A kind of Lactobacillus paracasei and its application in degrading PBAT
By providing the T1-9 strain of C. paracetaccharin, the problems of low degradation efficiency and few bacterial species resources of PBAT biodegradable plastics are solved, and the effect of efficient degradation of PBAT plastics is achieved, reducing environmental pressure.
Patent Information
- Application Number
- CN202410252179.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-01
- Publication Date
- 2025-04-29
- Estimated Expiration
- 2044-03-01
AI Technical Summary
In the prior art, PBAT biodegradable plastic has low degradation efficiency and few effective bacterial strain resources, which limits its application scope.
A strain of C. paracetium T1-9 was provided. This strain is isolated and fermented food, has high lipase activity and good growth activity, and can effectively degrade PBAT plastic.
Through the application of the T1-9 strain of C. paracetaccacia, the degradation process of PBAT plastics was significantly accelerated, the pressure on landfill was reduced, and the safety and application range of degradants were improved.
Smart Images
Figure SMS_1 
Figure SMS_2 
Figure HDA0004733539430000011
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei and an application thereof in degrading PBAT. Background Art
[0002] With the increasing efforts in environmental protection, various parts of the country have begun to restrict the use of traditional plastics. In this case, biodegradable plastics show certain biodegradability, making it a new development trend in the plastics industry.
[0003] Biodegradable thermoplastic plastic (PBAT) is an aliphatic-aromatic copolyester (polybutylene adipate-terephthalate), which has both the excellent processability of aromatic polyester and the degradability of aliphatic polyester. The terephthalic acid group in its structure gives it good stability and mechanical properties. PBAT has good mechanical properties, thermoplasticity and biodegradability, making it widely used in food packaging, biomedicine, agricultural film, industrial composting and other fields.
[0004] The biodegradation mechanism of PBAT can be roughly divided into three stages: microbial colonization-enzymatic hydrolysis-microbial utilization, and the degradation rate depends on the growth ability of the microorganism and the activity of the polyester hydrolase related to the strain. Therefore, although PBAT has some advantages in specific applications, there are still some problems: First, its degradation efficiency is not high in actual use. The high aromatic hydrocarbon content in PBAT will lead to its high resistance to microbial biological attacks. Therefore, although biodegradable plastics can be degraded, the degradation time may be relatively long, especially in landfills. Complete degradation may take several years or even longer; second, there are currently few studies on the biodegradation of PBAT plastics, and there are not many effective bacterial resources. They are basically concentrated on Pseudomonas, Micromonas and Bacillus isolated from the soil. The strains have low safety and are mainly targeted at the degradation of PBAT mulch, which greatly limits the scope of application of the strains. Summary of the invention
[0005] In view of the problem that there are few effective bacterial strains for the biodegradation of PBAT in the natural environment, the present invention provides a new strain of Lactobacillus paracasei T1-9 that can degrade PBAT. The strain is separated from fermented food, is an edible strain, is harmless to the environment, can be applied to daily PBAT waste plastics, accelerates the degradation of PBAT plastics, and reduces the pressure on the land caused by landfill waste. The strain has high growth activity and high lipase activity in an environment containing PBAT film.
[0006] Specifically, the present invention proposes the following technical solutions:
[0007] In a first aspect, the present invention provides a Lacticaseibacillus paracasei T1-9 strain, characterized in that the strain is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC NO: M 2024064.
[0008] In a second aspect, the present invention provides a bacterial agent, characterized in that it comprises the Lacticaseibacillus paracasei T1-9 strain.
[0009] In a third aspect, the present invention provides a degradation agent, characterized in that it comprises, by weight, 0.5-1.5 parts of Lactobacillus paracasei T1-9 powder, 13-15 parts of gelatin, 6.5-8 parts of carbon source and 2-2.5 parts of nitrogen source.
[0010] Preferably, the degradation agent is characterized in that the carbon source is selected from one or more of glucose, sucrose, lactose, maltose and glycerol.
[0011] Preferably, the degradation agent is characterized in that the nitrogen source is selected from one or more of soy peptone, tryptone, ammonium sulfate and yeast powder.
[0012] In a fourth aspect, the present invention provides a method for preparing the degradation agent, characterized in that the method comprises mixing Lactobacillus paracasei T1-9 powder, gelatin, carbon nitrogen and a nitrogen source according to weight parts.
