WO2022179268A1 - 芽孢杆菌nk101及其在降解塑料中的应用 - Google Patents

芽孢杆菌nk101及其在降解塑料中的应用 Download PDF

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WO2022179268A1
WO2022179268A1 PCT/CN2021/139199 CN2021139199W WO2022179268A1 WO 2022179268 A1 WO2022179268 A1 WO 2022179268A1 CN 2021139199 W CN2021139199 W CN 2021139199W WO 2022179268 A1 WO2022179268 A1 WO 2022179268A1
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bacillus
plastic
polyolefin
biodegradable plastic
microparticle
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French (fr)
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谷尚昆
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毕节市尚昆塑料制品有限公司
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Priority to US18/449,736 priority Critical patent/US20230391979A1/en

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    • C08J3/226—Compounding polymers with additives, e.g. colouring using masterbatch techniques using a polymer as a carrier
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    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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    • C08J2323/00—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers
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    • C08J2323/00—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers
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    • C08J2423/00—Characterised by the use of homopolymers or copolymers of unsaturated aliphatic hydrocarbons having only one carbon-to-carbon double bond; Derivatives of such polymers
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    • C08J2483/00—Characterised by the use of macromolecular compounds obtained by reactions forming in the main chain of the macromolecule a linkage containing silicon with or without sulfur, nitrogen, oxygen, or carbon only; Derivatives of such polymers
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
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    • Y02W30/00—Technologies for solid waste management
    • Y02W30/50—Reuse, recycling or recovery technologies
    • Y02W30/62—Plastics recycling; Rubber recycling

Definitions

  • the invention relates to the technical field of microorganisms, in particular to a novel Bacillus NK101 and its application in degrading plastics.
  • Plastic products bear the notoriety of white pollution, but they are also indispensable supplies for human civilized life, and they are also the mother of industry. Therefore, while the world is calling for the solution of white pollution, biodegradable materials are springing up like mushrooms.
  • Biodegradable plastics refer to a class of plastics that are degraded by the action of naturally occurring microorganisms such as bacteria, molds (fungi) and algae.
  • the ideal biodegradable plastic is a polymer material with excellent performance, which can be completely decomposed by environmental microorganisms after being discarded, and finally be inorganicized to become an integral part of the carbon cycle in nature.
  • Paper is a typical biodegradable material
  • synthetic plastic is a typical polymer material. Therefore, biodegradable plastics are polymer materials that have the properties of both “paper” and “synthetic plastics”.
  • the purpose of the present invention is to provide a Bacillus NK101 with excellent properties, and the application of the Bacillus NK101 in degrading plastics.
  • Bacillus NK101 Bacillus subtilis SK01 (Bacillus subtilis SK01), preserved in China Center for Type Culture Collection, deposit number CCTCC NO: M2020812.
  • the ability to decompose organic substances is particularly strong: amylolytic enzymes, proteolytic enzymes, lipolytic enzymes, cellulolytic enzymes, and other types of decomposing enzymes with high activity.
  • the invention also provides the application of the bacillus in degrading plastics.
  • Plastics mainly refer to polyolefin plastics.
  • Polyene plastics are used as carriers of various microbial enzymes produced by Bacillus NK101. When plastic products are used, their physical properties will gradually age, and they will be buried in soil or in compost.
  • the microbial enzymes produced by Bacillus NK101 are released from polyene plastics, thereby inducing the growth and proliferation of environmental indigenous bacteria on the body of plastic products, resulting in accelerated aging of plastic products, and then being swallowed and metabolized by metazoans in the environment. For the purpose of reducing decomposition, this effect is referred to as the "bio-lunch" of the bio-energy anti-corrosion carrier.
  • the amount of Bacillus in the plastic is 0.05-0.2% (0.5-2kg/1000kg) of the total weight of the plastic.
  • the present invention also provides a Bacillus microparticle capsule, which is composed of the above-mentioned Bacillus NK101 coated in a coating film, and the coating film material is lactic acid or chitosan, and methyl silicone oil.
  • the dosage of each component is: Bacillus 49-53%, lactic acid or chitosan 45-47%, methyl silicone oil 1-4%.
  • the present invention also provides a method for preparing the above-mentioned Bacillus microparticle capsules, which comprises the following steps: mixing, drying, grinding, sieving, and fine grinding, adding methylsilicone oil and kneading the Bacillus NK101 and the coating material to form a coating film covering the Bacillus of microparticle capsules.
