Antibacterial pediococcus pentosaceus cell-free supernatant and application thereof in pork preservation
By using cell-free supernatant of Pediococcus pentosaceus YZJC001, the problems of chemical preservative toxicity and biofilm enhancement in food cleaning have been solved, thus achieving safe preservation and extended shelf life of pork.
Patent Information
- Application Number
- CN202511234404.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-01
- Publication Date
- 2025-11-21
AI Technical Summary
In existing technologies, the extensive use of chemical preservatives is toxic to the human body, while the presence of biofilms increases the difficulty of food cleaning and disinfection. Pathogenic bacteria such as Serratia can cause meat products to spoil and deteriorate, affecting food safety.
Cellless supernatant of Pediococcus pentosaceus YZJC001 isolated from local Yangzhou pickles was prepared by fermentation and centrifugation. This cellless supernatant was used to inhibit the growth of Serratia marcescens, Salmonella cytomegalovirus, Staphylococcus aureus and Listeria monocytogenes, and was applied to pork preservation.
It significantly inhibits the growth of pathogenic bacteria, extends the shelf life of pork, enhances food safety, and provides an environmentally friendly food preservative solution.
Smart Images
Figure CN120989170A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of food preservation, specifically an antibacterial cell-free supernatant of Pediococcus pentosaceus and its application in pork preservation. Background Technology
[0002] As people's living standards improve, they are paying more attention to the safety of meat products. Besides foodborne microorganisms such as Listeria monocytogenes, Salmonella, and Staphylococcus aureus, which frequently contaminate meat and meat products during slaughter or production, Serratia marcescens also poses a significant threat. Serratia marcescens efficiently metabolizes proteins and carbohydrates in pork, producing biogenic amines (such as cadaverine and putrescine), sulfides, and green pigments, leading to meat becoming slimy, emitting off-odors, and exhibiting discoloration (such as greening), accelerating sensory decay. Consuming this contaminated meat or meat products can easily cause food poisoning, with symptoms ranging from relatively mild gastroenteritis (abdominal pain, diarrhea, vomiting, fever) to life-threatening systemic infections, seriously affecting human health.
[0003] Biofilm formation is a crucial factor in the development of microbial tolerance to environmental factors and antimicrobial agents. Pathogenic bacteria can enhance their tolerance to food processing environments by forming biofilms. This not only increases the burden of cleaning and disinfection in the food industry but also causes persistent contamination of processed foods, posing a significant risk factor for food contamination and foodborne illnesses. Currently, the main solutions for foodborne pathogen contamination and spoilage in meat products are the addition of antioxidants and preservatives. However, the extensive use of chemical preservatives can lead to the conversion of substances such as sodium nitrite, sodium nitrate, sodium sulfite, and sodium benzoate, which can be toxic to humans with long-term consumption.
[0004] The preservative effects of protective microorganisms, especially probiotics, are being increasingly explored. Protective microorganisms are active microorganisms that can be added to food to extend its shelf life and inhibit the growth of harmful microorganisms. Lactic acid bacteria, as natural antibacterial microorganisms, can inhibit the growth of pathogenic bacteria and disrupt biofilm structures through their fermentation broth via metabolites (such as organic acids, bacteriocins, cyclic peptides, and hydrogen peroxide). Existing experimental studies have also demonstrated that lactic acid bacteria and their metabolites can be considered an ideal class of natural preservatives, possessing the advantages of being green and safe. Summary of the Invention
[0005] The purpose of this invention is to provide an antibacterial cell-free supernatant of Pediococcus pentosaceus and its application in pork preservation. In this invention, the raw material is a lactic acid bacterium isolated from local Yangzhou pickles: Pediococcus pentosaceus YZJC001.
[0006] Technical solution: A cell-free supernatant of antibacterial Pediococcus pentosaceus, wherein Pediococcus pentosaceus YZJC001 was deposited on December 25, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.33185.
