The Impact of High Temperature on Microbial Communities in Pork and Duck Skin

Pork skin and duck skin are highly favored by consumers in China, and high-temperature processing methods are widely employed in cooking and food preparation. However, the influence of high-temperature treatment on the microbial communities within pork skin and duck skin remains unclear. In this study, a high-temperature treatment method simulating the cooking process was utilized to treat samples of pork skin and duck skin at temperatures ranging from 60 °C to 120 °C. The findings revealed that high-temperature treatment significantly altered the microbial communities in both pork skin and duck skin. Heat exposure resulted in a decrease in microbial diversity and induced changes in the relative abundance of specific microbial groups. In pork skin, high-temperature treatment led to a reduction in bacterial diversity and a decline in the relative abundance of specific bacterial taxa. Similarly, the relative abundance of microbial communities in duck skin also decreased. Furthermore, potential pathogenic bacteria, including Gram-positive and Gram-negative bacteria, as well as aerobic, anaerobic, and facultative anaerobic bacteria, exhibited different responses to high-temperature treatment in pork skin and duck skin. These findings highlighted the substantial impact of high-temperature processing on the composition and structure of microbial communities in pork skin and duck skin, potentially influencing food safety and quality. This study contributed to an enhanced understanding of the microbial mechanisms underlying the alterations in microbial communities during high-temperature processing of pork skin and duck skin, with significant implications for ensuring food safety and developing effective cooking techniques.


Introduction
Pork skin and duck skin are commonly used food ingredients with high consumption rates and frequency in China.Currently, China ranks first in terms of pig population, slaughter rate, and pork production, with an average annual per capita pork consumption of 40 kg [1].Pork skin, which constitutes approximately 10% of the total weight of a pig carcass, is a valuable by-product of pork production.It is nutritionally rich, particularly in collagen protein, which can be converted into gelatin with a mesh-like structure after cooking, providing physiological benefits to the human body [2,3].Pork skin has broad market prospects and application value.It can be added to sausages, hams [4], and other meat products to enhance their texture, or processed into specialty products such as pickled pork skin and fried pork skin [5].Moreover, delicious dishes made primarily from pork skin, such as braised pork skin, pork skin jelly, and pork skin and red date soup, are highly favored by people [6].Pork skin, which contains 2.5 times the protein content of pork and is rich in fat, is highly susceptible to microbial contamination during storage and transportation [7].Moreover, the processing of pork skin involves manual operations such as peeling and segmentation, further increasing the risk of microbial contamination [8].Coupled with the nutritional richness of pork skin, a diverse range of bacteria can easily form on its surface.When pork skin is contaminated by bacteria, under certain temperature conditions, bacteria on the surface of pork skin can rapidly multiply and proliferate, leading to poor sensory quality, spoilage, and a significant impact on the shelf life of pork skin and its products, resulting in substantial economic losses [9,10].
Duck skin is a by-product of duck slaughtering processes, valued for its nutritional content and its capacity to enhance properties such as color, texture, and flavor in meat products [11].In contrast to developed countries, China exhibits relatively limited utilization of poultry by-products, with the extent and depth of utilization varying among enterprises of differing scales [12][13][14][15].Historically, the primary focus for utilizing pork skin, duck skin, and chicken skin has been the extraction of collagen proteins [16].However, recent years have witnessed a substantial shift, with food companies and researchers emphasizing a more comprehensive approach to the development and utilization of animal skins to meet the demands of an increasingly discerning consumer base.This includes innovations like the creation of novel and diverse ham sausages [17,18].
In China, the poultry slaughtering and processing process involves a series of steps, including hanging poultry, stunning with electric shock, slaughter, bleeding, scalding, depilation, body surface inspection, gizzard removal, feather cleaning, decapitation, evisceration, visceral inspection [19,20], separation of carcass by-products, by-product processing, precooling inspection, high-pressure rinsing of carcasses, pre-cooling and decontamination, post-precooling inspection, and segmentation [21].The pre-cooling and decontamination stage constitutes a critical control point for microbial management during poultry meat processing.Effective pre-cooling can regulate the growth of microbial organisms in duck meat and related products.The microbial load of duck skin raw materials during the precooling stage is closely linked to the initial bacterial count during the thermal processing stage, and the reduction in both the variety and quantity of microorganisms in duck skin significantly influences the control of meat product safety [22].
Currently, heat treatment remains a common method for sterilization in the food industry.Depending on the temperature, meat products can be categorized as low, medium, or high-temperature processed, with a sterilization temperature range of 60 to 120 • C. Different temperatures yield varying sterilization effects [23].Different types of bacteria exhibit varying degrees of heat tolerance, which can lead to variations in the spoilage bacterial communities in meat products subjected to different heat treatments.High-throughput sequencing technologies include a step involving polymerase chain reaction (PCR), which cannot distinguish between DNA from live and dead cells, potentially resulting in unrealistically high diversity estimates or overestimation of the number of live cells.However, propidium monoazide (PMA) contains azido groups that, when exposed to light, can covalently cross-link with DNA from dead cells, thus inhibiting the amplification of DNA from dead cells.PMA is incapable of entering live cells and covalently cross-links only with DNA from dead cells.Leveraging this advantage, PMA molecules are used to treat the DNA from dead bacteria in duck skin and pork skin after heat sterilization [24][25][26], thereby obtaining the composition of the bacterial communities that remain alive in duck skin and pork skin.
This approach is of significant importance in guiding the selection of sterilization processes for heat-treated meat products, as well as in the prevention and prediction of potential spoilage microorganisms.