[0013] Preferably, the preparation method of the degradation agent is characterized in that the Lactobacillus paracasei T1-9 bacterial powder is obtained by adding a lyophilization protective agent to the bacterial mud obtained by fermentation and centrifugation of the Lactobacillus paracasei T1-9 strain and freeze-drying;
[0014] Preferably, the mass ratio of bacterial mud to freeze-drying protective agent is (0.5-1.5): (2.5-3.5);
[0015] Further preferably, the lyoprotectant comprises skim milk powder, trehalose, maltodextrin and mannitol;
[0016] More preferably, the lyoprotectant comprises, by mass percentage, 30-35% skim milk powder, 35-45% trehalose, 20%-30% maltose and 5-10% mannitol.
[0017] In a fifth aspect, the present invention provides a degradation agent prepared by the preparation method of the degradation agent.
[0018] In a sixth aspect, the present invention provides the use of the Lactobacillus paracasei T1-9 strain or the bacterial agent or the degrading agent in degrading PBAT plastic products.
[0019] Preferably, the use of the strain, bacterial agent or degradation agent in degrading PBAT plastic products is characterized in that the PBAT plastic products are PBAT agricultural films, packaging bags containing PBAT materials or packaging films containing PBAT materials;
[0020] Preferably, the PBAT plastic product is in the form of a film, powder or emulsion;
[0021] Further preferably, when the degradation agent degrades the PBAT plastic product, the degradation agent is mixed with water in a mass ratio of (0.8-1.2):(900-1100).
[0022] The beneficial effects of the present invention include:
[0023] The Lactobacillus paracasei T1-9 strain provided by the present invention is an edible fungus with high safety. It can be used to degrade daily PBAT plastic products, such as food-grade packaging bags, disposable tableware and medical biomaterials, and can accelerate the degradation of PBAT plastic products and reduce the pressure on land caused by landfill garbage. The Lactobacillus paracasei T1-9 strain can secrete lipase that degrades PBAT plastic products and has a high PBAT degradation efficiency.
[0024] The PBAT degrading agent containing Lactobacillus paracasei T1-9 bacterial powder provided by the present invention, the addition of gelatin can effectively promote the production of lipase by Lactobacillus paracasei T1-9 strain, and the carbon source and nitrogen source can effectively promote the metabolic activity of Lactobacillus paracasei T1-9 strain, further improving the degradation effect of the degrading agent on PBAT. The preparation method of the degrading agent is simple, environmentally friendly, and can effectively accelerate the degradation of PBAT plastic products.
[0025] Strain deposit information
[0026] The Lacticaseibacillus paracasei T1-9 strain provided by the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on January 9, 2024, with a deposit number of CCTCC NO: M2024064, a deposit address of Wuhan University, Wuhan, China, postal code: 430072, and a telephone number of 027-68754052. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 The OD values of the seven PBAT-degrading strains screened in Example 1 after incubation in a PBAT environment for 7 days are shown. 600 value.
[0028] Figure 2 Shown is the colony morphology of the Lactobacillus paracasei T1-9 strain in Example 2 on the MRS plate.
[0029] Figure 3 Shown is the degradation curve of PBAT film by Lactobacillus paracasei T1-9 strain in Example 3.
[0030] Figure 4 The figure shows the lipase activity of Lactobacillus paracasei T1-9 strain in Example 4 in different environments.
[0031] Figure 5 Shown are SEM images of the degradation effect of the PBAT membrane surface by the degradation agent of Lactobacillus paracasei T1-9 in Example 5-1 and the degradation agent of Lactobacillus paracasei CICC6108 in Comparative Example 1, (A) is a SEM image of the degradation effect of the PBAT membrane surface by the degradation agent of Lactobacillus paracasei CICC6108 in Comparative Example 1 (electron microscope scanner model Apreo 2, magnifications of 5000 times and 20000 times); (B) is a SEM image of the degradation effect of the PBAT membrane surface by the degradation agent of Lactobacillus paracasei T1-9 in Example 5-1 (electron microscope scanner model Apreo 2, magnifications of 5000 times and 20000 times).
[0032] Figure 6 Shown are the weight loss rates (%) of PBAT films under the action of different degradation agents in the examples and comparative examples. DETAILED DESCRIPTION
[0033] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings, but the present invention is not limited to the following technical solution.
[0034] Sources of biological materials
[0035] The Lactobacillus casei CICC6108 strain mentioned in Comparative Example 1 is a purchased standard strain purchased from the China Industrial Microbiological Culture Collection Center (CICC).
