Effect of three kinds of natural preservative cocktails on vacuum‐packed chilled pork

Abstract The aim of this study was to investigate the effects of three different natural preservatives on the microbial profile, the total volatile base nitrogen (TVB‐N), and biogenic amine contents of vacuum‐packed chilled pork during storage at 4°C. Solution A comprised of tea polyphenols, chitosan, spice extract, propolis, and nisin. Solution B comprised of clove extract, cassia bark extract, ginger juice, garlic juice, and lactobacillus fermentation solution. Solution C consisted of only lactobacillus fermentation solution. The results indicated that solution A was a good natural preservative with higher bacteria inhibitory effect and higher sensory score than B and C. Besides the effect on appealing color, solution B could inhibit microbial activity although its inhibition effect was not as good as solution A. Thus, solution A could be used as a good preservative in industry. Solution C could inhibit the initial growth of Pseudomonas and partially inhibited the growth of Enterobacteriaceae; however, the content of putrescine in the pork treated with solution C was as high as 30.14 ± 2.89 mg/kg after 21 days of storage at 4°C. Hence, solution C is not an ideal preservative for vacuum‐packed chilled pork.

its higher nutritive quality, it has a growing market share and has become mainstream of raw meat consumption Ngapo, Riendeau, Laberge, Leblanc, & Fortin, 2012). Thus, the chilled meat technologies play a key role in meat processing industry (Chang et al., 2019). However, owing to its nutrient-dense nature as well as its high moisture content, chilled meat is comparatively more susceptible to growth and proliferation of psychrophile bacteria versus frozen meat during the production, handling, and sales. This results in problems such as difficulty of market circulation, shorter shelf-life, and safety concerns (Casaburi, Piombino, Nychas, Villani, & Ercolini, 2015;Jiang et al., 2010). Therefore, how to prolong the preservation time and useful shelflife of chilled meat becomes an urgent topic.
In recent years, techniques to keep the quality of chilled pork during storage have been widely studied. There are a variety of options to extend the shelf-life of meat, such as microbial decontamination, smoking or radiation treatments (Kanatt, Chander, & Sharma, 2005;Kim et al., 2014), vacuum ( Blixt & Borch, 2002) or modified atmosphere packaging (Fraqueza & Barreto, 2009;Pérez-Rodríguez, Zamorano, Posada-Izquierdo, & García-Gimeno, 2014), and low temperature storage. As reported, combined preservation techniques based on the hurdle concept could achieve better effects by posing additional barriers to compensate for the shortcomings of different methods applied individually (Salcedo-Sandoval, Cofrades, Ruiz-Capillas, Carballo, & Jiménez-Colmenero, 2015;Xu, Huang, Huang, Xu, & Zhou, 2012). Furthermore, considering consumer aversion to the use of chemicals and antibiotics as food processing aids, the use of natural preservative compounds and their cocktails is of paramount interest among researchers.

| Chemicals and solutions
All microbiological media were from Shanghai Sangon Biotech Co., Ltd. All other chemicals were from Beijing Solarbio Science & Technology Co., Ltd., unless otherwise indicated.
Green tea polyphenol, chitosan, nisin, clove, cassia bark, and rosemary were purchased from Linyi Lumeng Food Co., Ltd. Aqueous extract of propolis was purchased from Shaanxi Laofengnong Biotechnology Co., Ltd. Fresh ginger and garlic were purchased from local supermarket in Taiyuan City. A strain of lactic acid bacteria (LAB) which could produce bacteriocin was provided by Biology

Institute of Shanxi.
The spice extract was prepared as follows. A volume of 300 ml water was added to 30 g spice, for instance rosemary, clove, and cassia bark, respectively. After heating to boiling and holding for 30 min, the mixture was filtrated. The volume of filtrate was made to 300 ml with sterile water and defined as spice extract, which was rosemary extract, clove extract, and cassia bark extract, respectively.
Ginger juice and garlic were prepared by homogenizing 10 g fresh ginger and garlic with 100 ml sterile water, respectively.
Lactic acid bacteria-fermented solution was prepared by incubating lactic acid bacteria with deMan Rogosa Sharpe (MRS) liquid medium to a log CFU/ml of 5.5.