Materials and Reagents
Fresh pork skins were sourced from a company located in Henan, China.The pork skins were collected following a standardized procedure.Similarly, fresh duck skin was obtained from a meat product company in Anyang, Henan Province, China, specifically using Beijing ducks.Both samples were handled and processed in accordance with the guidelines provided by the respective companies.NaCl, a key reagent used in the experiments, was purchased from Jiangtian Chemical Technology Co., Ltd., Tianjin, China.The DNA extraction kit was purchased from Beijing Tian En Ze Gene Technology Co., Ltd., Beijing, China.TransStart FastPfu DNA Polymerase was acquired from Beijing Tsingke Biotech Co., Ltd., Beijing, China.The 100 to 2000 bp DNA marker was obtained from Shanghai Sangon Biotech Co., Ltd., Shanghai, China.All other reagents and chemicals were purchased from Henan Bigen Biotechnology Co., Ltd., Zhengzhou, China and were of analytical grade.EX Taq enzyme was purchased from TaKaRa, Kusatsu, Shiga, Japan.Propidium Monoazide (PMA) was obtained from Biotium, Fremont, CA, USA.Dimethyl sulfoxide (DMSO) was purchased from Bigen Biotechnology Co., Ltd., Zhengzhou, China.

Sample Preparation and Grouping
Under sterile conditions, the pork skin and duck skin samples were divided into 24 random portions, each weighing 25 g.These portions were then placed in sterile homogenization bags and securely sealed with rubber bands.The sterile homogenization bags, each containing the pork skin and duck skin samples, were further divided into 3 groups, with 8 portions in each group.Each group was subjected to water bath heat sterilization at temperatures of 25, 60, 70, 80, 90, 100, 110, and 120 • C, following a standardized experimental procedure.
A needle-type meat center thermometer (range: 50-300 • C) was directly inserted into the duck and pig skin.When the center temperature of the sample reached the desired target temperature, a 10 min timer was initiated.Subsequent to the heat treatment, the samples were placed in a constant temperature incubator at 25 • C for 1 week to facilitate the enrichment of viable bacteria.
Following the 1-week incubation period, the samples were retrieved, and 225 mL of sterile saline solution was added to each sample.The samples were homogenized for 120 s using a stomacher.A 20 mL aliquot of the homogenized mixture was obtained by centrifugation at 17,226× g for 10 min to collect the bacteria.The supernatant was discarded, and the collected bacteria were resuspended in 500 µL of sterile saline solution (0.9% NaCl) and set aside.

PMA Treatment
To prepare the PMA (Propidium Monoazide) treatment, 1 mg of PMA was dissolved in 200 µL of 20% (v/v) dimethyl sulfoxide (DMSO) to create a stock solution of 5 µg/µL, which was stored at −20 • C in a light-protected environment.For experimental use, the PMA stock solution was diluted tenfold to generate a working PMA solution.
Using a transparent 1.5 mL centrifuge tube, 10 µL of the PMA working solution was added to 500 µL of the bacterial solution, resulting in a final mass concentration of 10 µg/mL.The mixture was then incubated in the dark at room temperature for 5 min.Subsequently, the sample was exposed to a 650 W halogen light source for 5 min, positioned approximately 20 cm away from the sample tube, which was placed horizontally on ice to prevent overheating.The sample tube was gently shaken during this process to ensure uniform illumination.After light-induced cross-linking, the sample was centrifuged at 17,226× g for 10 min to collect the bacteria.

