Antimicrobial property of recombinant Lactolisterin BU in vitro and its initial application in pork refrigerated storage

Lactolisterin BU is a novel bacteriocin identified from Lactococcus lactis in 2017. It exhibits antimicrobial activity against food spoilage and foodborne pathogens. In this study, Lactolisterin BU was expressed in Pichia pastoris (P.pastoris) and isolated from the supernatant of yeast culture for the first time. It was found to exhibit a broad antimicrobial spectrum and rapid bactericidal activity against foodborne bacterial pathogens, both gram-positive and gram-negative ones, with minimum inhibition concentrations ranging within 10–60 μg/mL. The recombinant Lactolisterin BU (rLactolisterin BU) also had an antioxidant effect and was resistant to heating, acid–base, and high-dose-saline treatments and barely had any hemolytic activity or cytotoxicity. Moreover, rLactolisterin BU effectively suppressed the growth of bacterial pathogens; suppressed the increases in pH, total volatile basic nitrogen (TVB-N), and thiobarbituric acid reactive substances (TBARS) of pork samples; and maintained a high quality of fresh pork during storage at 4 ℃. Furthermore, rLactolisterin BU effectively inhibited the growth of three kinds of bacteria in a pork-spoilage model. Taken together, rLactolisterin BU could be a promising preservative for food storage.


Introduction
Pork and pork products are essential foods for people around the world. They provide high-value nutrition, including all the essential amino acids, lipids, vitamins, and minerals. However, microbial-pathogen contamination always occurs during processing, storage, transportation, distribution, and retailing [1]. Notably, consumers are paying increased attention to food quality and safety [2], especially to nutritional values, food-processing procedures, storage conditions, and shelf-lives. Although the traditional storage methods such as using low temperature, vacuum packaging, and chemical preservatives can prolong the storage time of pork to some extent, they may damage the sensory properties or introduce food-hazard risks during storage [3]. Thus, new storage methods and antimicrobial agents are urgent to develop for meat preservation. In recent years, bio-preservation technology has emerged as a potential method with high efficiency and no chemical preservatives. It uses natural metabolic products from various organisms in the earth to ensure food security by preventing foodpathogen reproduction [4]. In particular, antimicrobial peptides (AMPs) are gaining widespread attention in related to food preservation owing to its low molecular weight, extensive bactericidal performance, stable bioactivity, and lack of residue and drug resistance [5]. AMPs are generally spread in various organisms, including microorganisms, plants, invertebrates, and vertebrates [6]. At present, more than 2700 types of AMPs have been isolated, and their sequences can be found in the Antimicrobial Peptide Database (http:// aps. unmc. edu/ AP/). Many AMPs including nisin [7], mytichitin-CB [8], and MccJ25(G12Y) [9], which originate from Lactococcus lactis, Mytilus coruscu, and E. coli, respectively, reportedly have the potential to be substituted for chemical preservatives for inhibiting the growth of foodborne pathogens in various foods, including meat, yogurt, and fruit drinks. Therefore, the application of these AMPs provided a promising way to improve food safety and quality by inhibiting food pathogens/spoilage microorganisms.
Lactolisterin BU with 42 amino acids was identified from Lactococcus lactis in 2017 and found to exhibit potent antimicrobial activity against food-spoilage bacteria, such as Staphylococcus aureus, Bacillus spp., Listeria monocytogenes, and Streptococci. These features suggest that Lactolisterin BU may be applied as a new type of food preservative in the food industry [10]. However, natural Lactolisterin BU AMPs exist in low abundance, which is a drawback for its massive production and application. The application of Lactolisterin BU in food has also not yet been investigated.
To further explore the function of Lactolisterin BU, the current work aimed to generate rLactolisterin BU from Pichia pastoris system by recombinant-DNA engineering method, to evaluate its in vitro antimicrobial activity and biostability, and to investigate the effect of rLactolisterin BU when used as a food preservative in pork-preservation models.