  • the present invention also provides a biodegradable plastic master batch, which is made from the above-mentioned Bacillus microparticle capsules, polyolefin, calcium carbonate, dispersant and zinc stearate through banburying and granulation.
  • the dosage of each component is: 0.3-1% of Bacillus microparticle capsule, 12-16% of polyolefin, 80-87% of calcium carbonate, 2% of dispersant , Zinc stearate 1%.
  • the present invention also provides a method for preparing the above-mentioned biodegradable plastic masterbatch, comprising the following steps:
  • Bacillus microparticle capsule, polyolefin, calcium carbonate, dispersant and zinc stearate are stirred and mixed to obtain mixture;
  • the banburying and fusing temperature is 170-202°C, and the time is 25-35 minutes; the granulation temperature of the screw extruder is 190-202°C, and the time is 2-5 minutes. Its activity is not lost or reduced in this warm state.
  • the present invention also provides a biodegradable plastic, which is made of the above-mentioned biodegradable plastic master batch and polyolefin material, and the addition amount of the biodegradable plastic master batch is 5-50% of the total weight of the biodegradable plastic.
  • the present invention has the following beneficial effects:
  • the Bacillus NK101 of the present invention can produce a variety of enzymes, has a strong ability to decompose organic matter, and is suitable for degrading plastics and other purposes.
  • Bacillus microparticle capsules are coated with lactic acid coating film or chitosan coating film, so that it can withstand 340°C/0.35hr high temperature under extremely anaerobic and high temperature environments, such as dry heat, and the components are not destroyed. To ensure that it can degrade when making biodegradable plastics in the future.
  • Biodegradable plastic masterbatch is used for the preparation of finished degradable plastics.
  • the masterbatch can isolate the damage of Bacillus (NK101) microbial enzymes caused by high temperature, reduce the specific gravity difference with polyene plastics, and the shelf life can reach more than 1 year.
  • NK101 Bacillus
  • the microbial enzymes produced by Bacillus NK101 can be slowly degraded in compost, soil burial environment (humidity, temperature) for 153 days, the total degradation of organic solids is 49.11%, sunlight exposure (photodegradation) or soaking in water, excluding the above environment,
  • the storage period of the finished polyolefin plastics added with Bacillus (NK101) microbial enzymes is consistent with that of non-degradable plastics, which can meet the needs of various polyolefin plastics.
  • Fig. 1 is a schematic diagram of the structure of biodegradable plastic masterbatch
  • Figure 2 is a 3D tunneling camera image of Bacillus NK101 in plastic products
  • Figure 3 is a 3D tunneling camera image of the plastic expansion and foaming after the microbial enzyme produced by Bacillus NK101 interacts with the plastic;
  • Figure 4 is a size display of Bacillus NK101 in plastic products (3D tunneling camera image);
  • Figure 5 is a graph of carbon dioxide release during the cellulose degradation of the 2012E0568 sample and the reference material.
  • Figure 6 is a graph of the biodegradation rate of the 2012E0568 sample and the reference material during cellulose degradation.
  • Bacillus NK101 primary strain used in the present invention is taken from withered straw, and the specific steps of its screening are as follows:
  • C source basal medium yeast extract 3g/L, peptone 10g/L, NaCl 5g/L, pH 7.2-7.4.
  • N source basal medium glucose 5g/L, NaCl 5g/L, pH7.2-7.4.
  • the selected strains were cultured in the C source basal medium, and glucose was used as the C source to prepare the medium containing different glucose concentrations.
  • the medium was divided into 25mL in a 250mL Erlenmeyer flask, and the influence of the glucose concentration on the spore formation of the primary strain was investigated.
  • N source basal medium peptone, yeast extract, corn steep liquor powder, soybean meal, soybean meal powder, peanut meal meal, fish meal and yeast powder 10.0g/L each; Nitrogen: 20.0 g/L each of urea, NH 4 Cl and (NH 4 ) 2 SO 4 , 25 mL of culture medium was divided into a 250 mL Erlenmeyer flask, and the effects of different nitrogen sources on the spore formation of the protist strain were investigated.
  • NaCl, CaCO 3 , MgSO 4 .7H 2 O, KH 2 PO 4 , K 2 HPO 4 , K 2 HPO 4 +KH 2 PO 4 were added to the medium with optimized carbon source and nitrogen source, respectively, to prepare the medium , 250mL conical flask was divided into 25mL medium, and the effect of different inorganic salts on the spore formation of the primary strain was investigated.