Bacterial Genomic DNA Extraction
In total, 225 mL of sterile physiological saline solution was added to the sterile homogenization bags containing the duck skin and pork skin samples.The mixture was homogenized for 120 s using a stomacher.After homogenization, a 20 mL aliquot of the homogenized mixture was collected by centrifuging at 12,000 rpm for 10 min.Subsequently, bacterial genomic DNA was extracted from the precipitate using a bacterial DNA extraction kit.

Sequence Bioinformatics Analysis
The amplified DNA was sent to Shanghai Meiji Biomedical Technology Co., Ltd., Shanghai, China for sequencing using the Illumina MiSeq platform.The obtained sequences were subjected to bioinformatics analysis using FLASH and Trimmomatic software (Version 0.38).

Analysis of Relative Abundance of Bacterial Genera at the Phylum Level in Duck Skin and Pork Skin
Figure 1 illustrates the microbial composition in the control groups (25 • C) of pork skin and duck skin.In pork skin, the dominant genus was Psychrobacter, constituting 39.8% of the microbiota, whereas in duck skin, Pseudomonas was the dominant genus, comprising 30.65% of the total microbial population.
In the high-temperature treatment of pork skin ranging from 60 • C to 120 • C, the predominant genus shifted significantly.At 60 • C, Bacillus emerged as a major genus, accounting for 51.7% of the relative abundance, accompanied by a lower presence of Pseudomonas at 8.32%.This dominance by Bacillus escalated at 70 • C, reaching an impressive 98.9% relative abundance.At 80 • C and 90 • C, Bacillus remained the dominant genus, with average relative abundances of 85.9% and 85.0%, respectively.LysiniBacillus emerged as the subdominant genus, with relative abundances of 26.3% and 30.9% at 80 • C and 90 • C, respectively.The influence of Bacillus waned at 100 • C, accounting for 8.3% of the relative abundance.However, at 110 • C and 120 • C, Bacillus regained dominance with relative abundances of 72.5% and 86.4%, respectively.In the high-temperature treatment of pork skin ranging from 60 °C to 120 °C, the predominant genus shifted significantly.At 60 °C, Bacillus emerged as a major genus, accounting for 51.7% of the relative abundance, accompanied by a lower presence of Pseudomonas at 8.32%.This dominance by Bacillus escalated at 70 °C, reaching an impressive 98.9% relative abundance.At 80 °C and 90 °C, Bacillus remained the dominant genus, with average relative abundances of 85.9% and 85.0%, respectively.LysiniBacillus emerged as the subdominant genus, with relative abundances of 26.3% and 30.9% at 80 °C and 90 °C, respectively.The influence of Bacillus waned at 100 °C, accounting for 8.3% of the relative abundance.However, at 110 °C and 120 °C, Bacillus regained dominance with relative abundances of 72.5% and 86.4%, respectively.
At lower temperatures, such as 60 °C, Bacillus species were present but not as dominant, allowing other genera, like Pseudomonas, to coexist.However, as the temperature escalated to 70 °C and beyond, Bacillus's competitive advantage became increasingly evident.The spore-forming ability of Bacillus species, which enables them to withstand harsh conditions, including high temperatures, likely contributed to their dominance in these environments.
The temporary decrease in Bacillus abundance at 100 °C may be attributed to the extreme conditions at this temperature, potentially affecting the spore formation and survival of certain Bacillus species.However, at 110 °C and 120 °C, Bacillus species rebounded, showcasing their adaptability and resilience and enabling them to reestablish dominance within the microbial community.
In the case of duck skin subjected to temperatures from 60 °C to 80 °C, the microbial composition exhibited a more diversified distribution without a single dominant genus.Several genera contributed to the composition.At 60 °C, the major genera included Pseudomonas (33.52%) and Acinetobacter (18.55%), along with lower abundances of Serratia (5.71%) and Clostridium sensu stricto 7 (6.32%).At 70 °C, the dominant genera comprised Acinetobacter (18.46%),Clostridium sensu stricto 18 (12.41%),Serratia (11.04%),Pseudomonas (10.17%), and Clostridium sensu stricto 7 (8.62%).The 80 °C and 90 °C treatment groups were characterized by the dominance of Clostridium sensu stricto 18, with relative abun- At lower temperatures, such as 60 • C, Bacillus species were present but not as dominant, allowing other genera, like Pseudomonas, to coexist.However, as the temperature escalated to 70 • C and beyond, Bacillus's competitive advantage became increasingly evident.The spore-forming ability of Bacillus species, which enables them to withstand harsh conditions, including high temperatures, likely contributed to their dominance in these environments.
The temporary decrease in Bacillus abundance at 100 • C may be attributed to the extreme conditions at this temperature, potentially affecting the spore formation and survival of certain Bacillus species.However, at 110 • C and 120 • C, Bacillus species rebounded, showcasing their adaptability and resilience and enabling them to reestablish dominance within the microbial community.
At 110 • C, Bacillus demonstrates its exceptional resilience and becomes the overwhelmingly dominant genus, comprising nearly the entire microbial community.This dominance can be attributed to Bacillus's ability to form spores and endure extreme temperatures.In contrast, at 120 • C, the microbial community undergoes a shift, with Bacillus still present but sharing dominance with other genera.The co-dominance of Bacillus and Clostridium sensu stricto 18 and the emergence of Clostridium sensu stricto 7 at 120 • C suggest a more complex microbial response to the extreme conditions.

