Application of pediococcus pentosaceus ACX 0180 in preparation of biofilm inhibitor
By using the biofilm inhibitor prepared by Pelococcus pentosaccharide ACX 0180, the problem of Staphylococcus aureus forming biofilm on the surface of food processing equipment was solved, and the efficient inhibition effect under low temperature conditions was achieved, ensuring food safety and extending the shelf life.
Patent Information
- Application Number
- CN202510300117.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-14
- Publication Date
- 2025-06-20
AI Technical Summary
The prior art is difficult to effectively inhibit the biofilm formed by Staphylococcus aureus on the surface of food processing equipment, and the biofilm is resistant to antibiotics and antibacterial agents, resulting in major challenges in food safety.
Biofilm inhibitors prepared with P. pentosacci ACX 0180, which exhibited superior effects over traditional chemical inhibitors by significantly inhibiting the reproduction of Staphylococcus aureus and preventing its formation of biofilms on the surface of stainless steel.
The antibacterial agent exhibits the best inhibitory effect under low temperature conditions of 4℃, can quickly and efficiently remove biofilms, is safe and non-toxic, environmentally friendly, and does not produce harmful residues. It is suitable for food storage and transportation, extends the shelf life of food and reduces the risk of cross-contamination.
Smart Images
Figure CN120167458A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microbial technology, and particularly relates to the application of Pediococcus pentosaceus ACX 0180 in the preparation of a biofilm inhibitor. Background Art
[0002] Staphylococcus aureus is a Gram-positive coccus widely present in the natural environment and is also one of the common foodborne pathogenic bacteria. Under suitable conditions, this bacterium can produce enterotoxin, causing food poisoning. The ways it contaminates food are diverse, including food processing personnel, raw material contamination, and processing equipment. Staphylococcus aureus has strong salt tolerance and heat resistance, and can reproduce rapidly in foods with high protein and starch content. In addition, the biofilm formed by it can adhere to the surface of food processing equipment and is difficult to remove through conventional cleaning and disinfection. The biofilm is composed of extracellular polymers, providing a protective barrier for microorganisms against external stressors (such as chemical disinfectants), thereby enhancing the bacteria's resistance to antibiotics and antibacterial agents. Research shows that 60% of foodborne diseases are related to biofilms, making the control of biofilms an important challenge in the field of food safety.
[0003] In recent years, with the increasing attention to food safety and health, traditional chemically synthesized antibacterial agents have been gradually restricted, and finding safe and efficient natural food antibacterial agents has become a research hotspot. Lactic acid bacteria have attracted attention due to their protease, lipase, amylase activities, and the antibacterial effects of bacteriocins. Among them, Pediococcus pentosaceus has gradually become the focus of future food antibacterial due to its safe and efficient antibacterial ability. However, current research is mostly in the experimental theory stage, lacking industrialized and systematic production methods. In addition, the formation and removal of biofilms have always been difficult problems in the food processing industry. Since biofilm cells may develop resistance to antibiotic disinfectants, the continuous use and inefficiency of these disinfectants further highlight the necessity of finding new control strategies. Summary of the Invention
[0004] The present invention aims to provide the application of Pediococcus pentosaceus ACX 0180 in the preparation of a biofilm inhibitor. The biofilm inhibitor prepared by using Pediococcus pentosaceus ACX 0180 has strong antibacterial ability and can effectively inhibit the biofilm formed by Staphylococcus aureus on the stainless steel surface during the processing of rice and flour products.
[0005] To solve the above technical problems, the technical solution adopted by the present invention is as follows:
[0006] Application of Pediococcus pentosaceus ACX 0180 in the preparation of a biofilm inhibitor.
[0007] Preferably, the Pediococcus pentosaceus ACX 0180 was deposited at the China Center for Type Culture Collection on November 06, 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is: CCTCC M20232133.
[0008] Preferably, the biofilm inhibitor is an inhibitor that inhibits the formation of biofilms by foodborne pathogenic bacteria on the surface of stainless steel.