  • the effects of inoculum amount and aeration amount on the number of fermented bacteria and spore formation were investigated by single factor test.
  • Set the pH of the medium to 7.0 divide 25mL of medium into a 250mL conical flask, inoculate the strains with a spore rate of more than 90% screened in the previous steps, so that the number of spores in the shake flask is 10 4 , 10 5 , 10 6 , and 10 , respectively. 7 orders of magnitude, shake flask culture for 24 hours, and investigate the effect of inoculum on the formation of Bacillus spores.
  • 25mL and 50mL of culture medium were divided, and the effect of aeration on bacterial growth and spore formation was investigated.
  • Bacillus NK101 strain that can produce acidic biological enzymes under the condition of organic matter activation was obtained. It can produce various proteases (especially alkaline proteases), saccharification enzymes, lipases, amylases according to environmental nutrient sources. .
  • Bacillus NK101 was implanted in polymer plastics, and the plastics were affected by acid-base or temperature changes in the environment or ultraviolet radiation, resulting in physical deterioration.
  • the Bacillus NK101 implanted in plastic polymers was released due to the deterioration of the carrier. (For example: after the cracked food packaging is broken, the food is parasitized by mold and moldy), the released Bacillus NK101 rapidly spreads and grows with the organic matter in the environment as the nutrient source. The types of organic matter in the environment are different, so the transformed organisms Enzymes belong to diversity.
  • the organic matter of the plastic high-scoring that was originally used as the carrier of Bacillus NK101 enters the nutritious food chain synchronously, and is finally degraded into carbon dioxide, inorganic salts and water.
  • the Bacillus NK101 can obtain the properties of high temperature resistance and promoting plastic foaming under the condition of being protected by excipients.
  • Bacillus subtilis SK01 Bacillus subtilis SK01.
  • Bacillus NK101 microorganism (weight percent of solids 49-53%) and chitosan (weight percent of solids 45-47%) are mixed and dried at 43-45°C at low temperature ⁇ grinding ⁇ 1250-1500 mesh sieving ⁇ fine grinding (to sub-micron level, 10 -8 m) ⁇ add methyl silicone oil (1-4% by weight of solids) and knead ⁇ complete coating ⁇ Bacillus microparticle capsules.
  • Chitosan is obtained from shrimp and crab shells after acid-dissolving, and can also be directly used to buy commodities. It has the characteristics of high temperature resistance and high temperature formation of polymers. After mixing with Bacillus NK101, it can effectively protect Bacillus NK101 at 340 Its activity is not lost below °C.
  • the function of adding methylsilicone oil for mixing is to prevent Bacillus NK101 and chitosan from being mixed with low temperature drying ⁇ grinding ⁇ sieving ⁇ fine grinding.
  • the decomposition temperature of methyl silicone oil is around 316°C, and the decomposition temperature also increases as the amount of methyl group replaced by propyl group increases. When the propyl content is 30%, the decomposition temperature reaches 400°C.
  • chitosan polymer is used as a parasitic carrier and then coated with methyl silicone oil to achieve high dispersion and high performance. Cohesion and high temperature resistance.
  • Bacillus microparticle capsules can withstand 340°C/0.35hr high temperature under dry heat, and the components are not destroyed.
  • the mixture is sent to a high-temperature internal mixer, and is melted and fused for 25-35 minutes at a temperature of 170-202°C, and then extruded and granulated by a screw extruder at a temperature of 190-202°C for 2-5 minutes to obtain pellets.
  • the pellets are sequentially cooled by air supply, sieved, aggregated, weighed, and bagged to obtain biodegradable plastic masterbatches.
  • the microstructure of the biodegradable plastic masterbatch is in the form of airbag wrapping.
  • Bacillus self-absorbs gas, and the gas is blocked by the polymer due to banburying and melting to form airbag foam.
  • the air bag expands more and more, and the bacillus in the air bag floats in the gas.
  • the bacillus is close to the high temperature of the air bag wall, it instinctively moves to the lower temperature space.
  • Figure 2-4 From the 3D tunneling camera, you can see that the volume of the air bag is between 200 and 250 nm, and the volume of Bacillus is between 8 and 25 nm. Therefore, the gas in the air bag indirectly or directly protects the Bacillus to maintain at high temperature.