Analysis of Differences between Gram-Positive and Gram-Negative Bacteria
Gram-positive (Figure 2a) and Gram-negative (Figure 2b) column charts were employed to analyze and compare the relative abundance of various genera present in duck skin and pork skin samples.These visualizations facilitate the assessment of abundance variations among different genera and their distribution across diverse sample sets.Gram-Positive Bacteria (Figure 2a): Examining Figure 2a, it is evident that the dominant genus in both duck skin and pork skin samples within the control group (25 °C) is Vagococcus, with relative abundances of 17.07% and 24.89%, respectively.In duck skin, the relative abundance of Grampositive bacteria exhibits an upward trend as the temperature rises.Among the 60 °C heattreated duck skin samples, Gram-positive bacterial distribution is relatively uniform, with Clostridium sensu stricto 7 (8.99%)emerging as the dominant genus, followed by Vagococcus (6.27%).As the temperature continues to increase, the relative abundance of Grampositive bacteria in duck skin experiences a continuous escalation.The genus Bacillus demonstrates an initial surge followed by a decrease in relative abundance, with levels of 30.81%, 53.73%, 88.48%, and 69.45% at 90 °C, 100 °C, 110 °C, and 120 °C, respectively.In Gram-Positive Bacteria (Figure 2a): Examining Figure 2a, it is evident that the dominant genus in both duck skin and pork skin samples within the control group (25 • C) is Vagococcus, with relative abundances of 17.07% and 24.89%, respectively.In duck skin, the relative abundance of Gram-positive bacteria exhibits an upward trend as the temperature rises.Among the 60 • C heat-treated duck skin samples, Gram-positive bacterial distribution is relatively uniform, with Clostridium sensu stricto 7 (8.99%)emerging as the dominant genus, followed by Vagococcus (6.27%).
As the temperature continues to increase, the relative abundance of Gram-positive bacteria in duck skin experiences a continuous escalation.The genus Bacillus demonstrates an initial surge followed by a decrease in relative abundance, with levels of 30.81%, 53.73%, 88.48%, and 69.45% at 90 • C, 100 • C, 110 • C, and 120 • C, respectively.In contrast, the relative abundance of Gram-positive bacteria in pork skin gradually decreases with the elevation of temperature.The genus Bacillus exhibits an ascending pattern from 60 • C to 80 • C, reaching its zenith at 78.47% at 80 • C. Conversely, in heat-treated pork skin samples ranging from 90 • C to 120 • C, the abundance of Bacillus initiates a descent, reaching its nadir of 6.27% at 120 • C.
Gram-Negative Bacteria (Figure 2b): Figure 2b underscores that the dominant Gram-negative genera within the control group of duck skin include Citrobacter (20.63%) and Pseudomonas (13.01%).On the other hand, the control group of pork skin showcases prevailing genera Pseudomonas (20.73%) and Psychrobacter (14.23%).Upon evaluating the 60 • C heat-treated duck skin samples, the dominant genera are Citrobacter (17.91%) and Enlydrobacter (16.63%).With an increase in temperature, both the relative abundance of Citrobacter and Gram-negative bacteria in duck skin display a gradual decline.At 70 • C and 80 • C, the relative abundance of Citrobacter registers as 17.32% and 9.22%, respectively.Upon reaching 90 • C and 100 • C, the relative abundance of Gram-negative bacteria experiences a continued decrease, ultimately resulting in the absence of Citrobacter.The trend persists at 110 • C and 120 • C, where the relative abundance of Gram-negative bacteria also reaches negligible levels.Notably, in pork skin samples, the relative abundance of Gram-negative bacteria is non-existent in the heat-treated range of 60 • C to 90 • C. At 100 • C, the relative abundance of Psychrobacter accounts for 31.83%.The figure subsequently declines to 10.43% at 110 • C; however, at 120 • C, the relative abundance of Psychrobacter suddenly surges to 53.42%.
This comprehensive analysis utilizing Gram-positive and Gram-negative bacterial classifications provides valuable insights into the behavior of different bacterial genera in duck and pork skin samples under varying thermal conditions.The results underscore the temperature-sensitive dynamics of bacterial populations, highlighting their adaptation and response to heat treatments.These findings contribute to a deeper understanding of bacterial distribution and serve as a foundation for optimizing food safety strategies in both duck and pork processing.
Based on the analysis of Figure 2, it can be concluded that the relative abundance of Gram-negative bacteria is 0 in duck skin at 110 • C heat treatment and in pork skin from 60 • C to 90 • C heat treatment.The relative abundance of Gram-negative bacteria in duck skin decreases as the temperature increases, while in pork skin, the relative abundance of Gram-negative bacteria initially increases and then decreases, reaching the highest level at 120 • C.This indicates differences in the presence of Gram-negative bacteria between duck skin and pork skin samples.
Regarding Gram-positive genera, the dominant genera in the control groups of both duck skin and Pork skin are Vagococcus, while in the treated groups, the dominant genera are Bacillus for both duck skin and pork skin.The relative abundance of Gram-positive bacteria in duck skin samples increases continuously as the temperature rises, with the highest abundance of Bacillus at 110 • C. On the other hand, the relative abundance of Gram-positive bacteria in pork skin gradually decreases with increasing temperature, with the highest abundance of Bacillus at 80 • C. In the examination of Gram-negative genera, the prevailing genus identified in duck skin samples is Citrobacter, whereas in pork skin samples, a different trend emerges.Proper temperature control during food processing and cooking is of paramount importance for the survival and distribution of microorganisms.Varied temperature treatments can lead to shifts in the relative abundance of different bacterial types within food samples, consequently influencing the quality and safety of food products.The formulation of precise food processing strategies is imperative to ensure the integrity and safety of food items.