[0007] The method for preparing the cell-free supernatant of Pediococcus pentosaceus is as follows: Step 1) Solid plate culture: In a sterile laminar flow hood, Pediococcus pentosaceus ） YZJC001 was inoculated onto the surface of a solidified culture medium. After inoculation, it was placed in an anaerobic incubator at 37°C, inverted, and cultured overnight. Step 2), Liquid culture: Under aseptic conditions, a single colony was picked from a solid culture medium and inoculated into a liquid culture medium. The bottle was sealed with sealing film and incubated at 37°C for 36 h to obtain the *Pediococcus pentosaceus* fermentation broth. The *Pediococcus pentosaceus* fermentation broth was then adjusted to OD using a UV spectrophotometer. 600 =1.6; Step 3), Centrifugation: OD 600 The *Pediococcus pentosaceus* fermentation broth with a concentration of 1.6 was centrifuged, filtered to remove bacteria, and a cell-free supernatant of *Pediococcus pentosaceus* was obtained. This cell-free supernatant was then diluted with sterile water at a volume ratio of 4:6 to obtain a 40% cell-free supernatant of *Pediococcus pentosaceus*.
[0008] The cell-free supernatant of *Pediococcus pentosaceus* can inhibit *Serratia* and *Salmonella cytomegalovirus*. Salmonella The growth of Typhimurium, Staphylococcus aureus, and Listeria monocytogenes.
[0009] The aforementioned *Pediococcus pentosaceus* cell-free supernatant has a preservative effect on fresh pork, extending its shelf life and enhancing food safety. When applied to pork preservation, fresh pork is soaked in the *Pediococcus pentosaceus* YZJC001 cell-free supernatant for 10 minutes, then stored in a 4°C refrigerator. Preferably, the *Pediococcus pentosaceus* YZJC001 cell-free supernatant is a 40% concentration.
[0010] Beneficial effects: The lactic acid bacteria selected in this invention is Pediococcus pentosaceus YZJC001. Compared with other lactic acid bacteria, the cell-free supernatant of this bacterium can significantly inhibit Serratia and Salmonella cytomegalovirus. Salmonella Typhimurium (ATCC13311), Staphylococcus aureus (ATCC6538), and Listeria monocytogenes (ATCC19111) were found to have extended the shelf life and enhanced the safety of fresh pork when the cell-free supernatant of these bacteria was used to treat the pork, providing new ideas for the development of novel and environmentally friendly food preservatives. Attached Figure Description
[0011] Figure 1 Figure 1 shows the effect of different concentrations of cell-free supernatant from lactic acid bacteria on the growth of pathogenic bacteria. Figure 2 The effect of 40% cell-free supernatant (CFS) of Pediococcus pentosaceus YZJC001 on the total bacterial count of pork stored at 4℃ for different time periods is shown in the figure. Figure 3 The effect of 40% cell-free supernatant (CFS) of Pediococcus pentosaceus YZJC001 on TBA of pork stored at 4℃ for different times is shown in the figure. Figure 4 The effect of 40% cell-free supernatant (CFS) of Pediococcus pentosaceus YZJC001 on pH of pork stored at 4℃ for different times is shown in the figure. Figure 5 Images of pork preserved at 4°C for 1-7 days using cell-free supernatant (CFS) of 40% Pediococcus pentosaceus YZJC001. Detailed Implementation
[0012] The technical solution of the present invention will be described in detail below through embodiments, but the scope of protection of the present invention is not limited to the embodiments described.
[0013] Example 1 Step 1: Prepare cell-free supernatant of lactic acid bacteria, including Pediococcus pentosaceus. Cell-free supernatant of YZJC001 and Lactobacillus plantarum ATCC8014.
[0014] (1) Lactic acid bacteria strain samples: Pediococcus pentosaceus Pediococcus pentosaceusYZJC001 is deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCC No. 33185. Lactobacillus plantarum ATCC8014 was purchased.
[0015] (2) Culture medium preparation: Weigh an appropriate amount of MRS agar and dissolve it in deionized water. After high temperature and high pressure sterilization, cool it to a suitable temperature at room temperature (usually around 55℃). In a laminar flow hood, pour the culture medium into sterile petri dishes and wait for it to solidify (200mL of MRS agar medium can make 10 plates).
[0016] (3) Solid plate culture: In a sterile laminar flow hood, use an inoculation loop to pick up and dip the preserved lactic acid bacteria strain onto the surface of the solidified culture medium in three zones to ensure that the bacteria can be evenly distributed and form single colonies. Place the inoculated culture dish upside down in an anaerobic incubator at 37°C and incubate overnight.