  • the body temperature is constant, and the influence of temperature on Bacillus can be reduced or even destroyed by physical adjustment.
  • the biodegradable plastic masterbatch can reduce the specific gravity difference with polyolefin plastics.
  • the specific gravity of polyolefin plastics with Bacillus NK101 added is 0.919 to 0.926, which is higher than that of polyolefin plastics without Bacillus NK101 ( The specific gravity is 0.923 ⁇ 0.93) 0.04 lighter.
  • the plastics made from biodegradable plastic masterbatches can degrade slowly in compost, soil burial environment (humidity, temperature) for 153 days, the total degradation of organic solids is 49.11%, sunlight exposure (photodegradation) or immersion in water. , Excluding the above environment, the storage period of plastic finished products made of biodegradable plastic masterbatch is consistent with that of non-degradable plastics, and the storage period can reach more than 1 year.
  • Biodegradable plastic masterbatches can be used to manufacture polyolefin products, such as films, bags, blister products, and rigid plastics.
  • the amount of polyolefin materials used can be adjusted to 50-95% according to physical requirements.
  • Biodegradable plastic masterbatches use The amount ranges from 5 to 50%.
  • the numerical values and numerical range percentages in this embodiment are all weight percentages.
  • each two-year verification shows (certified by Beijing National Plastic Products Quality Supervision and Inspection Center, National Plastic Inspection [2013] C0292), adding biodegradable plastic masterbatch to polyolefin materials, if it reaches more than 5%, it has the ability to induce biological corrosion.
  • the performance of degrading polyolefin organic matter, the addition ratio is inversely proportional to the physical property requirements. The more the biodegradable plastic masterbatch is added, the stronger the biodegradability of the corrosion inducer, and the worse the lateral and longitudinal tensile force and extension force of the physical properties.
  • the plastic film thickness is 0.008mm
  • the masterbatch addition ratio is 5% as the base point. For each increase of 0.001mm, the addition ratio can be increased by 1-1.2%, and 50% is the highest addition limit.
  • the content of the finished polyolefin material is 54%, and the content of the biodegradable plastic masterbatch is 46%, and a 0.035mm film sample is made.
  • the polyolefin degradation rate of the polyolefin material (calcium carbonate is inorganic) reaches 90.9% in 153 days.
  • the reference material is cellulose.
  • Figure 5 is a graph of carbon dioxide release during the cellulose degradation of the 2012E0568 sample and the reference material.
  • the biodegradable plastic masterbatch erodes the biodegradable polyolefin polymer, and its main principle is to implant the deep-processed Bacillus NK101 into the polyolefin material. , Extrusion, injection, film formation or bag making at high temperature, the components are not destroyed. When the bag and film are buried in soil or compost after use, Bacillus NK101 grows and reproduces, and the produced microbial enzymes induce indigenous bacteria to gather and accumulate.
  • test results of lead, cadmium, mercury, hexavalent chromium, polybrominated biphenyls (PBB), and polybrominated diphenyl ethers (PBDE) for products with biodegradable plastic masterbatches comply with the EU RoHS Directive 2002/96/EC Reformulation Directive 2011/ 65/EU Annex II limit requirements.
  • test results are as follows:
  • the packaging bag with biodegradable plastic masterbatch has a length of 700 ⁇ 5mm, a width of 510 ⁇ 5mm, the sutures are bilateral, and the number of monofilaments within 100mm is not less than 47.
  • Applicable temperature of packaging bag ⁇ 100°C.
  • Moisture-proof performance of packaging bags 3-day compressive strength ratio ⁇ 90%.
  • Single-filament tensile test results single-filament tensile qualified rate ⁇ 80%.
  • Biodegradable plastic masterbatch is different from food-based materials (biodegradable plastics based on natural substances such as starch currently mainly include the following products: polylactic acid (PLA), polyhydroxyalkanoate (PHA), starch plastics, biological Engineering plastics, bio-universal plastics: polyolefin and polyvinyl chloride, etc.), with the mechanism of eroding biodegradable polyolefin polymers, biodegradable plastic products can be produced without any changes, additions or modifications in the production process and equipment. Affordable cost has strong market competitiveness.

