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

Abstract

The present disclosure relates to an enzyme solution having biodegradable resin degradability and useful for evaluating the biodegradability of a biodegradable resin, and a method for evaluating the biodegradability of a biodegradable resin using the same.

Description

Technical Field

[0001] Cross-references to related applications

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

[0003] This disclosure relates to enzyme solutions having biodegradability of biodegradable resins and being used to assess the biodegradability of biodegradable resins, as well as methods for assessing the biodegradability of biodegradable resins using the same. Background Technology

[0004] Conventional petroleum-based plastics do not decompose naturally and cause serious environmental pollution. Therefore, biodegradable polymers that are biodegradable in nature, such as polylactide (PLA), poly(butylene adipate-copolymer-terephthalate) (PBAT), and polybutylene succinate (PBS), have attracted attention as alternatives to petroleum-based plastics.

[0005] Biodegradability assessment is essential when developing such biodegradable resins. However, existing biodegradability tests take weeks to months depending on various conditions such as soil, compost, or the ocean, and require expensive, large-scale equipment.

[0006] Therefore, there is a need to develop assessment methods that can evaluate the biodegradability of biodegradable resins in a shorter time period using simpler methods. Summary of the Invention

[0007] Technical issues

[0008] One object of the present invention is to provide an enzyme solution that is degradable to biodegradable resins and can more easily and quickly predict the biodegradability of biodegradable resins when exposed to the natural environment.

[0009] Furthermore, an object of the present invention is to provide a method for assessing the biodegradability of biodegradable resins using the enzyme solution.

[0010] Technical solution

[0011] According to one embodiment of this disclosure, an enzyme solution is provided comprising: a hydrolase, said hydrolase comprising lipase and keratinase; and a phosphate-buffered saline solution.

[0012] According to another embodiment of this disclosure, a method for assessing the biodegradability of a biodegradable resin is provided, comprising the steps of: i) contacting an enzyme solution containing a hydrolase and a phosphate-buffered saline solution with the biodegradable resin, wherein the hydrolase comprises a lipase and a keratinase; and ii) observing the degradation state of the biodegradable resin over time.

[0013] Beneficial effects

[0014] The enzyme solution disclosed herein possesses biodegradability of biodegradable resins and can be effectively used to assess the biodegradability of biodegradable resins. According to the biodegradability assessment method for biodegradable resins using the enzyme solution of this disclosure, the biodegradability of biodegradable resins can be assessed simply and rapidly without the need for expensive, large-scale equipment. Furthermore, according to the biodegradability assessment method for biodegradable resins, the biodegradability of biodegradable resins under various natural environmental conditions such as soil, compost, and ocean can be easily predicted. Attached Figure Description

[0015] Figure 1 The results of the biodegradability assessment of poly(butylene adipate-copoly-terephthalate) compound 1 (PBAT C1) and poly(butylene adipate-copoly-terephthalate) compound 2 (PBAT C2) in soil / compost environment carried out in (3) of Example A-1 are shown.

[0016] Figure 2 The results of the biodegradability tests of PBAT C1, PBAT C2 and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) under industrial composting conditions, measured according to ISO 14855-1, are shown.

[0017] Figure 3 The results of the biodegradability assessment of poly(butylene succinate) (PBS) and poly(butylene adipate succinate) (PBSA) in a marine environment, carried out in (3) of Example A-2, are shown.

[0018] Figure 4 The results of biodegradability tests of PBS and PBSA under marine conditions, measured according to ASTM D6691, are shown.

[0019] Figure 5 The results of the biodegradability assessment of the PBAT / TPS and PBAT / PLA compounds in a soil / compost environment, conducted in (3) of Example B-1, are shown.

[0020] Figure 6 The results of the biodegradability assessment of the PBAT / TPS compound, PBAT / PLA compound, and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example B-1 in a soil environment according to ISO 17556 are shown.

[0021] Figure 7 The results of the biodegradability assessment of PBS and PBSA under marine conditions, carried out in (3) of Example B-2, are shown.

[0022] Figure 8 This is a schematic diagram illustrating the method of adding enzyme solution to samples in the biodegradability assessment of Examples C-1 and C-2.

[0023] Figure 9 The results of the biodegradability assessment of PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2) and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example C-1 in an industrial composting environment are shown according to ISO 14855.

[0024] Figure 10 The results of the biodegradability assessment of PBAT compound 1 and PBAT compound 2 in soil / compost environments are shown for (1) Example C-1, (2) Comparative Example C-1 and (3) Comparative Example C-2.

[0025] Figure 11 The results of the biodegradability assessment of P3HP 1, P3HP 2 and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) used in Example C-2 in a marine environment are shown according to ASTM D6691.

[0026] Figure 12 The results of the biodegradability assessment of P3HP 1 and P3HP 2 in a marine environment, carried out in (4) of Example C-2, are shown.

[0027] Figure 13 This is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the present disclosure.

[0028] Figure 14 This is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the present disclosure.

[0029] Figure 15 This is a graph illustrating the difference in image brightness over time obtained according to one embodiment of the present disclosure.

[0030] Figure 16 This is a graph showing the first and second derivative functions obtained according to one embodiment of the present disclosure.

[0031] Figure 17 The results of the biodegradability assessment in home composting of PBSA compound 1 (PBSA C1), PBSA compound 2 (PBSA C2), PBSA compound 3 (PBSAC3), PBSA compound 4 (PBSA C4) and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) are shown according to ISO 14855-1. Detailed Implementation

[0032] The terminology used in this specification is for describing exemplary embodiments only and is not intended to limit the invention. Unless the context clearly indicates otherwise, singular expressions include plural expressions. In this specification, terms such as “comprising,” “providing,” or “having” are intended to specify the presence of implemented features, steps, components, or combinations thereof, and it should be understood that they do not preclude the possibility of the presence or addition of one or more other features, steps, components, or combinations thereof.

[0033] Since the present invention can be modified in various ways and can take many forms, specific embodiments will be illustrated and described in detail below. However, this is not intended to limit the invention to the specific forms disclosed, and it should be understood to include all changes, equivalents, or alternatives that are included within the spirit and technical scope of the invention.

[0034] In this disclosure, "biodegradable resin" or "biodegradable polymer" means a polymer that is broken down by microorganisms into natural byproducts such as water, carbon dioxide, nitrogen, biomass and inorganic salts.

[0035] The contents of this disclosure will be described in detail below.

[0036] enzyme solution

[0037] According to one embodiment of this disclosure, an enzyme solution is provided comprising: a hydrolase, said hydrolase comprising lipase and keratinase; and a phosphate-buffered saline solution.

[0038] The enzyme solution disclosed herein has biodegradability to biodegradable resins and can be effectively used to assess the biodegradability of biodegradable resins over a short period of time.

[0039] In particular, by adjusting the type and / or content of hydrolytic enzymes contained in the enzyme solution, various environments such as soil, composting, and marine environments can be achieved, thereby predicting the biodegradability of biodegradable resins under various environmental conditions.

[0040] As a solvent for preparing an enzyme solution according to one embodiment of this disclosure, phosphate-buffered saline (PBS) can be used.

[0041] As an example, phosphate-buffered saline (PBS) can 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.

[0042] In one embodiment, the lipase may include a lipase derived from one or more microorganisms selected from Pseudomonas Cepacia, Rhizopus Oryzae, Aspergillus niger, and Aspergillus Oryzae.

[0043] In one embodiment, the lipase may include lipases from *Pseudomonas cepacia* and / or lipases from *Rhizopus oryzae*.

[0044] In addition, keratinase may include keratinase derived from one or more microorganisms selected from Humicola Insolens, Aspergillus oryzae, Fusarium solani, and Pseudomonas putida.

[0045] In one embodiment, the keratinase may include keratinase from specific saprophytic fungi and / or keratinase from Aspergillus oryzae.