[0017] (4) Liquid culture: Prepare 200 mL of MRS liquid culture medium and autoclave it. Under aseptic conditions, use a 10 μL pipette tip to pick a single colony from the solid culture medium and inoculate it into the liquid culture medium. Seal the bottle mouth with sealing film and incubate at 37℃ for 36 h. Adjust the OD of the two lactic acid bacteria fermentation broth to OD using a UV spectrophotometer. 600 =1.6.
[0018] (5) Dispense 200 mL of lactic acid bacteria culture into 50 mL centrifuge tubes, balancing the tubes to ensure the error is less than 0.01 g. Centrifuge at 8000 rpm for 10 min. Place the sample symmetrically into the centrifuge rotor, start the centrifuge according to the centrifuge's operating instructions, and wait for it to reach the set speed and temperature. During centrifugation, carefully observe the centrifuge's operating status and any abnormalities (such as noise, vibration, etc.). After centrifugation, filter the supernatant after centrifugation using a 0.22 mm filter membrane to remove bacteria, and keep the filtered, sterile, cell-free lactic acid bacteria supernatant for later use.
[0019] (6) The cell-free supernatant (CFS) of Pediococcus pentosaceus YZJC001 and the cell-free supernatant of Lactobacillus plantarum ATCC8014 were finally obtained.
[0020] Step 2: Isolation and culture of pathogenic bacteria.
[0021] 2.1 Isolation of Serratia marcescens Serratia marcescens was screened from soil samples taken at Yangzhou University's Yangzijiang Campus. The specific screening method was as follows: (1) Take 5 g of soil, add 90 mL of distilled water, shake on a shaker at 120 rpm for 20 min, and take the upper liquid as the bacterial suspension. Inoculate with 5% of the bacterial suspension into beef extract peptone liquid medium and culture at 37℃ and 120 rpm for 12-18 h. (2) The culture medium was serially diluted with physiological saline. After dilution, 100 mL of 10 -4 10 -5 and 10 -6 The diluted solution was spread onto beef extract peptone solid medium, and three replicates were performed for each gradient.
[0022] (3) The plated agar was incubated at 37°C for 12–18 h. Single colonies were picked and streaked twice on the same medium to purify the bacteria. The 16S rRNA gene of the strain was amplified using universal bacterial primers 27F and 1492R. The sequence of universal bacterial primer 27F was 5'-AGAGTTTGATCMTGGCTCAG-3', and the sequence of universal bacterial primer 1492R was 5'-GGTTACCTTGTTACGACTT-3'. PCR was performed using 20 μL of reaction mixture with Taq DNA Polymerase (Takara). The PCR conditions were: pre-denaturation at 94°C for 3 min; amplification phase of 35 cycles, performed at 94°C for 30 s; 55°C for 30 s; 72°C for 1 min. The PCR products were analyzed by agarose gel electrophoresis and sent to General Biotechnology for sequencing. Using SeqMan, the sequence was compared with previously published bacterial 16S rRNA sequences in the NCBI database, and the naming was correct.
[0023] The sequencing results of the isolated Serratia marcescens bacterial 16S rRNA sequence are as follows: "Contig 1" (1,1387) Contig Length: 1387 bases Average Length / Sequence: 843 bases Total Sequence Length: 1687 bases Top Strand: 1 sequence Bottom Strand: 1 sequence Total: 2 sequences 2.2 Culture of pathogenic bacteria (1) Source of strains: Serratia, Salmonella cytogenes ATCC13311, Staphylococcus aureus ATCC6538, and Listeria monocytogenes ATCC19111 were deposited in the laboratory of the School of Tourism and Culinary Arts, Yangzhou University. Salmonella cytogenes ATCC13311, Staphylococcus aureus ATCC6538, and Listeria monocytogenes ATCC19111 were purchased.
[0024] (2) Preparation of solid culture medium: Weigh an appropriate amount of LB agar and dissolve it in deionized water. After high temperature and high pressure sterilization, cool it to a suitable temperature (about 55℃) at room temperature. In a clean bench, pour the culture medium into sterile petri dishes and wait for it to solidify (200mL of LB agar medium can be poured into 10 plates).
[0025] (3) Solid plate culture: In a sterile laminar flow hood, use an inoculation loop to pick up and dip the inoculated pathogenic bacterial strain onto the surface of the solidified culture medium in three zones to ensure that the bacteria are evenly distributed and form single colonies. Place the inoculated culture dish upside down in a 37°C incubator and incubate overnight.
