Enzyme solution and method for evaluating biodegradability of biodegradable resin using same

Abstract

The present invention relates to: an enzyme solution which has the ability to decompose a biodegradable resin and can be useful for evaluating the biodegradability of the biodegradable resin; and a method for evaluating the biodegradability of the biodegradable resin using the enzyme solution.

Description

Enzyme solution and method for evaluating biodegradability of biodegradable resin using the same

[0001] Cross-citation with related application(s)

[0002] This application claims the benefit of priority to Korean Patent Application Nos. 10-2024-0119312, 10-2024-0119313, and 10-2024-0119314, filed September 3, 2024, and Korean Patent Application No. 10-2024-0183657, filed December 11, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to an enzyme solution having a biodegradable resin decomposition ability and being useful for evaluating the biodegradability of a biodegradable resin, and a method for evaluating the biodegradability of a biodegradable resin using the enzyme solution.

[0004]

[0005] Conventional petroleum-based plastics do not decompose naturally, causing serious environmental pollution. Therefore, biodegradable polymers, such as polylactide (PLA), poly(butylene adipate-co-terephthalate) (PBAT), and polybutylene succinate (PBS), which can be biodegraded in nature, are attracting attention as alternatives to petroleum-based plastics.

[0006] Biodegradability assessment is essential in developing these biodegradable resins. However, existing biodegradability tests are conducted over several weeks or months, depending on the specific conditions—soil, compost, or ocean—and require expensive, large-scale equipment.

[0007] Therefore, there is a need to develop an evaluation method that can evaluate the biodegradability of biodegradable resins in a simpler manner and in a short period of time.

[0008]

[0009] The present invention aims to provide an enzyme solution having a decomposition ability for biodegradable resins and enabling a simpler and faster prediction of the degree of biodegradation of biodegradable resins when exposed to a natural environment.

[0010] In addition, the present invention aims to provide a method for evaluating the biodegradability of a biodegradable resin using the enzyme solution.

[0011]

[0012] According to one embodiment of the present invention, an enzyme solution is provided, which comprises a hydrolytic enzyme including lipase and cutinase; and a phosphate buffered saline solution.

[0013] According to another embodiment of the present invention, a method for evaluating the biodegradability of a biodegradable resin is provided, comprising the steps of: i) contacting a biodegradable resin with an enzyme solution containing a hydrolytic enzyme including lipase and cutinase; and phosphate buffered saline; and ii) observing a degradation pattern of the biodegradable resin over time.

[0014]

[0015] The enzyme solution of the present invention has the ability to decompose biodegradable resins and can be usefully used to evaluate the biodegradability of biodegradable resins. According to the method for evaluating the biodegradability of biodegradable resins of the present invention using the enzyme solution, the biodegradability of biodegradable resins can be evaluated simply and quickly without expensive, large-scale equipment. In addition, according to the method for evaluating the biodegradability of biodegradable resins, the biodegradability of biodegradable resins can be easily predicted under various natural environmental conditions such as soil, compost, and the ocean.

[0016]

[0017] Figure 1 shows the results of biodegradability evaluation in a soil / compost environment of poly(butylene adipate-co-terephthalate) compound 1 (PBAT C1) and poly(butylene adipate-co-terephthalate) compound 2 (PBAT C2) performed in (3) of Example A-1.

[0018] Figure 2 shows the results of a biodegradability test under industrial composting conditions of PBAT C1, PBAT C2, and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) measured according to the ISO 14855-1 standard.

[0019] Figure 3 shows the results of biodegradability evaluation in a marine environment of poly(butylene succinate) (PBS) and poly(butylene succinate adipate) (PBSA) performed in (3) of Example A-2.

[0020] Figure 4 shows the results of biodegradability tests of PBS and PBSA under marine conditions measured according to the ASTM D6691 standard.

[0021] Figure 5 shows the results of biodegradability evaluation of PBAT / TPS compound and PBAT / PLA compound in soil / compost environment performed in (3) of Example B-1.

[0022] Figure 6 shows the results of biodegradability evaluation in a soil environment according to the ISO 17556 standard of the PBAT / TPS compound and PBAT / PLA compound and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example B-1.

[0023] Figure 7 shows the results of biodegradability evaluation of PBS and PBSA under marine conditions performed in (3) of Example B-2.

[0024] Figure 8 is a schematic diagram showing a method of dripping an enzyme solution onto a test piece in the biodegradability evaluation of Examples C-1 and C-2.

[0025] Figure 9 shows the results of biodegradability evaluation in an industrial compost environment according to the ISO 14855 standard of PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example C-1.

[0026] Figure 10 (1) shows the results of biodegradability evaluation of PBAT compound 1 and PBAT compound 2 of Example C-1, (2) of Comparative Example C-1, and (3) of Comparative Example C-2 in a soil / compost environment.

[0027] Figure 11 shows the results of biodegradability evaluation in a marine environment according to the ASTM D6691 standard of P3HP 1, P3HP 2, and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example C-2.

[0028] Figure 12 shows the results of biodegradability evaluation of P3HP 1 and P3HP 2 in a marine environment performed in (4) of Example C-2.

[0029] Figure 13 is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the invention.

[0030] Figure 14 is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the invention.

[0031] FIG. 15 is a graph showing the difference in image brightness over time obtained according to one embodiment of the invention.

[0032] FIG. 16 is a graph showing a first-order differential function and a second-order differential function obtained according to one embodiment of the invention.

[0033] Figure 17 shows the results of biodegradability evaluation in home compost of PBSA compound 1 (PBSA C1), PBSA compound 2 (PBSA C2), PBSA compound 3 (PBSA C3), PBSA compound 4 (PBSA C4) and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) measured according to the ISO 14855-1 standard.

[0034]

[0035] The terminology used herein is for the purpose of describing exemplary embodiments only and is not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly indicates otherwise. In this specification, it should be understood that the terms "comprise," "include," or "have" indicate the presence of a feature, step, component, or combination thereof, but do not preclude the possibility of the presence or addition of one or more other features, steps, components, or combinations thereof.

[0036]

[0037] The present invention is susceptible to various modifications and takes various forms. Specific embodiments are illustrated and described in detail below. However, this is not intended to limit the present invention to specific disclosed forms, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention.

[0038]

[0039] In the present invention, “biodegradable resin” or “biodegradable polymer” means a polymer that is decomposed by microorganisms into natural byproducts such as water, carbon dioxide, nitrogen, biomass, and inorganic salts.

[0040]

[0041] Hereinafter, the present invention will be described in detail.

[0042]

[0043] enzyme solution

[0044]

[0045] According to one embodiment of the present invention, an enzyme solution is provided, comprising a hydrolytic enzyme including lipase and cutinase; and a phosphate buffered saline solution.

[0046]

[0047] The enzyme solution of the present invention has a biodegradable resin decomposition ability and can be usefully used to evaluate the biodegradability of a biodegradable resin in a short period of time.

[0048]

[0049] In particular, by controlling the type and / or content of the hydrolytic enzyme included in the enzyme solution, various environments such as a soil environment, a compost environment, and a marine environment can be implemented, thereby predicting the biodegradability of the biodegradable resin under each environmental condition.

[0050]

[0051] As a solvent for preparing an enzyme solution according to one embodiment of the present invention, phosphate-buffered saline (PBS) can be used.

[0052]

[0053] For example, the phosphate-buffered saline (PBS) may contain 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4 as a mixture of ultrapure grade phosphate buffer and saline adjusted to pH 7.4.

[0054]

[0055] In one embodiment, the lysate may include a lysate derived from one or more microorganisms selected from the group consisting of Pseudomonas Cepacia, Rhizopus Oryzae, Aspergillus niger, and Aspergillus Oryzae.

[0056]

[0057] In one embodiment, the lysate may comprise a lysate of Pseudomonas Cepacia and / or a lysate of Rhizopus Oryzae.

[0058]

[0059] In addition, the cutinase may include a cutinase derived from one or more microorganisms selected from the group consisting of Humicola Insolens, Aspergillus Oryzae, Fusarium solani, and Pseudomonas putida.

[0060]

[0061] In one embodiment, the cutinase may comprise a cutinase from Humicola Insolens and / or a cutinase from Aspergillus Oryzae.

[0062]

[0063] In one embodiment, the lipase may comprise a lipase from Pseudomonas Cepacia, and the cutinase may comprise a cutinase from Humicola Insolens.

[0064]

[0065] When evaluating the biodegradability of biodegradable resins in soil and compost (industrial or domestic compost) environments, it is preferable to include the liphase of Pseudomonas Cepacia and the liphase of Rhizopus Oryzae. In addition, the liphase of Aspergillus niger and / or the liphase of Aspergillus Oryzae may be further included.