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Abstract

一种芽孢杆菌NK101及其在降解塑料中的应用，该芽孢杆菌NK101保藏于中国典型培养物保藏中心，保藏编号CCTCC NO:M2020812，具有较强的降解塑料的能力，可用于制备可降解塑料，能够隔离高温对芽孢杆菌NK101的破坏，缩小与聚烯类塑料的比重差异，保存期可达1年以上，确保芽孢杆菌NK101产生的微生物酶在堆肥、土壤掩埋的环境中153天有机固体物总降解量49.11％、阳光照射(光降解)或浸泡水中可缓速降解，排除以上的环境，添加芽孢杆菌NK101的聚烯类塑料成品的保存期与无降分解塑料是一致的，可应付各种聚烯类塑料的需求。

Description

芽孢杆菌NK101及其在降解塑料中的应用 技术领域
本发明涉及微生物技术领域，具体涉及一种新型的芽孢杆菌NK101，及其在降解塑料中的应用。
背景技术
塑料制品背负着白色污染的恶名，但也是人类文明生活不可或缺的用品，更是工业之母，因此在全球大声疾呼解决白色污染的同时，生物降解材料如雨后春笋般百家争鸣。
生物降解塑料是指一类由自然界存在的微生物如细菌、霉菌(真菌)和藻类的作用而引起降解的塑料。理想的生物降解塑料是一种具有优良的使用性能、废弃后可被环境微生物完全分解、最终被无机化而成为自然界中碳素循环的一个组成部分的高分子材料。“纸”是一种典型的生物降解材料，而“合成塑料”则是典型的高分子材料。因此，生物降解塑料是兼有“纸”和“合成塑料”这两种材料性质的高分子材料。
要大规模使用，最终需要以成本为第一考虑重点，目前生物降解塑料的研发成果还无法做到低成本、可工业化生产的程度。
发明内容
本发明的目的在于提供一种特性优异的芽孢杆菌NK101，以及该芽孢杆菌NK101在降解塑料中的应用。
本发明技术方案详述如下：
芽孢杆菌NK101，菌株名称为枯草芽孢杆菌SK01(Bacillus subtilis SK01)，保藏于中国典型培养物保藏中心，保藏编号CCTCC NO:M2020812。
该菌株的生物学特性如下：
(1)该株芽孢杆菌菌体增殖快速，与其他菌种并存时具有繁殖优势。
(2)其耐温度特性如下：
1)活跃温湿度：温度15～85℃/湿度30～100％；
2)休眠湿温度：温度零下60℃～14℃、86℃～119℃/湿度30～100％；
3)死亡温湿度：温度零下60℃以下、120℃以上/湿度30～100％；
4)死亡温湿度：温度340℃/湿度0.01～0.2％/20秒。
Figure PCTCN2021139199-appb-000001
(3)孢子抗逆境能力强，在活跃温湿度孢子出芽快速。
(4)有机物质分解力特强：具有高活性的淀粉分解酶、蛋白质分解酶、脂肪分解酶、纤维素分解酶、及其它种类的分解酶。
(5)针对腐败菌、恶臭菌及病源菌的抑制力特高：对金黄色葡萄球菌(含MRSA)、沙门氏菌、大肠菌(含O157:H7)、赤痢菌、肠炎弧菌、绿脓菌、退伍军人杆菌等重要病原细菌；镰刀菌(Fusarium)、格孢菌Alternarium、Botrytis、Pestalotia等植物病原性真菌具有拮抗能力。
本发明还提供了该芽孢杆菌在降解塑料方面的应用。塑料主要指聚烯类塑料。
聚烯类塑料作为芽孢杆菌NK101产生的各类微生物酶的载体，当塑料制品被使用后，物性渐近老化，待埋入土壤或处于堆肥中，通过带有酸性水分的浸湿以及表面氧化，芽孢杆菌NK101产生的微生物酶便从聚烯类塑料中释出，从而诱发环境土著菌在塑料制品本体上生长扩繁，造成塑料制品的加速老化，再经过环境中的后生动物吞食代谢，最终达到降分解的目的，此作用简称为生物能反蚀载体“生物便当”。
优选的，做上述应用时，塑料中芽孢杆菌的添加量为塑料总重的0.05～0.2％(0.5～2kg/1000kg)。