Analysis of Differences in Aerobic, Anaerobic, and Facultative Anaerobic Bacteria
Aerobic Bacteria (Figure 3a): where the initial reduction in aerobic bacteria is succeeded by an increase.This shift coincides with the transition from the Micrococcus genus to Psychrobacter as the dominant genus.In the control group of duck skin, Pseudomonas dominates with the highest relative abundance (13.01%), followed by Alcaligenes (11.27%).Throughout the heat treatment process, Enhydrobacter abundance steadily declines between 60 °C and 90 °C, displaying slight recovery at 100 °C before dropping to undetectable levels at 110 °C and 120 °C.
Conversely, the control group of pork skin displays higher relative abundances of Pseudomonas (20.74%) and Psychrobacter (14.23%).Following heat treatment, pork skin samples are characterized by Micrococcus dominance at 60 °C (20.24%) and 70 °C (11.73%).A decline occurs at 80 °C, with a subsequent increase at 90 °C.Notably, the abundance of aerobic bacteria initially decreases, followed by a subsequent increase, peaking at 120 °C (53.42%).This increase corresponds with the shift from Micrococcus to Psychrobacter as the dominant genus.The comparison of aerobic bacteria in duck skin and pork skin samples at different storage temperatures reveals distinctive trends.In duck skin, the abundance of aerobic bacteria progressively diminishes with rising temperatures, eventually reaching negligible levels at 110 • C and 120 • C. Conversely, pork skin samples exhibit a nuanced pattern, where the initial reduction in aerobic bacteria is succeeded by an increase.This shift coincides with the transition from the Micrococcus genus to Psychrobacter as the dominant genus.
In the control group of duck skin, Pseudomonas dominates with the highest relative abundance (13.01%), followed by Alcaligenes (11.27%).Throughout the heat treatment process, Enhydrobacter abundance steadily declines between 60 • C and 90 • C, displaying slight recovery at 100 • C before dropping to undetectable levels at 110 • C and 120 • C.
A decline occurs at 80 • C, with a subsequent increase at 90 • C. Notably, the abundance of aerobic bacteria initially decreases, followed by a subsequent increase, peaking at 120 • C (53.42%).This increase corresponds with the shift from Micrococcus to Psychrobacter as the dominant genus.
Anaerobic Bacteria (Figure 3b): The comparison of anaerobic bacteria in duck skin and pork skin samples exposes variations.Overall, pork skin demonstrates lower anaerobic bacterial abundance.In duck skin, the abundance of anaerobic bacteria experiences an initial rise, followed by a decline, with peak levels recorded at 80 • C.
In the control group of duck skin, Peptoniphilus dominates with a relative abundance of 4.31%.Notable transformations occur in the anaerobic bacteria composition during heat treatment.At 60 • C, Clostridium sensu stricto 7 prevails (8.99%), followed by a shift to Lachnoclostridium (19.92%) at 70 • C. At 80 • C, anaerobic bacteria distribution evens out, with Hathewaya exhibiting higher abundance (13.74%).The dominant phylum shifts to Anaerosalibacter at 90 • C and 100 • C, with relative abundances of 31.09% and 24.68%, respectively.At 120 • C, Clostridium sensu stricto 7 resurfaces as the dominant genus with a relative abundance of 26.25%.
Contrarily, the control group of pork skin demonstrates lower anaerobic bacteria abundance, with no prevailing genus.Under distinct heat treatment conditions, anaerobic bacteria levels remain consistently low.An initial decrease is succeeded by a sharp increase, followed by subsequent decreases.At 90 • C, Clostridium sensu stricto 13 experiences a sudden surge, becoming the dominant genus (11.88%).Anaerobic bacteria abundance reaches zero at 100 • C, slightly recovers at 110 • C, and maintains a minor resurgence at 120 • C.
Facultative Anaerobic Bacteria (Figure 3c): The abundance of facultative anaerobic bacteria in duck skin decreases progressively with increasing temperature, ultimately reaching absence at 110 • C. In pork skin, facultative anaerobic bacteria initially increase and peak at 80 • C (65.97%), subsequently decreasing.The decrease persists at 110 • C and 120 • C, maintaining levels lower than those in the control group.
In the control group of duck skin, Citrobacter dominates with a relative abundance of 20.63%, followed by Vagococcus at 11.33%.With rising temperatures, Citrobacter abundance gradually decreases at 60 • C, 70 • C, and 80 • C (17.91%, 17.32%, 9.22%, respectively).At 90 • C, the dominant genus shifts to Bacillus.As temperatures reach 100 • C and 110 • C, Bacillus abundance decreases to 1.00% and 0%, respectively.At the highest temperature of 120 • C, Bacillus abundance slightly recovers to 0.17%.Conversely, Citrobacter abundance in duck skin decreases with increasing temperature, yielding Bacillus as the dominant genus with continuous decline.
For the control group of pork skin, Vagococcus prevails with a relative abundance of 17.62%, followed by Leucobacter at 6.20%.As temperatures rise, Bacillus becomes the dominant genus, peaking at 65.97% at 80 • C.However, at 90 • C and 100 • C in pork skin samples, Bacillus abundance diminishes to 63.90% and 42.36%, respectively.In pork skin samples at 110 • C and 120 • C, Bacillus abundance continues to decline to 13.43% and 5.71%, respectively.