[0066]

[0067] When evaluating the biodegradability of biodegradable resins in soil and compost (industrial or domestic compost) environments, it is preferable to include cutinase from Humicola Insolens and cutinase from Aspergillus Oryzae. In addition, cutinase from Fusarium solani and / or cutinase from Pseudomonas putida may be further included.

[0068]

[0069] When evaluating the biodegradability of biodegradable resins in a marine environment, it is preferable to include the lysate of Pseudomonas Cepacia. In addition, the lysate of Aspergillus niger and / or the lysate of Aspergillus Oryzae may be further included.

[0070]

[0071] When evaluating the biodegradability of biodegradable resins in a marine environment, it is preferable to include cutinase from Humicola Insolens and cutinase from Aspergillus Oryzae. In addition, cutinase from Fusarium solani and / or cutinase from Pseudomonas putida may be further included.

[0072]

[0073] Meanwhile, the enzyme solution may further contain additional enzymes in addition to the lipase and cutinase. As such additional enzymes, one or more selected from the group consisting of α-amylase, cellulase, proteinase K, protease, acetyl-CoA carboxylase (ACCase), and alcalase may be used.

[0074]

[0075] Among the above additional enzymes, α-amylase can be applied to resins containing starch such as TPS and CMS, cellulase can be applied to resins containing cellulose such as CMC, and proteinase K, protease, and alkalise can be applied to resins containing polylactic acid.

[0076]

[0077] In one embodiment, the α-amylase may be α-amylase of Bacillus Licheniformis and / or α-amylase of Aspergillus oryzae; the cellulase may be cellulose of Trichoderma reesei and / or cellulase of Aspergillus sp.; the proteinase K may be proteinase K of Tritiracium album; the protease may be protease of Bacillus licheniformis; and the alkaline enzyme may be alkaline enzyme of Bacillus licheniformis. The above enzymes may all be used in simulating soil and compost environments or marine environments.

[0078]

[0079] In one embodiment, the enzyme solution for evaluating the biodegradability of a biodegradable resin in soil and compost (industrial or home compost) environments comprises a lipase of Pseudomonas Cepacia, a lipase of Rhizopus Oryzae, a cutinase of Humicola Insolens, a cutinase of Aspergillus Oryzae, and optionally further comprises an α-amylase of Bacillus Licheniformis.

[0080]

[0081] In one embodiment, the enzyme solution for evaluating the biodegradability of a biodegradable resin in a marine environment comprises a lipase from Pseudomonas Cepacia and a cutinase from Humicola Insolens, and optionally may further comprise a cutinase from Aspergillus Oryzae.

[0082]

[0083] The amount of each enzyme included in the above enzyme solution can be appropriately selected depending on the environmental conditions to be simulated and the biodegradable resin to be evaluated.

[0084]

[0085] However, it is preferable that the total amount of hydrolytic enzyme per 1 ml of the enzyme solution is 400 units or more, or 800 units or more, or 1,000 units or more, or 2,000 units or more, or 3,000 units or more, or 4,000 units or more, or 6,000 units or more, or 8,000 units or more, and 80,000 units or less, or 60,000 units or less, 40,000 units or less, or 30,000 units or less, or 20,000 units. If the amount of hydrolytic enzyme in the solution is too small, the decomposition of the biodegradable resin may be slow, making it difficult to quickly evaluate biodegradability. On the other hand, if the amount of hydrolytic enzyme in the solution is too large, the biodegradable resin decomposes too quickly, making it difficult to determine the relative difference, making it unsuitable for evaluating biodegradability.

[0086]

[0087] Specifically, when evaluating the biodegradability of biodegradable resins in soil and compost environments, cutinase may be 10 units or more, or 20 units or more, or 100 units or more, or 200 units or more, or 500 units or more, or 1,000 units or more, or 2,000 units or more, but not more than 40,000 units, or not more than 30,000 units, or not more than 20,000 units, or not more than 15,000 units, or not more than 10,000 units, or not more than 8,000 units, or not more than 4,000 units, and lipase may be 10 units or more, or 20 units or more, or 800 units or more, or 1,500 units or more, or 1,600 units or more, or 3,000 units or more, but not more than 40,000 units, or not more than 30,000 units, or It may be 20,000 units or less, or 15,000 units or less, or 10,000 units or less, or 8,000 units or less, or 4,000 units or less.

[0088]

[0089] In the above enzyme solution, the enzyme unit ratio of lipase:cutinase may be 0.5:1 or more, 0.8:1 or more, or 1:1 or more, and 4:1 or less, 3:1 or less, 2.5:1 or less, 2:1 or less, 1.8:1 or less, or 1.5:1 or less.

[0090]

[0091] When evaluating the biodegradability of biodegradable resins in soil and compost environments, the enzyme unit ratio of lipase:cutinase may be 0.5:1 or more, or 0.8:1 or more, but 2:1 or less, 1.8:1 or less, or 1.5:1 or less.

[0092]

[0093] Specifically, when evaluating the biodegradability of a biodegradable resin in a soil and compost environment, the amount of lipase per unit of cutinase may be 0.5 units or more, or 0.8 units or more, but 2 units or less, or 1.8 units or less, or 1.5 units or less.

[0094]

[0095] Meanwhile, when simulating soil and compost environments, if additional enzymes are included, α-amylase may be 100 units or more, or 200 units or more, but 3,000 units or less, or 1,500 units or less; cellulase may be 700 units or more, or 1,400 units or more, but 3,400 units or less, or 1,700 units or less; and proteinase K, protease, and alkalise may be 0.75 units or more, or 1.5 units or more, but 5 units or less, or 2.5 units or less, respectively.

[0096]

[0097] When evaluating the biodegradability of a biodegradable resin in a marine environment, cutinase may be 10 units or more, or 20 units or more, or 100 units or more, or 200 units or more, or 500 units or more, or 1,000 units or more, or 2,000 units or more, but not more than 40,000 units, or not more than 20,000 units, or not more than 16,000 units, or not more than 10,000 units, or not more than 8,000 units, or not more than 6,000 units, or not more than 3,000 units, and lipase may be 10 units or more, or 20 units or more, or 800 units or more, or 1,500 units or more, or 1,600 units or more, or 3,000 units or more, but not more than 40,000 units, or not more than 30,000 units, or not more than 20,000 units, or It may be 15,000 units or less, or 10,000 units or less, or 8,000 units or less, or 4,000 units or less.

[0098]

[0099] When evaluating the biodegradability of a biodegradable resin in a marine environment, the enzyme unit ratio of lipase:cutinase may be 0.5:1 or more, 0.8:1 or more, or 1:1 or more, but 4:1 or less, 3:1 or less, or 2.5:1 or less.

[0100]

[0101] Specifically, when evaluating the biodegradability of a biodegradable resin in a marine environment, the amount of lipase per unit of cutinase may be 0.5 units or more, 0.8 units or more, or 1 unit or more, or 1.5 units or more, but 4 units or less, or 3 units or less, or 2.5 units or less.

[0102]

[0103] When simulating a marine environment, when including additional enzymes, α-amylase may be 100 units or more, or 200 units or more, but 3,000 units or less, or 1,500 units or less; cellulase may be 700 units or more, or 1,400 units or more, but 3,400 units or less, or 1,700 units or less; proteinase K, protease, and alkalise may be 0.75 units or more, or 1.5 units or more, but 5 units or less, or 2.5 units or less, respectively.

[0104]

[0105] The unit “unit” of the above hydrolase is the enzyme unit or international unit of enzyme activity, also expressed as U or IU. 1 unit of enzyme refers to the amount of enzyme that converts 1 μmol of substrate into product per minute under the enzyme’s optimal reaction conditions (temperature, pH, substrate concentration).

[0106]

[0107] The above reaction optimal conditions vary depending on the enzyme. For example, the reaction optimal temperatures for the above-mentioned lipase, cutinase, and additional enzymes range from 25°C to 58°C. The pH ranges from 4 to 9, and may vary depending on conditions (e.g., 4 to 9 for soil conditions, 7.9 to 8.2 for marine conditions, and 6 to 9 for compost conditions).

[0108]

[0109] Method for evaluating the biodegradability of biodegradable resins

[0110]

[0111] According to one embodiment of the present invention, a method for evaluating the biodegradability of a biodegradable resin is provided, comprising the steps of: i) contacting a biodegradable resin with an enzyme solution containing a hydrolytic enzyme including lipase and cutinase; and phosphate buffered saline; and ii) observing a degradation pattern of the biodegradable resin over time.