本发明还提供了一种芽孢杆菌微粒胶囊，由上述芽孢杆菌NK101包覆在衣膜中构成，所述衣膜材料为乳酸或壳聚糖，和甲基硅油。
优选的，上述芽孢杆菌微粒胶囊，以干燥固体形态重量百分比计，各成分用量为：芽孢杆菌49～53％，乳酸或壳聚糖45～47％，甲基硅油1～4％。
本发明还提供了上述芽孢杆菌微粒胶囊的制备方法，是将芽孢杆菌NK101和衣膜材料经混合、干燥、研磨、过筛、精磨后添加甲基硅油混炼，形成衣膜包覆芽孢杆菌的微粒胶囊。
本发明还提供了一种生物降解塑料母粒，是由上述芽孢杆菌微粒胶囊、聚烯 烃、碳酸钙、分散剂和硬脂酸锌经密炼造粒制成。
优选的，上述生物降解塑料母粒，以干燥固体形态重量百分比计，各成分用量为：芽孢杆菌微粒胶囊0.3～1％，聚烯烃12～16％、碳酸钙80～87％，分散剂2％，硬脂酸锌1％。
本发明还提供了上述生物降解塑料母粒的制备方法，包括以下步骤：
(1)将芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌搅拌混匀，得混合物；
(2)混合物经密炼机进行密炼熔合后，螺杆挤出机造粒；
(3)送风冷却，过筛，得产品。
优选的，上述制备方法中，步骤(2)中密炼熔合温度为170～202℃，时间25～35分钟；螺杆挤出机造粒的温度190～202℃，时间2～5分钟。在此温状态下其活性并不被灭失或减少。
本发明还提供了一种生物降解塑料，由上述生物降解塑料母粒和聚烯烃材料制成，生物降解塑料母粒的添加量为生物降解塑料总重量的5～50％。
与现有技术相比，本发明具有以下有益效果：
本发明的芽孢杆菌NK101能够产生多种酶类，对有机质的分解能力较强，适用于降解塑料等用途。
芽孢杆菌微粒胶囊使用乳酸衣膜、或壳聚糖衣膜进行包覆，使其在极度厌氧和高温的环境下，如干热情况下能承受340℃/0.35hr高温，成分不被破坏。确保其后续制作生物可降解塑料时能够发挥降解作用。
生物降解塑料母粒用于制备成品可降解塑料，该母粒能够隔离高温对芽孢杆菌(NK101)微生物酶的成分破坏，缩小与聚烯类塑料的比重差异，保存期可达1年以上，确保芽孢杆菌NK101产生的微生物酶在堆肥、土壤掩埋的环境中(湿度、温度)153天有机固体物总降解量49.11％、阳光照射(光降解)或浸泡水中可缓速降解，排除以上的环境，添加芽孢杆菌(NK101)微生物酶的聚烯类塑料成品的保存期与无降分解塑料是一致的，可应付各种聚烯类塑料的需求。
保藏信息：
培养物名称：枯草芽孢杆菌SK01，拉丁文名称：Bacillus subtilis SK01；
保藏机构：中国典型培养物保藏中心，地址：中国.武汉.武汉大学；
保藏日期：2020年12月2日，保藏编号CCTCC NO:M2020812。
附图说明
图1为生物降解塑料母粒结构示意图；
图2为芽孢杆菌NK101在塑料制品中的3D穿隧式摄像图；
图3为芽孢杆菌NK101产生的微生物酶与塑料作用后使塑料膨胀发泡的3D穿隧式摄像图；
图4为芽孢杆菌NK101在塑料制品中的尺寸展示(3D穿隧式摄像图)；
图5为2012E0568试样和参比材料纤维素降解过程中二氧化碳释放量曲线图。
图6为2012E0568试样和参比材料纤维素降解过程中生物分解率曲线图。
具体实施方式
实施例1 芽孢杆菌NK101的获取及特性检测
本发明所用的芽孢杆菌NK101原生菌株取自枯干稻草，其筛选具体步骤如下：
1、芽孢杆菌NK101培养基优化、培养条件的筛选
(1)C源基础培养基：酵母膏3g/L，蛋白胨10g/L，NaCl 5g/L，pH7.2-7.4。
(2)N源基础培养基：葡萄糖5g/L，NaCl 5g/L，pH7.2-7.4。
培养筛选出来的菌株在C源基础培养基中，以葡萄糖为C源，配制含不同葡萄糖浓度的培养基，250mL三角瓶内分装培养基25mL，考察葡萄糖浓度对原生菌株芽孢形成的影响。