[0046] In one embodiment, the lipase may include a lipase from *Pseudomonas cepacia*, and the keratinase may include a keratinase from *Pseudomonas spp.*

[0047] When assessing the biodegradability of biodegradable resins in soil and compost (industrial or household compost) environments, it is preferable to include lipases from *Pseudomonas cepacia* and *Rhizopus oryzae* as lipases. Alternatively, lipases from *Aspergillus niger* and / or *Aspergillus oryzae* may be included.

[0048] When assessing the biodegradability of biodegradable resins in soil and compost (industrial or household compost) environments, it is preferable to include cutinases from specific *Pyrophyllus* species and *Aspergillus oryzae* species as cutinases. In addition, cutinases from *Fusarium solani* and / or *Pseudomonas putida* species may also be included.

[0049] When assessing the biodegradability of biodegradable resins in a marine environment, it is preferable to include lipases from *Pseudomonas cepacia* as the lipase. Alternatively, lipases from *Aspergillus niger* and / or *Aspergillus oryzae* may be included.

[0050] When assessing the biodegradability of biodegradable resins in a marine environment, it is preferable to include keratinase from specific *Pseudomonas* species and *Aspergillus oryzae* species as keratinase. In addition, keratinase from *Fusarium solani* and / or *Pseudomonas putida* species may also be included.

[0051] In addition to lipase and keratinase, the enzyme solution may also contain other enzymes. Such other enzymes may be selected from at least one of α-amylase, cellulase, proteinase K, proteolytic enzymes, acetyl-CoA carboxylase (ACCase), and alkaline protease.

[0052] Among other enzymes, α-amylase can be used on resins containing starch such as TPS and CMS, cellulase can be used on resins containing cellulose such as CMC, and proteinase K, proteolytic enzymes and alkaline proteases can be used on resins containing polylactic acid.

[0053] In one embodiment, α-amylase from Bacillus licheniformis and / or α-amylase from Aspergillus oryzae can be used as α-amylase; cellulase from Trichoderma reesei and / or cellulase from Aspergillus sp. can be used as cellulase; proteinase K from Candida albicans can be used as proteinase K; proteolytic enzymes from Bacillus licheniformis can be used as proteolytic enzymes; and alkaline proteases from Bacillus licheniformis can be used as alkaline proteases. All of these enzymes can be used in simulated soil and composting environments or marine environments.

[0054] In one embodiment, the enzyme solution used to evaluate the biodegradability of the biodegradable resin in soil and compost (industrial or household compost) environments comprises lipases from *Pseudomonas cepacia*, lipases from *Rhizopus oryzae*, cutinases from *Potassium oxysporum* and *Aspergillus oryzae*, and may optionally also contain α-amylases from *Bacillus licheniformis*.

[0055] In one embodiment, the enzyme solution used to evaluate the biodegradability of the biodegradable resin in a marine environment comprises lipase from *Pseudomonas cepacia* and cutinase from *Pseudomonas spp.*, and may also optionally contain cutinase from *Aspergillus oryzae*.

[0056] The amounts of various enzymes contained in the enzyme solution can be appropriately selected based on the environmental conditions to be simulated and the biodegradable resin being evaluated.

[0057] However, the total amount of hydrolytic enzyme per 1 ml of enzyme solution is preferably 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 is slowed down, making rapid biodegradability assessment difficult. Conversely, if the amount of hydrolytic enzyme in the solution is too large, the biodegradable resin decomposes too quickly, making it difficult to determine relative differences, which is unsuitable for biodegradability assessment.

[0058] Specifically, when assessing the biodegradability of biodegradable resins in soil and compost environments, the keratinase can 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, and 40,000 units or less, or 30,000 units or less, 20,000 units or less, or 15,000 units or less, or 10,000 units or less, or 8,000 units or less. The amount of lipase may be 0 units or less, or 4,000 units or less, and the amount of 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, or 40,000 units or less, or 30,000 units or less, or 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.

[0059] In an enzyme solution, the ratio of lipase to keratinase enzyme units can be 0.5:1 or greater, 0.8:1 or greater, or 1:1 or greater, as well as 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.

[0060] When assessing the biodegradability of biodegradable resins in soil and compost environments, the lipase:keratase ratio can be 0.5:1 or greater, or 0.8:1 or greater, or 2:1 or less, 1.8:1 or less, or 1.5:1 or less.

[0061] Specifically, when it is desired to assess the biodegradability of biodegradable resins in soil and compost environments, the amount of lipase per unit of keratinase may be 0.5 units or more, or 0.8 units or more, or 2 units or less, or 1.8 units or less, or 1.5 units or less.

[0062] Additionally, when additional enzymes are included during soil and compost environment simulations, α-amylase may be 100 units or more, or 200 units or more, and 3,000 units or less, or 1,500 units or less; cellulase may be 700 units or more, or 1,400 units or more, and 3,400 units or less, or 1,700 units or less; proteinase K, proteolytic enzymes, and alkaline protease may each be 0.75 units or more, or 1.5 units or more, and 5 units or less, or 2.5 units or less.

[0063] When assessing the biodegradability of biodegradable resins in a marine environment, the keratinase can 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, and 40,000 units or less, or 20,000 units or less, or 16,000 units or less, or 10,000 units or less, or 8,000 units or less, or 6,000 units or less. Fewer, or 3,000 units or less, 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, and 40,000 units or less, or 30,000 units or less, or 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.

[0064] When assessing the biodegradability of biodegradable resins in a marine environment, the lipase:keratase ratio can be 0.5:1 or greater, 0.8:1 or greater, or 1:1 or greater, and 4:1 or less, 3:1 or less, or 2.5:1 or less.

[0065] Specifically, when it is desired to assess the biodegradability of biodegradable resins in a marine environment, the amount of lipase per unit of keratinase can be 0.5 units or more, 0.8 units or more, or 1 unit or more, or 1.5 units or more, or 4 units or less, or 3 units or less, or 2.5 units or less.

[0066] When additional enzymes are included during marine environment simulation, α-amylase may be 100 units or more, or 200 units or more, and 3,000 units or less, or 1,500 units or less; cellulase may be 700 units or more, or 1,400 units or more, and 3,400 units or less, or 1,700 units or less; proteinase K, proteolytic enzymes, and alkaline protease may each be 0.75 units or more, or 1.5 units or more, and 5 units or less, or 2.5 units or less.

[0067] The unit of a hydrolase is the enzyme unit or the international unit of enzyme activity, also expressed as U or IU. One enzyme unit refers to the amount of enzyme that converts 1 μmol of substrate into product in 1 minute under the corresponding enzyme's optimal reaction conditions (temperature, pH, substrate concentration).

[0068] Optimal reaction conditions vary depending on the enzyme. For example, the optimal reaction temperature for the lipases, keratinases, and other enzymes mentioned above is in the range of 25°C to 58°C. The pH ranges from 4 to 9 and can be varied depending on the conditions (e.g., 4 to 9 for soil conditions, 7.9 to 8.2 for marine conditions, and 6 to 9 for compost conditions).

[0069] Methods for assessing the biodegradability of biodegradable resins

[0070] According to one embodiment of this disclosure, a method for assessing the biodegradability of a biodegradable resin is provided, comprising the steps of: i) contacting an enzyme solution containing a hydrolase and a phosphate-buffered saline solution with the biodegradable resin, the hydrolase comprising a lipase and a keratinase; and ii) observing the degradation state of the biodegradable resin over time.

[0071] The biodegradation of biodegradable resins follows a process in which the polymer is hydrolyzed into oligomers or monomers, which are then metabolized by microorganisms. Therefore, the faster the rate of hydrolysis, the first step, the faster the biodegradation occurs.

[0072] The evaluation method of this disclosure focuses on this point, and according to this evaluation method, the relative biodegradability of biodegradable resins can be easily and quickly assessed by measuring the hydrolysis rate of biodegradable resins using an enzyme solution containing hydrolytic enzymes containing lipase and keratinase.