[0026] (4) Liquid culture: Prepare liquid culture medium (broth), dispense it into test tubes in 5 mL portions and autoclave to ensure sterility. Under sterile conditions, use a 10 μL pipette tip to pick a single colony from the solid culture medium and inoculate it into the liquid culture medium and incubate at 37°C for 12 h.
[0027] Step 3: Test the antibacterial effect of cell-free supernatants of different lactic acid bacteria on pathogenic bacteria.
[0028] (1) Culture medium preparation: Pour the melted solid LB culture medium into a petri dish and let it solidify to serve as the bottom layer.
[0029] (2) Inoculation of bacterial suspension: The OD of the cultured pathogenic bacteria suspension was detected using an ultraviolet spectrophotometer. 600 OD detected 600 The bacterial culture was diluted to OD using LB. 600 =0.1, spread the bacterial solution evenly on LB medium.
[0030] (3) Place Oxford cups: After the bacterial layer solidifies, use sterile tweezers to place the sterilized Oxford cups vertically on the surface of the culture medium (3 cups per dish, with spacing).
[0031] (4) Adding samples: Add 150 mL of the two cell-free supernatants (prepared in step one) to each Oxford cup.
[0032] (5) Incubation: Incubate the petri dish at 37°C for 12 hours.
[0033] (6) Results observation: Measure the diameter of the inhibition zone (accurate to mm), record and analyze the results.
[0034] (7) The results and analysis are as follows: Table 1 shows the antibacterial effects of cell-free supernatants of different lactic acid bacteria on pathogenic bacteria. The experimental group was compared with the control group using an independent samples t-test (* indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001).
[0035] Table 1. Results of the antibacterial effects of cell-free supernatants of different lactic acid bacteria against pathogenic bacteria.
[0036] Based on the inhibition zone results of cell-free supernatants of *Lactobacillus plantarum* ATCC8014 and *Pediococcus pentosaceus* YZZC001 against *Serratia marcescens*, *Salmonella cytomegaloides* ATCC13311, *Staphylococcus aureus* ATCC6538, and *Listeria monocytogenes* ATCC19111, *Pediococcus pentosaceus* YZZC001 showed significantly greater inhibitory effects on all four pathogenic bacteria compared to *Lactobacillus plantarum* ATCC8014 (Table 1).
[0037] Step 4: Test the effect of different concentrations of cell-free supernatant of Pediococcus pentosaceus YZJC001 on pathogenic bacteria.
[0038] The OD of the cultured pathogenic bacteria solution was detected using an ultraviolet spectrophotometer. 600 OD detected 600 The bacterial culture was diluted to OD using LB. 600 =0.4 is used for subsequent experiments.
[0039] Preparation method of TSB medium for cell-free supernatant of Pediococcus pentosaceus YZJC001 at different concentrations: The cell-free supernatant of Pediococcus pentosaceus YZJC001 prepared in step one was 100%. The cell-free supernatant was diluted to 80% with sterile water, and then serially diluted ten times. These cell-free supernatants of different concentrations were added to 2×TSB in a 1:1 ratio to obtain TSB containing 40%, 20%, 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.15625%, and 0.078125% cell-free supernatant.
[0040] 4.1 Effects of different concentrations of cell-free supernatant of Pediococcus pentosaceus YZJC001 on the growth of pathogenic bacteria (1) Experimental groups: Experimental group: In pathogenic bacteria (OD) 600=0.4) Inoculate at a ratio of 1:100 into TSB medium containing cell-free supernatant of Pediococcus pentosus YZJC001 at different concentrations.
[0041] Control group: Pathogenic bacteria (OD) 600 =0.4) Inoculate at a ratio of 1:100 into an equal volume of TSB medium containing cell-free supernatant of Pediococcus pentosus YZJC001.
[0042] The control group of the experimental group: TSB medium containing the same volume of cell-free supernatant of Pediococcus pentosus YZJC001 at different concentrations.
[0043] The blank control group consisted of an equal volume of TSB culture medium solution.
