Antimicrobial property of Pichia pastoris‐derived natto peptide against foodborne bacteria and its preservative potential to maintain pork quality during refrigerated storage

Abstract Pork spoilage caused by foodborne bacteria contamination always leads to substantial economic loss in the meat industry. The toxicity and drug resistance of chemical preservatives have raised public concerns about their safety and stability. In this study, natto peptide from Pichia pastoris was prepared using DNA recombinant technology. It showed an excellent antibacterial effect against Gram‐positive and ‐negative bacteria, with minimum inhibitory concentrations (MICs) ranging from 6 to 30 μg/ml. Of note, natto peptide exhibited low cytotoxicity and hemolytic activity. The application of natto peptide on pork during refrigerated storage dramatically decreased the growth of Staphylococcus spp., Escherichia spp., and Pseudomonas spp. The bactericidal properties remained in force when natto peptide was used in pork models contaminated with artificial bacteria. Moreover, the application of natto peptide (90 μg/ml) inhibited the increase in pH variation and drip loss, decreased the generation of total volatile basic nitrogen (TVB‐N) and thiobarbituric acid reactive substances (TBARS), and maintained a high sensory quality score during pork storage. These results implied that P. pastoris‐derived natto peptide could extend the storage time of pork, and it has the potential to be a promising antiseptic biopreservative to replace chemical preservatives.

such as Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa, pork and its products could easily spoil and cause substantial economic loss during processing, transportation, and storage (Gram et al., 2002;Zhou et al., 2010). Thus, microorganism pollution is a serious problem in pork preservation to maintain quality and safety.
Various methods have been used in the pork preservation area, including physical and chemical strategies (Gogliettino et al., 2019;Lee et al., 2021;Papadochristopoulos et al., 2021), to inhibit the growth of foodborne microorganisms. Unfortunately, pork spoilage with microorganism pollution could still not be totally eliminated. Chemical preservatives, including sodium benzoate and potassium sorbate (Erickson & Doyle, 2017), have been used in food storage, such as beverages, meat, and cereal foods, for decades. However, the toxicity and drug resistance of chemical preservatives and abuse in their application have raised public concerns about their safety and stability (Eijlander et al., 2011).
An increasing number of researchers have focused their attention on the development of new kinds of antimicrobial agents. Antimicrobial peptides (AMPs), which are generally small-molecular-weight peptides containing 3-200 amino acids derived from a wide range of organisms, including prokaryotic and eukaryotic organisms, exhibited broad antimicrobial spectrum against bacteria, fungi, and parasites (Zasloff, 2002).
Moreover, AMPs showed no drug resistance and possessed stable physical and chemical properties (Wang et al., 2019;Xu & Lai, 2015). Therefore, they have been recognized as promising antibiotic substitutes. Nisin, which is a typical AMP that exerts potent antimicrobial activity, has been used in food storage as a safe and efficient biopreservative all around the world for decades (Gharsallaoui et al., 2016).
Natto is a traditional Japanese fermented soybean product that contains numerous valuable nutrients, including vitamins, isoflavone, lecithin, and nattokinase . It is considered a healthy food. Nattokinase is a member of the subtilisin family, which is composed of 275 amino acids and possesses therapeutic potential for the treatment of thrombovascular diseases, hypertension, Alzheimer's disease, and vitreoretinal disorders (Dabbagh et al., 2014). In a previous study (Kitagawa et al., 2017), Shinichi Yokota's group identified a natto peptide consisting of 45 amino acid residues from natto extract. It shared 97.8% homology with the C-terminal region of nattokinase produced by Bacillus subtilis.
Furthermore, the natto-derived peptide showed a narrow antimicrobial effect against Streptococcus pneumoniae and B. subtilis only.
The predicted structural characteristics of the peptide were similar to those of cathelicidin family AMPs, which exhibited a broad antimicrobial spectrum (Margherita et al., 2002;Sang & Blecha, 2008).
Meanwhile, the source of peptide is a healthy food with medical function. Therefore, natto peptide may be a promising antimicrobial agent that could be used in food preservation. DNA recombinant technology was used to solve the problems of extraction from natural products to improve the antimicrobial property and productivity of the peptide. Pichia pastoris is a classical expression host that produces recombinant proteins compared with prokaryotic hosts, such as E. coli and B. subtilis (Ahmad et al., 2014;Karbalaei et al., 2020).
Many bioactive peptides have been produced in P. pastoris by the DNA recombinant technology, as for the advantages of posttranslational modifications such as protein folding and glycation (Karbalaei et al., 2020). Many AMPs have been reported to express P. pastoris, and their antimicrobial activity and spectrum improved compared with those of natural extracted products, including Mytichitin-A (Meng et al., 2016), Padef (Meng et al., 2017), Hispidalin (Meng et al., 2019), and Lactolisterin BU .
This study aimed to improve the antimicrobial property of natto peptide by using DNA recombinant technology with P. pastoris system and analyze the antimicrobial effect of the purified natto peptide. The antimicrobial application of purified natto peptide in pork preservation was initially evaluated by investigating the growth of bacterial pathogens, pH, total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substance (TBARS), and sensory evaluation of pork samples during storage. DH5α strain, and pPICZα-A plasmid were purchased from Invitrogen Company. They were used for gene cloning and protein expression. All other chemicals were obtained from Solarbio, except those described.