[0073] Furthermore, various environments such as soil, compost, and the ocean can be simulated by altering the composition of the enzymes contained in the enzyme solution. Therefore, according to the aforementioned biodegradability assessment method, the relative biodegradability of biodegradable resins exposed to various natural environmental conditions can be predicted rapidly and easily.

[0074] The biodegradability assessment method for biodegradable resins according to one embodiment of this disclosure can be applied to a variety of resins requiring biodegradability assessment. Examples of biodegradable resins that can be the target of assessment include, but are not limited to, polylactide (PLA), poly(butylene adipate-copolymer-terephthalate) (PBAT), polybutylene succinate (PBS), polycaprolactone (PCL), polyhydroxybutyrate (PHB), thermoplastic starch, etc., and by applying the biodegradability assessment method of this disclosure to any resin expected to be biodegradable, the relative biodegradation rate in the natural environment can be easily and rapidly predicted.

[0075] According to the described biodegradability assessment method, the biodegradability of a resin can be evaluated within hours to days. Therefore, the described biodegradability assessment method can be effectively used when a simple biodegradability test is required for a short period of time, such as during the development phase of a biodegradable resin.

[0076] The temperature and pressure conditions of the method for assessing the biodegradability of biodegradable resins can be appropriately selected according to the natural environmental conditions to be simulated. As an example, the method for assessing the biodegradability of biodegradable resins of this disclosure can be carried out at atmospheric pressure (760±20 Torr) in a temperature range of 20°C to 30°C, or 30°C to 40°C, or 40°C to 60°C.

[0077] The composition of the enzyme solution used in the method for assessing the biodegradability of biodegradable resins is as described above.

[0078] Specifically, the biodegradability assessment method for biodegradable resins can be performed according to the following methods 1 to 3.

[0079] a. Method 1

[0080] In method 1, step i) can be performed by adding an enzyme solution dropwise onto a resin sample containing biodegradable resin.

[0081] In addition, step ii) can be performed by measuring the change in the decomposition area of ​​the resin sample over time.

[0082] Specifically, method 1 may include: preparing an enzyme solution containing lipase and keratinase; adding the enzyme solution dropwise onto a resin sample containing biodegradable resin; and measuring the change in the decomposition area of ​​the resin sample over time.

[0083] The resin sample contains a biodegradable resin for which biodegradability measurement is required, and its shape is not limited, but two-dimensional sheet-like shapes such as films or sheets are preferred because the degradation state after the addition of enzyme solution can be easily identified.

[0084] Resin samples may consist solely of biodegradable resins or biodegradable resin compounds for which biodegradability measurements are required.

[0085] There are no restrictions on the size of the film or sheet resin sample, but for example, it is preferable to have a width and length of 5 cm or more, or 10 cm or more, or within the range of 20 cm or less.

[0086] The thickness of the film or sheet-type resin sample can preferably be 250 μm or less, or 100 μm or less, or 20 μm or more, or 50 μm or more.

[0087] After preparing the enzyme solution and resin sample as described above, the enzyme solution was dropped onto the resin sample, and the biodegradability was assessed by observing the degradation state of the resin sample over time.

[0088] There are no particular limitations on the method of adding the enzyme solution to the resin sample, but it is preferable to use a pipette or similar device to add it to at least one or more points on the sample surface, and to set the amount of enzyme solution added at each point to be at the level of 3 μl to 5 μl. In this case, to reduce the error in the biodegradability assessment, two or more points for adding the enzyme solution can be used.

[0089] The points used for adding enzyme solution should be separated from the edge of the sample by at least 20 mm, and when adding at two or more points, a distance of at least 10 mm should be ensured between each point so that the collapse points caused by resin decomposition during the evaluation process do not overlap with each other.

[0090] The biodegradability of biodegradable resins is assessed by measuring the area of ​​collapse that occurs on the sample over time, starting immediately after the enzyme solution is dropped onto the sample as described above.

[0091] Resins with faster hydrolysis rates typically exhibit higher biodegradability, and the formation and expansion of collapse area occur rapidly during biodegradability assessments. Therefore, by using this method to simultaneously test two or more biodegradable resins, the relative biodegradability of each resin in the natural environment can be predicted.

[0092] The temperature and pressure conditions during enzyme solution addition and decomposition area measurement can be appropriately selected according to the natural environmental conditions to be simulated. As an example, the biodegradability assessment method for biodegradable resins of this disclosure can be carried out at atmospheric pressure (760 ± 20 Torr) in a temperature range of 20°C to 60°C, for example, 20°C to 30°C, 30°C to 40°C, or 40°C to 60°C.

[0093] According to the method for assessing the biodegradability of biodegradable resins, the biodegradability of the resin can be assessed within hours to days. The assessment time can vary depending on the size and weight of the biodegradable resin sample, the composition of the enzyme solution, etc., but will not exceed 14 days.

[0094] b. Method 2

[0095] In method 2, step i) can be performed by immersing a resin sample containing biodegradable resin in an enzyme solution.

[0096] Specifically, method 2 may include: preparing an enzyme solution containing lipase and keratinase; immersing a resin sample containing biodegradable resin in the enzyme solution; and observing the degradation state of the impregnated resin sample over time.

[0097] The resin sample contains a biodegradable resin for which biodegradability measurement is required, and its shape is not limited; various shapes such as film and sheet can be used. Film is preferred because the degradation state can be rapidly identified when immersed in a hydrolysis solution.

[0098] In this regard, the thickness of the membrane sample can preferably be 250 μm or less, or 100 μm or less, or 20 μm or more, or 50 μm or more.

[0099] After preparing the enzyme solution and resin sample as described above, the resin sample was immersed in the enzyme solution, and the biodegradability was evaluated by observing the degradation state of the immersed resin sample over time.

[0100] There is no limit to the amount of enzyme solution used, and it should be used in an amount that allows the resin sample to be completely impregnated.

[0101] That is, the amount of enzyme solution used can be appropriately adjusted according to the concentration of the enzyme solution and the size and / or weight of the resin sample. For example, when using an enzyme solution containing 4,000 to 40,000 units of hydrolase per 1 ml, the enzyme solution can be applied at a rate of 10 × 10 mm. 2For resin samples with a thickness of 20 μm to 250 μm, use amounts of 100 μl or more, or 250 μl or more, or 1000 μl or less, or 750 μl or less, or 500 μl or less. However, the amount of enzyme solution used is not limited to these, and the appropriate amount should be selected and used according to the concentration of the hydrolase in the hydrolase, or the shape, size, weight, etc. of the resin sample.

[0102] Because biodegradable resin samples are hydrolyzed over time in enzyme solutions, their weight gradually decreases, and the collapse and fragmentation of the sample can be visually observed. Therefore, "observation of degradation status" can be performed by observing changes over time using various methods, such as periodically measuring changes in the weight of the resin sample or visually observing the collapse of the resin sample.

[0103] By simultaneously testing two or more biodegradable resins using the methods described above, the relative biodegradability of each resin in the natural environment can be predicted. Generally, resins with faster hydrolysis rates have higher biodegradability, and therefore, biodegradability can be relatively assessed by comparing the hydrolysis rates of two or more biodegradable resins.

[0104] The temperature and pressure conditions for immersing the resin sample in the enzyme solution and observing the degradation state of the resin sample can be appropriately selected according to the natural environmental conditions to be simulated. As an example, the biodegradability assessment method for biodegradable resins of this disclosure can be performed at atmospheric pressure (760 ± 20 Torr) in a temperature range of 20°C to 60°C, for example, 20°C to 30°C, 30°C to 40°C, or 40°C to 60°C.