Expression and purification of 6 × His-rLactolisterin BU
Positive transformants were collected, placed in BMGY medium, and cultured at 28 °C and 250 rpm/min for 24 h. The yeast culture was then inoculated into fresh BMGY medium at a ratio of 5% and cultured at 28 °C and 250 rpm/min. When the optical density (OD) of 600 nm reached 8-10, the cultured cells were collected by centrifugation at 4000 rpm/min and washed twice with sterilized deionization water before culturing in fresh BMGY medium at 28 °C and 250 rpm/min. Then, 0.22 μm-membrane-filtered 100% methanol was added into cultures every 24 h until reaching 144 h of induction with a final concentration of 1% methanol. The cultured suspension was collected by centrifugation at 6000 rpm/ min, and the supernatant was collected for affinity purification and Tricine-SDS-PAGE analysis. Then, the 6 × His-Lactolisterin BU was bound to the Ni-NTA column after equilibration with binding buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, and 5 mM imidazole; pH 7.4), and then the unbound proteins were washed away by washing buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, and 60 mM imidazole, pH 7.4). The purified 6 × His-rLactolisterin BU was separated using elution buffer (20 mM NaH 2 PO 4 , 500 mM NaCl, and 500 mM imidazole; pH 7.4) and then stored at − 80 °C after lyophilization.

Tricine-SDS-PAGE and silver staining
Tricine-SDS-PAGE assay was carried out as previously described with minor modifications [11]. We used 20.0% of separating gels and 4.0% stacking gels to separate the small-molecular-weight proteins in the samples, with the voltage set to 60 V before all samples entered the separating gels. Then, the voltage was increased to 120 V until the proteins ran to the gel bottom. Afterwards, the gels were stained with a silver staining kit (G7210, Solarbio, Beijing, China) following the manufacturer's instructions and captured with a gel imager (CLINX GenoSens 1800, Shanghai, China).

Minimum inhibition concentration (MIC) assay
The bacterial growth-inhibition effect was determined as previously described [12]. In a typical procedure, rLactolisterin BU from P. pastoris was adjusted with PBS to a series of concentrations ranging within 0-60 μg/mL and incubated with tested bacterial cultures (S. aureus ATCC 25923, S. enteriditis ATCC 10467, E. coli H7:O157 ATCC 35150, E.coli ATCC 25922, B. subtilis AHU 1035, P. aeruginosa ATCC 27853 and L. monocytogenes ATCC 21633) at mid-log phase with concentrations of about 5 × 10 5 CFU/mL for 14 h at 37 °C. PBS buffer served as a negative control. The purified Lactolistrin BU from Lactococcus lactis were diluted with PBS to a serious of concentrations as described in the previous study [10], which were used to perform the comparative trial with the rLactolisterin BU from P. pastoris. Then, the optical value at 600 nm was measured with a microplate reader (HBS-1096A, Detie, Nanking, Jiangshu, China). All experiments were performed three times.

Time-killing kinetic assay
To determine the time-killing kinetics of rLactolisterin BU, E. coli H7:O157 ATCC 35150 was used to count the colonies with different treatments and to create the timekilling curve as previously described [13,14]. In a typical procedure, the E. coli colony was collected and initially cultured in LB medium at 37 °C and 200 rpm/min for 12 h. After incubating the cell suspensions in LB medium at a ratio of 1:100 (v/v) and in various concentrations of rLactolisterin BU (0 × , 0.5 × , 1 × , 2 × , and 4 × MIC) until the bacterial cell cycle reached the mid-log growth phase. The post-incubation samples, collected at different time points (0, 30, 60, 90, 120, 150, and 180 min), were spread onto the solid LB medium and cultured at 37 °C for 12 h before counting the viable colonies. The LB medium and gentamicin (50 µg/mL) treated bacteria suspensions served as negative and positive controls, respectively. All assays were performed in triplicate.