| Raw materials
Chilled pork samples (dorsal muscle) were obtained from a local supermarket in Taiyuan City, China. The tendons and velums were removed. Then, the separated pork was cut into small pieces of 100 g around under sterile conditions in a Clean Bench (SW-CJ-1FD, Shanghai Boxun Industry ＆ Commerce Co., Ltd.).

| Experimental design
Pork blocks were randomly separated into four groups in the experiment, and there were 30 pork blocks in each group. The pork blocks soaked in sterile distilled water, and preservation solutions A, B, and C were defined as control group and experimental groups 1, 2, and 3, respectively. After soaked for 1 min, the pork blocks were drained for 2 ～ 5 min until water dripping ceased. Then, the pork blocks were vacuum packaged using a vacuum packing machine (Jinan Xunjie Packing Machinery Co., Ltd.) and kept in refrigerator at 4°C. The vacuum packaging tube was made of nylon/polyethylene with an oxygen permeating rate of 90 ml m −1 24 hr −1 atm −1 , under 23°C and 60% relative humidity. Analyses were performed for bacterial count, total volatile basic nitrogen (TVB-N) value, and biogenic amines at 0, 7, 14, and 21 days (starting 3 hr after they were put into the refrigerator).

| Microbial analysis
Total bacterial count was measured according to China national food safety standard GB4789.2-2016 and expressed in Log 10 CFU/g (CFU, colony-forming unit) . The detailed procedure was as follows: Samples (25 g) were taken aseptically from the pork blocks and cut up with steam sterilized scissors. The cut chips were suspended in 225 ml sterile normal saline and agitated for 30 min in a shaker (HYG-A, Taicang experimental equipment factor, China). An aliquot of 1 ml supernatant as well as two subsequent decimal dilutions was spread on the surface of agar medium. Numbers were determined on plate count agar (PCA) for total microbe counts at 30°C for 48 hr, streptomycin thallous acetate actidione (STAA) agar After incubation of inoculated Petri dishes at appropriate temperatures, the counts of characteristic colonies were enumerated, calculated, and displayed in CFU.

| Determination of biogenic amines
Lean meat was selected from the pork blocks, soaked in 0.4 M perchloride acid, and homogenized in a mixer (Sorvall Omni) for 2 min at 9,000 g. Biogenic amines were extracted and centrifugated at 6,800 g for 10 min at 4°C. The supernatant was derivatized with dansyl chloride, and the contents of tyramine, putrescine, cadaverine, histamine, spermine, tryptamine, and spermidine were determined using an HPLC instrument (Shimadzu LC-10A) on a C18 reverse-phase column, following the method described by Latorre-Moratalla et al., with some modifications (Latorre-Moratalla et al., 2009). The detection condition was as follows: Excitation and detection wavelength were 340 nm and 440 nm, respectively; elution rate was 1.0 ml/min, and derivation solution flow rate was 0.5 ml/min.

| Color measurement
The chilled pork samples from each treatment were subjected to color measurement using a TC-P2 full-automatic color difference meter (Beijing Xinaoyike Photoelectric Technology Co., Ltd). Before measurement, the colorimeter was calibrated with a white tile. Color coordinates including L * (lightness), a * (redness/greenness), and b * (yellowness/blueness) were measured. Three measurements were performed on each sample.

| Sensory analysis
Sensory analysis including aroma, flavor, juiciness, tenderness, and color of chilled pork was carried out by the methods for sensory evaluation of meat (Bradley et al., 2011;Brenesselova et al., 2015). Sensory examination was performed by six trained evaluators on day 21 of storage. The chilled pork samples were examined by panelists for color and elasticity. After the samples were boiled for 30 min, every panelist examined the chilled meat for the indexes of aroma, flavor, juiciness, and tenderness. Each sample was coded with a random letter. The descriptors were measured using a 5-point intensity line scale for each index; for instance, number 5 indicated the highest quality of evaluated parameter and the number 0 indicated the lowest quality.