[0105] According to the method for assessing the biodegradability of biodegradable resins, the biodegradability of the resin can be assessed within hours to days. The assessment time can vary depending on the size and weight of the biodegradable resin sample, the composition of the enzyme solution, etc., but will not exceed 14 days.

[0106] c. Method 3

[0107] In method 3, step i) involves preparing a sample by spraying a biodegradable resin solution onto a culture medium, followed by adding an enzyme solution dropwise onto the sample.

[0108] Step ii) can be performed by observing the changes in the transparent area formed on the sample over time.

[0109] Specifically, method 3 may include: preparing a sample by spraying a biodegradable resin solution onto a culture medium; adding an enzyme solution containing lipase and keratinase to the sample; and measuring the change of the transparent area formed on the sample over time.

[0110] The culture medium is a means of supporting a resin solution containing a biodegradable resin and providing a moist environment in which enzymes can function, and is preferably a material that is not reactive to the biodegradable resin and enzyme solution.

[0111] The materials for the culture medium can be selected based on the type of biodegradable resin and the composition of the enzymes contained in the enzyme solution. For example, the culture medium can be a solid medium containing agar and / or gelatin, but is not limited to this.

[0112] Unlike culture media used to cultivate microorganisms, this culture medium may consist only of agar and / or gelatin and distilled water without additives such as sugars and peptides.

[0113] Such culture media are preferably sterilized before use. As an example, the culture medium can be sterilized by treating it at 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 for the culture medium is not limited to this, and a sterilization method known for each material can be appropriately selected.

[0114] The biodegradable resin solution consists of a biodegradable resin and a solvent for assessing biodegradability.

[0115] There are no particular limitations on the solvent, as long as it can uniformly disperse the biodegradable resin. As an example, at least one selected from chloroform, chloromethane, carbon tetrachloride, benzene, dimethyl sulfoxide, dimethylformamide, toluene, and xylene can be used.

[0116] The content (solid content) of biodegradable resin in the biodegradable resin solution can be 0.5% by weight or higher, or 1% by weight or higher, or within the range of 3% by weight or lower, or 2% by weight or lower. When these ranges are met, droplets of the biodegradable resin solution can be uniformly applied to the culture medium at an appropriate concentration, and thus, an environment conducive to the function of hydrolytic enzymes can be created, which is preferred.

[0117] There are no particular limitations on the method of spraying biodegradable resin solutions, and conventionally known spraying methods for polymer solutions can be appropriately selected. For example, spraying devices such as sprayers, atomizers, spray guns, or electric spraying devices can be used.

[0118] After spraying the biodegradable resin solution onto the culture medium, it is dried to allow all the solvent in the biodegradable resin solution to evaporate. If biodegradability assessment is performed without solvent evaporation, enzyme activity may be very low, making successful assessment difficult. Therefore, drying is performed by adjusting the temperature, pressure, and time, taking into account the solvent used, the type of biodegradable resin, and the characteristics of the culture medium. Taking chloroform as an example, the culture medium sprayed with the biodegradable resin solution can be dried by placing it at room temperature (25°C) and atmospheric pressure (0.1 MPa) for 5 to 20 minutes, preferably 10 to 20 minutes.

[0119] The biodegradable resin solution can be sprayed to achieve an average thickness of 0.6 µm or greater, or 0.8 µm or greater, or within the range of 1.5 µm or less on the culture medium. When the biodegradable resin is applied at the appropriate thickness as described above, it is suitable for observing changes over time after the addition of the enzyme solution.

[0120] Next, the biodegradability assessment was initiated by dropping an enzyme solution containing lipase and keratinase as hydrolytic enzymes onto a culture medium (sample) that had been sprayed with a biodegradable resin solution.

[0121] At this point, various environments, such as soil, compost, and marine environments, can be simulated by adjusting the type and / or content of the hydrolytic enzymes in the enzyme solution. The enzyme composition of the enzyme solution used to simulate each condition is as described above.

[0122] There are no particular limitations on the method of adding the enzyme solution to the culture medium (sample) sprayed with biodegradable resin solution, but it is preferable to use a pipette or the like to add it to at least one or more points on the surface of the sample (the surface on which the biodegradable resin solution is applied), and to set the amount of enzyme solution added to each point to be at the level of 3 μl to 5 μl. In this case, in order to reduce the error in the biodegradability assessment, two or more points for adding the enzyme solution can be used.

[0123] There are no particular restrictions on the location on the sample where the enzyme solution is added, but preferably, it should be separated from the edge of the sample by at least 20 mm, and when added at two or more points, a distance of at least 20 mm should be ensured between each point so that the transparent areas resulting from resin decomposition during the evaluation process do not overlap. Depending on the sample, a wide range of weak transparent areas may form. When using such samples, the enzyme solution is added so as to be separated from the edge of the sample by at least 40 mm, and when added at two or more points, a distance of at least 40 mm should be ensured between each point so that the transparent areas do not overlap. In this way, the drop points of the enzyme solution on the sample can be determined taking into account the characteristics of the sample.

[0124] The transparent region is the transparent portion created when water-insoluble biodegradable resin is broken down into water-soluble molecules by hydrolytic enzymes. The biodegradability of biodegradable resin is assessed by measuring the change in the area of ​​the transparent region over time, or the difference in the degree to which the transparent region becomes transparent over time, immediately after the enzyme solution is added to the sample as described above.

[0125] Generally, resins with faster hydrolysis rates exhibit higher biodegradability, and the formation and expansion of transparent regions occur rapidly during biodegradability assessment. Therefore, by using this method to simultaneously test two or more biodegradable resins, the relative biodegradability of each resin in the natural environment can be predicted.

[0126] The temperature and pressure conditions during enzyme solution addition and decomposition area measurement can be appropriately selected according to the natural environmental conditions to be simulated. As an example, the biodegradability assessment method for biodegradable resins of this disclosure can be carried out at atmospheric pressure (760 ± 20 Torr) in a temperature range of 20°C to 30°C, or 30°C to 40°C, or 40°C to 60°C.

[0127] According to the described method for assessing the biodegradability of biodegradable resins, the biodegradability of the resin can be assessed within hours to days. The assessment time can vary depending on the solids content of the biodegradable resin solution, the composition of the enzyme solution, etc., but will not exceed 7 days.

[0128] d. Image analysis of biodegradable resin samples

[0129] Meanwhile, in the biodegradability assessment method for biodegradable resins, step ii) observing the degradation state of the biodegradable resin over time may include: a) obtaining an image of the resin sample based on the elapsed unit time; and b) assessing the biodegradability by the change in the image of the resin sample over time.

[0130] First, images of the biodegradable resin sample in contact with the enzyme solution are acquired over a certain period of time (unit time). The unit time can be, for example, about several seconds, several minutes, or several hours, and this can be set differently depending on the composition of the sample or the hydrolytic enzyme.

[0131] If method 1 or 3 is followed, step a) is preferably performed under conditions that prevent the added enzyme solution from evaporating. When taking images, it is preferable to maintain the same color balance, illuminance, shutter speed, lens brightness, and other shooting conditions to improve the accuracy of the analysis.

[0132] In this case, real-time image measurement devices can be used for images, and for example, JuLI such as NanoEntek can be used. TM The device for the stage is described, but this disclosure is not limited thereto.

[0133] Next, biodegradability was assessed by observing changes in the resin samples over time. Over time, hydrolytic enzymes hydrolyze the resin in samples in contact with the enzyme solution, resulting in changes in appearance.

[0134] For example, in the areas where hydrolysis occurs, the resin thickness of the sample becomes thinner, and compared to the areas where hydrolysis does not occur, a brighter area (transparent area) can be formed in terms of appearance.

[0135] Therefore, by measuring and analyzing the changes in this bright area, the degree of biodegradation of the resin in the corresponding sample can be determined.

[0136] However, in order to analyze it accurately, it is necessary to quantify and standardize the shape of the bright area.