Identification and Analysis of Potential Pathogenic Bacteria
In recent times, concerns regarding food safety due to potential pathogenic bacteria have heightened public awareness.This study investigates the relative abundance of potential pathogenic bacteria in duck skin and pork skin samples across a spectrum of temperatures (Figure 4).Notably, this analysis sheds light on the dynamic behavior of these bacteria under varying thermal conditions.
Heat Treatment Effects: Throughout the heat treatment process, the once-dominant genus Citrobacter among potential pathogenic bacteria in duck skin exhibited a consistent decline from 60 °C to 80 °C, culminating in absence at 120 °C.This absence was replaced by Clostridium sensu stricto 7 (26.25%).For pork skin, Bacillus emerged as the dominant genus among potential pathogenic bacteria across temperatures from 60 °C to 90 °C, constituting 53.11%, 69.69%, 76.92%, and 69.63%, respectively.A shift occurred at 100 °C, where Bacillus and Psychrobacter displayed analogous abundance levels at 31.65% and 31.83%,respectively.The shift continued at 120 °C, with Psychrobacter becoming the dominant genus (53.42%) while Bacillus declined.
An intricate examination of Figure 5 uncovers potential pathogenic bacteria in duck skin, encompassing Pseudomonas, Proteus, Acinetobacter, Brochothrix, Serratia, Clostridium sensu stricto 5, Erysipelothrix, Myroides, Escherichia-Shigella, Providencia, Bacteroides, Citrobacter, and Peptoniphilus.For pork skin, potential pathogenic bacteria comprise Pseudomonas, Psychrobacter, Acinetobacter, Proteus, Serratia, Brochothrix, Staphylococcus, Clostridium sensu stricto 5, Peptoniphilus, and Enterococcus.While these bacteria share the classification of potential pathogens owing to their capability to induce diseases, their specific pathogenic traits and conditions can exhibit variability due to multifarious factors.Thorough research and analysis are essential to categorize them as opportunistic or foodborne pathogens.An intricate examination of Figure 5 uncovers potential pathogenic bacteria in duck skin, encompassing Pseudomonas, Proteus, Acinetobacter, Brochothrix, Serratia, Clostridium sensu stricto 5, Erysipelothrix, Myroides, Escherichia-Shigella, Providencia, Bacteroides, Citrobacter, and Peptoniphilus.For pork skin, potential pathogenic bacteria comprise Pseudomonas, Psychrobacter, Acinetobacter, Proteus, Serratia, Brochothrix, Staphylococcus, Clostridium sensu stricto 5, Peptoniphilus, and Enterococcus.While these bacteria share the classification of potential pathogens owing to their capability to induce diseases, their specific pathogenic traits and conditions can exhibit variability due to multifarious factors.Thorough research and analysis are essential to categorize them as opportunistic or foodborne pathogens.
Temperature-Dependent Abundance Changes: A significant finding pertains to the shifts in relative abundance of potential pathogenic bacterial genera in both duck skin and pork skin as temperature fluctuates.Remarkably, potential pathogenic bacteria in pork skin maintain relatively elevated abundance levels, contrary to the trend seen in duck skin where their relative abundance diminishes with escalating temperature.Among them, the abundance of potential pathogenic bacteria in pork skin remained at a relatively high level, while the relative abundance of potential pathogenic bacteria in duck skin decreased as the temperature increased.Especially in the high-temperature treatment group, the relative abundance of Bacillus in pork skin decreased and was replaced by Psychrobacter.This suggests that although high-temperature treatment has a certain inhibitory effect on the survival and proliferation of Bacillus, preventive measures against Psychrobacter should be formulated during the processing and production of pork skin.This investigation highlights the nuanced behavior of potential pathogenic bacteria within duck and pork skin samples across diverse temperature ranges.The findings shed light on the intricate interactions between microbial community structures and their respective niches within the pork and duck skin environments, influenced by the environmental factor of temperature.These findings underscore the imperative need for the development of tailored food safety strategies and production protocols.The temperature-dependent dynamics of potential pathogenic bacteria emphasize the importance of understanding and managing microbial ecology in food processing and safety protocols.Temperature-Dependent Abundance Changes: A significant finding pertains to the shifts in relative abundance of potential pathogenic bacterial genera in both duck skin and pork skin as temperature fluctuates.Remarkably, potential pathogenic bacteria in pork skin maintain relatively elevated abundance levels, contrary to the trend seen in duck skin where their relative abundance diminishes with escalating temperature.Among them, the abundance of potential pathogenic bacteria in pork skin remained at a relatively high level, while the relative abundance of potential pathogenic bacteria in duck skin decreased as the temperature increased.Especially in the high-temperature treatment group, the relative abundance of Bacillus in pork skin decreased and was replaced by Psychrobacter.This suggests that although high-temperature treatment has a certain inhibitory effect on the survival and proliferation of Bacillus, preventive measures against Psychrobacter should be formulated during the processing and production of pork skin.This investigation highlights the nuanced behavior of potential pathogenic bacteria within duck and pork skin samples across diverse temperature ranges.The findings shed light on the intricate interactions between microbial community structures and their respective niches within the pork and duck skin environments, influenced by the environmental factor of temperature.These findings underscore the imperative need for the development of tailored food safety strategies and production protocols.The temperature-dependent dynamics of potential pathogenic bacteria emphasize the importance of understanding and managing microbial ecology in food processing and safety protocols.