[0009] Preferably, the biofilm inhibitor is an inhibitor that inhibits the formation of biofilms by foodborne pathogenic bacteria on the surface of stainless steel during the processing of rice and flour products.
[0010] Preferably, the foodborne pathogenic bacterium is Staphylococcus aureus.
[0011] Preferably, the content of the active ingredient of the biofilm inhibitor in rice and flour products is 17.35 mg / mL.
[0012] Preferably, the active ingredient of the biofilm inhibitor is a polypeptide, and the content of the polypeptide is 17.35 mg / mL.
[0013] The present invention also provides a biofilm inhibitor, comprising the Pediococcus pentosaceus ACX 0180.
[0014] The present invention also provides a preparation method of the biofilm inhibitor, comprising the following steps:
[0015] S1. Inoculate the Pediococcus pentosaceus strain ACX 0180 into a culture medium and culture it until the stationary phase to obtain a bacterial suspension;
[0016] S2. Centrifuge the bacterial suspension obtained in S1 to discard the bacterial sludge, and take the supernatant;
[0017] S3. Filter the supernatant through a filter membrane to obtain the biofilm inhibitor.
[0018] Preferably, in S1, the Pediococcus pentosaceus strain ACX 0180 is inoculated into MRS broth and cultured at 37°C for 16 h until the stationary phase.
[0019] Preferably, in S2, the bacterial suspension obtained in S1 is centrifuged at 25°C and 5000 r / min for 5 min to discard the bacterial sludge and take the supernatant.
[0020] Preferably, in S3, the supernatant is filtered through a 0.22 μm filter membrane.
[0021] Compared with the prior art, the present invention has the following advantages and technical effects:
[0022] The present invention provides the application of Pediococcus pentosaceus ACX 0180 in the preparation of a biofilm inhibitor. This strain can significantly inhibit the reproduction of Staphylococcus aureus and prevent it from forming biofilms on the surface of food processing equipment, showing better effects than traditional chemical antibacterial agents. In particular, the inhibitory effect is optimal under the low temperature condition of 4 °C, which is suitable for the food storage and transportation links. The antibacterial agent of the present invention can be prepared quickly, has the ability to efficiently remove biofilms, is safe, non-toxic, environmentally friendly, and does not produce harmful residues. It shows excellent stability in practical applications, highly fits the food cold chain transportation conditions, can effectively reduce microbial contamination, extend the shelf life of food, and reduce the risk of cross-contamination. The present invention provides a new strategy for safe, efficient, and environmentally friendly microbial control in the food industry, with significant industrial application advantages and broad market prospects.
[0023] The technical solution of the present invention will be further described in detail below through the accompanying drawings and examples. Description of the Drawings
[0024] Figure 1 It is a statistical chart of the antibacterial ability of the strain in Example 1;
[0025] Figure 2 It is a statistical chart of the acid production ability of Pediococcus pentosaceus ACX 0180 at different time points in Example 2;
[0026] Figure 3 It is the growth situation of the strain of the biofilm inhibitor provided in Example 2 at different temperatures. Among them, Figure 3 A in it is the growth situation of the strain at 4 °C, Figure 3 B in it is the growth situation of the strain at 15 °C, Figure 3 C in it is the growth situation of the strain at 37 °C;
[0027] Figure 4 It is the removal situation of the Staphylococcus aureus biofilm on the stainless steel sheet of the biofilm inhibitor provided in Example 2 at different temperatures. Among them, Figure 4 A in it is the removal situation of the antibacterial agent on the biofilm of Staphylococcus aureus at 4 °C, Figure 4 B in it is the removal situation of the antibacterial agent on the biofilm of Staphylococcus aureus at 15 °C. Detailed Embodiments
[0028] The technical solution of the present invention will be further described below through the accompanying drawings and examples.
[0029] Unless otherwise defined, the technical terms or scientific terms used in the present invention shall have the ordinary meanings understood by those of ordinary skill in the field to which the present invention belongs.