[0137] According to one example, step b) may include: b-1) obtaining the brightness difference between the image of the resin sample obtained based on the elapsed unit time and the image immediately following the addition of the hydrolytic enzyme; b-2) expressing the image brightness difference as a function of the elapsed unit time; and b-3) assessing biodegradability by analyzing the function.

[0138] That is, in the obtained image, the brightness of the area where the hydrolase was added is measured, and the change in brightness over time is determined. Then, the difference between the brightness measured at a specific elapsed time and the brightness immediately after the addition of the hydrolase is obtained.

[0139] If the brightness difference is compared as a function of time, the degree of brightness change over time can be determined. Furthermore, based on the pattern of brightness change, it can be assumed that a specific time point, such as the time when the second derivative of the function reaches its maximum value, is the time when the acceleration of biodegradation of the biodegradable resin reaches its maximum value and is also the time when the transparent area begins to form.

[0140] The time at which the second derivative of a function reaches its maximum value is an inflection point in the brightness function over time, and also a point where the rate of change in brightness changes, which can be viewed as a point where the rate of biodegradation changes. Therefore, if the time elapsed at the point where the second derivative of the function reaches its maximum value is relatively early, the biodegradation rate is relatively fast; conversely, if the time elapsed at the point where the second derivative of the function reaches its maximum value is relatively slow, the biodegradation rate is relatively slow, and thus the biodegradability of the resin can be assessed.

[0141] In the following embodiments, implementations of the present disclosure will be described in more detail. However, the following embodiments are merely illustrative of implementations of the present disclosure, and the content of the present disclosure is not limited to the following embodiments.

[0142] [Example]

[0143] Example A-1: ​​Biodegradability assessment in soil / compost environments

[0144] (1) Sample preparation

[0145] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded at a barrel temperature of 180°C to 200°C, a feed rate of 30 kg / h to 50 kg / h, and 300 rpm to prepare a resin composition in pellet form.

[0146] Subsequently, the granulated resin composition was formed to a thickness of 0.05 mm using a single screw extruder (Blown Film M / C, 19 pi, L / D=25) at an extrusion temperature of 170°C to prepare a blown film. At this time, the blow-up ratio was 1.8 and the linear velocity was 5 m / min.

[0147] Subsequently, it was cut into a membrane shape with a width * length = 5 cm * 5 cm and a thickness of 0.05 mm, and then inserted into a fixed frame and used as a sample.

[0148] Samples of each of the following polymer compounds were prepared using the methods described above.

[0149] a. Poly(butylene adipate-copolymer-terephthalate) compound 1 (manufacturer: LG Chem, raw materials: PBAT and PLA)

[0150] b. Poly(butylene adipate-copolymer-terephthalate) compound 2 (manufacturer: LG Chem, raw materials: PBAT, PLA and additives)

[0151] (2) Preparation of enzyme solution

[0152] Enzyme solutions were prepared by mixing the following two types of lipases and two types of keratinases with 1 ml of 1X phosphate-buffered saline (PBS).

[0153] Rhizopus oryzae lipase 10,000 units

[0154] 10,000 units of Pseudomonas cepacia lipase

[0155] Aspergillus oryzae cutinase 10,000 units

[0156] 10,000 units of specific humic mold keratinase

[0157] 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 the same composition of PBS is used in all the following examples.

[0158] (3) Biodegradability assessment

[0159] Biodegradability was assessed using the following method at 58°C and 760 mmHg.

[0160] 5 μl of enzyme solution (2) was added dropwise to the sample prepared in (1). The size of the area lost due to enzyme decomposition of the sample was measured after 27 hours and 45 hours. Figure 1 ).

[0161] (4) Results Analysis

[0162] Figure 2 The biodegradability test results of poly(butylene adipate-copoly-terephthalate) compound 1 (PBAT C1), poly(butylene adipate-copoly-terephthalate) compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) under industrial composting conditions, measured according to ISO 14855-1, are shown. (Refer to...) Figure 2 It can be determined that the biodegradability of PBAT C1 without additives is higher than that of PBAT C2.

[0163] Figure 1 The results of the biodegradability assessment of (3) are shown in the first row, and the photos were taken 27 hours (27HPI) and 45 hours (47HPI) after the enzyme solution was dropped onto the sample. Figure 1The second row is a photograph showing only the lost portion of the photograph in the first row, to determine the area of ​​the sample lost due to hydrolytic enzymes, and the lost area is described below.

[0164] Reference Figure 1 It can be determined that the biodegradability of additive-free PBAT C1 is higher than that of PBAT C2, which is consistent with the above. Figure 2 The results are consistent with those in the previous section.

[0165] Example A-2: Biodegradability assessment in marine environments

[0166] (1) Sample preparation

[0167] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded at a barrel temperature of 180°C to 200°C, a feed rate of 30 kg / h to 50 kg / h, and 300 rpm to prepare a resin composition in pellet form.

[0168] Subsequently, the granulated resin composition was formed to a thickness of 0.05 mm using a single screw extruder (Blown Film M / C, 19 pi, L / D=25) at an extrusion temperature of 170°C to prepare a blown film. At this time, the blow-up ratio was 1.8 and the linear velocity was 5 m / min.

[0169] Subsequently, it was cut into sections with a width * length = 1 cm * 1 cm and a thickness of 0.05 mm to prepare membrane samples.

[0170] Samples of each of the following polymers were prepared using the methods described above.

[0171] a. Poly(butylene succinate) (Manufacturer: PTT MCC, Raw material: PBS)

[0172] b. Poly(butylene adipate) (Manufacturer: LG Chem, Raw material: PBSA)

[0173] (2) Preparation of enzyme solution

[0174] Enzyme solutions were prepared by mixing one type of lipase and two types of keratinase with 1 ml of 1X phosphate-buffered saline (PBS) and 36 mg of sea salt.

[0175] 10,000 units of Pseudomonas cepacia lipase

[0176] Aspergillus oryzae cutinase 10,000 units

[0177] 10,000 units of specific humic mold keratinase

[0178] (3) Biodegradability assessment

[0179] Biodegradability was assessed using the following method at 30°C and 760 mmHg.

[0180] 5 μl of enzyme solution (2) was added dropwise to the sample prepared in (1). After 7 days, the size of the area lost due to enzyme decomposition of the sample was measured. Figure 3 ).

[0181] (4) Results Analysis

[0182] Figure 3 The results of the biodegradability assessment for (3) are shown. Figure 3 In this context, DPI (day post-inoculation) refers to the number of days elapsed, and 10 DPI means that it is a photograph taken 10 days after the enzyme solution was added to the sample.

[0183] The following table 1 and Figure 4 The results of the biodegradability tests conducted on the polymers in a. and b. above, measured according to ASTM D6691, under marine conditions are shown. Each sample was tested twice, and the test count is described as “n” following each description. That is, “cellulose 1” and “cellulose 2” refer to the test results performed on the same cellulose, where “cellulose 1” refers to the first test result and “cellulose 2” refers to the second test result. The values ​​in Table 1 indicate the degradation rate (%) of the corresponding resin after each number of days.

[0184] [Table 1]

[0185]

[0186] Figure 3 The first row shows photos taken 10 days (10 DPI) after the enzyme solution was added to the sample. Figure 3 The second row is a photograph showing only the lost portion of the photograph in the first row, to determine the area of ​​the sample lost due to hydrolytic enzymes, and the lost area is described below.

[0187] Reference Figure 3 It can be determined that the biodegradability of PBSA is higher than that of PBS, which is consistent with the above. Figure 4 The results are consistent with those in the previous section.

[0188] Example B-1: Biodegradability assessment in soil / compost environments

[0189] (1) Sample preparation

[0190] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded at a barrel temperature of 180°C to 200°C, a feed rate of 30 kg / h to 50 kg / h, and 300 rpm to prepare a resin composition in pellet form.