Conclusions
This study elucidates the alterations in the microbial composition in response to different temperature conditions in duck and pork skin.Notably, high-temperature treatment of pork skin results in a prevalence of Bacillus species, while duck skin demonstrates a more evenly distributed bacterial population.Furthermore, the influence of temperature is evident in the relative abundance of Gram-positive and Gram-negative bacteria, as well as aerobic and anaerobic bacteria.Moreover, it is evident that temperature has a significant impact on the presence of potentially pathogenic bacteria, underscoring the critical role of temperature control in ensuring food safety.Subsequent research endeavors should aim to delve deeper into the underlying mechanisms governing temperature-induced shifts in microbial dynamics and explore the roles of microbial consortia within

Conclusions
This study elucidates the alterations in the microbial composition in response to different temperature conditions in duck and pork skin.Notably, high-temperature treatment of pork skin results in a prevalence of Bacillus species, while duck skin demonstrates a more evenly distributed bacterial population.Furthermore, the influence of temperature is evident in the relative abundance of Gram-positive and Gram-negative bacteria, as well as aerobic and anaerobic bacteria.Moreover, it is evident that temperature has a significant impact on the presence of potentially pathogenic bacteria, underscoring the critical role of temperature control in ensuring food safety.Subsequent research endeavors should aim to delve deeper into the underlying mechanisms governing temperature-induced shifts in microbial dynamics and explore the roles of microbial consortia within skin environments.