Biofilm formation assay
Crystal violet staining assay was used to investigate the biofilm inhibition effect of rLactolisterin BU as described previously [15], and E. coli H7:O157 ATCC 35150 was applied as the tested strain. Gentamicin was used as positive control, and PBS was used as blank control.

Cell culture and cytotoxicity assay
The cytotoxicity of rLactolisterin BU on mammalian cells was determined using MTT method as previously described [14]. In a standard procedure, mouse RAW264.7 cells were cultured in different concentrations of rLactolisterin BU at 37 °C in 5% CO 2 atmosphere for 24 h. The post-incubation cell suspensions were mixed with MTT solutions and incubated for 3 h before removing the supernatant. Then, the formazan crystals were dissolved in DMSO, and the OD at 570 nm was measured using a microplate reader (HBS-1096A, Detie, Nanking, China). The percentage of cell viability was calculated according to the formula: cell viability (%) = OD sample/OD control. All experiments were performed in triplicate.

Hemolytic assay
The hemolytic activity of rLactolisterin BU was measured as previously described [14]. Blank and 100% positive controls were prepared with rabbit erythrocytes suspended in PBS or 1% Triton X-100, respectively. All assays were performed in triplicate.

Inhibition-zone assay
Inhibition-zone assay was applied to determine the antimicrobial activity of rLactolisterin BU under various treatment conditions through the growth-inhibition effect on E. coli as previously described [14]. In a typical procedure, a single colony of E. coli H7:O157 ATCC 35150 was picked, cultured in LB medium at 37 ℃ and 200 rpm/min for 12 h, and transferred into fresh LB medium at a ratio of 1:100. The bacterial cultures were then spread on solid LB-medium plates when cell growth reached the mid-log phase (1 × 10 5 to 5 × 10 5 CFU/mL). Then, the rLactolisterin BU samples dissolved in PBS buffer at various pH values (2, 4, 6, 8, and 10) or salt concentrations (20, 40, 60, 80, and 100 mM), heated at various temperatures (4 ℃, 25 ℃, 37 ℃, 65 ℃, and 90 ℃), or digested with different proteinases (papain, pepsin, trypsin, and proteinase K) were prepared and added to the punched wells of the plates. After 14-16 h of incubation, the incubation zones were captured and analyzed with image J software. All assays were performed in three times.

Antioxidant-activity assay
The hydroxyl and superoxide anion radical-scavenging effects were used to determine the antioxidant ability of rLactolisterin BU by using a Hydroxyl Free-Radical Scavenging Capacity Assay Kit (BC1325, Solarbio, Beijing, China) and Superoxide Anion Detection Kit (BC1290, Solarbio, Beijing, China) as described by the manufacturer's instructions. Furthermore, the DPPH and ABTS + test methods were performed as described in our previous study [13]. All assays were carried out in triplicate.

Treatment with various concentrations of rLactolisterin BU in pork models
Fresh pork legs were purchased from a local supermarket of Binzhou, China. The lean-pork meat slices (5 ± 0.1 g) were prepared as previously described with minor modifications [8]. In a typical procedure, the pork meat was handled using a knife to remove the fat, tissues, and porcine cortical skins aseptically. Then, the slices were soaked in rLactolisterin BU with different concentrations (0, 20, 40, and 60 μg/mL) for 10 min before 10-day storage experiments at 4 ℃. The sensory properties and bacterial count were then determined every 24 h. All experiments were carried out in triplicate.

Microbiological analysis
The slices with various treatments were diluted with saline solution (1% w/w) at a ratio of 1:10 and homogenized for 5 min every 24 h for 10 days to determine the bacterial viability. For total viable count (TVC), platecount agar was used to incubate the decimal homogenates' dilutions at 37 ℃ for 48 h. For S. aureus count, mannitol-salt agar was used to incubate the decimal homogenates' dilutions at 37 ℃ for 48 h. Similarly, Violet Red bile agar was used for E. coli count at 37 ℃ for 48 h. For L. monocytogenes count, McBride agar base was used to incubate the decimal homogenates' dilutions at 30 ℃ for 48 h. All assays were carried out in triplicate.