[0191] Subsequently, the granulated resin composition was formed to a thickness of 0.05 mm using a single screw extruder (Blown Film M / C, 19 pi, L / D=25) at an extrusion temperature of 170°C to prepare a blown film. At this time, the blow-up ratio was 1.8 and the linear velocity was 5 m / min.

[0192] Subsequently, it was cut into sections with a width * length = 1 cm * 1 cm and a thickness of 0.05 mm to prepare membrane samples.

[0193] Samples of each of the following polymer compounds were prepared using the methods described above.

[0194] a. Poly(butylene adipate-copolymer-terephthalate) / thermoplastic starch compounds (LG Chem, PBAT / TPS compounds)

[0195] b. Poly(butylene adipate-copolymer-terephthalate) / polylactic acid compounds (LG Chem, PBAT / PLA compounds)

[0196] (2) Preparation of enzyme solution

[0197] Enzyme solutions were prepared by mixing the following two types of lipase, two types of keratinase, and one type of α-amylase with 1 ml of 1X phosphate-buffered saline (PBS).

[0198] 1000 units of Rhizopus oryzae lipase

[0199] 3000 units of Pseudomonas cepacia lipase

[0200] Aspergillus oryzae cutinase 2000 units

[0201] 1500 units of specific humic mold keratinase

[0202] Bacillus licheniformis α-amylase 1500 units

[0203] (3) Biodegradability assessment

[0204] Biodegradability was assessed using the following method at 37°C and 760 mmHg.

[0205] 0.5 ml of enzyme solution (2) was placed into one well of a 24-well plate and two samples (1) were soaked in it. Thereafter, the pattern of sample collapse and fragment detachment was visually observed every 1 to 3 days.

[0206] (4) Results Analysis

[0207] Figure 5 The results of the biodegradability assessment for (3) are shown. Figure 5 In this context, DPI (days post-inoculation) refers to the number of days elapsed. 0 DPI is a photograph taken immediately after the sample is immersed in the enzyme solution, and 2 DPI is a photograph taken 2 days after immersion.

[0208] Table 2 and below Figure 6 The results of the biodegradability tests on the polymer compounds in a. and b. above, measured according to ISO 17556, under soil conditions are shown. Each sample was tested twice, and the test counts are described as “_n” following each description. That is, “cellulose_1” and “cellulose_2” are the results of the experiments performed on the same cellulose, where “cellulose_1” refers to the first experimental result and “cellulose_2” refers to the second experimental result. The values ​​in Table 2 indicate the degradation rate (%) of the corresponding resin after each number of days.

[0209] [Table 2]

[0210]

[0211] Reference Figure 5 In compound a. PBAT / TPS, it was confirmed that pores formed and began to collapse after 4 days of impregnation, with a significant portion of the sample edges collapsing by day 7. On the other hand, in compound b. PBAT / PLA, since no significant collapse was observed even by day 7 of impregnation, it can be determined that compound a. PBAT / TPS has relatively high biodegradability compared to compound b. PBAT / PLA, consistent with Table 2 and... Figure 6 The results are consistent with those measured according to the standard test method.

[0212] Example B-2: Biodegradability assessment in marine environments

[0213] (1) Sample preparation

[0214] A biodegradable resin was fed into a twin-screw extruder (die diameter = 32 mm) and extruded at a barrel temperature of 180°C to 200°C, a feed rate of 30 kg / h to 50 kg / h, and 300 rpm to prepare a resin composition in pellet form.

[0215] Subsequently, the granulated resin composition was formed to a thickness of 0.05 mm using a single screw extruder (Blown Film M / C, 19 pi, L / D=25) at an extrusion temperature of 170°C to prepare a blown film. At this time, the blow-up ratio was 1.8 and the linear velocity was 5 m / min.

[0216] Subsequently, it was cut into sections with a width * length = 1 cm * 1 cm and a thickness of 0.05 mm to prepare membrane samples.

[0217] Samples of each of the following polymers were prepared using the methods described above.

[0218] a. Poly(butylene succinate) (Manufacturer: PTT MCC, Raw material: PBS)

[0219] b. Poly(butylene adipate) (Manufacturer: LG Chem, Raw material: PBSA)

[0220] (2) Preparation of enzyme solution

[0221] Enzyme solutions were prepared by mixing one type of lipase and two types of keratinase with 1 ml of 1X phosphate-buffered saline (PBS) and 36 mg of sea salt.

[0222] 10,000 units of Pseudomonas cepacia lipase

[0223] Aspergillus oryzae cutinase 10,000 units

[0224] 10,000 units of specific humic mold keratinase

[0225] (3) Biodegradability assessment

[0226] Biodegradability was assessed using the following method at 30°C and 760 mmHg.

[0227] 0.5 ml of enzyme solution (2) was placed into one well of a 24-well plate and two samples (1) were soaked in it. After 7 days, the pattern of sample collapse and fragment detachment was visually observed.

[0228] (4) Results Analysis

[0229] Figure 7 The results of the biodegradability assessment for (3) are shown. Figure 7 In this context, DPI (days post-inoculation) refers to the number of days elapsed. 0 DPI is the photo taken immediately after the sample is immersed in the enzyme solution, and 7 DPI is the photo taken 7 days after immersion.

[0230] Reference Figure 7It can be determined that a significant portion of the b. polybutylene adipate succinate (PBSA) samples collapsed after 7 days of immersion. On the other hand, in the a. polybutylene adipate succinate (PBA) samples, since no significant collapse was observed, it can be determined that b. polybutylene adipate succinate (PBSA) has relatively high biodegradability compared to a. polybutylene adipate succinate (PBA). This is consistent with Table 1 and... Figure 4 The results are consistent with those measured according to the standard test method.

[0231] Example C-1, Comparative Example C-1 and Comparative Example C-2: Biodegradability Assessment in Soil / Compost Environments

[0232] (1) Preparation of culture medium

[0233] Circular 1% agar plates with a diameter of 90 mm were prepared using the method for preparing culture medium in petri dishes.

[0234] The 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 then autoclaved at 121°C and 0.1 MPa for 15 minutes. The sterilized solution was dispensed in 10 ml portions into sterile 90 mm diameter petri dishes to prepare the culture medium.

[0235] (2) Preparation of resin solution

[0236] 1) Preparation of PBAT compound 1 solution

[0237] Compound 1 of PBAT was prepared by mixing 79.2 wt% polybutylene adipate-copolymer-terephthalate (PBAT, LG Chem), 8.8 wt% polylactic acid (PLA, TotalEnergies Corbion), and 12 wt% calcium carbonate (CaCO3). Each of the resins used was sterilized with 70% ethanol.

[0238] PBAT compound 1 was dissolved in 20 ml of chloroform at a weight of 1% to prepare a PBAT compound 1 solution.

[0239] 2) Preparation of PBAT compound 2 solution

[0240] Compound 2 of PBAT was prepared by mixing 79.2 wt% polybutylene adipate-copolymer-terephthalate (PBAT, LG Chem), 8.8 wt% meso-PLA (Bio Valore), and 12 wt% calcium carbonate (CaCO3). Each of the resins used was sterilized with 70% ethanol.

[0241] PBAT compound 2 was dissolved in 20 ml of chloroform at a weight of 1% to prepare a PBAT compound 2 solution.

[0242] (3) Preparation of enzyme solution

[0243] Enzyme solutions (stock solutions) of Examples C-1, Comparative Examples C-1 and C-2 were prepared. Additionally, a 1 / 10 dilution of the stock solution was prepared.