[0030] Source of test materials:
[0031] Preparation of TSA plate medium: Tryptone (15 g / L), agar (15 g / L), sodium chloride (5 g / L), soybean papain hydrolyzate (5 g / L). Weigh 40 grams of this product, add it to 1000 ml of distilled water, heat to boiling for dissolution, dispense, and sterilize at 121 °C under high pressure for 15 minutes for standby.
[0032] Preparation of TSB liquid medium: Tryptone (17 g / L), soy peptone (3 g / L), sodium chloride (5 g / L), dipotassium hydrogen phosphate (2.5 g / L), glucose (2.5 g / L). Weigh 30 grams of this product, add it to 1000 ml of distilled water, heat to boiling for dissolution, dispense, and sterilize at 121 °C under high pressure for 15 minutes for standby.
[0033] Preparation of MRS liquid medium: Glucose (20 g / L), yeast powder (4 g / L), peptone (10 g / L), beef powder (5 g / L), sodium acetate (5 g / L), dipotassium hydrogen phosphate (2 g / L), magnesium sulfate (0.2 g / L), ammonium citrate tribasic (2 g / L), manganese sulfate (0.05 g / L), Tween 80 (1 mL / L). Weigh 48.3 grams of this product, add it to 1000 ml of distilled water, heat to boiling for dissolution, dispense, and sterilize at 121 °C under high pressure for 15 minutes for standby.
[0034] Preparation of MRS plate medium: Glucose (20 g / L), yeast powder (4 g / L), peptone (10 g / L), beef powder (5 g / L), sodium acetate (5 g / L), dipotassium hydrogen phosphate (2 g / L), magnesium sulfate (0.2 g / L), ammonium citrate tribasic (2 g / L), manganese sulfate (0.05 g / L), Tween 80 (1 mL / L), agar powder (15 g / L). Weigh 63.3 grams of this product, add it to 1000 ml of distilled water, heat to boiling for dissolution, dispense, and sterilize at 121 °C under high pressure for 15 minutes for standby.
[0035] The old starter was obtained by purchasing through commercial channels.
[0036] In the present invention, unless otherwise specified, other test materials and instrument equipment are all conventional test materials in the art and can be obtained by purchasing through commercial channels.
[0037] Example 1
[0038] Strain screening
[0039] 12 Pediococcus pentosaceus strains were isolated and purified from the old starter, subcultured 3 times and experimented. Solid streak culture was carried out and cultured at 37 °C for 24 h. The size of the inhibition zone was observed by the pre-added bacterial liquid pour plate method. The specific steps are as follows:
[0040] (1) Streak Staphylococcus aureus and Pediococcus pentosaceus from the frozen glycerol tubes stored at -70°C onto TSA plate medium and MRS plate medium respectively. Pick single colonies and transfer them to TSB liquid medium and MRS liquid medium respectively. Culture at 37°C for 24 h, then inoculate into fresh liquid medium at an inoculation amount of 1% (v / v) and culture until the stationary phase.
[0041] (2) Place Oxford cups on the culture dishes in advance. Pour 20 mL of TSA medium with Staphylococcus aureus onto the culture dishes. After it solidifies, remove the Oxford cups.
[0042] (3) Add the Pediococcus pentosaceus bacterial liquid cultured for 24 h (concentration 10 9 CFU / mL) to the TSA punched plate, using MRS liquid medium as a blank control. Measure the size of the inhibition zone by the cross-cross method and select the Pediococcus pentosaceus with the strongest antibacterial ability according to the results. The results are as Figure 1 .
[0043] It can be seen from Figure 1 that the Pediococcus pentosaceus ACX 0180 has the strongest antibacterial ability.