| MIC
The antimicrobial effect of natto peptide was determined using microbroth dilution assay (Dong et al., 2018). In brief, various concentrations of the natto peptide (0-120 μg/ml) were incubated with bacterial cultures (6 × 10 5 CFU/ml) for 14-16 h at 37°C to determine the minimum inhibitory concentration (MIC), which refers to no obvious colonies that occurred when the cultures were incubated overnight. Phosphate-buffered saline (PBS) buffer and gentamicin were used as negative and positive controls. All experiments were performed three times.

| Time-killing curves
Time-killing curves were obtained as described previously to evaluate the bactericidal rates of natto peptide .
Concisely, the natto peptide at final concentrations of 1×, 2×, and 4× MICs was incubated with E. coli O157, which was cultured to midlog phase. Then, the culture samples were harvested at different time intervals for colony counting. PBS buffer and gentamicin were used as negative and positive controls. All the experiments were performed in triplicate.

| Biofilm formation
Crystal violet staining method was performed as described previously to evaluate the biofilm inhibition effect of natto peptide , and E. coli O157 was used as the tested strain. Butyl paraben was used as positive control, and Mueller-Hinton (MH) broth was used as blank control.

| Hemolysis
The hemolytic effect of natto peptide against rabbit erythrocytes was investigated, as described previously (Liu et al., 2020). PBS and 1% Triton X-100 were used as blank and positive controls, respectively. In brief, blood cells were initially mixed with anticoagulant dipotassium EDTA, the erythrocytes were isolated by centrifugation at 1500g for 15 min and washed three times with PBS buffer. Then, the erythrocytes were resuspended with PBS at a ratio of 1:20 and incubated at 37°C with various concentrations of the natto peptide (0-200 μg/ml) for 1 h. After centrifugation at 1500g for 10 min at room temperature, the supernatants were transferred to a new 96well microplate to determine the absorbance value at 540 nm by using a microplate reader (HBS-1096A; Detie). All assays were performed three times.

| Cytotoxicity
The cytotoxicity of natto peptide toward RAW264.7 cells was determined by the methyl-thiazol-diphenyltetrazolium (MTT) method, as described in our previous study (Xue et al., 2020). Briefly, the RAW264.7 cells were cultured in Rosewell Park Memorial Institute (RPMI)-1640 medium containing 10% fetal bovine serum (FBS), 1% penicillin, and 1% streptomycin at 37°C in a 5% CO 2 atmosphere environment. The diluted natto peptide with different concentrations (0-200 μg/ml) was incubated with RAW264.7 cells in 96-well plates at 37°C in a 5% CO 2 atmosphere. Then, the MTT solutions (1 mg/ ml) were added in the cell suspensions for another 4 h of incubation in the dark at 37°C. After discarding the supernatants, the formed formazan crystals were resolved with dimethyl sulfoxide (DMSO) and the absorbance value at 570 nm was determined by using a microplate reader (HBS-1096A; Detie). The cell viability was calculated as the following: cell viability (%) = OD sample/OD control × 100%.
All the experiments were performed three times.