[0112]

[0113] Biodegradation of biodegradable resins involves the hydrolysis of polymers into oligomers or monomers, which are then metabolized by microorganisms. Therefore, the faster the initial hydrolysis step, the faster biodegradation occurs.

[0114]

[0115] The evaluation method of the present invention is based on this point, and according to the evaluation method, the relative biodegradability of a biodegradable resin can be easily and quickly evaluated by measuring the hydrolysis rate of the biodegradable resin using an enzyme solution containing a hydrolytic enzyme including lipase and cutinase.

[0116]

[0117] Furthermore, by varying the composition of the enzymes contained in the enzyme solution, various environments, such as soil, compost, and the ocean, can be simulated. Accordingly, the biodegradability assessment method described above allows for quick and easy prediction of the relative biodegradability of biodegradable resins when exposed to various natural environmental conditions.

[0118]

[0119] The method for evaluating the biodegradability of a biodegradable resin according to one embodiment of the present invention can be applied to various resins requiring biodegradability evaluation. Examples of biodegradable resins that can be evaluated include, but are not limited to, polylactide (PLA), poly(butylene adipate-co-terephthalate) (PBAT), polybutylene succinate (PBS), polycaprolactone (PCL), polyhydroxybutyric acid (PHB), thermoplastic starch, etc., and the method for evaluating the biodegradability of the present invention can be applied to all resins expected to have biodegradability to easily and quickly predict the relative biodegradation rate in a natural environment.

[0120]

[0121] According to the above biodegradability evaluation method, the biodegradability of a resin can be evaluated within a few hours to a few days. Therefore, the above biodegradability evaluation method can be useful in situations where a simple, short-term biodegradability test is required, such as during the development stage of a biodegradable resin.

[0122]

[0123] The temperature and pressure conditions of the biodegradability evaluation method of the biodegradable resin may be appropriately selected depending on the natural environmental conditions to be simulated. For example, the biodegradability evaluation method of the biodegradable resin of the present invention may be performed under atmospheric pressure (760±20 torr) at a temperature range of 20°C to 30°C, or 30°C to 40°C, or 40°C to 60°C.

[0124]

[0125] The composition of the enzyme solution used in the biodegradability evaluation method of the above biodegradable resin is as described above.

[0126]

[0127] The method for evaluating the biodegradability of the above biodegradable resin can be specifically performed as in Method 1 to Method 3 below.

[0128]

[0129] a. Method 1

[0130]

[0131] In method 1, step i) can be performed by dropping the enzyme solution onto a resin test piece containing a biodegradable resin.

[0132]

[0133] Additionally, the above step ii) can be performed by measuring the change in the decomposition area of ​​the resin test piece over time.

[0134]

[0135] Specifically, method 1 may include the steps of preparing an enzyme solution containing lipase and cutinase; dropping the enzyme solution onto a resin test piece containing a biodegradable resin; and measuring a change in the decomposition area of ​​the resin test piece over time.

[0136]

[0137] The above resin test piece contains a biodegradable resin for which biodegradability measurement is required, and the shape is not limited, but a two-dimensional plate-shaped specimen such as a film or sheet is preferable because it allows for easy identification of the decomposition pattern after dripping of an enzyme solution.

[0138]

[0139] The above resin test piece may be composed only of a biodegradable resin or biodegradable resin compound for which biodegradability measurement is required.

[0140]

[0141] The size of the above film or sheet-shaped resin test piece is not limited, but for example, it may be preferable that the width and length be 5 cm or more, or 10 cm or more, and 20 cm or less.

[0142]

[0143] The above film or sheet-shaped resin test piece may preferably have a thickness of 250 μm or less, or 100 μm or less, and 20 μm or more, or 50 μm or more.

[0144]

[0145] After preparing the enzyme solution and resin test piece as described above, the enzyme solution is dripped onto the resin test piece, and the decomposition pattern of the resin test piece is observed over time to evaluate the biodegradability.

[0146]

[0147] The method for dripping the enzyme solution onto the resin test piece is not particularly limited, but it is preferable to drip at least one point on the surface of the test piece using a pipette or the like, and to set the amount of enzyme solution dripped onto each point to 3 to 5 μl. At this time, in order to reduce errors in biodegradability evaluation, the number of points for dripping the enzyme solution may be two or more.

[0148]

[0149] The point where the enzyme solution is dripped should be at least 20 mm away from the edge of the test piece, and when dripping at two or more points, the distance between each point should be at least 10 mm to prevent the collapse points created by the decomposition of the resin during the evaluation process from overlapping each other.

[0150]

[0151] The biodegradability of a biodegradable resin is evaluated by measuring the collapse area generated on the test piece over time immediately after the enzyme solution is dripped onto the test piece as described above.

[0152]

[0153] Generally, resins with a faster hydrolysis rate exhibit higher biodegradability, and the rapid creation and expansion of the collapse area occurs during the biodegradability evaluation. Therefore, by simultaneously testing two or more biodegradable resins using the above method, the relative biodegradability of each resin in a natural environment can be predicted.

[0154]

[0155] The temperature and pressure conditions for measuring the droplet and decomposition area of ​​the enzyme solution may be appropriately selected depending on the natural environmental conditions to be simulated. For example, the method for evaluating the biodegradability of the biodegradable resin of the present invention may be performed under atmospheric pressure (760±20 torr) at a temperature ranging from 20°C to 60°C, for example, from 20°C to 30°C, or from 30°C to 40°C, or from 40°C to 60°C.

[0156]

[0157] According to the above-described method for evaluating the biodegradability of a biodegradable resin, the biodegradability of the resin can be evaluated within several hours to several days. The evaluation time may vary depending on the size and weight of the biodegradable resin test piece, the composition of the enzyme solution, etc., but does not exceed 14 days.

[0158]

[0159] b. Method 2

[0160]

[0161] In method 2, step i) can be performed by immersing a resin test piece containing a biodegradable resin in the enzyme solution.

[0162]

[0163] Specifically, method 2 may include the steps of preparing an enzyme solution containing lipase and cutinase; immersing a resin test piece containing a biodegradable resin in the enzyme solution; and observing the decomposition pattern of the immersed resin test piece over time.

[0164]

[0165] The above resin test piece contains a biodegradable resin for which biodegradability measurement is required. The form is not limited, and various forms such as film and sheet forms can be used. Among these, the film form is more preferable because the decomposition pattern can be quickly determined when immersed in a hydrolysis solution.

[0166]

[0167] In this respect, it may be preferable that the film-shaped test piece has a thickness of 250 μm or less, or 100 μm or less, and 20 μm or more, or 50 μm or more.

[0168]

[0169] After preparing the enzyme solution and resin test piece as described above, the resin test piece is immersed in the enzyme solution, and the decomposition pattern of the immersed resin test piece is observed over time to evaluate the biodegradability.

[0170]

[0171] The amount of the above enzyme solution used is not limited, and is used in an amount that allows the resin test piece to be completely immersed.

[0172]

[0173] That is, the amount of enzyme solution used can be appropriately adjusted depending on the concentration of the enzyme solution and the size and / or weight of the resin test piece. For example, when using an enzyme solution containing 4,000 to 40,000 units of hydrolytic enzyme per ml, a 10 x 10 mm resin test piece with a thickness of 20 to 250 μm is used. 2The enzyme solution may be used in an amount of 100 μl or more, or 250 μl or more, and 1000 μl or less, or 750 μl or less, or 500 μl or less. However, the amount of the enzyme solution used is not limited thereto, and an appropriate amount is selected and used depending on the concentration of the hydrolytic enzyme in the hydrolytic enzyme, or the shape, size, weight, etc. of the resin test piece.

[0174]

[0175] As the biodegradable resin specimen is hydrolyzed over time in the enzyme solution, the weight of the specimen gradually decreases, and the specimen disintegrates into multiple pieces, which can be visually observed. Therefore, the above-mentioned "observation of the decomposition pattern" can be performed in various ways, such as measuring the weight change of the resin specimen at regular intervals or visually observing the disintegration of the resin specimen.

[0176]

[0177] By simultaneously testing two or more biodegradable resins using the above method, the relative biodegradability of each resin in a natural environment can be predicted. Generally, resins with faster hydrolysis rates also exhibit higher biodegradability. Therefore, comparing the hydrolysis rates of two or more biodegradable resins allows for a relative assessment of their biodegradability.

[0178]

[0179] The temperature and pressure conditions of the step of immersing the resin specimen in the enzyme solution and the step of observing the decomposition pattern of the resin specimen can be appropriately selected according to the natural environmental conditions to be simulated. For example, the method for evaluating the biodegradability of the biodegradable resin of the present invention can be performed at a temperature range of 20°C to 60°C, for example, 20°C to 30°C, or 30°C to 40°C, or 40°C to 60°C, under atmospheric pressure (760±20 torr).