[0044] Example 2
[0045] A method for preparing a biofilm inhibitor using the Pediococcus pentosaceus ACX 0180, comprising the following steps:
[0046] S1. Inoculate the seed liquid of the Pediococcus pentosaceus strain ACX 0180 screened in Example 1 into MRS liquid medium at an inoculation amount of 1% and culture at a constant temperature of 37°C for 60 h. Measure its OD 600nm value and pH value every 2 h in the first 24 h and every 12 h later. Determine that the Pediococcus pentosaceus reaches the stationary phase at 16 h, and at this time, its acid production ability is relatively strong. The results are as Figure 2 , and obtain a bacterial suspension;
[0047] S2. Centrifuge the bacterial suspension obtained in S1 at 25°C and 5000 r / min for 5 min to discard the bacterial sludge, and take the supernatant;
[0048] S3. Filter the supernatant through a 0.22 μm filter membrane to obtain a biofilm inhibitor. Among them, the active ingredient of the biofilm inhibitor is a polypeptide, and the content of the polypeptide is 17.35 mg / mL.
[0049] It can be seen from Figure 2It can be seen that with the increase of culture time, the number of Pediococcus pentosaceus ACX 0180 gradually increased and began to enter the logarithmic growth phase at about 4 hours, and reached the stable phase after 16 hours of culture, and the growth slowed down. During the growth lag phase and logarithmic growth phase of Pediococcus pentosaceus ACX 0180, the pH value in the culture medium has been decreasing, from 6.38 to 4.20, and stabilized after 16 hours, proving that the strain has a strong and stable acid production ability.
[0050] The effect of the biofilm inhibitor provided in the above Example 2 was verified:
[0051] 1. Study the optimal antibacterial preparation conditions of the biofilm inhibitor provided in the above Example 2 and determine the temperature conditions for the best effect in practical applications. The specific steps are as follows:
[0052] (1) After the Staphylococcus aureus cultured to the stable phase was centrifuged at 4°C and 10,000 rpm for 5 min, the supernatant was discarded, and an equal amount of PBS solution with a pH of 7.2 was added to wash the bacteria. This was repeated three times and the supernatant was removed by centrifugation again to obtain bacterial sludge;
[0053] (2) culturing Pediococcus pentosaceus for 2 h, 10 h, and 16 h to obtain bacterial suspensions in the delayed phase, logarithmic phase, and stable phase, respectively, and then centrifuging the Pediococcus pentosaceus at 5000 r / min for 5 min to obtain a supernatant, and filtering the supernatant through a 0.22 μm filter membrane to obtain a sterile cell-free supernatant;
[0054] (3) The cell-free supernatant and MRS liquid medium were mixed with PBS solution with a pH of 7.2 in a ratio of 1:1 and added to a vortex to mix thoroughly. The mixture was diluted to 10 4 CFU / mL. The bacterial solution was cultured at 4℃, 15℃, and 37℃, and the counts were performed at regular intervals, and the results were plotted as growth curves. Figure 3 .
[0055] Depend on Figure 3It can be seen the effects of the cell-free supernatants of Pediococcus pentosaceus in different periods on the growth of Staphylococcus aureus. The results show that the cell-free supernatant of Pediococcus pentosaceus in the lag phase has good antibacterial effects at 4°C and 15°C in the first 12 h, and the antibacterial ability decreases after 12 h, and there is almost no antibacterial effect at 37°C. The cell-free supernatant of Pediococcus pentosaceus in the logarithmic phase has good antibacterial effects at 4°C, 15°C, and 37°C in the first 12 h, and the antibacterial ability decreases after 12 h. The cell-free supernatant of Pediococcus pentosaceus in the stationary phase has good antibacterial ability against Staphylococcus aureus within 24 h at 4°C, 15°C, and 37°C, and has good antibacterial ability in the first 12 h at 37°C, and the antibacterial ability decreases after 12 h. It is proved that the cell-free supernatant of Pediococcus pentosaceus in the stationary phase has better antibacterial effects than those in the lag phase and the logarithmic phase, and the antibacterial effect is better under low-temperature conditions.