Microorganisms 2023 , 14 Figure 1 .
Figure 1.Displays the analysis of the relative abundance of microbial genera at the species level in duck skin (DS) and pork skin (PS).

Figure 1 .
Figure 1.Displays the analysis of the relative abundance of microbial genera at the species level in duck skin (DS) and pork skin (PS).

Figure 2 .
Figure 2. Presents the analysis of Gram-positive (a) and Gram-negative (b) microbial communities.

Figure 2 .
Figure 2. Presents the analysis of Gram-positive (a) and Gram-negative (b) microbial communities.

Figure 3 .
Figure 3.Comparison of aerobic (a), anaerobic (b), and facultative anaerobic (c) bacterial quantities in the microbial composition of duck skin and pork skin.

Figure 3 .
Figure 3.Comparison of aerobic (a), anaerobic (b), and facultative anaerobic (c) bacterial quantities in the microbial composition of duck skin and pork skin.

Figure 4 .
Figure 4. Analysis of the relative abundance of potential pathogenic bacteria.

Figure 4 .
Figure 4. Analysis of the relative abundance of potential pathogenic bacteria.Heat Treatment Effects: Throughout the heat treatment process, the once-dominant genus Citrobacter among potential pathogenic bacteria in duck skin exhibited a consistent decline from 60 • C to 80 • C, culminating in absence at 120 • C.This absence was replaced by Clostridium sensu stricto 7 (26.25%).For pork skin, Bacillus emerged as the dominant genus among potential pathogenic bacteria across temperatures from 60 • C to 90 • C, constituting 53.11%, 69.69%, 76.92%, and 69.63%, respectively.A shift occurred at 100 • C, where Bacillus and Psychrobacter displayed analogous abundance levels at 31.65% and 31.83%,respectively.The shift continued at 120 • C, with Psychrobacter becoming the dominant genus (53.42%) while Bacillus declined.An intricate examination of Figure5uncovers potential pathogenic bacteria in duck skin, encompassing Pseudomonas, Proteus, Acinetobacter, Brochothrix, Serratia, Clostridium sensu stricto 5, Erysipelothrix, Myroides, Escherichia-Shigella, Providencia, Bacteroides, Citrobacter, and Peptoniphilus.For pork skin, potential pathogenic bacteria comprise Pseudomonas, Psychrobacter, Acinetobacter, Proteus, Serratia, Brochothrix, Staphylococcus, Clostridium sensu stricto 5, Peptoniphilus, and Enterococcus.While these bacteria share the classification of potential pathogens owing to their capability to induce diseases, their specific pathogenic traits and conditions can exhibit variability due to multifarious factors.Thorough research and analysis are essential to categorize them as opportunistic or foodborne pathogens.Temperature-Dependent Abundance Changes: A significant finding pertains to the shifts in relative abundance of potential pathogenic bacterial genera in both duck skin and pork skin as temperature fluctuates.Remarkably, potential pathogenic bacteria in pork skin maintain relatively elevated abundance levels, contrary to the trend seen in duck skin where their relative abundance diminishes with escalating temperature.Among them, the abundance of potential pathogenic bacteria in pork skin remained at a relatively high level, while the relative abundance of potential pathogenic bacteria in duck skin decreased as the temperature increased.Especially in the high-temperature treatment group, the relative abundance of Bacillus in pork skin decreased and was replaced by Psychrobacter.This suggests that although high-temperature treatment has a certain inhibitory effect on the survival and proliferation of Bacillus, preventive measures against Psychrobacter should be formulated during the processing and production of pork skin.This investigation highlights the nuanced behavior of potential pathogenic bacteria within duck and pork skin samples across diverse temperature

Microorganisms 2023 , 14 Figure 5 .
Figure 5. Relative abundance changes of surface microbiota based on the genus level.

Figure 5 .
Figure 5. Relative abundance changes of surface microbiota based on the genus level.