| Data analyses
Samples were analyzed in triplicate, and the results were shown as mean ± standard deviation. All the experimental data were analyzed with SPSS software (8.0 edition). One-way analysis of variance was used to evaluate statistical significance of differences between four groups of meat with different treatments. The level of significance was p < .05.

| Microbial analyses
The data for counts of total bacteria, Pseudomonas, Enterobacteriaceae, total lactic acid bacteria, Brochothrix thermosphacta, and psychrophiles in different groups during the storage at 4 ± 1°C are shown in Figures 1-6.
The changes in the total microbe counts of chilled pork treated with natural preservation solution and stored at 4°C Total bacterial counts (TBC) of control samples increased during the storage. It reached to 7.01 ± 0.27 log CFU/g on day 21. Similar results were recorded in the work of Zhao et al (Zhao et al., 2015), who stored vacuum-packed chilled pork at 0°C. In their work, the TBC was 6.50 ± 0.34 log CFU/g on day 21. The total microbe counts decreased by 2 log CFU/g on the surface of the pork treated with preservation solution A stored at 4°C for 3 hr (Figure 1). The TBC in group 1 varied slowly during the storage period and increased to 5.18 ± 0.25 log CFU/g on day 21. Significant difference of TBC between group 1 and control group was observed (p < .01). Before the 14th day, the TBCs in groups 2 and 3 were a little higher than that of control group, but no significant difference was obtained(p > .05).
However, after the 14th day, the TBCs in groups 2 and 3 were much lower than that of control group(p < .05). The values in groups 2 and 3 were 5.04 ± 0.24 and 5.56 ± 0.25 log CFU/g, respectively, on the 21st day. The reason for the above phenomenon is that lactic acid bacteria in preservation solutions B and C were active, resulting in a higher total microbial count in groups 2 and 3 from the dominant bacteria colony of lactic acid bacteria at the initial storage (Pipek et al., 2006). But due to the competitive advantage of lactic acid bacteria, the propagation of other bacteria was inhibited, resulting in a lower total microbial count than that of control group at the end of the storage period. As shown in Figure 1, all the three preserva- implying that chilled pork began to decay. There were at least two conditions responsible for the decay of control group. One reason was that no preservation solution was used in control group, and the other one was related to the oxygen permeability of the package bag. The O 2 permeation rate of the tube used in the experiment was 2 ml m −2 h −1 atm −1 at 23°C, while each Pseudomonas cell just needs 9.7 × 10 -8 μl oxygen per day for growth and propagation (Slabbert & Grabow, 1986).
Therefore, 480 ml oxygen permeated each day could amply meet the requirements of 5 × 10 8 cfu/ml Pseudomonas. At the initial stage, respiration action of the pork would exhaust most of them. With time, the pork stopped respiration and the oxygen permeated was mainly used by Pseudomonas which resulted in the counts increase in the control group at late stage. Figure 3, preservation solution A exhibited obvious inhibitory effect on Enterobacteriaceae. The number of group 1 was more than 2 log CFU/g lower than that of control group at the initial stage and increased slowly during the whole storage period. On the 21st day, the log CFU/g value of group 1 was 5.0 ± 0.3 log CFU/g, which was lower than and significantly different (p < .01) from the value of control group. Preservation solutions B and C also showed some inhibitory effects on Enterobacteriaceae, but much weaker than A. On the 21st day, the log CFU/g values of groups 2 and 3 were 6.415 ± 0.31 log CFU/g and 6.146 ± 0.40 log CFU/g, respectively, which were much lower than that of control group (p < .05). These results indicate that preservation solution A had stronger inhibitory effect on Enterobacteriaceae than B and C.

As shown in
As shown in Figure 4, the counts of lactic acid bacteria of groups 2 and 3 on the 1st day were up to 2 log CFU/g higher than that of control group due to the presence of active bacteria. However, the counts did not increase further until the 14th day, and the values reached to 7.00 Log CFU/g on the 21st day, which was much higher than that of control group. Preservation solution A showed significant inhibitory effect on lactic acid bacteria in chilled pork with the initial counts 2 log CFU/g lower than that of control group (p < .01). This finding is consistent with the report by Jiang et al. investigated the bacterial communities of vacuum-packed chilled pork during storage using PCR-denaturing gradient gel electrophoresis (DGGE), who found that the composition of the microbes varied at different stages of storage, and Lactobacillus was the dominant species at the end of storage (Jiang et al., 2010).

