The effects of thermal treatment on the bacterial community and quality characteristics of meatballs during storage

Abstract Thermal treatment is a widely applied food processing technology in the meat industry due to its convenience. However, the interpretation of the changes in the bacterial community and quality properties in the thermal processed meat products have not been well established. Therefore, the effects of thermal treatment on the quality characteristics and bacterial communities in meatballs during storage at 4°C were investigated, which will provide a more comprehensive understanding of the influence of thermal treatment on the meat quality. Thermal treatment (121°C, 15 min) decreased the initial total viable bacterial counts by 2.1 log CFU/g and the diversity of the initial bacterial communities in meatballs. Compared with the thermal treatment group, a significantly more rapidly increasing trend of total volatile basic nitrogen and a decreasing trend of pH were observed in the control group. At the end of storage, the bacterial community was dominated by Streptococcus, Acinetobacter and Pseudomonas in the thermal treatment meatballs, whereas Pseudomonas, Pantoea, and Serratia. dominated the bacterial community of the control group. Besides, the predicted metabolic pathways revealed high levels of carbohydrate, amino acid, and lipid metabolism in the control group. This finding could contribute to a deep understanding of the influence of thermal treatment on the meat quality. Moreover, these results could provide a theoretical foundation for the development of alternative and novel nonthermal processing technologies for use in the meat industry.

the most commonly used one (Lyng et al., 2019;Misra & Jo, 2017;Wu et al., 2020). Thermal processing is a unit operation in which foods are heated at a sufficiently high temperature for specific amount of time to destroy vegetative microbial cells, spores and enzymes (Fellows, 2017). However, the texture, taste, flavor, and nutritional value of meat products are significantly deteriorated because of the thermal processing treatment ( Barbosa-Cánovas et al., 2014).
Studies have been performed to analyze the effects of thermal processing on the flavor profile, shelf-life, texture, and sensory characteristics of meat and meat products (Barbosa-Cánovas et al., 2014;Wang et al., 2019). For example, Wang et al. (2019) observed a significantly higher peroxide value in a water bath thermal sterilization group than that detected in the control group, which indicated that lipid oxidation in the vacuum-packaged braised beef was accelerated via water bath thermal sterilization. In addition, Song et al. (2019) showed that thermal processing at 121°C could prolong the shelflife but deteriorate the flavor properties of salted duck. In thermal processing of meat products, the change in food structure and loss of texture is common; additionally, lipid oxidation is a major cause of rancidity and off-flavors during subsequent storage (Misra & Jo, 2017). It is well established that the initial total bacterial counts significantly decreased via thermal processing treatment, whereas the changes of specific bacterial genus and bacterial metabolism in meat products have not been investigated. Currently, some studies have investigated the effect of processing treatments on the bacterial communities in meat and meat products (Han et al., 2020;Nieminen et al., 2012;Wang, Qin, et al., 2020). However, relatively little work has been done on the influence of thermal processing treatment on the bacterial communities in meat and meat products.
And the influence of thermal processing on the bacterial metabolism in meat products during storage has not been well detailed.
In the contemporary world, emerging technologies like high hydrostatic pressure, pulsed electric field, ultrasound, ionizing radiation, and cold atmospheric plasma have shown the potential in achieving industrial application in foods (Hernández-Hernández et al., 2019). However, microbial inactivation of these nonthermal processing technology can be influenced by the initial number of microorganisms, environmental influences, food composition, exposure time, the characteristics of microorganisms present in that food (Horita et al., 2018;Song et al., 2009). Hence, conventional thermal processing processed significant advantages in microbial inactivation than those in nonthermal processing technologies, and thermal treatment is still dominant in food preservation field .
Investigations of the effect of thermal treatment on the bacterial communities may provide the theoretical foundation for developing alternative and novel nonthermal processing technologies for use in the meat industry. Therefore, the effects of thermal processing treatment (121°C 15 min) on the quality characteristics and bacterial communities in meatballs during storage at 4°C were investigated, which will provide a more comprehensive understanding of the influence of thermal processing treatment on the meat quality and safety.