[0180]

[0181] According to the above-described method for evaluating the biodegradability of a biodegradable resin, the biodegradability of the resin can be evaluated within several hours to several days. The evaluation time may vary depending on the size and weight of the biodegradable resin test piece, the composition of the enzyme solution, etc., but does not exceed 14 days.

[0182]

[0183] c. Method 3

[0184]

[0185] In method 3, step i) is performed by spraying a biodegradable resin solution on a medium to prepare a test piece, and then dropping the enzyme solution onto the test piece.

[0186] The above step ii) can be performed by observing the change in the clear zone formed on the test piece over time.

[0187]

[0188] Specifically, method 3 may include the steps of preparing a test piece by spraying a biodegradable resin solution on a medium; the step of dripping an enzyme solution containing lipase and cutinase onto the test piece; and the step of measuring a change in a clear zone formed on the test piece over time.

[0189]

[0190] The above medium is a means for supporting a resin solution containing a biodegradable resin and providing a wet environment in which an enzyme can function, and a material that is not reactive to the biodegradable resin and enzyme solution is preferable.

[0191]

[0192] The material of the medium may be selected depending on the type of biodegradable resin and the composition of the enzyme contained in the enzyme solution. For example, the medium may be a solid medium containing agar and / or gelatin, but is not limited thereto.

[0193]

[0194] Unlike a medium for microbial culture, the above medium may be composed only of agar and / or gelatin and distilled water, without additives such as sugars or peptides.

[0195]

[0196] It is desirable to sterilize these media before use. For example, the media can be sterilized by treating them under conditions of a temperature of 100°C to 150°C and a pressure of 0.05 MPa to 0.3 MPa for 10 to 60 minutes. However, the sterilization method of the media is not limited to this, and a known sterilization method for each material can be appropriately selected.

[0197]

[0198] The biodegradable resin solution is composed of a biodegradable resin, which is the target for evaluating biodegradability, and a solvent.

[0199]

[0200] The above solvent is not particularly limited as long as it can uniformly disperse the biodegradable resin, and for example, one or more selected from the group consisting of chloroform, methyl chloride, carbon tetrachloride, benzene, dimethyl sulfoxide, dimethylformamide, toluene, and xylene may be used.

[0201]

[0202] The content (solid content) of the biodegradable resin in the biodegradable resin solution may be in the range of 0.5 wt% or more, or 1 wt% or more, and 3 wt% or less, or 2 wt% or less. When the above range is satisfied, droplets of the biodegradable resin solution can be uniformly applied on the medium at an appropriate concentration, thereby creating an environment favorable for the action of the hydrolytic enzyme, which is preferable.

[0203]

[0204] The method for spraying the biodegradable resin solution is not particularly limited, and any known spraying method for polymer solutions can be appropriately selected. For example, spraying devices such as sprayers, sprayers, airbrushes, and electrospray devices can be used.

[0205]

[0206] After spraying the above biodegradable resin solution onto the medium, the medium is dried so that all solvent in the biodegradable resin solution can evaporate. If the biodegradability evaluation is conducted without the solvent evaporating, the enzyme activity may be greatly reduced, making it difficult to conduct a smooth evaluation. Therefore, the temperature, pressure, and time are adjusted for drying in consideration of the solvent used, the type of biodegradable resin, the characteristics of the medium, etc. For example, when chloroform is used as the solvent, the medium onto which the biodegradable resin solution has been sprayed can be dried by leaving it at room temperature (25°C) and atmospheric pressure (0.1 MPa) for 5 to 20 minutes, preferably 10 to 20 minutes.

[0207]

[0208] The spraying of the biodegradable resin solution may be performed such that the average thickness of the biodegradable resin applied to the substrate is 0.6 μm or more, or 0.8 μm or more, and 1.5 μm or less. When the biodegradable resin is applied to an appropriate thickness as described above, it is suitable for observing changes over time after dripping the enzyme solution.

[0209]

[0210] Next, the biodegradability evaluation is initiated by dropping an enzyme solution containing lipase and cutinase as hydrolytic enzymes onto a medium (test piece) onto which a biodegradable resin solution has been sprayed.

[0211]

[0212] At this time, various environments, such as soil environments, compost environments, and marine environments, can be simulated by adjusting the type and / or content of hydrolytic enzymes contained in the enzyme solution. The enzyme composition of the enzyme solution to simulate each condition is as described above.

[0213]

[0214] The method for dripping the enzyme solution onto the medium (test piece) onto which the biodegradable resin solution has been sprayed is not particularly limited, but it is preferable to drip the enzyme solution onto at least one point on the surface of the test piece (the surface onto which the biodegradable resin solution has been applied) using a pipette or the like, and the amount of enzyme solution dripped onto each point is 3 μl to 5 μl. At this time, in order to reduce the error in evaluating the biodegradability, the number of points onto which the enzyme solution is dripped may be 2 or more.

[0215]

[0216] The position at which the enzyme solution is dripped on the test piece is not particularly limited, but it is preferably at least 20 mm away from the edge of the test piece, and when dripping at two or more points, the distance between each point should be at least 20 mm to prevent the clear zones created by the decomposition of the resin during the evaluation process from overlapping. Depending on the sample, a faint clear zone may be formed widely. When using such a sample, the enzyme solution is dripped at least 40 mm away from the edge of the test piece, and when dripping at two or more points, the distance between each point should be at least 40 mm to prevent the clear zones from overlapping. In this way, the dripping points of the enzyme solution on the test piece can be determined in consideration of the characteristics of the sample.

[0217]

[0218] The above clear zone is a transparent area created when a water-insoluble biodegradable resin is broken down into water-soluble molecules by a hydrolytic enzyme. The biodegradability of the biodegradable resin is assessed by measuring the change in the area of ​​the clear zone or the degree to which the clear zone becomes transparent over time, starting immediately after the enzyme solution is applied to the test piece, as described above.

[0219]

[0220] Generally, resins with a faster hydrolysis rate exhibit higher biodegradability, and the formation and expansion of clear zones occur more rapidly during biodegradability evaluation. Therefore, by simultaneously testing two or more biodegradable resins using the above method, the relative biodegradability of each resin in a natural environment can be predicted.

[0221]

[0222] The temperature and pressure conditions for measuring the droplet and decomposition area of ​​the enzyme solution may be appropriately selected depending on the natural environmental conditions to be simulated. For example, the method for evaluating the biodegradability of the biodegradable resin of the present invention may be performed under atmospheric pressure (760±20 torr) at a temperature range of 20°C to 30°C, or 30°C to 40°C, or 40°C to 60°C.

[0223]

[0224] According to the above-described method for evaluating the biodegradability of a biodegradable resin, the biodegradability of the resin can be evaluated within several hours to several days. The evaluation time may vary depending on the solids content of the biodegradable resin solution, the composition of the enzyme solution, etc., but does not exceed seven days.

[0225]

[0226] d. Image analysis of biodegradable resin specimens

[0227]

[0228] Meanwhile, in the method for evaluating the biodegradability of the biodegradable resin, ii) the step of observing the decomposition pattern of the biodegradable resin over time may include a) a step of obtaining an image of the resin test piece over time; and b) a step of evaluating the biodegradability through changes in the image of the resin test piece over time.

[0229]

[0230] First, images of the biodegradable resin specimen in contact with the enzyme solution are acquired over a specified period of time (unit of time). The unit of time may be, for example, several seconds, several minutes, or several hours, and can be set differently depending on the composition of the specimen or hydrolytic enzyme.

[0231]

[0232] If using Method 1 or 3, it may be desirable to conduct the experiment in step a) under conditions that prevent evaporation of the dripped enzyme solution. When capturing images, it is desirable to maintain the same color balance, illumination, shutter speed, lens brightness, and other shooting conditions to increase the accuracy of the analysis.

[0233]

[0234] At this time, the image can be measured using real-time image measurement equipment, for example, NanoEntek's JuLI TM Equipment such as a stage may be used, but the present invention is not necessarily limited thereto.

[0235]

[0236] Next, biodegradability is assessed by examining changes in the image of the resin specimen over time. Over time, the resin specimen exposed to the enzyme solution undergoes hydrolysis by hydrolytic enzymes, resulting in changes in its appearance.

[0237]

[0238] For example, in the area where hydrolysis occurs, the resin thickness of the test piece may become thinner, and a region (clear zone) that is brighter in appearance may be formed compared to the area where hydrolysis does not occur.

[0239]

[0240] Therefore, by measuring and analyzing the changes in this bright area, we can determine the degree of biodegradation of the resin in the specimen.