| Stability
Inhibition-zone assay was used to determine the stability of natto peptide against E. coli O157, as described in a previous study . In brief, 6 μg/ml of natto peptide with different treatments, including dissolution in diverse pH (pH = 2, 4, 6, 8, and 10) or salinity (50, 100, 200, 300, 400, and 500 mM) buffers; incubation at various temperatures (4, 25, 37, 65, and 90°C); and digestion with papain, pepsin, trypsin, and proteinase K solutions, was used to determine the pH, salt, and thermal and protease stabilities. Untreated peptides and PBS were used as positive and negative controls, respectively. All experiments were conducted three times.

| Antioxidant-activity assay
The antioxidant effect of natto peptide was determined by investigating the scavenging capacities of 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ), hydroxyl and superoxide anion free radicals, as described previously . For the DPPH assay, the natto peptide was incubated with DPPH buffer which was dissolved in 95% methanol in a dark environment for 30 min, then the absorbance value at 517 nm was measured by using a microplate reader. In the ABTS + assay, the natto peptide was incubated with an ABTS + working solution in dark for 10 min, then the absorbance value at 734 nm was determined by using a microplate reader. Moreover, the ABTS + working solution was prepared by using potassium persulfate solution (2.6 mM) to dilute the ABTS + stock solution (7 mM) at a ratio of 1:1 and incubated in dark for 12 h before determination.
The hydroxyl and superoxide anion radical scavenging abilities were determined by using a Hydroxyl Free Radical Scavenging Capacity Assay Kit (BC1325; Solarbio) and a Superoxide Anion Detection Kit (BC1290; Solarbio), according to the manufacturer's instructions. All determinations were repeated three times.

| Pork preparation and treatment with natto peptide
Fresh-pork legs were bought from a local butcher (Binzhou, China) 24 h postmortem. The connective tissue and fat of the pork were removed before cutting into even slices (5 ± 0.1 g). Afterwards, the slices were soaked in 0, 30, 60, and 90 μg/ml of natto peptide for 20 min. Then, the slices were drained and placed in sterilized polyvinyl chloride trays, sealed using a polyethylene film, and stored at 4°C in a refrigerator for 8 days. Meanwhile, 50 μg/ml of nisin and 100 μg/ ml of butyl paraben were used as positive controls to analyze the aseptic effects of natto peptide, including microbial growth, TVB-N, lipid oxidation, pH, drip loss, and sensory quality during storage.

| Microbial growth analysis
The microbial growth of pork was determined by colony count, as described previously (Meng et al., 2021). In brief, after the pork sample (5 g) was homogenized with 0.9% sterilized saline buffer (25 ml), 100 μl of the 10× diluent was spread on plate-count agar, mannitolsalt agar, violet red bile agar, and centrimide (CN) agar, and incubated at a suitable temperature for 48 h. All experiments were performed three times.

| Microbiological-challenge tests
Escherichia coli O157 ATCC 35150, S. aureus ATCC 25923, and P. aeruginosa ATCC 902 were used to pollute the fresh pork and construct a pork spoilage model, which was used to further evaluate the antimicrobial effect of natto peptide. Five grams of pork samples was inoculated with 100 μl of bacteria (2.0 × 10 5 CFU (colony-forming units)/piece) by infusion before the natto peptide was coated on the samples. Then, the pork samples were placed in sterilized polyvinyl chloride trays, sealed using a polyethylene film, and stored at 37°C for 24 h. Thereafter, 5 g of pork sample was homogenized with 0.9% sterilized saline buffer (25 ml), and 100 μl of the 10× diluent was spread on the corresponding agar and incubated at 37°C for 48 h.
The bacterial colony count was illustrated as logarithms of colonyforming units per gram (Log CFU/g). All assays were performed three times.