Sensory quality
Quality-index analysis was applied to determine the sensory qualities as previously described [15]. The key factors affecting pork-meat quality including appearance, odor, texture, and overall acceptance were evaluated by nine experienced assessors (approved by the Institutional Review Board (IRB), No. IRB-BZXYSP20210501) within a score scale ranging from 1 to 10, in which the score range of 8-10 represents the best sensory quality, 6-8 represents ordinary good sensory quality, 4-6 represents poor sensory quality with slight spoilage, and 0-4 represents disgusting sensory quality with obvious spoilage.

Thiobarbituric acid reactive substance (TBARS) assay
The TBARS method is extensively applied to determine lipid-oxidation products (malondialdehyde, MDA) in meat as previously described [8]. In a typical procedure, 2 g of meat sample was homogenized with 20 mL of 5% trichloroacetic acid and passed through a filter paper to discard the insoluble residues. Then, 5 mL of the filtered solution was mixed with 5 mL of 2-thiobarbituric acid (2.88 g/L) by using a shaker at 200 rpm for 10 min before treatment in a 100 ℃ water bath for 15 min. After cooling to room temperature, the OD of the solution at 532 nm was evaluated with a spectrophotometer (T-6 series UV-Vis spectrophotometer, Feile, Nanking, China). TBARS values were measured from a standard curve of MDA solution.

Microbiological-challenge tests
Pork-meat spoilage models were constructed independently with three types of foodborne pathogens including S. aureus ATCC 25923, E. coli H7:O157 ATCC 35150, and L. monocytogenes ATCC 221633 to investigate the potential of rLactolisterin BU as a bio-preservative during meat storage. For individual bacteria, the prepared meat slices (5 ± 0.1 g) were inoculated with 100 μL of 2.0 × 10 5 CFU/ piece of bacteria by infusion, and then various concentrations of rLactolisterin BU (0, 20, 40, and 60 μg/mL) were used to analyze the preservative effect. Slices inoculated with bacteria but without rLactolisterin BU served as a control. Bacterial count was then calculated as described in section "Microbiological analysis". All assays were carried out in triplicate.

Statistical analysis
GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) was used to analyze the data by one-way analysis of variance (ANOVA), and Tukey's test. P < 0.05 was

Expression and purification of rLactolisterin BU from P. pastoris
The production scheme of rLactolisterin BU from P. pastoris is shown in Fig. 1A. The rLactolisterin BU were secreted in the supernatant of the P. pastoris containing pPICZα-A-Lactolisterin BU vector after induction with methanol, which was analyzed by using Tricine-SDS-PAGE assay as shown in Fig. 1B. The purified rLactolisterin BU was confirmed by Tricine-SDS-PAGE analysis as shown in Fig. 1C. Lactolisterin BU, a type of bacteriocin, was initially isolated from L. lactis subsp. lactis bv. diacetylactis BGBU1-4 in 2017 [14]. Lactolisterin BU offers various metabolites with potent antimicrobial activity and potential applications in the food industry, but its purification from L. lactis is not economical and efficient. Accordingly, P. pastoris aroused our interest because yeast could produce large amounts of functional recombinant proteins and perform many eukaryotic post-translational modifications, including protein folding and glycation, and the DNA manipulation, transformation and identification were easily to be achieved [11,12]. Moreover, many AMPs including bovine lactoferricin peptide [16], Microcin J25 [17], Mytichitin-A [11], Hispidalin [18], and mytichitin-CB [8], have been successfully expressed in P. pastoris and exhibited potent antimicrobial activity and high yields. In a previous study, Lactolisterin BU has been purified by reversed-phase high-performance liquid chromatography, but the production rate is unclear [10].
To further improve the efficiency of Lactolisterin BU preparation, we used protein-affinity purification method, which is generally considered as low cost and high throughput. Meanwhile, His-tag is widely used for protein expression because it has a low molecular weight and does not easily affect the protein structure and characters compared with MBP-tag and GST-tag. The requirement of His-tag fusion protein purification was also easy to meet. Therefore, in the current study, rLactolisterin BU was produced from P. pastoris and purified with a Ni-NTA column for subsequent experiments.