[0244] <Example C-1>

[0245] 1 ml of 1X phosphate-buffered saline (PBS)

[0246] 1000 units of Rhizopus oryzae lipase

[0247] 3000 units of Pseudomonas cepacia lipase

[0248] Aspergillus oryzae cutinase 2000 units

[0249] 1500 units of specific humic mold keratinase

[0250] <Comparative Example C-1>

[0251] 1 ml of 1X phosphate-buffered saline (PBS)

[0252] 1500 units of specific humic mold keratinase

[0253] <Comparative Example C-2>

[0254] 1 ml of 1X phosphate-buffered saline (PBS)

[0255] 3000 units of Pseudomonas cepacia lipase

[0256] (4) Biodegradability assessment

[0257] On the agar plate of (1), 6 ml of the PBAT compound 1 solution prepared in (2) 1) 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" sample with an average thickness of 0.7 μm. The average thickness of PBAT compound 1 was determined by using an optical profilometer (model name: NewView). TMThe roughness of the sample was obtained by measuring it before and after spraying the PBAT compound 1 solution (8300, Zygo corp.).

[0258] In the same manner, the PBAT compound 2 solution prepared in (2) was sprayed onto a separate agar plate and dried to prepare a "PBAT compound 2" sample with an average thickness of 0.7 μm.

[0259] like Figure 8 As shown, on each sample, the enzyme solution stock solution and 1 / 10 dilution of Example C-1 (3) were each added at four points in portions of 5 μl. Subsequently, the area and transparency of the transparent area resulting from the decomposition of the resin sprayed on the culture medium by the enzyme solution were visually measured over time.

[0260] The relative biodegradability of the enzyme solutions of Comparative Example C-1 and Comparative Example C-2 was evaluated in the same manner as above.

[0261] The entire biodegradability assessment was conducted at 28°C and atmospheric pressure (760±20 Torr).

[0262] (5) Results Analysis

[0263] Figure 9 This is an assessment of the biodegradability of PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) in an industrial composting environment, measured according to ISO 14855 (determination of the final aerobic biodegradability of plastic materials under controlled composting conditions – by analysis of released carbon dioxide). (Refer to...) Figure 9 It can be determined that PBAT compound 2 has higher biodegradability than PBAT compound 1.

[0264] Figure 10 The results of the biodegradability assessment of (4) are the sample photographs taken 4 hours and 30 minutes after the addition of the hydrolytic enzyme. Figure 10 The images were taken with a black screen added to the sample, and the parts that appear black are actually transparent areas. The results of Example C-1 are represented by (1), the results of Comparative Example C-1 by (2), and the results of Comparative Example C-2 by (3).

[0265] Reference Figure 10 (1) When the enzyme solution of Example C-1 was added and the same amount of time had elapsed, it was determined that the transparent area (the part that appears black) of the sample of PBAT compound 2, which has excellent biodegradability, was clearer than that of the sample of PBAT compound 1.

[0266] However, refer to Figure 10 Based on (2) and (3), it can be determined that no significant difference was observed between PBAT compound 1 and PBAT compound 2 in the cases of enzyme solution containing only keratinase in Comparative Example C-1 and enzyme solution containing only lipase in Comparative Example C-2.

[0267] Therefore, based on the above experimental results, it can be determined that by using the enzyme solution of this disclosure for assessing the biodegradability of biodegradable resins, the relative biodegradability of biodegradable resins in soil / compost environments can be predicted simply and reliably.

[0268] Example C-2: Biodegradability assessment in marine environments

[0269] (1) Preparation of culture medium

[0270] The culture medium was prepared in the same manner as in (1) of Example C-1.

[0271] (2) Preparation of resin solution

[0272] 1) Preparation of P3HP1 solution

[0273] A solution of P3HP 1 was prepared by dissolving poly-3-hydroxypropionic acid (P3HP, LGChem) sterilized with 70% ethanol and a weight-average molecular weight (Mw) of 120,000 g / mol in 20 ml of chloroform at a weight of 1%.

[0274] 2) Preparation of P3HP2 solution

[0275] P3HP (LG Chem) sterilized with 70% ethanol and a weight-average molecular weight (Mw) of 70,000 g / mol was dissolved in 20 ml of chloroform at a concentration of 1% by weight to prepare P3HP 2 solution.

[0276] (3) Preparation of enzyme solution

[0277] An enzyme solution (stock solution) is prepared by mixing one type of lipase and one type of keratinase with 1 ml of 1X phosphate-buffered saline (PBS). Additionally, a 1 / 10 dilution of the stock solution is prepared.

[0278] 3000 units of Pseudomonas cepacia lipase

[0279] 1500 units of specific humic mold keratinase

[0280] (4) Biodegradability assessment

[0281] The biodegradability of P3HP 1 resin and P3HP 2 resin was evaluated using the resin solutions of (2) and the enzyme solution of (3) in the same manner as in (4) of Example C-1.

[0282] (5) Results Analysis

[0283] Figure 11 This is an assessment of the biodegradability of P3HP 1, P3HP 2, and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) in a marine environment, measured according to ASTM D6691 standard. (See reference...) Figure 11 It can be determined that P3HP2 has higher biodegradability than P3HP1.

[0284] Figure 12 The results of the biodegradability assessment (4) are photographs of the samples taken 18 hours after the addition of the hydrolytic enzyme. When the same amount of time has elapsed, it can be determined that the transparent area of ​​the P3HP 2 sample, which has excellent biodegradability, is clearer than that of the P3HP 1 sample.

[0285] Therefore, based on the above experimental results, it can be determined that by using the enzyme solution of this disclosure for assessing the biodegradability of biodegradable resins, the relative biodegradability of biodegradable resins in marine environments can be predicted simply and reliably.

[0286] Example D-1: Biodegradability Assessment and Image Analysis

[0287] (1) Preparation of culture medium

[0288] Circular 1% agar plates with a diameter of 90 mm were prepared using the method for preparing culture medium in petri dishes.

[0289] The 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 then autoclaved at 121°C and 0.1 MPa for 15 minutes. The sterilized solution was dispensed in 10 ml portions into sterile 90 mm diameter petri dishes to prepare the culture medium.

[0290] (2) Preparation of biodegradable resin solution

[0291] A resin solution was prepared by completely dissolving the biodegradable resin in 20 ml of chloroform at a concentration of 1% by weight.

[0292] (3) Preparation of enzyme solution

[0293] Enzyme solutions were prepared by mixing the following two types of lipase, two types of keratinase, and one type of α-amylase with 1 ml of 1X phosphate-buffered saline (PBS).

[0294] 1000 units of Rhizopus oryzae lipase

[0295] 3000 units of Pseudomonas cepacia lipase

[0296] Aspergillus oryzae cutinase 2000 units

[0297] 1500 units of specific humic mold keratinase

[0298] Bacillus licheniformis α-amylase 1500 units

[0299] (4) Biodegradability assessment and image acquisition

[0300] On the agar plate of (1), spray 1 ml to 3 ml of the resin solution prepared in (2) evenly and dry at room temperature (25°C) for about 10 minutes to allow the solvent to evaporate, thereby preparing a sample with a thickness of 0.7 μm.

[0301] Seal the agar plate and mount it on the JuLI image measurement / analysis device. TM On a stage (manufactured by NanoEntek). The enzyme solution prepared in (3) was dropped onto a biodegradable resin layer on an agar plate. Using JuLI TM The stage acquires real-time images at certain time intervals while keeping the image acquisition (capturing) conditions constant.

[0302] The measurement conditions are summarized in the table below.

[0303] [Table 3]

[0304]

[0305] (5) Image analysis

[0306] By analyzing the images obtained above, the brightness of the image immediately following enzyme addition and the brightness of images taken after each elapsed time were determined, and the average brightness difference was plotted against the elapsed time. The curve obtained by plotting was fitted with a sigmoid function, and the time elapsed when the second derivative of the function reached its maximum value was determined.

[0307] Figure 13 This is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the present disclosure.