[0241]

[0242] However, in order to analyze this accurately, it is necessary to quantify and standardize the shape of the bright area.

[0243]

[0244] According to an example, the step b) may include step b-1) of obtaining the difference in brightness between each image of the resin test piece obtained over a unit of time and the image immediately after the hydrolytic enzyme is applied; step b-2) of expressing the image brightness difference value as a function of the unit of time; and step b-3) of evaluating the biodegradability through the function analysis.

[0245]

[0246] That is, in the obtained image, the brightness of the area where the hydrolytic enzyme was dripped is measured, and the change in brightness over time is confirmed. Then, the difference between the brightness measured at a specific elapsed time and the brightness immediately after the hydrolytic enzyme was dripped is calculated.

[0247]

[0248] Comparing this brightness difference as a function of time allows us to determine the degree of brightness change over time. Furthermore, depending on the nature of the brightness change, a specific point in time, for example, the time at which the second derivative of the function reaches its maximum, can be assumed to be the time at which the biodegradable resin's biodegradation acceleration reaches its maximum value, and also the time at which the clear zone begins to form.

[0249]

[0250] The time at which the second derivative of the above function is maximum is a kind of inflection point in the brightness function according to elapsed time, and is the point at which the brightness change rate changes, which can be seen as the point at which the biodegradation rate changes. Accordingly, if the 'elapsed time' at which the 'second derivative of the above function is maximum' is relatively fast, the biodegradation rate can be assumed to be relatively fast, and if the 'elapsed time' at which the 'second derivative of the above function is maximum' is relatively slow, the biodegradation rate can be assumed to be relatively slow, and accordingly, the biodegradability of the resin can be evaluated.

[0251]

[0252] Hereinafter, embodiments of the present invention will be described in more detail in the following examples. However, the following examples are merely illustrative of embodiments of the present invention, and the content of the present invention is not limited by the following examples.

[0253]

[0254] [Example]

[0255] Example A-1: ​​Evaluation of biodegradability in soil / compost environment

[0256] (1) Preparation of test specimens

[0257] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded under conditions of a barrel temperature of 180°C to 200°C, a feed rate of 30 to 50 kg / hr, and 300 rpm, thereby manufacturing a resin composition in the form of pellets.

[0258] Afterwards, a blown film was manufactured by molding the pellet-shaped resin composition into a thickness of 0.05 mm at an extrusion temperature of 170°C using a single screw extruder (Blown Film M / C, 19 pi, L / D=25). At this time, the blown-up ratio was 1.8 and the line speed was 5 m / min.

[0259] Afterwards, it was cut into a film shape with a width*length = 5 cm*5 cm and a thickness of 0.05 mm, and then placed in a fixed frame to be used as a test piece.

[0260]

[0261] Test specimens of each of the following polymer compounds were prepared using the above method.

[0262] a. Poly(butylene adipate-co-terephthalate) compound 1 (Manufacturer: LG Chem, Raw materials: PBAT and PLA)

[0263] b. Poly(butylene adipate-co-terephthalate) compound 2 (Manufacturer: LG Chem, Raw materials: PBAT, PLA, and additives)

[0264]

[0265] (2) Preparation of enzyme solution

[0266] An enzyme solution was prepared by mixing the following two types of lipase and two types of cutinase in 1 ml of 1X Phosphate-Buffered Saline (PBS).

[0267] LipaseRhizopus Oryzae10,000 units

[0268] LipasePseudomonas Cepacia10,000 units

[0269] CutinaseAspergillus Oryzae10,000 units

[0270] CutinaseHumicola Insolens10,000 units

[0271]

[0272] The above PBS is a mixture of ultrapure grade phosphate buffer and saline solution adjusted to pH 7.4, containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na2HPO4, and 2 mM KH2PO4, and PBS of the same composition was used in all the examples below.

[0273]

[0274] (3) Biodegradability evaluation

[0275] Biodegradability evaluation was performed under the conditions of 58 ℃ and 760 mmHg using the following method.

[0276] 5 μl of the enzyme solution of (2) was dropped onto the test piece prepared in (1) above. After 27 hours and 45 hours, the size of the lost area resulting from the decomposition of the test piece by the enzyme solution was measured (Fig. 1).

[0277]

[0278] (4) Results Analysis

[0279] Figure 2 shows the results of a biodegradability test under industrial compost conditions of poly(butylene adipate-co-terephthalate) compound 1 (PBAT C1), poly(butylene adipate-co-terephthalate) compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697), measured according to the ISO 14855-1 standard. Referring to Figure 2, it can be confirmed that the biodegradability of PBAT C1 without additives is higher than that of PBAT C2.

[0280]

[0281] Figure 1 shows the results of the biodegradability evaluation of (3) above. The first row is a photograph taken 27 hours after the enzyme solution was dripped onto the test piece (27 HPI) and 45 hours after the enzyme solution was dripped onto the test piece (47 HPI). The second row of Figure 1 is a photograph showing only the lost portion of the first row of photographs in order to confirm the area of ​​the test piece lost by the hydrolytic enzyme, and the lost area is written below it.

[0282]

[0283] Referring to Fig. 1, it can be confirmed that the biodegradability of PBAT C1 without additives is higher than that of PBAT C2, which is consistent with the results of Fig. 2 above.

[0284]

[0285] Example A-2: Evaluation of biodegradability in a marine environment

[0286] (1) Preparation of test specimens

[0287] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded under conditions of a barrel temperature of 180°C to 200°C, a feed rate of 30 to 50 kg / hr, and 300 rpm, thereby manufacturing a resin composition in the form of pellets.

[0288] Afterwards, a blown film was manufactured by molding the pellet-shaped resin composition to a thickness of 0.05 mm at an extrusion temperature of 170°C using a single screw extruder (Blown Film M / C, 19 pi, L / D=25). At this time, the blown-up ratio was 1.8 and the line speed was 5 m / min.

[0289] Afterwards, a film-type test piece was manufactured by cutting it into a width x length = 1 cm x 1 cm and a thickness of 0.05 mm.

[0290]

[0291] Test specimens for each of the following polymers were prepared using the above method.

[0292] a. Poly(butylene succinate) (Manufacturer: PTT MCC, Raw material: PBS)

[0293] b. Poly(butylene succinate adipate) (Manufacturer: LG Chem, Raw material: PBSA)

[0294]

[0295] (2) Preparation of enzyme solution

[0296] An enzyme solution was prepared by mixing one type of lipase and two types of cutinase in 1 ml of 1X Phosphate-Buffered Saline (PBS) and 36 mg of sea salt.

[0297] LipasePseudomonas Cepacia10,000 units

[0298] CutinaseAspergillus Oryzae10,000 units

[0299] CutinaseHumicola Insolens10,000 units

[0300]

[0301] (3) Biodegradability evaluation

[0302] Biodegradability evaluation was performed under the conditions of 30 ℃ and 760 mmHg using the following method.

[0303] 5 μl of the enzyme solution of (2) was dropped onto the test piece prepared in (1) above. After 7 days, the size of the lost area resulting from the decomposition of the test piece by the enzyme solution was measured (Fig. 3).

[0304]

[0305] (4) Results Analysis

[0306] Figure 3 shows the results of the biodegradability evaluation of (3) above. In Figure 3, DPI (days post-inoculation) refers to the number of days elapsed, and 10 DPI means that the photo was taken 10 days after the enzyme solution was dripped onto the test piece.

[0307]

[0308] Table 1 and Fig. 4 below show the results of biodegradability tests under marine conditions, measured according to the ASTM D6691 standard, for the polymers a and b. above. The tests were repeated twice for each specimen, and the number of test runs is indicated as “n” after each legend. That is, “Cellulose1” and “Cellulose2” are the results of tests conducted on the same cellulose, respectively, with “Cellulose1” indicating the results of the first test, and “Cellulose2” indicating the results of the second test. The numbers in Table 1 indicate the degradation rate (%) of the corresponding resin after each period of time.

[0309]

[0310]

[0311]

[0312] The first row of Fig. 3 shows photographs taken 10 days after the enzyme solution was dripped onto the test piece (10 DPI). The second row of Fig. 3 shows photographs that show only the lost portion of the first row of photographs to confirm the area of ​​the test piece lost due to the hydrolytic enzyme, and the lost area is indicated below it.

[0313]

[0314] Referring to Fig. 3, it can be confirmed that the biodegradability of PBSA is higher than that of PBS, which is consistent with the results of Fig. 4 above.