Antimicrobial activity
The MIC values are listed in Table 1. P. pastoris derived rLactolisterin BU showed a broad and enhanced antibacterial effect against gram-negative and gram-positive strains, with values ranging within 6-40 μg/mL. Of note, L. lactis derived Lactolisterin BU showed antimicrobial effect against gram-positive strains including L. monocytogenes ATCC 221633, B. subtilis AHU 1035 and S. aureus ATCC 25923 with values ranging within 5-35 μg/ mL, but P. pastoris derived rLactolisterin BU exhibited antimicrobial activity against gram-negative strains including E. coli H7:O157 ATCC 35150 (MIC = 12 μg/ mL), S. enteritidis ATCC 10467 (MIC = 20 μg/mL), and P. aeruginosa ATCC 27853 (MIC = 60 μg/mL), whereas L. lactis derived Lactolisterin BU did not show any antimicrobial effect against those strains. The antibacterial rate of P. pastoris derived rLactolisterin BU was determined by a time-killing kinetic assay against E. coli H7:O157 Results of biofilm-formation assay (Fig. 2B) indicated that various concentrations of rLactoliserin BU dramatically decreased the biofilm formation in relation to the antibiotic resistance of bacteria. Furthermore, Lactoliserin BU originating from L. lactis was not sensitive to gramnegative bacteria, including E. coli, S. enteritidis, and P. aeruginosa. By contrast, rLactoliserin BU expressed in P. pastoris displayed a broad antibacterial spectrum. These results were in accordance with some recombinant peptides obtained from P. pastoris such as EntP::EntHF peptide [19], Hispidalin [18], Mytichitin-A [11], and defensins (including NZ2114 and its derivatives) [20].
They showed improved antimicrobial activity because the correct post-translational modifications such as Oand N-linked glycosylation and disulfide-bond formation were easy to achieve in the P. pastoris system [21]. Additionally, the results of antibacterial rate indicated that the antibacterial effects of rLactoliserin BU were similar to those of gentamycin with bactericidal rather than bacteriostatic effects in time-and dose-dependent manners. Thus, rLactoliserin BU was potential to replace antibiotics or other chemical preservatives for food preservation.

Cytotoxicity and hemolytic activity
The safety properties such as the cytotoxicity and hemolytic activity of rLactoliserin BU should be examined before its application as a food preservative. The MTT assay was used to evaluate the cytotoxicity of rLactoliserin BU, and results suggested that rLactoliserin BU (0-256 μg/mL) had no cytotoxicity to RAW264.7 cells, which are a classical type of macrophages related to immunoreaction in mouse (Fig. 3A). Moreover, hemolytic assay of rLactoliserin BU (0-256 μg/mL) was performed to determine its hemolytic activity. Results (Fig. 3B) indicated that rLactoliserin BU had low hemolytic activity and could be used in fresh meat with a small amount of blood. Therefore, rLactoliserin BU can replace chemical preservatives as a bio-preservative for meat storage.

Stability of rLactoliserin BU
To further assess the application of rLactolisterin BU in the food industry, the thermostability, pH resistance, proteinase resistance, and saline resistance of rLactoliserin BU in relation to its antibacterial activity were determined by inhibition-zone assay. As shown in Fig. 4A-C, the inhibition diameters of rLactolisterin BU treated with heat (4-90 ℃), extreme pH (2-10), and various concentrations of NaCl (50-500 mM) were similar to those of the control, suggesting that antibacterial activity was not affected. However, rLactolisterin BU was not tolerant to proteinase digestion, as shown in Fig. 4D, and antibacterial activity decreased compared with that of the control. These results agreed with the L. lactis derived Lactolisterin BU and most bacteriocins, including BAC-IB17 [22], BaCf3 [23], and plantacyclin B21AG [24], which were thermostable. These results showed that rLactolisterin BU could be used in food products processed with high temperature, addition of acid or base, and salinization.