[0036] Unless otherwise specified, the various reagents and instruments used in the examples and comparative examples of the present invention are conventional commercially available products. The sources of experimental materials and instrument information used in the present invention are shown in Table 1 below:
[0037] Table 1
[0038]
[0039]
[0040] In order to better understand the technical solution of the present invention, the technical solution of the present invention is described in detail below in conjunction with specific embodiments.
[0041] Example 1 Screening of strains capable of degrading PBAT membrane
[0042] Lactobacillus paracasei T1-9 was isolated from milk lumps made by herders in Jiacha County, Shannan City, Tibet Autonomous Region. The specific isolation, purification and identification methods are as follows:
[0043] Isolation and purification of strains: Take 5g of milk lumps and add them to 45mL of sterile saline, and shake at 37℃ for 30min. Take the sample solution for gradient dilution, apply it on MRS medium, and culture it at 37℃ for 48h. Pick typical colonies with different morphologies and separate and purify them three times on MRS medium plates. Pick strains for Gram staining and catalase test, select Gram-positive and catalase-negative strains for glycerol storage, and a total of 40 strains were isolated and screened.
[0044] Screening of PBAT-degrading strains: After the screened strains were activated, they were inoculated into MRS liquid culture medium at a 2% inoculum and cultured at 37°C until the logarithmic growth phase. 0.1g of PBAT membrane was sterilized (treated with 2% SDS solution for 4h, rinsed with sterile water, sterilized with 75% ethanol for 30min, rinsed with sterile water, and air-dried for 15min under a laminar flow cabinet irradiated with ultraviolet light), added to the culture medium, and incubated in a 37°C incubator at 100rpm for 7d. The optical density was measured at 600nm using a UV-visible spectrophotometer to monitor the growth of the culture. The results showed that among the 40 strains tested, 7 strains showed excellent growth activity in the PBAT environment, among which T1-9 had the best growth activity, with an OD of 0. 600 is 0.82 (see Figure 1 ).
[0045] Example 2 Identification of strains
[0046] 16SrDNA identification of strains: The culture fluid of T1-9 was operated according to the instructions of the genome extraction kit, and the strain was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') (SEQ ID NO.1) and 1492R (5'-GGTTACCTTGTTACGACTT-3') (SEQ ID NO.2). The amplification conditions were: 94°C pre-denaturation for 3min, 94°C denaturation for 30s, 55°C annealing for 30s, 72°C extension for 1min, 30 cycles, 72°C extension for 5min, and 4°C storage. The PCR product was analyzed by 1% agarose gel electrophoresis, and the 16S rDNA sequence of the strain was measured as follows: SEQ ID NO.3:
[0047] CTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAACGAGTTCTCGTTGATGATCGGTGCTTGCACCGAGATTCAACATGGAACGAGTGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCTTAAGTGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAGATCCAAGAACCGCATGGTTCTTGGCTGAAAGATGGCGTAAGCTATCGCTTTTGGATGGACCCGCGGCGTATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGATGATACGTAGCCGAACTGAGAGGTTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGCAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTGGAGAAGAATGGTCGGCAGAGTAACTGTTGTCGGCGTGACGGTATCCAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCCTCGGCTTAACCGAGGAAGCGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAATGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCTTTTGATCACCTGAGAGATCAGGTTTCCCCTTCGGGGGCAAAATGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATGACTAGTTGCCAGCATTTAGTTGGGCACTCTAGTAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAGACCGCGAGGTCAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGACTGTAGGCTGCAACTCGCCTACACGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCGAAGCCG
[0048] The measured 16S rDNA gene sequence was compared in NCBI, homology analysis was performed using BLAST, developmental tree was established using MEGA 7.0 software, and combined with morphological analysis, the strain was identified as Lacticaseibacillus paracasei, which was deposited in the China Center for Type Culture Collection (CCTCC) on January 9, 2024, with the deposit number: CCTCC NO: M2024064. Figure 2 The figure shows the colony morphology of Lacticaseibacillus paracasei T1-9 strain on MRS plate. The colonies are milky white, smooth and opaque, and have neat edges.