| Sampling and storage
Meatballs (ingredients: pork; horse hoof; shallot; and spices) with an average weight of 180 g/bag were collected from a local meat products processing company. Among the meatballs, seventy-six bags of meatballs were treated at 121°C for 15 min using a sterilization kettle (SH 800; Jinding Instruments) after they were packaged, which were assigned to the thermal processing group, while the other samples were assigned to the control group. All samples were transferred into chilled insulated boxes and transported to the laboratory within 3 hr. Then, all the samples were stored at 4°C for up to 21 days and analyzed at days 0, 7, 14, and 21.

| pH measurements
The pH of the meatball samples was measured according to the method described by Huang et al. (2020) with some appropriate modifications. Briefly, a 3.0 g sample of meatballs was mixed with 30 ml of an ice-cold solution (pH = 7.0) comprising 5 mM sodium iodoacetate and 150 mM potassium chloride. The mixtures were

| Total volatile basic nitrogen (TVB-N) assays
The TVB-N of meatballs was determined according to the method described by Gharibzahedi andMohammadnabi (2017) andHuang et al. (2020) with some appropriate modifications. Briefly, 10 g of minced meatball sample was homogenized in 100 ml of distilled water at room temperature for 30 min. After filtering, 10 ml of the resulting supernatant was mixed with 10 ml of MgO (10 g/L) and then distilled using a nitrogen apparatus (K1160, HaiNeng Instruments).
The TVB-N of meatball was expressed as mg of N per 100 g of meat.

| Total viable counts (TVC)
The total viable counts (TVC) for each sample were obtained using the pour plate technique according to the China National Food

Safety Standard methods (Food Microbiology Examination-Aerobic
Plate Count; GB 4789.2-2016). Briefly, 25 g of meatball sample was aseptically transferred to a sterile stomacher bag and homogenized in 225 ml of sterile saline for 2 min in a stomacher (BagMixer 400 VW, Interscience Co.). After performing 1:10 serial dilutions, 1 ml of the suspension from each dilution was inoculated onto plate count agar (PCA, Beijing Luqiao) and incubated at 37°C for 48 hr to allow the total viable counts to be determined. The results were expressed as decimal logarithms of colony-forming units per gram (LogCFU/g).

| Characterization of bacterial communities
2.5.1 | DNA extraction and 16S rRNA amplicon sequencing Total bacterial genomic DNA was extracted from meatball samples according to the method described by Zhang et al. (2017) with some modifications. Briefly, 10 ml of homogenates (obtained in section 2.4) were centrifuged at 13,000 × g for 10 min. Subsequently, the pelleted sediment was resuspended in 1 ml of a sterile 0.9% NaCl solution and transferred to centrifuge tubes. DNA was then extracted from the pel- Mix (New England Biolabs), 1 µl of primer, 2 µl of template DNA, and PCR grade water. Thermal cycling consisted of an initial denaturation at 98°C for 1 min followed by 30 cycles of denaturation at 98°C for 10 s, primer annealing at 50°C for 30 s, and extension at 72°C for 60 s, with a final extension step at 72°C for 5 min. The PCR products were mixed in identical ratios and purified using a GeneJET Gel Extraction kit (Thermo Scientific).

| Statistical analysis
The data are presented as the means and standard deviations.
Differences in mean values for TVB-N, pH, and TVC were analyzed using one-way ANOVA implemented in IBM SPSS Statistics 20.

| pH
The pH changes in the thermal processing and control group during refrigerated storage are shown in Figure 2a. The initial pH values of thermal processing and control group were 6.505 ± 0.02 and 6.510 ± 0.01, respectively, which demonstrated that thermal processing treatment has little influence on the initial pH of the F I G U R E 1 Experimental design and workflow of sample collection meatballs in this study. Wang, Shi, et al. (2020) investigated the effect of different thermal temperatures on the pH of Dezhou-braised chicken and observed that heat treatment did not significantly alter the initial pH of samples, which was consistent with our results.
Although the pH in both the thermal processing and control group decreased gradually throughout the entire storage period, the pH in the control group was significantly lower than that observed in the thermal processing group at the end of storage (p < .05). The reason for this result may be that more acid-forming bacteria were present in the control group, which may have led to a lower pH of the meatballs. The above findings indicated that the use of thermal treatment (121°C, 15 min) could delay the pH decrease in meatballs during storage. Song et al. (2019) studied the pH changes in untreated and thermal processed salted duck during 4°C storage, observing that thermal treatment (121°C, 30 min) could diminish the decreasing rate of pH in the salted ducks, which was in accordance with our findings.