[0315]

[0316] Example B-1: Evaluation of biodegradability in soil / compost environment

[0317] (1) Preparation of test specimens

[0318] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded under conditions of a barrel temperature of 180°C to 200°C, a feed rate of 30 to 50 kg / hr, and 300 rpm, thereby manufacturing a resin composition in the form of pellets.

[0319] Afterwards, a blown film was manufactured by molding the pellet-shaped resin composition to a thickness of 0.05 mm at an extrusion temperature of 170°C using a single screw extruder (Blown Film M / C, 19 pi, L / D=25). At this time, the blown-up ratio was 1.8 and the line speed was 5 m / min.

[0320] Afterwards, a film-type test piece was manufactured by cutting it into a width x length = 1 cm x 1 cm and a thickness of 0.05 mm.

[0321]

[0322] Test specimens of each of the following polymer compounds were prepared using the above method.

[0323] a. Poly(butylene adipate-co-terephthalate) / Thermoplastic Starch Compound (LG Chem, PBAT / TPS Comp'd)

[0324] b. Poly(butylene adipate-co-terephthalate) / polylactic acid compound (LG Chem, PBAT / PLA Comp'd)

[0325]

[0326] (2) Preparation of enzyme solution

[0327] An enzyme solution was prepared by mixing the following two types of lipase, two types of cutinase, and one type of alpha amylase in 1 ml of 1X Phosphate-Buffered Saline (PBS).

[0328] LipaseRhizopus Oryzae1000 units

[0329] LipasePseudomonas Cepacia3000 units

[0330] CutinaseAspergillus Oryzae2000 units

[0331] CutinaseHumicola Insolens1500 units

[0332] α-amylaseBacillus Licheniformis1500 units

[0333]

[0334] (3) Biodegradability evaluation

[0335] Biodegradability evaluation was performed under the conditions of 37 ℃ and 760 mmHg using the following method.

[0336] 0.5 ml of the enzyme solution of (2) above was added to one well of a 24-well plate, and two test pieces of (1) above were immersed. Thereafter, the test pieces were visually observed every 1 to 3 days to determine whether they were disintegrating and falling off into pieces.

[0337]

[0338] (4) Results Analysis

[0339] Figure 5 shows the results of the biodegradability evaluation of (3) above. In Figure 5, DPI (days post-inoculation) refers to the number of days elapsed, with 0 DPI being a photo taken immediately after the test piece was immersed in the enzyme solution, and 2 DPI being a photo taken two days after immersion.

[0340]

[0341] Table 2 and Fig. 6 below show the results of biodegradability tests under soil conditions, measured according to the ISO 17556 standard, for the polymer compounds a and b. above. The tests were repeated twice for each specimen, and the test number is indicated as “_n” after each legend. That is, “Cellulose_1” and “Cellulose_2” are the results of tests conducted on the same cellulose, respectively, with “Cellulose_1” indicating the result of the first test, and “Cellulose_2” indicating the result of the second test. The numbers in Table 2 indicate the decomposition rate (%) of the corresponding resin after each date.

[0342]

[0343]

[0344]

[0345]

[0346] Referring to Fig. 5, a. PBAT / TPS compound began to collapse with small holes formed after 4 days of immersion, and it can be seen that the edges of the test piece were significantly collapsed on the 7th day. On the other hand, b. PBAT / PLA compound did not show any noticeable collapse of the test piece even after 7 days of immersion, confirming that a. PBAT / TPS compound has a relatively high biodegradability compared to b. PBAT / PLA compound, which is also consistent with the results of Table 2 and Fig. 6 measured according to the standard test method.

[0347]

[0348] Example B-2: Evaluation of biodegradability in a marine environment

[0349] (1) Preparation of test specimens

[0350] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded under conditions of a barrel temperature of 180°C to 200°C, a feed rate of 30 to 50 kg / hr, and 300 rpm, thereby manufacturing a resin composition in the form of pellets.

[0351] Afterwards, a blown film was manufactured by molding the pellet-shaped resin composition to a thickness of 0.05 mm at an extrusion temperature of 170°C using a single screw extruder (Blown Film M / C, 19 pi, L / D=25). At this time, the blown-up ratio was 1.8 and the line speed was 5 m / min.

[0352] Afterwards, a film-type test piece was manufactured by cutting it into a width x length = 1 cm x 1 cm and a thickness of 0.05 mm.

[0353]

[0354] Test specimens for each of the following polymers were prepared using the above method.

[0355] a. Poly(butylene succinate) (Manufacturer: PTT MCC, Raw material: PBS)

[0356] b. Poly(butylene succinate adipate) (Manufacturer: LG Chem, Raw material: PBSA)

[0357]

[0358] (2) Preparation of enzyme solution

[0359] An enzyme solution was prepared by mixing one type of lipase and two types of cutinase in 1 ml of 1X Phosphate-Buffered Saline (PBS) and 36 mg of sea salt.

[0360] LipasePseudomonas Cepacia10,000 units

[0361] CutinaseAspergillus Oryzae10,000 units

[0362] CutinaseHumicola Insolens10,000 units

[0363]

[0364] (3) Biodegradability evaluation

[0365] Biodegradability evaluation was performed under the conditions of 30 ℃ and 760 mmHg using the following method.

[0366] 0.5 ml of the enzyme solution of (2) above was added to one well of a 24-well plate, and two test pieces of (1) above were immersed. After 7 days, the test pieces were observed to disintegrate and fall off with the naked eye.

[0367]

[0368] (4) Results Analysis

[0369] Figure 7 shows the results of the biodegradability evaluation of (3) above. In Figure 7, DPI (day post-inoculation) refers to the number of days elapsed, with 0 DPI being a photo taken immediately after the test piece was immersed in the enzyme solution, and 7 DPI being a photo taken 7 days after immersion.

[0370]

[0371] Referring to Fig. 7, b. Polybutylene succinate adipate (PBSA) can be confirmed to have significantly disintegrated the test piece after 7 days of immersion. On the other hand, a. Polybutylene succinate (PBA) did not disintegrate significantly in the test piece, confirming that b. Polybutylene succinate adipate (PBSA) has a relatively higher biodegradability than a. Polybutylene succinate (PBA). This is also consistent with the results of Table 1 and Fig. 4 measured according to the standard test method.

[0372]

[0373] Example C-1, Comparative Example C-1, and Comparative Example C-2: Evaluation of biodegradability in soil / compost environment

[0374] (1) Manufacturing of badges

[0375] A circular 1% agar plate with a diameter of 90 mm was prepared using a method for preparing a petri dish medium.

[0376] A solution was prepared by adding 10 g of agar (manufacturer: BD Difco, product name: Bacto Agar, product number: 214010) and 1000 ml of distilled water to a 2000 ml Erlenmeyer flask. The solution was placed in an autoclave and sterilized at 121°C and 0.1 MPa for 15 minutes. 10 ml of the sterilized solution was dispensed into sterile round petri dishes with a diameter of 90 mm to prepare a medium.

[0377]

[0378] (2) Preparation of resin solution

[0379] 1) Preparation of PBAT compound 1 solution

[0380] PBAT compound 1 was prepared by mixing 79.2 wt% poly-butylene adipate-co-terephthalate (PBAT, LG Chem Co.); 8.8 wt% polylactic acid (PLA, TotalEnergies Corbion Co.); and 12 wt% calcium carbonate (CaCO3). Each of the above resins was used after being sterilized with 70% ethanol.

[0381] The above PBAT compound 1 was dissolved in 20 ml of chloroform at 1 wt% to prepare a PBAT compound 1 solution.

[0382]

[0383] 2) Preparation of PBAT compound 2 solution

[0384] PBAT compound 2 was prepared by mixing 79.2 wt% of poly-butylene adipate-co-terephthalate (PBAT, LG Chem Co.); 8.8 wt% of meso-polylactic acid (meso-PLA, Bio Valore Co.); and 12 wt% of calcium carbonate (CaCO3). Each of the above resins was used after being sterilized with 70% ethanol.

[0385] The above PBAT compound 2 was dissolved in 20 ml of chloroform at 1 wt% to prepare a PBAT compound 2 solution.

[0386]

[0387] (3) Preparation of enzyme solution

[0388] Enzyme solutions (stock solutions) of Example C-1, Comparative Example C-1, and Comparative Example C-2 were prepared. In addition, a 1 / 10 dilution of the stock solution was prepared.