| Measurement of pH and drip loss
The pH values of the pork were measured in accordance with the National Standard of People's Republic of China (GB 5009.237-2016) by using a pH meter (REX PHSJ-5, Shanghai, China). Drip loss (%) was calculated using the following formula: drip loss (%) = (initial weight of pork − final weight of pork)/initial weight of pork × 100%. All assays were performed in triplicate.

| Measurement of TVB-N
The TVB-N value of pork samples was determined in accordance with the National Standard of People's Republic of China (GB 5009.237-2016) by using Kjeldahl assay. In brief, 5 g of pork sample was completely homogenized with 25 ml of distilled water for 30 min. After filtering with a filter paper, the homogenate was mixed with 10 g/L of MgO at a ratio of 1:1 (v/v) and distilled. Then, the distillate was resolved in an equal volume of boric acid, which contained 0.1% methyl red and 0.1% bromocresol green. Finally, hydrochloric acid (0.01 mol/L) was used to titrate the solution and determine the TVB-N value. All assays were performed in triplicate.

| TBARS assay
Thiobarbituric acid reactive substances assay was performed to investigate the process of lipid oxidation in pork by determining the malondialdehyde (MDA) product content. Concisely, 5 grams of pork sample was homogenized with 50 ml of 7.5% trichloroacetic acid buffer before filtering with a filter paper. Then, 5 ml of the filtered buffer was reacted with an equal volume of 2-thiobarbituric acid (0.22 mol/L) at 100°C for 15 min and cooled to room temperature. Afterwards, the optical density at 532 nm of the reactant was measured using an ultraviolet-visible (UV-Vis) spectrophotometer (Nanjing Feile Instrument Co., Ltd.). The TBARS value was determined using the standard curve of 1,1,3,3-tetramethoxypropane and described as milligram (mg) MDA equivalents per 100 g of pork.
All assays were performed in triplicate.

| Sensory quality
Sensory quality, including color, odor, texture, and overall acceptance, was investigated using quality index analysis, as described previously . Ten experienced evaluators were selected to train and assess the sensory quality of pork samples in individual chambers, and the samples were scored on a scale of 1-10 (8-10 = excellent and highly acceptable, 6-8 = good and acceptable, 4-6 = poor and unacceptable, and 0-4 = very poor and very unacceptable).

| Statistical analysis
All results were analyzed using GraphPad Prism 7.0 (GraphPad Software) by one-way analysis of variance (ANOVA) and Duncan's multiple range test. Meanwhile, all data in the manuscript are expressed as mean ± standard derivation, and the results were defined as statistically different when p < .05.

| MIC of natto peptide
The MIC of natto peptide was performed using 13 types of general foodborne bacteria, including Gram-positive and -negative strains.
As shown in Table 1, natto peptide exhibited a broad antimicrobial effect against both bacteria, and the minimum concentrations differed among different bacterial strains, with MICs ranging from 6 to 30 μg/ml.

| Time-killing curve of natto peptide
A time-killing curve of natto peptide against E. coli O157 was plotted to determine the antimicrobial rate of natto peptide. As shown in decreased to 67%, 43%, 19%, and 11%, respectively.

| Cell cytotoxicity and hemolytic activity of natto peptide
As shown in Figure 1c, the cell survival rate at various concentrations of natto peptide had no significant difference (p > .05), compared with that of the control. Moreover, the hemolytic activity of natto peptide was <1.8% even at a concentration of 200 μg/ml (Figure 1d), which showed nearly no hemolytic effect.

| Stability of natto peptide
The stability of natto peptide in extreme external conditions, including thermostability, acid-base, and proteinase and saline resistance, was determined. As shown in Figure 1e-g, the antimicrobial activity of natto peptide was not affected in different temperatures (4-90°C; p > .05), pH values (p > .05), and protease digestions (p > .05).
However, it was impaired with the increase in the concentration of NaCl (Figure 1h; p < .0001), indicating that the natto peptide was not resistant to high-concentration saline.