[0049] Example 3 Degradation experiment of PBAT film by Lactobacillus paracasei T1-9 strain The Lactobacillus paracasei T1-9 strain was activated and then inoculated into MRS liquid culture medium at a 2% (v / v) inoculation amount, and cultured at 37° C. to the logarithmic growth phase, followed by the addition of 0.1 g of PBAT membrane, and the PBAT membrane was surface sterilized (2% SDS solution treatment for 4 h, sterile water rinsing, 75% ethanol sterilization for 30 min, sterile water rinsing, and air-dried for 15 min under a laminar flow cabinet irradiated with ultraviolet light), and 0.1 g of surface-sterilized PBAT membrane was added to MRS culture medium (50 mL) without the strain, and incubated under the same conditions as a blank control, and continued to incubate at 37° C., 100 rpm for 21 days, and samples were taken on the 5th, 7th, 10th, 14th and 21st days, respectively, and the incubated PBAT membrane was washed with 2% sodium dodecyl sulfate solution for 2 h, then rinsed with distilled water, dried in an oven at 40° C. for 2 d, and then weighed to obtain the accurate weight of the PBAT membrane. The percentage of PBAT film weight loss was used to express the degree of biodegradation of the PBAT film, and the weight loss rate of the film was calculated using the following formula (1):
[0050] Weight loss percentage (%) = (W0-W1) / W0×100(1)
[0051] Wherein, W0 and W1 are the weight (g) of the PBAT film before degradation and the weight (g) after degradation, respectively.
[0052] The results are as follows Figure 3 As shown in the figure, the co-culture of Lactobacillus paracasei T1-9 strain and PBAT membrane for 21 consecutive days can make the weight loss rate of PBAT membrane reach 10.5%. Although the blank group has weight loss, it is far lower than the effect of Lactobacillus paracasei T1-9 strain, indicating that in addition to natural hydrolysis, PBAT is also biodegraded by Lactobacillus paracasei T1-9 strain.
[0053] Example 4: Lipase activity of Lactobacillus paracasei T1-9 strain in PBAT membrane environment In order to evaluate the lipase production ability of Lactobacillus paracasei T1-9 strain, three groups of experiments were designed.
[0054] Experimental Group 1: The Lactobacillus paracasei T1-9 strain was activated and inoculated into 50 mL of MRS liquid culture medium at a 2% inoculum (v / v), cultured at 37°C until the logarithmic growth phase, 0.1 g of sterilized PBAT membrane was added to the culture medium, and then incubated in an incubator at 37°C with shaking at 100 rpm for 14 days, and the supernatant was collected.
[0055] Experimental Group 2: The Lactobacillus paracasei T1-9 strain was activated and inoculated into 50 mL of MRS liquid culture medium containing 1.4% gelatin at a 2% inoculum (v / v). The subsequent experimental steps were the same as those of Experimental Group 1.
[0056] Blank group: The Lactobacillus paracasei T1-9 strain was activated and inoculated into 50 mL MRS liquid culture medium at a 2% inoculum (v / v) without adding PBAT membrane. The subsequent culture conditions were the same as those of experimental group 1.
[0057] Each group had three parallels. The cultured bacterial suspension was transferred to a conical centrifuge tube and centrifuged at 4°C and 4000 x g for 15 min. The supernatant was collected and filtered with a 0.45 μm filter to obtain the final supernatant for analysis. The lipase activity was detected using a lipase (LPS) activity assay kit. The results are shown in Figure 4 As shown, when cultured in the PBAT membrane environment, the lipase activity of the supernatant of the culture fluid of Lactobacillus paracasei T1-9 strain was 4U / mL. When 1.4% gelatin was added and then co-cultured with the PBAT membrane, the lipase activity of the supernatant of the culture fluid of Lactobacillus paracasei T1-9 strain reached 4.8U / mL, both of which were higher than 1.18U / mL of the blank group, indicating that in the PBAT environment, Lactobacillus paracasei T1-9 strain can degrade the PBAT membrane by secreting a large amount of lipase, and the addition of gelatin can promote the secretion of lipase from Lactobacillus paracasei T1-9.