[0389]

[0390] <Example C-1>

[0391] 1X Phosphate-Buffered Saline (PBS) 1 ml

[0392] LipaseRhizopus Oryzae1000 units

[0393] LipasePseudomonas Cepacia3000 units

[0394] CutinaseAspergillus Oryzae2000 units

[0395] CutinaseHumicola Insolens1500 units

[0396]

[0397] <Comparative Example C-1>

[0398] 1X Phosphate-Buffered Saline (PBS) 1 ml

[0399] CutinaseHumicola Insolens1500 units

[0400]

[0401] <Comparative Example C-2>

[0402] 1X Phosphate-Buffered Saline (PBS) 1 ml

[0403] LipasePseudomonas Cepacia3000 units

[0404]

[0405] (4) Biodegradability evaluation

[0406] On the agar medium of the above (1), 6 ml of the PBAT compound 1 solution prepared in 1) of the above (2) was evenly sprayed and dried at room temperature (25°C) for about 10 minutes to allow the solvent to evaporate, thereby preparing a “PBAT compound 1” test piece having an average thickness of 0.7 μm of PBAT compound 1. The average thickness of the PBAT compound 1 was measured by measuring the roughness of the test piece before and after spraying the PBAT compound 1 solution using an optical profiler (model name: NewView TM 8300, Zygo corp.) was measured and obtained.

[0407] In the same manner, the PBAT compound 2 solution prepared in 2) of (2) was sprayed onto a separate agar medium and dried to prepare a “PBAT compound 2” test piece having an average thickness of 0.7 μm.

[0408] On each of the above test pieces, as shown in Fig. 8, the original enzyme solution and the 1 / 10 diluted solution of Example C-1 of (3) were dropped at four points, each in an amount of 5 μl. Thereafter, the area of ​​the clear zone created by the decomposition of the resin sprayed on the medium by the enzyme solution and the degree of clarity were visually measured over time.

[0409] The relative biodegradability of each test piece was evaluated using the same method as above for the enzyme solution of Comparative Example C-1 and the enzyme solution of Comparative Example C-2.

[0410] The entire process of the above biodegradability evaluation was performed at 28°C and atmospheric pressure (760±20 torr).

[0411]

[0412] (5) Results Analysis

[0413] Figure 9 shows the results of biodegradability evaluation in an industrial compost environment of PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697), measured according to ISO 14855 (Determination of the ultimate aerobic biodegradability of plastic materials under controlled composting conditions - Method by analysis of evolved carbon dioxide). Referring to Figure 9, it can be confirmed that PBAT compound 2 has a higher biodegradability than PBAT compound 1.

[0414]

[0415] Figure 10 is a photograph of a test piece taken 4 hours and 30 minutes after the hydrolytic enzyme was dripped, showing the results of the biodegradability evaluation of (4) above. Figure 10 was taken with a black screen placed under the test piece, and the black portion is the clear zone. The results of Example C-1 are indicated as (1), the results of Comparative Example C-1 as (2), and the results of Comparative Example C-2 as (3).

[0416]

[0417] Referring to (1) of Fig. 10, when the enzyme solution of Example C-1 was dripped and the same amount of time elapsed, it can be confirmed that the clear zone (black portion) of the test piece of PBAT compound 2, which has excellent biodegradability, appeared more distinctly than that of the test piece of PBAT compound 1.

[0418] However, referring to (2) and (3) of Fig. 10, it can be confirmed that no significant difference between PBAT compound 1 and PBAT compound 2 can be observed with the enzyme solution of Comparative Example C-1 containing only cutinase and the enzyme solution of Comparative Example C-2 containing only lipase.

[0419]

[0420] Accordingly, from the above experimental results, it can be confirmed that the method for evaluating the biodegradability of a biodegradable resin using the enzyme solution of the present invention can easily and reliably predict the relative biodegradability of a biodegradable resin in a soil / compost environment.

[0421]

[0422] Example C-2: Evaluation of biodegradability in a marine environment

[0423] (1) Manufacturing of badges

[0424] A medium was prepared using the same method as (1) of Example C-1.

[0425]

[0426] (2) Preparation of resin solution

[0427] 1) Preparation of P3HP 1 solution

[0428] Poly-3-hydroxypropionic acid (P3HP, LG Chem) having a weight average molecular weight (Mw) of 120,000 g / mol and disinfected with 70% ethanol was dissolved in 20 ml of chloroform at 1 wt% to prepare a P3HP 1 solution.

[0429]

[0430] 2) Preparation of P3HP 2 solution

[0431] P3HP (LG Chem) with a weight average molecular weight (Mw) of 70,000 g / mol, disinfected with 70% ethanol, was dissolved in 20 ml of chloroform at a concentration of 1 wt% to prepare a P3HP 2 solution.

[0432]

[0433] (3) Preparation of enzyme solution

[0434] An enzyme solution (stock solution) was prepared by mixing one type of lipase and one type of cutinase below in 1 ml of 1X Phosphate-Buffered Saline (PBS). In addition, a 1 / 10 dilution of the stock solution was prepared.

[0435] LipasePseudomonas Cepacia3000 units

[0436] CutinaseHumicola Insolens1500 units

[0437]

[0438] (4) Biodegradability evaluation

[0439] Using each resin solution of (2) and enzyme solution of (3), the biodegradability of P3HP 1 and P3HP 2 resins was evaluated using the same method as (4) of Example C-1.

[0440]

[0441] (5) Results Analysis

[0442] Figure 11 shows the results of biodegradability evaluation in a marine environment of P3HP 1, P3HP 2, and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) measured according to the ASTM D6691 standard. Referring to Figure 11, it can be confirmed that P3HP 2 has a higher biodegradability than P3HP 1.

[0443]

[0444] Figure 12 is a photograph of a test piece taken 18 hours after the hydrolytic enzyme was dripped, showing the results of the biodegradability evaluation of (4) above. After the same amount of time had elapsed, it can be confirmed that the clear zone of the test piece of P3HP 2, which has excellent biodegradability, is more distinct than that of the test piece of P3HP 1.

[0445] Accordingly, from the above experimental results, it can be confirmed that the method for evaluating the biodegradability of a biodegradable resin using the enzyme solution of the present invention can easily and reliably predict the relative biodegradability of a biodegradable resin in a marine environment.

[0446]

[0447] Example D-1: Biodegradability Evaluation and Image Analysis

[0448] (1) Manufacturing of badges

[0449] A circular 1% agar plate with a diameter of 90 mm was prepared using a method for preparing a petri dish medium.

[0450] A solution was prepared by adding 10 g of agar (manufacturer: BD Difco, product name: Bacto Agar, product number: 214010) and 1000 ml of distilled water to a 2000 ml Erlenmeyer flask. The solution was placed in an autoclave and sterilized at 121°C and 0.1 MPa for 15 minutes. 10 ml of the sterilized solution was dispensed into sterile round petri dishes with a diameter of 90 mm to prepare a medium.

[0451]

[0452] (2) Preparation of biodegradable resin solution

[0453] A resin solution was prepared by completely dissolving the biodegradable resin in 20 ml of chloroform at a concentration of 1 wt%.

[0454]

[0455] (3) Preparation of enzyme solution

[0456] An enzyme solution was prepared by mixing the following two types of lipase, two types of cutinase, and one type of alpha amylase in 1 ml of 1X Phosphate-Buffered Saline (PBS).

[0457] Lipase Rhizopus Oryzae 1000 units

[0458] Lipase Pseudomonas Cepacia 3000 units

[0459] Cutinase Aspergillus Oryzae 2000 units

[0460] Cutinase Humicola Insolens 1500 units

[0461] α-amylase Bacillus Licheniformis 1500 units

[0462]

[0463] (4) Biodegradability evaluation and image acquisition

[0464] On the agar medium of the above (1), 1 to 3 ml of the resin solution prepared in the above (2) was evenly sprayed and dried at room temperature (25°C) for about 10 minutes to allow the solvent to evaporate, thereby preparing a test piece with a thickness of 0.7 μm.

[0465] Seal the agar plate and use the JuLI image measurement / analysis equipment. TM It was mounted on a stage (NanoEntek product). The enzyme solution prepared in (3) above was dropped onto the biodegradable resin layer on the agar medium. JuLI TM Using the stage, real-time images were acquired at regular time intervals while maintaining constant image acquisition (shooting) conditions.

[0466] The measurement conditions are summarized in the table below.

[0467]

[0468] Sample No. Resin agar injection amount Resin injection amount Enzyme solution concentration (ratio) Interval 1 PBAT C 10.6ml 4ml 12 2 min 2-1 PBAT C 20.7ml 4ml 13 9 min 2-2 PBAT C 10.7ml 4ml 13 9 min 3-1 PBSA C 10.7ml 2ml 1 / 10 dilution 11 min 3-2 PBSA C 20.7ml 2ml 1 / 10 dilution 11 min 3-3 PBSA C 30.7ml 2ml 1 / 10 dilution 11 min 3-4 PBSA C 40.7ml 2ml 1 / 10 dilution 11 min

[0469] (5) Image analysis

[0470] The images obtained above were analyzed to determine the brightness of the image immediately after enzyme instillation and the brightness of the images captured after each elapsed time, and the average value of the brightness difference was plotted against the elapsed time. The curve obtained through plotting was fitted to a sigmoid function, and the elapsed time at which the second derivative of the function reached a maximum was determined.