| Antioxidant activity of natto peptide
As shown in Figure 2a

| Microbial growth analysis
The antibacterial function of natto peptide during pork refrigerated storage was determined using the colony counting method, including the growth of total bacteria, Staphylococcus spp., Escherichia spp., and Pseudomonas spp. As shown in Figure 3a-

| pH and drip loss
The pH variation of pork during storage is shown in Figure 4a. The natto peptide and butyl paraben treatment groups inhibited the increase in pH compared with the control group. However, the pork treated with nisin showed an obvious increase in pH during storage similar to the control group. In addition, the drip loss in all treatment groups increased with similar tendency, as shown in Figure 4b. A no-

| TVB-N
As shown in Figure 4c

| Sensory quality
The quality changes in pork acceptance during storage were determined by sensory evaluation. As shown in Table 3, the sensory scores of pork significantly reduced with the extension of storage time in the control group, whose sensory scores were <4 on day 4. On the contrary, the sensory scores of natto peptide treatment groups were decreased slightly on day 4 (p < .05), which showed significant differences compared with control. According to the evaluation criteria, various concentrations (30, 60, and 90 μg/ml) of natto peptide treated groups maintained the sensory scores of pork to acceptable (>5) at days 5, 6, and 7, respectively. In addition, the
Abbreviations: Log CFU, logarithms of colony-forming units per gram.
natto peptide showed a broad antibacterial spectrum, including Gram-positive and -negative strains, as shown in Table 1. However, the naturally obtained natto peptide only presented a narrow antibacterial activity against S. pneumoniae and B. subtilis groups (Kitagawa et al., 2017). The time-killing curve indicated that natto peptide could sterilize E. coli in 90 min, suggesting that it possesses bacteriostatic and bactericidal activities. Meanwhile, the biofilm formation was related to the drug resistance of bacteria (De Zoysa et al., 2015). The results showed that 1× MIC of natto peptide inhibited the biofilm formation below 50%, and the antibacterial effect was dose dependent. Nevertheless, the detailed antimicrobial mechanism of natto peptide still needs to be investigated in further studies.
The applicability of natto peptide on different external conditions similar to foods, such as pork, during processing, transportation, and storage was investigated by determining the stability in extreme conditions, the cytotoxicity on mammalian cells, and hemolytic and antioxidant activities. The analysis results on the stability of natto peptide showed that high temperature, strong acid/base, and proteinase digestion treatment could not affect the antimicrobial activity of natto peptide, except for NaCl treatment, which attenuated the antimicrobial activity to lower than 50% when the NaCl concentration was increased from 200 to 500 mM. AMPs perform bactericidal activity by mutual attraction and attachment between the negatively charged appearances of bacteria and cationic sites of the peptides (Plant et al., 2010). Thus, the decreased antimicrobial activity of natto peptide under a high concentration of NaCl could be attributed to the competition between the peptides and Na + . Moreover, natto peptide showed a prominent antioxidant effect, indicating that it could be used as an antioxidant agent in pork storage to prevent the oxidation process that seriously deteriorates pork quality. All of these results implied that natto peptide could be potentially used as a pork preservative for processing, transportation, or storage in various external environments.
Bacterial count was used to analyze the inhibitory function of natto peptide to further evaluate its antiseptic properties against the typical foodborne microorganisms (Staphylococcus spp., Escherichia spp., and Pseudomonas spp.) in pork during refrigerated storage. Considering that bacterial growth is the main cause of spoilage, which leads to deterioration of quality and safety, TVC is an essential parameter to judge the antiseptic characters of preservatives. According to the results of total bacterial analyses, 90 μg/ml of natto peptide showed prominent antiseptic effect and decreased the total viable counts (TVCs) of microorganisms during pork refrigerated storage. It exerted a better antibacterial effect with less addition than 100 μg/ml of butyl paraben, which is a conventional chemical preservative accepted in food antiseptic field, with broad antimicrobial spectrum. These results were identical to those of some AMPs, such as housefly pupae peptide (Wang et al., 2010), mytichitin-CB (Meng et al., 2021), Lactolisterin BU , and MccJ25(G12Y) (Corbalán et al., 2021), which have been investigated in pork chilled storage. Pseudomonas spp. and Escherichia spp.