Preservation solutions A, B, and C could all inhibit the initial growth
of Brochothrix thermosphacta in chilled pork as shown in Figure 5. The initial inhibitory effect of preservation solutions A and B on Brochothrix thermosphacta was significant, while C was insignificant. During the whole storage period, the counts increased slowly in group 1 and remained lower than that of the control group. However, the log CFU/g values of groups 2 and 3 increased rapidly and reached to the same level with the control group from day 7. Compared with the total microbe counts and Pseudomonas counts, the number of Brochothrix thermosphacta in all experimental groups increased more slightly and the values were only 3.11 ± 0.22 log CFU/g in group 1 and around 4.0 log CFU/g in the other three groups by day 21, which implied Brochothrix thermosphacta was not the predominant bacteria in the chilled pork.
The change in psychrophiles in chilled pork treated with preservation solutions was presented in Figure 6. The initial log CFU/g values of all treated samples were lower than that of the control group. This tendency lasted from day 1 to day 21, and the log CFU/g values of psychrophiles in groups 1, 2, and 3 were 3.98 ± 0.27, 5.00 ± 0.34, and 4.54 ± 0.37 log CFU/g, respectively, which were lower than the 5.80 ± 0.41 log CFU/g of the control group (p < .05).

| Physical and chemical indexes
Total volatile basic nitrogen (TVB-N) content is an important reference index for evaluating pork freshness (Huang, Zhao, Chen, & Zhang, 2014). The experimental results of TVB-N value and biogenic amines of all groups stored at 4 ± 1°C are shown in Figure 7 and Table 1, respectively. Figure 7 than 15.00 mg/100 g with the level of group 1 being the lowest at 11.19 ± 0.83 mg/100 g. These results indicate that all the preservation solutions achieved higher freshness retention of chilled pork, and in this regard, preservation solutions A and B were more effective than solution C. These TVB-N analyses results were consistent with the total microbe counts data. For example, when the total microbe counts of the control group reached to the highest value (7.01 ± 0.27 log CFU/g), the TVB-N value attained the highest value (22.90 ± 1.47 mg/100 g) on day 21 which was mainly due to accelerated protein degradation and accumulated biogenic amines by a large number of bacteria . Table 1, the content of the biogenic amines in groups 1, 2, and control group was relatively low except spermidine.

As indicated in
For example, histamine even could not be detected. The contents of putrescine, spermine, and tryptamine in group 1 were significantly lower than that of the control group (p < .05), while the contents of cadaverine and tyramine were also lower but not significant (p > .05). As to group 3, except putrescine (30.14 ± 2.89 mg/kg), the contents of other biogenic amines were relatively low. The content of spermidine in group 3 was 6 times less than that of group control.
Hitherto, neither tyramine nor tryptamine was detected. As previously reported, putrescine and cadaverine were released during the decomposition of amino acid, and putrescine was produced mainly due to protein degradation by Pseudomonas (Krizek, Smith, & Phebus, 1995). The content of putrescine in group 3 reached a high value of 30.14 ± 2.89 mg/kg. This high value may be related with Pseudomonas considering the high value of Pseudomonas in group 3 (6.886 ± 0.33 log CFU/g) at the end of storage. Another reason for the high content of putrescine in group 3 may be related with lactic acid bacteria. The pork blocks in group 3 were soaked with lactic acid bacteria fermentation solution, and the counts of lactic acid bacteria reached the highest on day 21 of storage. Biogenic amines could be accumulated from reactions catalyzed by amino acid decarboxylase of lactic acid bacteria (Poveda, Ruiz, Sesena, & Palop, 2017).
Meat color measurements showed that the L * values in groups 2 and 3 were significantly higher than that in groups control and 1 (p < .05), while a * and b * did not show significant changes in the four groups (p > .05) ( Table 1).