[0471] Figure 13 is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the invention.

[0472] Referring to Figure 13, among the areas where biodegradable resin is located, an “observation area” can be confirmed in which the white biodegradable resin is hydrolyzed in the center of the area and appears brighter than the surrounding area, and an “exclusion area” can be confirmed in which other residues generated in the process of dripping an enzyme onto the resin, not formed by hydrolysis through the reaction of an enzyme and the resin, interact with the resin and appear as a dark background color.

[0473]

[0474] That is, depending on the degree of hydrolysis progress, the brightness of the biodegradable resin area becomes brighter, and when hydrolysis passes a certain point, an "exclusion zone" is created where it becomes darker. Therefore, the exclusion zone needs to be excluded from the evaluation for accurate evaluation, and in addition, if the brightness is exceptionally large compared to the surrounding area, it also needs to be excluded from the evaluation.

[0475]

[0476] Figure 14 is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the invention.

[0477] More specifically, the left drawing in Fig. 14 is an image taken immediately after the hydrolytic enzyme droplet was applied, and is post-processed to exclude the exclusion region (black circle) from the observation area. The middle drawing in Fig. 14 is an image taken a unit of time after the hydrolytic enzyme droplet was applied, and is also post-processed to exclude the exclusion region from the observation area. It can be confirmed that the brightness of the observation area is somewhat brighter than that in the left drawing. The right drawing in Fig. 14 shows the calculated difference in brightness between the left and middle drawings.

[0478]

[0479] By processing as described above, the existence of a brightened area and the difference in brightness can be confirmed.

[0480] The average value of the brightness difference obtained above was calculated, plotted against the elapsed time when the image was captured, and fitted in the form of a sigmoid function.

[0481]

[0482] FIG. 15 is a graph showing the difference in image brightness over time and the fitted function obtained according to one embodiment of the invention.

[0483]

[0484] The form of the sigmoid function used for fitting in Fig. 15 is as follows. At this time, The unit of time is minutes (min). is the fitting constant obtained through fitting, is a natural constant (Euler's Number).

[0485]

[0486] And, the first and second derivatives of this function are as follows, respectively.

[0487] ,

[0488]

[0489] FIG. 16 is a graph showing a first-order differential function and a second-order differential function obtained according to one embodiment of the invention.

[0490]

[0491] Referring to Figure 16, the second derivative function shows a maximum value in the measurement range, which can be interpreted as the point at which the brightness change of the image progresses the fastest (the point at which the acceleration of change of the biodegradable resin has the largest value), and this point can be designated as the point at which hydrolysis of the resin occurs and a clear zone begins to form.

[0492] Obtain the second derivative function and the elapsed time when the second derivative is at its maximum value. After checking, it was organized in the table below.

[0493]

[0494] Sample No. Time (min) 14402-16002-210403-15943-23193-31763-4143

[0495] Referring to Table 4 above, the time at which the clear zone starts to occur for each experiment can be confirmed, and when this is compared under the same experimental conditions, i.e. by interval, it can be seen that the clear zone starts to occur for sample No. 2-1 (PBAT C2) earlier than for No. 2-2 (PBAT C1), and when this is calculated inversely, it can be confirmed that the biodegradability of No. 2-1 (PBAT C2) is about 70% better than that of No. 2-2 (PBAT C1). In the case of PBSA, the time at which the clear zone starts to occur gradually gets faster as you go from No. 3-1 to No. 3-4, and the biodegradability of PBSA is the best for No. 3-4 (PBSA C4), followed by No. 3-3 (PBSA C3), No. 3-2 (PBSA C2), and No. 3-1 (PBSA C1).

[0496]

[0497] Figure 9 shows the results of biodegradability evaluation in a soil environment of PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) measured according to ISO 14855 (Determination of the ultimate aerobic biodegradability of plastic materials under controlled composting conditions-Method by analysis of evolved carbon dioxide). Referring to Figure 9, it can be confirmed that PBAT compound 2 (PBAT C2) has a higher biodegradability than PBAT compound 1 (PBAT C1).

[0498]

[0499] Figure 17 shows the results of biodegradability evaluation in home compost of PBSA compound 1 (PBSA C1), PBSA compound 2 (PBSA C2), PBSA compound 3 (PBSA C3), PBSA compound 4 (PBSA C4) and cellulose (manufacturer: Sigma-Aldrich, product number: 310697), measured according to the ISO 14855-1 standard. Referring to Figure 17, it can be confirmed that PBSA C4 > PBSA C3 > PBSA C2 > PBSA C1 have high biodegradability in the following order.

[0500]

[0501] From the above results, it can be seen that the results of the image analysis of the present invention are consistent with the results of evaluating the biodegradability of biodegradable resin according to the ISO standard.

Claims

1. A hydrolytic enzyme including lipase and cutinase; and a phosphate buffered saline solution. Enzyme solution.

2. In paragraph 1, The ratio of enzyme units of lipase:cutinase is 0.5:1 to 4:1, Enzyme solution.

3. In paragraph 1 or 2, The total amount of hydrolytic enzyme in 1 ml of the above enzyme solution is 400 units to 80,000 units. Enzyme solution.

4. In any one of paragraphs 1 to 3, The above-mentioned life phase is a life phase of one or more microorganisms selected from the group consisting of Pseudomonas Cepacia, Rhizopus Oryzae, Aspergillus niger, and Aspergillus Oryzae. Enzyme solution.

5. In any one of paragraphs 1 to 4, The above cutinase is a cutinase of one or more microorganisms selected from the group consisting of Humicola Insolens, Aspergillus Oryzae, Fusarium solani, and Pseudomonas putida. Enzyme solution.

6. In any one of paragraphs 1 to 5, The above life phase includes the life phase of Pseudomonas Cepacia, The above cutinase includes the cutinase of Humicola Insolens, Enzyme solution.

7. In any one of paragraphs 1 to 6, The life phase includes the life phase of Pseudomonas Cepacia and the life phase of Rhizopus Oryzae, Cutinase, including cutinase from Humicola Insolens and cutinase from Aspergillus Oryzae. Enzyme solution.

8. In any one of paragraphs 1 to 7, Including the life phase of Pseudomonas Cepacia, Cutinase, including cutinase from Humicola Insolens and cutinase from Aspergillus Oryzae. Enzyme solution.

9. In any one of paragraphs 1 to 8, Further comprising at least one enzyme selected from the group consisting of α-amylase, cellulase, proteinase K, protease, acetyl-CoA carboxylase, and alkalise. Enzyme solution. 10.i) a step of contacting a biodegradable resin with an enzyme solution containing a hydrolytic enzyme including lipase and cutinase; and phosphate buffered saline; and ii) a step of observing the decomposition pattern of the biodegradable resin over time; Method for evaluating the biodegradability of biodegradable resins.

11. In paragraph 10, The above step i) is performed by dropping the enzyme solution onto a resin test piece containing a biodegradable resin. Method for evaluating the biodegradability of biodegradable resins.

12. In paragraph 10, The above step i) is performed by immersing a resin test piece containing a biodegradable resin in the enzyme solution. Method for evaluating the biodegradability of biodegradable resins.

13. In paragraph 10, The above step i) is performed by spraying a biodegradable resin solution on a medium to prepare a test piece, and then dripping the enzyme solution onto the test piece. The above step ii) is performed by observing the change in the clear zone formed on the test piece over time. Method for evaluating the biodegradability of biodegradable resins.

14. In any one of paragraphs 10 to 13, Step ii) above, a) a step of obtaining an image of the biodegradable resin over a period of time; and b) a step of evaluating biodegradability through image changes of the resin test piece over time; Method for evaluating the biodegradability of biodegradable resins.

15. In paragraph 14, Step b) above, b-1) A step of calculating the difference in brightness between the image of each resin test piece obtained over a period of time and the image immediately after the hydrolytic enzyme is applied; b-2) a step of representing the image brightness difference value as a function of the unit time elapsed; and b-3) Including a step of evaluating biodegradability through the above function analysis, Method for evaluating the biodegradability of biodegradable resins.

16. In paragraph 15, The step b-3) above is to check the time at which the second derivative of the function is at its maximum value and evaluate it as the biodegradability of the biodegradable resin. Method for evaluating the biodegradability of biodegradable resins.

Images