adversely affected the quality of pork during chilled storage, and they were too stubborn to be eliminated by conventional measures, including freezing, surface dehydration, and simulated spray chilling. The performance of nisin in the present study was barely satisfactory, which could be attributed to its poor antibacterial effect The inhibition of bacteria growth could reduce the production of volatile alkaline nitrogen molecules such as amine, which might be the reason why natto peptide decreased the pork pH during chilled storage (Cao et al., 2019;Ruan et al., 2019). The natto peptide dramatically decreased the drip loss of pork during storage compared with control group which was related to the maintenance of meat freshness. The concentration of TVB-N in pork reflected its fresh content, which was produced mainly owing to proteolysis by the spoilage bacteria (Bekhit et al., 2021). Natto peptide treatment decreased the generation of TVB-N and increased the storage days in a dose-dependent manner compared with the control. Therein, highdose (90 μg/ml) natto peptide exhibited the lowest concentration of TVB-N among the three treatment groups, including nisin, butyl paraben, and natto peptide. These results could be explained by the excellent bactericidal property of natto peptide, which was similar to those of some biopreservatives such as mytichitin-CB (Meng et al., 2021), nisin (Gharsallaoui et al., 2016), and green tea aqueous extract (GTAE; Montaño-Sánchez et al., 2020). Furthermore, the lipid oxidation-formed byproducts, including aldehydes and other fetid decomposition products, during pork storage could disrupt the sensory qualities and lead to spoilage (Xiong et al., 2020). The results of TBARS assay showed that natto peptide also decreased the generation of TBARS in pork. However, nisin and butyl paraben displayed no significant difference in TBARS with control, which could be attributed to the antioxidant function of natto peptide investigated in this study. Fasseas group confirmed that oregano oil could be used as an antioxidant to inhibit the fat oxidation during meat refrigeration, which had antibacterial and antioxidant activity in vitro (Fasseas et al., 2008). Previous studies have elucidated that AMPs with scavenging capacities of free radicals were also conducive to the prevention of lipid oxidation, indicating that natto peptide could be used as an antioxidant agent to help pork retain its freshness (Meng et al., 2021). The sensory quality of pork is a critical factor for the acceptance of consumers. The microbial growth, lipid oxidation, pH variation, and drip loss during chilled storage would affect the sensory quality of pork (Siripatrawan & Noipha, 2012). The sensory evaluation results were consistent with those of physicochemical analyses as expected, that is, natto peptide extended the shelf life of pork and delayed the occurrence of deterioration during pork refrigerated storage.

| CON CLUS IONS
This study indicated that the natto peptide prepared from P. pastoris by the DNA recombinant technology exhibited a broader antibacterial spectrum, including Gram-positive and -negative strains, and enhanced bactericidal property, with MICs ranging from 6 to 30 μg/ml. The addition of natto peptide during freshpork refrigerated storage predominantly decreased the growth of Staphylococcus spp., Escherichia spp., and Pseudomonas spp. In addition, the bactericidal properties remained in force when the natto peptide was used in pork models artificially contaminated with bacteria. Moreover, the application of natto peptide (90 μg/ ml) significantly inhibited the increase in pH variation and drip loss, decreased the generation of TVB-N and TBARS, and maintained a high sensory quality score during pork storage at 4°C.
These results implied that P. pastoris-derived natto peptide could significantly extend the shelf life of pork, and it has a potential to be a promising antiseptic biopreservative to replace chemical preservatives during pork refrigerated storage.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest. Funding acquisition (equal); Resources (equal); Software (equal).

AUTH O R CO NTR I B UTI
Tao Wu: Funding acquisition (equal); Resources (equal); Software (equal).

DATA AVA I L A B I L I T Y S TAT E M E N T
Data available on request from the authors.