcus/Micrococcus and mold/yeast was the slowest, with the total number of colonies being about 10² CFU/mL. In the third layer, the total number of Thermokill Soxhlet colonies was close to 10⁴ CFU/mL, followed by the number of Enterobacteriaceae colonies, which was significantly higher than that of Pseudomonas, and the total number of Staphylococcus/Micrococcus and mold/yeast colonies was close to 10¹ CFU/mL. In the fourth layer, the total number of Thermokill Soxhlet colonies was close to 10³ CFU/mL, and the number of Enterobacteriaceae colonies was second only to Thermokill Soxhlet. The number of Lactobacillus species was higher than the number of Pseudomonas, and the total number of mold/yeast colonies was more than 10¹ CFU/mL, while the total number of Staphylococcus/Micrococcus colonies was less than 10¹ CFU/mL.

It can be inferred from Figure 4 that when cooled pork is stored at 10 ± 2ºC, the ability of some dominant spoilage bacteria to penetrate the 4 layers of cooled pork is as follows: Thermokill Soxhlet>Enterobacteriaceae>Lactic Acid bacteria>Pseudomonas>Yeast/Yeast>Staphylococcus/Micrococcus.

Growth and decline law and comparative analysis of spoilage microorganisms in cooled and segmented pork at 20 ± 2ºC

Figure 5 shows that as the storage time increases, the total number of bacteria and various spoilage microorganisms in cooled pork stored at 20 ± 2ºC also increase, with minimal growth in the first 12 hours and a period of rapid growth afterward. The growth rate tends to slow down after 42 hours. Thermocidae has the highest initial number and maintains a high number throughout the storage period. Enterobacteriaceae grows rapidly and has the second-highest number after Thermocidae. Lactic acid bacteria have a higher initial number but grow slightly slower than Enterobacteriaceae. The initial number of Pseudomonas is significantly smaller than that of the other bacteria but has a high growth rate in storage, ranking second only to lactic acid bacteria. Mold/yeast growth is slower, while Staphylococcus/Micrococcus has the lowest number and growth rate compared to the other bacteria, including Thermocida, Enterobacteriaceae, Lactic Acid Bacteria, and Pseudomonas species. Therefore, it can be concluded that Thermocida, Enterobacteriaceae, Pseudomonas, and Lactic Acid Bacteria are the dominant spoilage bacteria in cooled pork stored at 20 ± 2ºC (Figure 5).

Figure 5: The growth of spoilage microorganism in chilled pork stored at 20 ± 2ºC.

Actually, the information you provided is about chilled pork stored at 20 ± 2ºC, not cooled pork. It is important to note that there is a difference between “chilled” and “cooled” pork. Chilled pork refers to meat that has been cooled to a temperature just above freezing, typically between 0 and 2ºC, while cooled pork refers to meat that has been cooled to a higher temperature, usually between 4 and 10 ºC.

The study you mentioned showed that Enterobacteriaceae and Pseudomonas increased in proportion during storage of chilled pork at 20 ± 2ºC, while the proportion of Thermokill Soxhlet lactic acid bacteria, Staphylococcus/Micrococcus, and mold/yeast decreased. This suggests that Enterobacteriaceae and Pseudomonas are dominant spoilage bacteria in chilled pork under these conditions, with lactic acid bacteria also playing a role in spoilage. It is important to note that the dominant spoilage bacteria may differ under different storage conditions, such as temperature and packaging, and that proper storage and handling of meat is necessary to prevent spoilage and foodborne illness (Table 5).

Table 5: The change of microflora constitution of chilled pork storage at 20 ± 2ºC.

The microbial changes in the 4-layer part of pork during storage at 20 ± 2 ºC. From the results, it appears that the microbial growth tends to stabilize at the 30^th hour of storage. The first layer of cooled pork had the highest number of microorganisms, with Thermokill Soxhlet showing the most rapid growth rate and highest number of colonies. The second and third layers also showed high numbers of colonies of Enterobacteriaceae and Lactobacillus species. In contrast, the fourth layer had relatively lower numbers of colonies of all microorganisms except for Thermokill Soxhlet.

The results suggest that Thermokill Soxhlet is the most important spoilage bacteria in all four layers of meat, while Enterobacteriaceae and lactic acid bacteria are also significant spoilage microorganisms. Pseudomonas, Staphylococcus/Micrococcus, and mold/yeast had lower total colony numbers and weaker proliferation capacity at 20 ± 2ºC for 36 hours. Overall, the study provides important insights into the microbial changes in pork during storage at ambient temperatures, which can help in the development of better preservation strategies to prevent spoilage and improve food safety (Figure 6).

Figure 6: The spoilage microorganism quantity in 4-layer chilled meat at 20 ± 2°C.

Chilled meat is a nutrient-rich food that is susceptible to microbial contamination starting from the slaughtering process. Segmentation further exacerbates the secondary microbial contamination of chilled pork [8]. During subsequent storage and sales, variations in temperature, environmental hygiene, and cross-contamination by personnel can accelerate the proliferation of microorganisms. Microbial contamination and proliferation are the main causes of spoilage in chilled meat. When the bacterial count on the surface of chilled meat exceeds 10⁷ CFU/cm², it begins to deteriorate, and these spoilage characteristics are closely related to the biological and decay characteristics of the spoilage microorganism’s themselves [9]. Different types of spoilage microorganisms have varying growth and reproduction characteristics under different environmental conditions. They are influenced by factors such as nutrition, temperature, pH, competition, and coexistence, which produce unique microbial effects [10].