[0308] Reference Figure 13In the area where the biodegradable resin is located, an "observation area" can be identified in which the white biodegradable resin in the center of the corresponding area is hydrolyzed and appears in a lighter color than the periphery. In the "observation area", an "exclusion area" can be identified in which other residues generated during the process of adding the enzyme to the resin, rather than those formed by hydrolysis through the reaction of the enzyme with the resin, form a dark background color through interaction with the resin.

[0309] That is, as hydrolysis proceeds, the brightness of the biodegradable resin region becomes brighter, and then, if hydrolysis exceeds a certain point, an "exclusion region" appears that actually becomes darker. Therefore, for accurate assessment, it is necessary to exclude the exclusion region from the assessment, as well as in other cases where the brightness differs abnormally from the surrounding area.

[0310] Figure 14 This is an image showing the degree of biodegradation of a biodegradable resin obtained according to one embodiment of the present disclosure.

[0311] More specifically, in Figure 14 In the middle, the left image is a post-processed image taken immediately after the addition of the hydrolase, excluding the exclusion area (black circle) from the observation area. Figure 14 The middle image, taken after a certain time following the addition of the hydrolytic enzyme, has been post-processed to exclude the excluded area from the observation area, and it can be determined that the brightness of the observation area is slightly brighter than that of the left image. Figure 14 The right image in the diagram shows the calculated brightness difference between the left and middle images.

[0312] By processing as described above, the presence of the brightened area and the brightness difference can be determined.

[0313] The average value of the brightness difference obtained above is plotted relative to the time elapsed since the image was captured, and fitted with a Sigmoid function.

[0314] Figure 15 This is a graph showing the image brightness difference and fitting function over time obtained according to one embodiment of the present disclosure.

[0315] Figure 15 The Sigmoid function used for fitting is in the following form. Here, t is time in minutes (min), a, b, c, and d are fitting constants obtained through fitting, and e is the natural constant (Euler number).

[0316]

[0317] Furthermore, the first and second derivative functions of this function are as follows.

[0318] ,

[0319]

[0320] Figure 16 This is a graph showing the first and second derivative functions obtained according to one embodiment of the present disclosure.

[0321] Reference Figure 16 In the second derivative function, the maximum value appears within the measurement range. This point can be understood as the time point when the brightness change of the image proceeds most rapidly (the time point when the acceleration of the change in biodegradable resin reaches its maximum value), and this time point can be specified as the time point when the resin undergoes hydrolysis and the transparent area begins to appear.

[0322] After obtaining the second derivative function and determining the time t elapsed when the second derivative reaches its maximum value, these are summarized in the table below.

[0323] [Table 4]

[0324]

[0325] Referring to Table 4, the time at which the clear zone began to appear in each experiment can be determined. If compared under the same experimental conditions (i.e., by time interval), it can be seen that the clear zone began to appear earlier in sample 2-1 (PBAT C2) than in sample 2-2 (PBAT C1). Calculating reciprocally, it can be determined that the biodegradability of sample 2-1 (PBAT C2) is approximately 70% higher than that of sample 2-2 (PBAT C1). In the case of PBSA, it can be seen that the time at which the clear zone began to appear gradually became faster from sample 3-1 to sample 3-4, and regarding the biodegradability of PBSA, sample 3-4 (PBSA C4) had the best biodegradability, followed by sample 3-3 (PBSA C3), sample 3-2 (PBSA C2), and sample 3-1 (PBSA C1).

[0326] Figure 9 The results of the biodegradability assessment in a soil environment for PBAT compound 1 (PBAT C1), PBAT compound 2 (PBAT C2), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) are shown according to ISO 14855 (determination of the final aerobic biodegradability of plastic materials under controlled composting conditions – by analysis of released carbon dioxide). (Refer to...) Figure 9 It can be determined that PBAT compound 2 (PBAT C2) has higher biodegradability than PBAT compound 1 (PBATC1).

[0327] Figure 17 The results of the biodegradability assessment in home composting for PBSA compound 1 (PBSA C1), PBSA compound 2 (PBSA C2), PBSA compound 3 (PBSAC3), PBSA compound 4 (PBSA C4), and cellulose (manufacturer: Sigma-Aldrich, product number: 310697) are shown according to ISO 14855-1. (Refer to...) Figure 17 It can be determined that they have high biodegradability in the order of PBSA C4>PBSA C3>PBSA C2>PBSA C1.

[0328] The results above show that the image analysis results based on this disclosure are consistent with the biodegradability assessment results of biodegradable resins according to ISO standards.

Claims

1. An enzyme solution comprising: Hydrolytic enzymes, said hydrolytic enzymes comprising lipases and keratinases; and Phosphate buffered saline.

2. The enzyme solution according to claim 1, The ratio of lipase to keratinase units is 0.5:1 to 4:

1.

3. The enzyme solution according to claim 1 or 2, The total amount of hydrolytic enzyme in 1 ml of the enzyme solution is between 400 units and 80,000 units.

4. The enzyme solution according to any one of claims 1 to 3, The lipase mentioned therein is a lipase selected from one or more microorganisms, including Pseudomonas Cepacia, Rhizopus Oryzae, Aspergillus niger, and Aspergillus Oryzae.

5. The enzyme solution according to any one of claims 1 to 4, The keratinase mentioned therein is a keratinase selected from one or more microorganisms, including Humicola Insolens, Aspergillus oryzae, Fusarium solani, and Pseudomonas putida.

6. The enzyme solution according to any one of claims 1 to 5, The lipases mentioned include lipases from *Pseudomonas cepacia*, and The keratinase includes keratinase from specific humic fungi.

7. The enzyme solution according to any one of claims 1 to 6, The lipases include lipases from *Pseudomonas cepacia* and *Rhizopus oryzae*, and... The keratinase comprises keratinase from specific saprophytic fungi and keratinase from Aspergillus oryzae.

8. The enzyme solution according to any one of claims 1 to 7, The lipase includes a lipase from *Pseudomonas cepacia*, and The keratinase comprises keratinase from specific saprophytic fungi and keratinase from Aspergillus oryzae.

9. The enzyme solution according to any one of claims 1 to 8, It also contains one or more enzymes selected from α-amylase, cellulase, proteinase K, proteolytic enzyme, acetyl-CoA carboxylase, and alkaline protease.

10. A method for assessing the biodegradability of biodegradable resins, comprising: i) Contact an enzyme solution containing hydrolases and phosphate-buffered saline with a biodegradable resin, wherein the hydrolases contain lipases and keratinases; as well as ii) Observe the degradation status of the biodegradable resin over time.

11. The method for assessing the biodegradability of biodegradable resins according to claim 10, Wherein i) is performed by adding the enzyme solution dropwise onto a resin sample containing biodegradable resin.

12. The method for assessing the biodegradability of biodegradable resins according to claim 10, Wherein i) is carried out by immersing a resin sample containing biodegradable resin into the enzyme solution.

13. The method for assessing the biodegradability of biodegradable resins according to claim 10, Wherein i) a sample is prepared by spraying a biodegradable resin solution onto a culture medium, and then the enzyme solution is dropped onto the sample; and ii) This is done by observing the changes in the transparent areas formed on the sample over time.

14. The method for assessing the biodegradability of biodegradable resins according to any one of claims 10 to 13, Wherein ii) includes: a) Obtain an image of the biodegradable resin based on the elapsed unit time; as well as b) Assess biodegradability by observing changes in images of resin samples over time.

15. The method for assessing the biodegradability of biodegradable resins according to claim 14, Where b) includes: b-1) For each image of the resin sample obtained based on the elapsed unit time, obtain the brightness difference with the image immediately following the addition of the hydrolytic enzyme; b-2) Express the image brightness difference as a function of the elapsed unit of time; as well as b-3) Assess biodegradability by analyzing the function.

16. The method for assessing the biodegradability of biodegradable resins according to claim 15, (b-3) The biodegradability of the biodegradable resin is evaluated by determining the time when the second derivative of the function reaches its maximum value.

Images