| TVB-N
Total volatile basic nitrogen (TVB-N), which primarily comprises amines and ammonia, is widely used as an indicator of meat spoilage, resulting from the microbial degradation of protein and nonprotein nitrogenous compounds, such as amino acids and nucleotide catabolites (Liu et al., 2013). Figure 2b shows the effect of thermal treatment on the TVB-N in meatballs during 4°C storage. Compared with the control group, a significantly higher TVB-N value was observed in the thermal treatment group (p < .05), which indicated that the initial TVB-N in meatballs was significantly influenced by the thermal treatment. These results could be explained by the degradation reactions of proteins being accelerated by the thermal treatment process, which may have produced more amino acids and volatile bases in the thermal treatment group (Guo et al., 2016). The TVB-N values in both the thermal treatment and control groups displayed a continually increasing tendency during 4°C storage. Moreover, the control group exhibited a more dramatic increasing behavior, which may be due to more active bacterial metabolic activities in the control group. In cooked meat products, the increase in TVB-N is attributed to bacterial metabolic substances and degradation of protein and other nonprotein nitrogen-containing compounds, such as ammonia, monoethylamine, dimethylamine, and trimethylamine (Feng et al., 2016;.

| TVC
The initial TVC values of thermal treatment and control groups were 1.15 ± 0.15 and 3.41 ± 0.61 log CFU/g (Figure 2c), respectively, which demonstrated that the thermal treatment could kill and reduce approximately 2 log CFU/g in samples. Furthermore, bacteria grew more rapidly in the control group due to a significantly higher initial bacterial concentration. The TVC in the control group reached a maximum level of 7.14 ± 0.11 log CFU/g after 21 days of storage, whereas the TVC of the thermal treatment group was only 3.60 ± 0.13 log CFU/g. Wang et al. (2019) reported that the initial TVC value in vacuum-packaged braised beef was reduced by 1.6 log CFU/g using the thermal treatment (90°C, 30 min) method. In addition, Song et al., 2019) demonstrated that thermal treatment (121°C, 30 min) could decrease the initial TVC value by 1.6 log CFU/g in salted ducks. The results of this study also illustrated that the use of thermal treatment could effectively reduce the initial TVC of meat products during 4°C storage.

| Bacterial richness and diversity
Amplicon sequencing of the 16S rRNA gene could provide a comprehensive understanding of the diversity and abundance of bacterial communities in meatballs during refrigerated storage. A total of 1,020,024 high quality effective sequences were obtained by merging and filtering the raw sequences, which was equivalent to an average of 42,501 sequences for each sample. The sequence length in 24 samples was from 407.12-428.77 bp (Table 1). Coverage values for all samples were >99%, which demonstrated that the present sequencing results represented an accurate picture of the microorganisms in the samples. Effective sequences were clustered into 6,969 OTUs using a 97% similarity threshold. The alpha-diversity indexes are shown in Figure 3. At the beginning of storage, the thermal processing group showed a significantly lower richness value (Chao) than that observed in the control group (p < .05), which was primarily due to a section of microorganisms being damaged and apoptosis or a sub-lethally injured state was caused under the high tempera-  2015) confirmed that heat stress induces the death of sensitive species, which in turn could promote the proliferation of surviving species, reduce competition, and facilitate their access to resources. These findings could be explained by during 4°C storage, a more stable bacterial community developed in the thermal treatment group compared with that observed in the control group.