[0044] (2) Measurement method: Following the above grouping, 200 mL of the treated culture medium was inoculated into 96-well plates, ensuring three parallel control experiments per group; OD was measured every 2 hours. 600 The values were continuously monitored for 24 hours, and 24-hour long curves were plotted based on the obtained data to represent the concentrations of cell-free supernatant of different pathogenic bacteria corresponding to different concentrations of Pediococcus pentosaceus YZJC001.
[0045] (3) Processing of experimental results: Experimental group data: experimental group - blank group; Control group data: control group - blank group. Independent samples t-test was used to compare the experimental group and the control group (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).
[0046] (4) The results and analysis are as follows: Figure 1 The study investigated the effects of different concentrations of cell-free supernatant from *Pediococcus pentosaceus* YZJC001 on the growth of pathogenic bacteria. Specifically, (A) the effect of different concentrations of cell-free supernatant from *Pediococcus pentosaceus* YZJC001 on the growth of *Serratia marcescens*, (B) the effect of different concentrations of cell-free supernatant from *Pediococcus pentosaceus* YZJC001 on the growth of *Salmonella marcescens*, (C) the effect of different concentrations of cell-free supernatant from *Pediococcus pentosaceus* YZJC001 on the growth of *Serratia marcescens*, and (D) the effect of different concentrations of cell-free supernatant from *Pediococcus pentosaceus* YZJC001 on the growth of *Listeria monocytogenes*.
[0047] Combined with the experimental data from the control group, it can be seen that the inhibitory effect of cell-free supernatant of *Pediococcus pentosaceus* YZJC001 on pathogenic bacteria is concentration-dependent, with higher concentrations showing more significant inhibitory effects. Among them, 40% cell-free supernatant of *Pediococcus pentosaceus* YZJC001 showed the most significant inhibitory effect, essentially inhibiting the growth of *Serratia marcescens*, *Salmonella cytomegalovirus* ATCC13311, *Staphylococcus aureus* ATCC6538, and *Listeria monocytogenes* ATCC19111. Figure 1 ).
[0048] 4.2 Effects of different concentrations of cell-free supernatant from Pediococcus pentosaceus YZJC001 on biofilm formation of pathogenic bacteria (1) Experimental groups: Experimental group: Pathogenic bacteria (OD) 600 =0.4) Inoculate at a ratio of 1:100 into TSB medium containing cell-free supernatant of Pediococcus pentosus YZJC001 at different concentrations.
[0049] Control group: Pathogenic bacteria (OD) 600 =0.4) Inoculate at a ratio of 1:100 into an equal volume of TSB medium containing cell-free supernatant of Pediococcus pentosus YZJC001.
[0050] The control group of the experimental group: TSB medium containing the same volume of cell-free supernatant of Pediococcus pentosus YZJC001 at different concentrations.
[0051] The blank control group consisted of an equal volume of TSB culture medium solution.
[0052] (2) Biofilm culture: According to the above grouping, 200 mL of the treated culture medium was inoculated into 96-well plates, and 3 parallel control experiments were ensured for each group; the plates were incubated at 25℃ and quantitative analysis was performed by crystal violet staining at 12 h, 24 h and 48 h respectively.
[0053] (3) Crystal violet staining method: Aspirate all bacterial culture from the wells and gently rinse the wells with 200 μL of sterile water to remove free bacteria and prevent them from affecting the final results. After rinsing, place the wells in a 55°C oven to dry for 5-10 min, then add 180 μL of methanol solution and fix for 5-10 min. Aspirate the solution and dry in the oven for another 5 min. After the methanol solution has completely evaporated, add 200 μL of 0.1% oxalic acid crystal violet staining solution for staining. Let stand for 5 min and then gently rinse the 96-well plate with a slow stream of water (Note: do not rinse directly into the wells, but rinse the edges of the plate). Rinsing is complete when no more purple dye flows out. After rinsing, dry for 5-10 min, then treat with 33% acetic acid for 2 min and measure the OD. 595 The biomass of the biofilm was calculated using the OD value. Optical density was measured after adding 33% acetic acid and treating for 2 min. Since BF has a maximum absorption peak at 595 nm, while the bacterial suspension has a maximum absorption peak at 600 nm, the OD value was chosen. 595 nm is used as an indicator to measure the amount of BF formation.
[0054] (4) Processing of experimental results: Experimental group data: Experimental group - control group Control group data: Control group - blank group SPSS 19.0 was used for statistical calculations and analysis. The Waller-Duncan test was used for analysis of variance (ANOVA). Independent samples t-tests were used to compare the experimental and control groups (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001).