[0056] 2. Apply the biofilm inhibitor provided in the above Example 2 to clean the biofilm in the simulated rice slurry stainless steel pipeline. The specific steps are as follows:
[0057] (1) Oscillate and culture Staphylococcus aureus at 37°C until the late exponential phase. After collection by centrifugation at 4°C and 10,000 rpm for 5 min, wash it twice with PBS solution with a pH of 7.2, and suspend it in an equal volume of PBS solution with a pH of 7.2 to prepare a bacterial suspension. Part of the suspension is cultured at 4°C under low-temperature conditions for 30 d, and part of the bacterial suspension is reserved for use;
[0058] (2) Wash the glutinous rice clean and soak it for four hours. Then, grind the rice and water into rice slurry in a blender at a ratio of 1:4 and reserve it. Mix it with the biofilm inhibitor provided in Example 2 at ratios of 1:1 and 3:1 to form cell-free supernatants with concentrations of 50% and 25% respectively;
[0059] (3) After diluting the bacterial solutions in the stationary phase and treated at 4°C for 30 d by two concentration gradients, drop 50 μL of the bacterial solution on a stainless steel sheet, and then add the glutinous rice slurry prepared in the above step (2) and the cell-free supernatants with concentrations of 50% and 25% respectively. After culturing for 3 h, 6 h, 9 h, 12 h, 24 h, and 48 h, wash it twice with PBS solution with a pH of 7.2. After wiping the surface of the stainless steel sheet clean with a sterile cotton swab, put it into 900 μL of physiological saline for counting and plot the growth curve of the results. The results are as Figure 4 .
[0060] From Figure 4It can be seen that the cell-free supernatant with a concentration of 50% has the strongest ability to remove Staphylococcus aureus biofilms on stainless steel sheets, and Staphylococcus aureus after low-temperature treatment forms biofilms more slowly in rice milk. Thus, it can be known that the bacteriostatic agent prepared from the supernatant of Pediococcus pentosaceus has an inhibitory effect on the formation of Staphylococcus aureus biofilms, and when combined with low temperature, it has a stronger inhibitory ability on biofilms.
[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that they can still modify or equivalently replace the technical solutions of the present invention, and these modifications or equivalent replacements cannot make the modified technical solutions deviate from the spirit and scope of the technical solutions of the present invention.
Claims
1. Application of Pediococcus pentosaceus ACX 0180 in the preparation of biofilm inhibitors.
2. The application according to claim 1, characterized in that: The Pediococcus pentosaceus ACX 0180 was deposited in the China Center for Type Culture Collection on November 6, 2023, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being: CCTCCM20232133.
3. The application according to claim 1, characterized in that: The biofilm inhibitor is an inhibitor for inhibiting the biofilm formed by food-borne pathogenic bacteria on the surface of stainless steel.
4. The use according to claim 1, characterized in that: The biofilm inhibitor is an inhibitor for inhibiting the biofilm formed by food-borne pathogenic bacteria on the surface of stainless steel during the processing of rice and flour products.
5. The use according to any one of claims 3 or 4, characterized in that: The foodborne pathogenic bacteria is Staphylococcus aureus.
6. The use according to claim 4, characterized in that: The content of the active ingredient of the biofilm inhibitor in the rice and flour products is 17.35 mg / mL.
7. A biofilm inhibitor, characterized in that The method comprises the Pediococcus pentosaceus ACX0180 according to any one of claims 1 or 2.
8. A method for preparing a biofilm inhibitor as claimed in claim 7, characterized in that: The following steps are involved: S1. Inoculate Pediococcus pentosaceus strain ACX 0180 into a culture medium and culture until the stable phase to obtain a bacterial suspension; S2, centrifuge the bacterial suspension obtained in S1, discard the bacterial sludge, and take the supernatant; S3. Filter the supernatant through a filter membrane to obtain a biofilm inhibitor.
9. The preparation method according to claim 7, characterized in that: In S1, Pediococcus pentosaceus strain ACX 0180 was inoculated into MRS liquid medium and cultured at 37°C for 16 h until the stationary phase.