| Composition of bacterial communities
Twenty-one phyla were identified in the sequencing analysis, including Proteobacteria, Firmicutes, Bacteroidetes, Actinobacteria, and Acidobacteria (Figure 5a). In the literature, Abendroth et al. (2018) reported a heat-resistant bacterial profile that consisted primarily of Firmicutes, Bacteroidetes, and Proteobacteria. Proteobacteria was the most predominant phyla in the thermal treatment and control groups, contributing 40.18%∼52.96% and 86.78%∼99.57% of the total OTUs, respectively. The relative abundance of Proteobacteria was higher in the control group than that observed in the thermal treatment group, whereas the relative abundances of Firmicutes, Bacteroidetes, and Actinobacteria were higher in the thermal treat-  These findings indicate that the initial bacterial communities in meatballs were significantly altered via the thermal treatment. The  Another reason for this result may be that the enzymes participating in the growth and proliferation of microorganisms in meatballs were inactivated during the thermal treatment ( Figure 4).
The relative abundance of Pseudomonas spp. was higher in the thermal treatment group than that observed in the control group on day 0. This result was consistent with the TVB-N determination results, where the thermal treatment group showed a significantly higher initial TVB-N value. The results of previous studies have indicated that the growth of Pseudomonas spp. during storage was associated with changes in the TVB-N in meat products (Balamatsia et al., 2007). In addition, Pseudomonas spp. have been shown to possess strong proteinase and amino acid metabolic abilities in meat products Wang, Zhang, et al., 2017). For the control group, despite a low relative abundance of Pseudomonas spp. observed in the initial samples, these levels rapidly increased during storage, reaching the highest value in the meatballs on day 7 (contributing to 90.92% of the total OTUs). Subsequently, the relative abundance of Pseudomonas spp. continually decreased, while that of Pantoea spp. and Serratia spp. continually increased after 7 days. This transition in the bacterial community favoring facultative anaerobes could be attributed to the oxygen-limiting conditions encountered in meat following the rapid proliferation and predominance of Pseudomonas spp. (Enfors & Molin, 1984). Pseudomonas spp. are strictly aerobic; however, oxygen in the plastic packaging bag is gradually reduced during storage. This condition was beneficial for the growth and proliferation of facultative anaerobic bacteria, such as Pantoea spp. and Serratia spp., which often contribute to meat spoilage (Gram et al., 2002;Tian et al., 2017

| Functional properties of the bacterial communities
To further investigate the changes in bacterial metabolism resulting from the thermal treatment, the relative abundances of various bacterial metabolic pathways were predicted and compared. As shown in Figure 6, metabolic pathways were abundant in the samples, suggesting that bacterial metabolism in meatballs was vigorous. Among of other secondary metabolites, carbohydrate, amino acid, lipid, and energy metabolism that were associated with bacterial activity and meat spoilage showed significant active in the control group on day 0 or day 7. This result may be due to the microorganisms participating in carbohydrate and amino acid metabolism being killed or inhibited by the thermal treatment, or possibly because the enzymes involved in the metabolic pathways mentioned above were inactivated during the thermal treatment ( Figure 6).
The abundances of metabolic pathways associated with biogenic amines and sulfide formation were significantly higher in the thermal F I G U R E 6 Differential phylogenetic distribution of the bacterial metabolic activities in the thermal treatment and control groups during 4°C storage. (C: Control group; T: Thermal treatment group-121°C for 15 min) treatment group on day 0, such as those involved in phenylalanine, tyrosine and tryptophan biosynthesis metabolism, which were associated with Enterobacteriaceae and Pseudomonas spp. (Curiel et al., 2011). This result was consistent with the results of TVB-N and the observed bacterial communities in meatballs. Furthermore, pathways involved in glyoxylate and dicarboxylate metabolism, nitrogen metabolism involving arginine, and proline metabolism related to the formation of biogenic and volatile amines were significantly abundant in the control group on day 7, a finding that was in accordance with the TVB-N results.

| CON CLUS ION
The findings of this study demonstrated that thermal treatment (121°C, 15 min) could significantly decrease bacterial community diversity and the growth of potential spoilage bacteria in meatballs.
In particular, thermal treatment could largely decrease the relative abundance of bacterial metabolic pathways in meatballs, such as carbohydrate, amino acid, and lipid metabolism to maintain the freshness of meatballs during storage at 4°C. This finding could contribute to a deep understanding of the influence of thermal treatment on the meat quality. Moreover, these results could provide a theoretical foundation for the development of alternative and novel nonthermal processing technologies for use in the meat industry.

CO N FLI C T O F I NTE R E S T
The authors declare that they do not have any conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.