[0055] (5) Combining the experimental results of the control group, it can be seen that: The cell-free supernatant of *Pediococcus pentosaceus* YZJC001 showed significant inhibitory effects on the biofilm of *Serratia marcescens* at 12 h, 24 h, and 48 h, with a concentration of 40% at all times. The cell-free supernatant of *Pediococcus pentosaceus* YZJC001 also showed significant inhibitory effects on the biofilm of *Salmonella cytomegaloides* at 12 h (0.625%), 24 h (5%), and 48 h (0.3125%). The cell-free supernatant of *Pediococcus pentosaceus* YZJC001 showed significant inhibitory effects on the biofilm of *Staphylococcus aureus* at 24 h (40%), but no significant inhibitory effects at 12 h and 48 h. The cell-free supernatant of *Pediococcus pentosaceus* YZJC001 also showed significant inhibitory effects on the biofilm of *Listeria monocytogenes* at 12 h (40%), but no significant inhibitory effects at 24 h and 48 h. As shown in Table 2, 40% cell-free supernatant of Pediococcus pentosaceus YZJC001 was selected for subsequent experiments.
[0056] Table 2. Time and concentration of cell-free supernatant of Pediococcus pentosaceus YZJC001 showing significant inhibitory effect on pathogenic bacteria
[0057] Step 5: Pork processing.
[0058] Based on the results of the effect of cell-free supernatant of *Pediococcus pentosaceus* YZJC001 on pathogenic bacteria, 40% cell-free supernatant of *Pediococcus pentosaceus* YZJC001 was selected for subsequent studies. The preparation method of 40% cell-free supernatant of *Pediococcus pentosaceus* YZJC001 was as follows: OD... 600 The fermentation broth of *Pediococcus pentosaceus* with a concentration of 1.6 was centrifuged and filtered to remove bacteria, resulting in a cell-free supernatant of *Pediococcus pentosaceus*. The cell-free supernatant of *Pediococcus pentosaceus* was diluted with sterile water at a volume ratio of 4:6 to obtain a 40% cell-free supernatant of *Pediococcus pentosaceus*.
[0059] (1) Cut fresh pork into 5 g pieces.
[0060] (2) Soak the cut pork in sterile water and mix well to bring the original bacteria on the surface of the pork closer together. After soaking for 30 seconds, wipe the surface of the pork dry with kitchen paper.
[0061] (3) The pork was divided into two groups. The experimental group was soaked in 40% cell-free supernatant of Pediococcus pentosaceus YZJC001 for 10 min and then dried with kitchen paper. The control group was treated with sterile water. The treated meat was placed in a refrigerator at 4℃. The total bacterial count and physicochemical properties (pH, thiobarbituric acid TBA, texture, and color difference) were measured at 1, 3, 5, and 7 days.
[0062] Step Six: Test the relevant indicators of the pork processed in Step Five.
[0063] 6.1 Microbiological Indicators Total colony count method: On a clean bench, weigh 5 g of meat sample from each of three parallel samples and place them in 45 mL of sterile physiological saline (0.85%). After sealing, homogenize at 10000 r / min for 1 min to obtain a uniform dilution for plate counting. Calculate the colony count using the formula below (log(log(b / b))). 10 (CFU / g)).
[0064] Equation (1) N—the number of colonies in the sample; ∑C—The sum of colony counts on the plate (including plates with colony counts within the appropriate range); n1 — Number of plates at the first dilution (lowest dilution factor); n ２ —Number of plates at the second dilution (highest dilution factor); d—Dilution factor (first dilution).
[0065] Total bacterial count results: Figure 2 The effect of 40% CFS on the total bacterial count of pork stored at 4℃ for different time periods was investigated. Independent samples t-tests were used to compare the experimental and control groups (* indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001). Figure 2 It can be seen that at 4℃, 40% cell-free supernatant (CFS) of Pediococcus pentosaceus YZJC001 has a more significant inhibitory effect on bacterial growth in pork compared with sterile water treatment, especially from the third day.