[0058] Example 5 Preparation of PBAT film degradation agent by Lactobacillus paracasei T1-9 strain
[0059] Example 5-1
[0060] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0061] (1) Activation of strains: The strains stored in the glycerol tubes were activated and inoculated into MRS broth at a 2% inoculum, and cultured at 37°C for 24 h;
[0062] (2) Preparation of fermentation broth: The fermentation broth activated in step (1) was inoculated into MRS medium at an inoculum size of 2% and cultured at 37° C. for 18 h;
[0063] (3) Preparation of bacterial powder: The fermentation liquid was centrifuged at 5000×g and 4°C for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain bacterial sludge, and then a freeze-dried protective agent treated at 90°C for 30 min was added. The mass ratio of bacterial sludge to freeze-dried protective agent was 1:3, and the mass percentages of the components of the freeze-dried protective agent were as follows: 30% skim milk powder, 35% trehalose, 30% maltose, and 5% mannitol. The mixture was pre-frozen in a -30°C refrigerator for 8 h, and the pre-frozen bacterial milk was freeze-dried in a vacuum freeze dryer for 48 h to obtain freeze-dried bacterial powder of Lactobacillus paracasei T1-9.
[0064] (4) Preparation of degradation agent: By weight, 1 part of Lactobacillus paracasei T1-9 powder, 14 parts of gelatin, 7 parts of carbon source, and 2 parts of nitrogen source are mixed to obtain the degradation agent. When used, the degradation agent is mixed with sterile water at a mass ratio of 1:1000.
[0065] Example 5-2
[0066] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0067] (1) Bacteria activation: Same as Example 5-1.
[0068] (2) Preparation of fermentation broth: Same as Example 5-1.
[0069] (3) Preparation of bacterial powder: The mass ratio of bacterial mud to lyophilization protective agent is 0.5:2.5, and the mass percentage of each component of the lyophilization protective agent is: 30% skim milk powder, 45% trehalose, 20% maltose, and 5% mannitol. Place it in a -30°C refrigerator for pre-freezing for 8 hours, and place the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the lyophilized bacterial powder of Lactobacillus paracasei T1-9.
[0070] (4) Preparation of degradation agent: By weight, 0.5 parts of Lactobacillus paracasei T1-9 powder, 13 parts of gelatin, 6.5 parts of carbon source, and 2.5 parts of nitrogen source are mixed to obtain the degradation agent. When used, the degradation agent is mixed with sterile water at a mass ratio of 1.2:1100.
[0071] Example 5-3
[0072] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0073] (1) Bacteria activation: Same as Example 5-1.
[0074] (2) Preparation of fermentation broth: Same as Example 5-1.
[0075] (3) Preparation of bacterial powder: The mass ratio of bacterial mud to lyophilization protective agent is 1.5:3.5, and the mass percentage of each component of the lyophilization protective agent is: 35% skim milk powder, 35% trehalose, 20% maltose, and 10% mannitol. Place it in a -30°C refrigerator for pre-freezing for 8 hours, and place the pre-frozen bacterial milk in a vacuum freeze dryer for 48 hours to obtain the freeze-dried bacterial powder of Lactobacillus paracasei T1-9.
[0076] (4) Preparation of degradation agent: By weight, 1.5 parts of Lactobacillus paracasei T1-9 powder, 15 parts of gelatin, 8 parts of carbon source, and 2.5 parts of nitrogen source are mixed to obtain the degradation agent. When used, the degradation agent is mixed with sterile water at a mass ratio of 0.8:900.
[0077] Example 6 Test on the Degradation Effect of Lactobacillus paracasei T1-9 Degradation Agent on PBAT Film
[0078] In order to evaluate the degradation effect of the degradation agent on PBAT, the PBAT film was cut into 5cm×5cm films, treated with 2% SDS solution for 4h, rinsed with sterile water, sterilized with 75% ethanol for 30min, rinsed with sterile water, air-dried for 15min under a laminar flow cabinet irradiated with ultraviolet light for sterilization, placed in a sterile culture dish, divided into 3 parts, and sprayed with the degradation agent of Lactobacillus paracasei T1-9 prepared in Example 5-1, Example 5-2 and Example 5-3 every day for 10 consecutive days. After 10 days, the PBAT film was taken out, washed with 2% sodium dodecyl sulfate solution for 2h, then rinsed with distilled water, dried in an oven at 40°C for 2d, and then weighed to obtain the accurate weight of the PBAT film. The percentage of weight loss of the PBAT film is used to express the degree of biodegradation of the PBAT film, and the weight loss rate of the film is calculated according to formula (1) (see Figure 6 ).
[0079] Another PBAT film after the degradation agent in Example 5-1 was treated for 10 days was soaked in 75% ethanol for 20 minutes, rinsed with deionized water 3 times, air-dried at 40°C for 12 hours, and then subjected to scanning electron microscopy (SEM) analysis. The results are shown in Figure 5 Middle (B). To facilitate imaging of the non-conductive PBAT film, a gold coating was applied prior to scanning imaging.