Antioxidant activity
To determine the antioxidant effect of rLactolisterin BU, the scavenging rates of hydroxyl, superoxide anion, DPPH, and ABTS + free radicals were measured. As shown in Fig. 5A-D, the IC 50 values of rLactolisterin BU on scavenging radicals of hydroxyl, superoxide anion, DPPH, and ABTS + were 25.0, 19.8, 30.6, and 29.0 μg/mL, respectively. A specific food preservative with antibacterial effects and antioxidant activity may show synergetic effect when used in food storage. Many AMPs including lactoferrin [25], cathelicidin [26], and mytichitin-CB [8]  are good preservatives having antibacterial and antioxidant activities, and they exert a synergetic effect as a food preservative. Therefore, the application of rLactolisterin BU as a food preservative might be show synergetic effect to enhance the applicability.

Bacteriological analyses
To examine the application of rLactoliserin BU in porkmeat storage, the TVC of bacteria contaminated naturally in meat samples were used to assess meat quality and safety during storage. Once the TVC of microorganisms in fresh pork meat exceeded a certain quantity, they became pathogenic microorganisms, causing deterioration during pork storage. Various concentrations (0, 20, 40, and 60 μg/mL) of rLactoliserin BU were used to evaluate the TVC of bacteria contaminated naturally during pork storage, and results are shown in Fig. 6A. The total bacterial growth was obviously inhibited by rLactoliserin BU in a concentration-dependent manner during storage compared with the control. However, the TVC in each treatment group exhibited a gentle growing trend during storage. Notably, the TVC in the control group was Different letters represent significant difference (P < 0.05) nearly 6.5 Log 10 CFU/g on the 10th day, 60 μg/mL rLactoliserin BU was effective in inhibiting bacterial growth during storage, and the TVC was only about 4.5 Log 10 CFU/g on the 10th day. The TVC of 20 μg/mL and 40 μg/ mL rLactoliserin BU treatment groups were 6.0 Log 10 CFU/g and 5.7 Log 10 CFU/g on the 10th day, respectively. To further investigate the antibacterial effect of rLactoliserin BU on common foodborne bacteria (Staphylococcus spp., Escherichia spp., and Listeria spp.) contaminated naturally during pork storage, bacterial counts were evaluated with different special selective media as described in the Material and methods section. As shown in Fig. 6B, the bacterial counts of Staphylococcus spp. in the control group were nearly 5.0 Log 10 CFU/g on the 10th day, but the rLactoliserin BU treatment groups (20,40, and 60 μg/ mL) had significantly decreased bacterial counts of 3.8 Log 10 CFU/g, 3.6 Log 10 CFU/g, and 3.5 Log 10 CFU/g on the 10th day. Similarly, the bacterial counts of Escherichia spp. in the control groups were nearly 4.0 Log 10 CFU/g on the 10th day as shown in Fig. 6C, but the rLactoliserin BU treatment groups (20,40, and 60 μg/mL) had significantly decreased bacterial counts of 3.5 Log 10 CFU/g, 2.9 Log 10 CFU/g, and 2.7 Log 10 CFU/g on the 10th day. For the Listeria spp. count (Fig. 6D), bacteria did not appear until the 6th day in the control and 20 μg/mL rLactoliserin BU treatment groups, but the 40 and 60 μg/mL rLactoliserin BU groups did not show any Listeria spp. colonies during the storage period. The meat-storage application of many AMPs including peptide from housefly pupae [27], mytichitin-CB [8], and Mcc J25(G12Y) [9] have been investigated. The results in this study are in agreement with those peptides and exhibited prominent efficiency in pork storage.