[0066] 6.2 Physicochemical Indicators: (1) Determination of thiobarbituric acid value (TBA value): For three parallel samples, take 2.5g of raw meat sample, cut it into pieces, add 12.5 mL of 7.5% trichloroacetic acid mixture (containing 0.1% EDTA), shake at 120 rpm for 30 min, filter twice with double-layer filter paper, take 5 mL of supernatant, add 5 mL of 0.02 mol / L TBA solution, keep warm in a 90 ℃ water bath for 40 min, take it out and cool for 1 h, centrifuge for 5 min, add 5 mL of chloroform to the supernatant and shake well, let it stand for layering, take the supernatant and measure the absorbance at 532 and 600 nm respectively, record the absorbance value, and calculate the TBA value.
[0067] TBA (mg / 100g) = (A 532 -A 600 Equation (2) is: 155 / 2.5×72.6×100 TBA value test results: Figure 3 The study investigated the effect of 40% CFS (Chemical Flavor Filtration) on the total fatty acid (TBA) content of pork stored at 4℃ for different time periods. TBA, or Thiobarbituric Acid Value, is an indicator used to measure the degree of fatty acid oxidation in fats and oils. A higher TBA value indicates a higher degree of fat oxidation and lower quality. For fresh meat products, a TBA value exceeding 0.25 mg / 100g is considered oxidative rancidity. Figure 3 It can be seen that at 4℃, the cell-free supernatant (CFS) of 40% Pediococcus pentosaceus YZJC001 had no significant difference in effect on TBA in pork compared with sterile water treatment.
[0068] (2) pH test method: For three parallel samples, accurately weigh 2.5 g of meat sample, cut it into pieces and put it in an Erlenmeyer flask, add 22.5 mL of distilled water, mix at 120 rpm for 30 min and then filter. Use a pH meter to measure the pH value of the filtrate.
[0069] pH test results: Figure 4 The effect of 40% CFS on pH at different times in pork stored at 4℃ was investigated. Fresh meat typically has a pH of 5.6-6.0. Initially, a pH between 5.6 and 6.0 indicates good preservation. When the pH rises to 6.0-6.4, it indicates the meat may have entered the initial stage of spoilage. At this stage, the number of microorganisms increases, and the surface of the meat may become slightly sticky and darken in color. When the pH exceeds 6.4, or even reaches above 7.0, the meat is usually severely spoiled. The proliferation of microorganisms produces an unpleasant odor, the texture becomes soft, the surface becomes severely sticky, and the meat loses its edible value. Figure 4It can be seen that at 4℃, the pH of the meat sample showed a trend of first decreasing and then increasing. On the fifth day, the pH of the control group was close to 6.5. The cell-free supernatant (CFS) of 40% Pediococcus pentosaceus YZJC001 had a lower overall pH in pork than that of sterile water treatment, indicating that it has better preservation properties for pork.
[0070] (3) Texture: Three parallel samples of the two groups of fresh pork were cut into 1 cm × 1 cm × 1 cm pieces. The hardness and elasticity were measured using a P / 36R probe. The speed before the test was 1 mm / s, the test speed was 1 mm / s, the speed after the test was 1 mm / s, the strain was 60%, and the triggering force was 5 g.
[0071] Texture test results: The effect of 40% CFS on the texture index of pork stored at 4℃ for different times is shown in Table 3.
[0072] Table 3. Effects of 40% CFS on texture parameters of pork stored at 4℃ at different times.
[0073] Elasticity refers to the ability of pork to return to its original shape after being deformed under stress, and is related to the elastin and collagen structure of muscle fibers. Decreased elasticity usually indicates damage to the muscle structure, a sign of poor preservation (such as microbial growth or enzymatic hydrolysis). Table 3 shows that at 4℃, 40% Pediococcus pentosaceus YZJC001 cell-free supernatant (CFS) significantly affected the elasticity of pork on the third and seventh days compared to aseptic water treatment. The experimental group exhibited better elasticity, indicating better preservation properties for pork.
[0074] Chewability reflects the energy required for teeth to chew pork samples into a swallowable state, essentially representing the pork's resistance to chewing. Excessive or insufficient chewability affects texture, and abnormal chewability during preservation may indicate meat deterioration. Table 3 shows that at 4℃, 40% Pediococcus pentosaceus YZJC001 cell-free supernatant (CFS) significantly affected the chewability of pork on the third day compared to aseptic water treatment. The experimental group exhibited better chewability, indicating better preservation properties for pork.