| Sensory indexes
The sensory evaluation of the samples on day 21 is presented in Table 2. The color of chilled pork in groups 1, 3, and the control was purple. This was the normal color of vacuum-packaged chilled pork and indicated that the muscle myoglobin existed mainly in reduced form (Hood & Riordan, 1973;Millar, Moss, & Stevenson, 1996). The color of pork is of major concern to the pork industry, and the optimum surface color of fresh pork should be reddish-pink (Mancini & Hunt, 2005). Among the 4 groups, the color of chilled pork in group 2 was a more attractive bright-red color with a highest lightness value (Table 2)  During low temperature storage, the main bacteria on chilled pork were Psychrophiles. The reason why they are adapted to low temperature is that the saturated fatty acids in their cytoplasm were transformed into unsaturated fatty acids during the evolutionary process (Nash & Grant, 1969). The unsaturated fatty acids can keep the fat of the cytoplasm membrane in a fluid state allowing membrane transportation of various substances, to ensure the survival, growth, and propagation of psychrophiles. Furthermore, the enzyme system of psychrophiles has also undergone evolutionary changes in structural and functional properties to enable high biocatalytic activity at low temperatures (Cavicchioli, Thomas, & Curmi, 2000). propolis showed antibacterial activity to L. monocytogenes, E. coli, P. fluorescens, and L. sake. (Zabaiou et al., 2017;Zhang, Wu, & Guo, 2016). As a natural green food preservative, nisin is the only bacteriocin approved by both U.S. FDA and the WHO so far (Zhang & Zhong, 2015). The combined effects of these components endowed solution A the most effective inhibitory activity against microbial growth. Therefore, preservation solution A is a good natural antimicrobial preservative solution.
Preservation solution B could also reduce the total microbe counts, as well as Pseudomonas, Enterobacteriaceae, and psychrophiles counts in chilled pork. However, compared with preservation solution A, solution B showed less inhibitory effect on the growth of lactic acid bacteria and B. thermosphacta. The TVB-N value and biogenic amine of chilled pork treated with solution B decreased, and the chilled pork presented a bright red, which is useful in practical application in the meat industry. Hence, solution B could also be used for natural preservation on vacuum-packaged chilled pork.

Solution C could inhibit the initial growth of Pseudomonas and
Enterobacteriaceae, and decrease TVB-N levels in the late stage of storage. However, the content of putrescine was relatively high in the late storage. Therefore, preservation C is not suitable for processing vacuum-packaged chilled pork.

| CON CLUS ION
The results of this study showed that the shelf-life of vacuumpacked chilled pork was affected by using different natural preservatives cocktails at 4°C. The storage of vacuum-packed chilled pork in three preservatives groups had a positive effect on the microbiological, biochemical, and sensory parameters compare to nontreated control meat. Though solution C could inhibit the initial growth of Pseudomonas and partially inhibited the growth of Enterobacteriaceae, the content of putrescine in the pork treated with solution C was as high as 30.14 ± 2.89 mg/kg after 21 days of storage at 4°C. Hence, Solution C is not an ideal preservative for vacuum-packed chilled pork. Solution A showed the highest bacteria inhibitory effect and received the highest score of sensory evaluation among the three preservatives. Besides the microbial inhibitory activity, solution B endowed the meat appealing color. Thus, both solution A and solution B could be expected to become a good preservative to be applied in the meat industry for improving shelf-life of vacuum-packed chilled pork.

This research was supported by the Science and Technology
Committee of Shanxi Province (2015021047)

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest, financial, or otherwise.

E TH I C A L A PPROVA L A N D CO N S E NT TO PA RTI CI PATE
The work reported in this manuscript did not involve use of human or animal subjects and, thus, did not require ethical approval and consent by the participating institutions.

H U M A N A N D A N I M A L R I G HT S
The study reported here did not require treatments for humans or animals; thus, human and animal rights concerns do not apply.

CO N S E NT FO R PU B LI C ATI O N
The manuscript was verified and approved by all the coauthors listed for publication in "Current Pharmaceutical Biotechnology."