Physicochemical analyses
To evaluate the freshness and physicochemical performance of meat, we considered pH, lipid oxidation, and TVB-N as the key parameters to determine the meat's physicochemical attributes. When proteins in meat are degraded, they generate volatile alkaline nitrogen molecules that cause the pH of pork meat to easily increase during storage [28][29][30]. In the present study, the pH of all pork-meat samples increased from the beginning to the end of the storage period. Compared with the control, the pH increase of pork-meat samples was effectively retarded in the rLactoliserin BU treatment groups (20,40, and 60 μg/mL) from the 2nd to the 10th day (Fig. 7A), and various concentrations of rLactoliserin BU did not show any difference throughout the storage period. These results may be related to the antibacterial effect of rLactoliserin BU, which delayed and attenuated the production of amines.
TVB-N refers to compounds such as trimethylamine, ammonia, and dimethylamine that are generated through the protein degradation caused by microorganisms during meat storage [31]. TVB-N is viewed as one of the most vital freshness indices to reveal the quality and safety of meat products, as stated by Chinese National Standard GB 2707-2016. As shown in Fig. 7B, the TVB-N of all pork-meat samples slowly increased in the  Fig. 7C, the TBARS of all samples increased during the storage period, whereas that of the rLactoliserin BU treatment groups constantly increased more slowly than did the control. The TBARS of the control was 0.68 mg/100 g at day 10, which was higher than that of the rLactoliserin BU treatment groups (mean = 0.58 mg/100 g), and the TBARS of meat samples treated with various concentrations of rLactoliserin BU showed no significant differences during storage. This result was in agreement with the antioxidant activity of rLactoliserin BU, which revealed that rLactoliserin BU can inhibit the lipid oxidation of pork meat and thus had the potential to be a promising preservative for meat storage.

Sensory analysis
Sensory analysis is a simple and intuitive method of evaluating the color, texture, odor, and overall acceptability of food quality [32]. In the current study, the sensory results are shown in Table 2. The sensory scores of all pork-meat samples decreased with prolonged storage time, and the sensory characteristics gradually deteriorated. Nevertheless, the scores of all groups on day 1 did not differ from that on day 0. Meanwhile, the score of the control significantly decreased in the next 2 days, and the score on day 4 was 4.0. which was unacceptable as the evaluation criteria stated. Conversely, the rLactoliserin BU treatment groups showed acceptable scores of 7, 8, and 8.5 on day 6 with 20, 40, and 60 μg/mL rLactoliserin BU addition. These results were in accordance with those of bacteriological and physicochemical investigations, indicating that 40 or 60 μg/mL rLactolisterin BU was suitable for pork storage.

Microbiological-challenge tests
Pork-meat samples treated with various concentrations of rLactolisterin BU were artificially contaminated with 5 × 10 5 CFU/g S. aureus ATCC 25923, E. coli H7:O157 ATCC 35150, and L. monocytogenes ATCC 221633 and stored at 4 ℃ to determine the potential of rLactoliserin BU as a preservative in a pork-spoilage model. As shown in Fig. 8A-C, after incubation for 24 h at 37 ℃ since inoculation, the growth of E. coli H7:O157 ATCC 35150, S. aureus ATCC 25923, and L. monocytogenes ATCC 221633 obviously decreased to 1.90, 2.62, and 3.08 Log 10 CFU/g by 60 μg/mL rLactolisterin BU in a dose-dependent manner. Owing to the abundant nutrient contents of pork and its products, they are easily contaminated by microorganisms including E. coli, L. monocytogenes, and S. aureus, thereby reducing the shelf life of pork [33]. Thus, new technologies such as bio-preservation, are receiving research interest. AMPs such as nisin [7], bacaucin-1 [34], and sakacin P [35] have been evaluated in pork, seafood, or other meat products, showing excellent effect in food-spoilage models. Results showed the potent antibacterial effect of rLactoliserin BU in a pork-spoilage model, in agreement with reported AMPs used in food storage and suggesting that rLactoliserin BU could be a promising preservative.