[0075] There were no significant differences in hardness, cohesion, and resilience between the two groups at different time points. The sudden increase in hardness on the seventh day may be due to moisture loss caused by the sealing bag not being properly sealed.
[0076] (4) Color difference: The colorimeter is calibrated with white and black calibration plates. Then, the L (brightness), a (redness), and b (yellowness) values of three parallel samples of two groups of fresh pork are measured and the average value is taken.
[0077] Texture test results: The effect of 40% CFS on the color difference index of pork stored at 4℃ for different times is shown in Table 4. Figure 5 Table 4 shows images of pork preserved for 1-7 days. As can be seen from Table 4, at 4℃, there were no significant differences in brightness (L), red-green component (a), and yellow-blue component (b) between pork treated with 40% lactic acid bacteria cell-free supernatant (CFS) and pork treated with sterile water.
[0078] Table 4. Effect of 40% CFS on color difference index of pork stored at 4℃ for different times.
[0079] In summary, this invention utilizes the natural metabolites of Pediococcus pentosaceus YZJC001 for pork preservation, and significantly inhibits the growth of Serratia marcescens, Salmonella cytomegalovirus ATCC13311, Staphylococcus aureus ATCC6538, and Listeria monocytogenes ATCC19111 in pork, without the use of chemical preservatives. The raw materials are easy to prepare, and the operation is simple, convenient, and quick.
[0080] As described above, although the invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the invention itself. Various changes in form and detail may be made without departing from the spirit and scope of the invention.
Claims
1. A cell-free supernatant of Pediococcus pentosaceus, characterized in that, The Pediococcus pentosaceus mentioned is Pediococcus pentosaceus YZJC001, which was deposited on December 25, 2024, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with accession number CGMCCNo.33185.
2. The cell-free supernatant of *Pediococcus pentosaceus* according to claim 1, characterized in that, The method for preparing the cell-free supernatant of Pediococcus pentosaceus is as follows: 1) Solid plate culture: In a sterile laminar flow hood, Pediococcus pentosaceus YZJC001 was inoculated onto the surface of a solidified culture medium. After inoculation, the medium was placed in an anaerobic incubator at 37°C, inverted, and incubated overnight. 2) Liquid culture: Under aseptic conditions, a single colony was picked from a solid culture medium and inoculated into a liquid culture medium. The bottle was sealed with sealing film and incubated at 37°C for 36 h to obtain the *Pediococcus pentosaceus* fermentation broth. The *Pediococcus pentosaceus* fermentation broth was then adjusted to OD using a UV spectrophotometer. 600 =1.6; 3) Centrifugation: The fermentation broth of *Pediococcus pentosacchari* was centrifuged and filtered to remove bacteria, resulting in cell-free supernatant of *Pediococcus pentosacchari*.
3. The cell-free supernatant of *Pediococcus pentosaceus* according to claim 1, characterized in that, The cell-free supernatant of *Pediococcus pentosaceus* inhibits the growth of *Serratia*, *Salmonella Typhimurium*, *Staphylococcus aureus*, and *Listeria monocytogenes*.
4. The application of the cell-free supernatant of Pediococcus pentosaceus as described in claim 1 or 2 in the preservation of pork.
5. The application according to claim 4, characterized in that, Fresh pork was soaked in cell-free supernatant of Pediococcus pentosaceus YZJC001 for a period of time and then placed in the refrigerator for preservation.
6. The application according to claim 5, characterized in that, Fresh pork was soaked in cell-free supernatant of Pediococcus pentosaceus YZJC001 for 10 minutes, and the refrigerator temperature was set to 4℃.
7. The application according to claim 5, characterized in that, The cell-free supernatant of Pediococcus pentosacchari YZJC001 is 40% cell-free supernatant of Pediococcus pentosacchari YZJC001. The method for preparing 40% cell-free supernatant of Pediococcus pentosaceus YZJC001 is as follows: OD 600 The fermentation broth of *Pediococcus pentosaceus* with a concentration of 1.6 was centrifuged and filtered to remove bacteria, resulting in a cell-free supernatant of *Pediococcus pentosaceus*. The cell-free supernatant of *Pediococcus pentosaceus* was diluted with sterile water at a volume ratio of 4:6 to obtain a 40% cell-free supernatant of *Pediococcus pentosaceus*.