[0080] Comparative Example 1
[0081] The Lactobacillus paracasei T1-9 strain in Example 5-1 was changed to the Lactobacillus paracasei CICC6108 strain, and the rest of the preparation method remained unchanged to prepare a Lactobacillus paracasei CICC6108 degrader. The Lactobacillus paracasei CICC6108 was an externally purchased standard strain (in preliminary experiments, the bacteria could grow in a PBAT membrane environment and had the ability to produce lipase). The prepared Lactobacillus paracasei CICC6108 degrader was tested for the degradation effect of the Lactobacillus paracasei CICC6108 degrader on the PBAT membrane according to the test method of Example 6.
[0082] The weight loss rate (%) of PBAT film under the action of Lactobacillus casei CICC6108 degradation agent is shown in Figure 6 The SEM images of the degradation effect of Lactobacillus casei CICC6108 on the surface of PBAT membrane are shown in Figure 5 (A). After 10 days of action of the degradation agent of Lactobacillus casei CICC6108, the surface of the PBAT film remained basically unchanged, and only small cracks and slight depressions appeared after further magnification. After 10 consecutive days of action of the degradation agent of Lactobacillus paracasei T1-9, multiple depressions appeared on the surface of the PBAT film, and obvious wide cracks or holes were visible after further magnification, indicating that the degradation agent of Lactobacillus paracasei T1-9 effectively biodegraded PBAT.
[0083] Comparative Example 2
[0084] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0085] (1) Bacteria activation: Same as Example 5-1.
[0086] (2) Preparation of fermentation broth: Same as Example 5-1.
[0087] (3) Preparation of bacterial powder: same as Example 5-1.
[0088] (4) Preparation of degradation agent: By weight, 1 part of Lactobacillus paracasei T1-9 powder, 10 parts of gelatin, 7 parts of carbon source, and 2 parts of nitrogen source are mixed to obtain the degradation agent. When used, the degradation agent is mixed with sterile water at a mass ratio of 1:1000.
[0089] The degradation agent of Lactobacillus paracasei T1-9 prepared in Comparative Example 2 was tested according to the test method of Example 6, and the percentage of weight loss of PBAT film was used to express the biodegradation degree of PBAT film. The weight loss rate of the film was calculated according to formula (1) (see Figure 6 ).
[0090] Comparative Example 3
[0091] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0092] (1) Bacteria activation: Same as Example 5-1.
[0093] (2) Preparation of fermentation broth: Same as Example 5-1.
[0094] (3) Preparation of bacterial powder: same as Example 5-1.
[0095] (4) Preparation of degradation agent: By weight, 1 part of Lactobacillus paracasei T1-9 powder, 14 parts of gelatin, 4 parts of carbon source, and 1 part of nitrogen source are mixed to obtain the degradation agent. When used, the degradation agent is mixed with sterile water at a mass ratio of 1:1000.
[0096] The degradation agent of Lactobacillus paracasei T1-9 prepared in Comparative Example 3 was tested according to the test method of Example 6, and the percentage of weight loss of PBAT film was used to express the biodegradation degree of PBAT film. The weight loss rate of the film was calculated according to formula (1) (see Figure 6 ).
[0097] Comparative Example 4
[0098] The preparation method of the Lactobacillus paracasei T1-9 degradation agent is carried out according to the following steps:
[0099] (1) Bacteria activation: Same as Example 5-1.
[0100] (2) Preparation of fermentation broth: Same as Example 5-1.
[0101] (3) Preparation of bacterial powder: The fermentation liquid was centrifuged at 5000×g and 4°C for 10 min, the supernatant was discarded, and the bacterial cells were washed twice with sterile physiological saline to obtain bacterial sludge, and then a freeze-dried protective agent treated at 90°C for 30 min was added. The mass ratio of bacterial sludge to freeze-dried protective agent was 1:5, and the mass percentages of the components of the freeze-dried protective agent were as follows: 33% skim milk powder, 40% trehalose, 25% maltose, and 8% mannitol. The mixture was pre-frozen in a -30°C refrigerator for 8 h, and the pre-frozen bacterial milk was freeze-dried in a vacuum freeze dryer for 48 h to obtain freeze-dried bacterial powder of Lactobacillus paracasei T1-9.