A study conducted by Jing, et al. revealed that the major spoilage bacteria in chilled meat were Pseudomonas (25%~26%), Enterobacteriaceae (19%~25%), Lactic acid bacteria (20%~21%), Micrococcus and Staphylococcus (12%~15%), and Cyclofilium thermodylidae (12%~13%) [3]. This finding differed from the results of the first detection ratio of Soxobacterium colonies in our study. Novakovic, et al. suggests that Pseudomonas is the dominant bacteria in cooling pork, while other scholars indicate that Zoobacterium, Moraxella, Enterobacteriaceae, and Soxobacter thermocidal can also be the dominant bacteria under different conditions [11,12]. The differences may be attributed to variations in production areas, meat processing enterprises, pork slaughtering environments, carcass segmentation equipment technology, and management measures of industry-related enterprises, resulting in initial contamination of different microorganisms and strain variations.

Experimental findings indicate that when stored at 2 ± 2ºC and 10 ± 2 ºC, the dominant spoilage bacteria in cooling pork are thermocidactylus, Enterobacteriaceae and Pseudomonas, with the highest proportion of Pseudomonas and Enterobacteriaceae [13]. While the initial number of lactic acid bacteria is higher, it is inhibited to some extent during storage. When stored at 20 ± 2 ºC, lactic acid bacteria grow more and become one of the dominant spoilage bacteria in addition to the above three. A study on the penetration of pork microorganisms at different levels of cooling and segmentation found that when stored at 2 ± 2ºC and 10 ± 2ºC, the proliferation of different species of spoilage microorganisms in the inner layer was as follows: Thermokill Soxhlet>Enterobacteriaceae>Lactic acid bacteria>Pseudomonas>Yeast/Yeast>Staphylococcus/Micrococcus. When stored at 20 ± 2ºC, the proliferation of different species of spoilage microorganisms was as follows: Thermokill Soxhlet>Enterobacteriaceae>Lactic acid bacteria>Yeast/Yeast>Pseudomonas>Staphylococcus/Micrococcus, mainly due to changes in oxygen levels affecting Enterobacteriaceae, lactic acid bacteria, and Pseudomonas [14].

Carcass contamination is the primary cause of cooled pork pollution, occurring when animals are first slaughtered and microorganisms are mainly distributed on the carcass surface. Subsequent segmentation breaks the carcass structural system, leading to secondary contamination of cooled pork. During the initial stages of segmentation, microorganisms are concentrated on the surface and have not yet penetrated the food's interior. However, further refinement of some cooled pork is necessary for production and consumer demand, leading to structural degradation and contamination of the inner layer [15]. Nevertheless, the overall number of microorganisms in the segmented cooled pork's interior is minimal or even sterile. Different types of spoilage microorganisms have distinct growth characteristics, adaptation abilities to cooling pork's surface and interior, and penetration rates from the surface to the inner layer. Facultative anaerobic microorganisms, such as Thermokill Soxhlet, Enterobacteriaceae, and lactic acid bacteria, have high proliferation rates in cooling pork. Pseudomonas is aerobic and proliferates at a relatively slow rate, while Pseudomonas aeruginosa is aerobic or facultative anaerobic with certain penetration abilities. Yeast/mold has slow reproduction rates but survive in hypoxic environments and have some penetration ability [16]. The proliferation rate of spoilage microorganisms is also influenced by factors such as oxygen partial pressure, nutrient utilization capacity, antagonism, and the composition of spoilage microorganisms in cooled pork [17].

Conclusion

This experimental study demonstrates that facultative anaerobic microorganisms have significant penetration ability, with growth occurring in each layer of cooling meat, and some microorganisms are in the late storage stages to grow in the inner layer of cooling meat. In general, the proliferation of Thermokill Soxhlet and Enterobacteriaceae is the highest, followed by lactic acid bacteria. Although one of the dominant spoilage microorganisms on the cooling meat surface, the number of yeast/mold proliferation is significantly lower than the above microorganisms. Enterprises should target sterilization to control the proliferation of cooled meat microorganisms and improve food safety.

Acknowledgments

We would like to express our sincere gratitude to all those who have contributed to the successful completion of this study on the growth, decline, and permeability of dominant microorganisms in chilled pork under different temperature conditions.

First and foremost, we extend our heartfelt appreciation to our research team members: Hai Dan, Zhang Yongshun, Huang Xianqing, Qiao Mingwu, Song Lianjun, Shen Yue, and Meng Ziheng, for their valuable inputs, tireless efforts, and teamwork throughout the study.

We also express our gratitude to the funding agencies, whose financial support made this research possible. We thank Science and Technology Innovation Team Support Program for Universities in Henan Province (23IRTSTHN023); and the Joint Postgraduate Training Base Project in Henan Province (YJS2022JD16) for their generous funding.

Our thanks also go to the technical staff and students at Henan Agricultural University, who provided assistance and support during the laboratory experiments. Finally, we would like to thank all the participants who provided their valuable time and samples for this study. Thank you all for your contributions to this research project.

Conflict of Interest

No conflict of interest declared.

Funding Sources

This work was supported by the Science and Technology Innovation Team Support Program for Universities in Henan Province (23IRTSTHN023); and the Joint Postgraduate Training Base Project in Henan Province (YJS2022JD16).

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