[0102] (4) Preparation of degradation agent: same as Example 5-1.
[0103] (4) Preparation of degradation agent: same as Example 5-1.
[0104] The degradation agent of Lactobacillus paracasei T1-9 prepared in Comparative Example 4 was tested according to the test method of Example 6, and the percentage of weight loss of PBAT film was used to express the biodegradation degree of PBAT film. The weight loss rate of the film was calculated according to formula (1) (see Figure 6 ).
[0105] from Figure 6 It can be seen that the degradation effect of the degradation agent of Lactobacillus casei CICC6108 on the PBAT film in Comparative Example 1 is the worst; when the degradation agent formula in Example 5-1, Example 5-2 and Example 5-3 is 0.5-1.5 parts of Lactobacillus paracasei, 13-15 parts of gelatin, 6.5-8 parts of carbon source, and 2-2.5 parts of nitrogen source, the prepared degradation agent has a higher degradation effect, and the effect is best under the formula of Example 5-1.
[0106] Although the above describes the specific implementation methods of the present invention, it does not limit the scope of protection of the present invention. Based on the technical solution of the present invention, various modifications or variations that can be made by those skilled in the art without creative work are still within the scope of protection of the present invention.
Claims
1. A Lacticaseibacillus paracasei T1-9 strain, characterized in that The strain is deposited in China Center for Type Culture Collection (CCTCC) with the deposit number being CCTCC NO:M 2024064.
2. A bacterial agent, characterized in that The invention comprises the Lacticaseibacillus paracasei T1-9 strain according to claim 1.
3. A degradation agent, characterized in that: By weight, the invention comprises 0.5-1.5 parts of Lactobacillus paracasei T1-9 powder containing the Lactobacillus paracasei T1-9 strain according to claim 1, 13-15 parts of gelatin, 6.5-8 parts of carbon source and 2-2.5 parts of nitrogen source.
4. The degradation agent according to claim 3, characterized in that The carbon source is selected from one or more of glucose, sucrose, lactose, maltose and glycerol.
5. The degradation agent according to claim 3 or 4, characterized in that The nitrogen source is selected from one or more of soybean peptone, tryptone, ammonium sulfate and yeast powder.
6. A method for preparing the degradation agent according to any one of claims 3 to 5, characterized in that: The method comprises mixing Lactobacillus paracasei T1-9 bacterial powder containing the Lactobacillus paracasei T1-9 strain according to claim 1, gelatin, a carbon source and a nitrogen source according to weight parts.
7. The method for preparing the degradation agent according to claim 6, characterized in that: The Lactobacillus paracasei T1-9 bacterial powder is obtained by adding a lyophilization protective agent to the bacterial mud obtained by fermentation and centrifugation of the Lactobacillus paracasei T1-9 strain according to claim 1 and freeze-drying.
8. The method for preparing the degradation agent according to claim 7, characterized in that: The mass ratio of bacterial mud to freeze-drying protective agent is (0.5-1.5): (2.5-3.5).
9. The method for preparing the degradation agent according to claim 7 or 8, characterized in that: Lyoprotectants include skim milk powder, trehalose, maltodextrin and mannitol.
10. The method for preparing the degradation agent according to claim 9, characterized in that: Calculated by mass percentage, the freeze-drying protective agent includes 30-35% skim milk powder, 35-45% trehalose, 20%-30% maltose and 5-10% mannitol.
11. A degradation agent prepared by the method for preparing a degradation agent according to any one of claims 6 to 10.
12. Use of the Lactobacillus paracasei T1-9 strain according to claim 1, the bacterial agent according to claim 2, or the degrading agent according to claim 3 or 11 in degrading PBAT plastic products.
13. The use of the strain, bacterial agent or degradation agent according to claim 12 in degrading PBAT plastic products, characterized in that: The PBAT plastic product is a PBAT agricultural film, a packaging bag containing a PBAT material, or a packaging film containing a PBAT material.
14. The use of the strain, bacterial agent or degradation agent according to claim 13 in degrading PBAT plastic products, characterized in that: PBAT plastic products are in the form of film, powder or emulsion.
15. Use of the strain, bacterial agent or degradation agent according to any one of claims 12 to 14 in degrading PBAT plastic products, characterized in that: When the degradation agent is used to degrade PBAT plastic products, the degradation agent is mixed with water in a mass ratio of (0.8-1.2):(900-1100).