以葡萄糖浓度为C源，在N源基础培养基中分别添加有机氮：蛋白胨、酵母膏、玉米浆粉、黄豆粉、黄豆饼粉、花生饼粉、鱼粉和酵母粉各10.0g/L；无机氮：尿素、NH 4Cl和(NH 4) 2SO 4各20.0g/L，250mL三角瓶内分装培养基25mL，考察不同氮源对原生菌株芽孢形成的影响。
在优化的碳源和氮源的培养基中，分别添加NaCl、CaCO 3，MgSO 4.7H 2O，KH 2PO 4、K 2HPO 4、K 2HPO 4+KH 2PO 4，配制培养基，250mL三角瓶内分装培养基25mL，考察不同无机盐对原生菌株芽孢形成的影响。
在优化的培养基中，通过单因子试验考察接种量、通气量对发酵菌数及芽孢形成的影响。设培养基pH为7.0，250mL三角瓶内分装培养基25mL，接种前述 步骤中筛选出来的芽孢率90％以上的菌株，使摇瓶中芽孢数量分别为10 4，10 5，10 6，10 7数量级，摇瓶培养24h，考察接种量对芽孢杆菌芽孢形成的影响。在250mL摇瓶中，分别分装培养基25mL和50mL，考察通气量对菌体生长和芽孢形成的影响。
经过多轮筛选，获得一株在有机物激活条件下能产生偏酸性生物酶的芽孢杆菌NK101，其能够依环境营养源生成多种蛋白酶(特别是碱性蛋白酶)、糖化酶、脂肪酶、淀粉酶。
经研究发现，该芽孢杆菌NK101被植入高分子塑料中，塑料受到环境中酸碱或温度变化或紫外线照射的影响，发生物性劣化，植入在塑料高分子的芽孢杆菌NK101因载体劣化而释放(例如：破裂的食品包装破裂后，食品受到霉菌寄生而发霉)，被释放的芽孢杆菌NK101以环境中有机物为营养源迅速扩繁生长，环境中的有机物种类不一，因此被转换形成的生物酶属于多样性，原作为芽孢杆菌NK101载体的塑料高分仔，其有机物同步进入营养食物链，最终被分降解为二氧化碳、无机盐、水。该芽孢杆菌NK101在受到辅料保护的情况下能够取得抗高温以及促成塑料发泡的特性。
该芽孢杆菌NK101菌株的保藏分类命名为枯草芽孢杆菌SK01。
实施例2 生物降解塑料制备及降解检测
1、芽孢杆菌微粒胶囊制备
芽孢杆菌NK101微生物(固体物重量百分比49～53％)和壳聚糖(固体物重量百分比45～47％)混合后经过43～45℃低温烘干→研磨→1250～1500目过筛→精磨(达到次微米级，10 -8m)→加入甲基硅油(固体物重量百分比1～4％)混炼→包覆完成→芽孢杆菌微粒胶囊。
壳聚糖取自虾蟹壳经过酸溶后取得，也可以直接够买商品，具有耐高温、高温下形成聚合物的特点，与芽孢杆菌NK101混合为一体后，可有效保护芽孢杆菌NK101在340℃以下其活性不被灭失。
加入甲基硅油混炼的作用是防止芽孢杆菌NK101和壳聚糖混合后经过低温烘干→研磨→过筛→精磨后其粉末达到次微米级(10 -8)的状态不会被挥发，甲基硅油分解温度在316℃左右，随着甲基被丙基取代量增加，分解温度亦增加。丙基含量为30％时，分解温度达到400℃。
因塑料制品其制备过程中需要提供170℃以上的高温才能熔解聚合，因此为保护其活性不被灭失，利用壳聚糖高分子作为寄生的载体再通过甲基硅油的包覆达到高分散、高聚合力、抗高温的特性。
芽孢杆菌微粒胶囊干热情况下能承受340℃/0.35hr高温，成分不被破坏。
2、生物降解塑料母粒制备
将芽孢杆菌微粒胶囊0.3～1％、聚烯烃树脂塑料12～15％、细度达到1250网目以上的碳酸钙粉75～81％、分散剂2％和硬脂酸锌1％投入到料桶中，搅拌混合均匀。
混合物送入高温密炼机，在170～202℃温度条件下密炼熔合25～35分钟，然后再经螺杆挤出机190～202℃、2～5min条件下挤出造粒，得料粒。
料粒依次经送风冷却、过筛、集料、秤重、装袋，得生物降解塑料母粒。
如图1所示，生物降解塑料母粒的微观结构为气囊包裹形态，在制备过程中，芽孢杆菌自体吸收气体，气体因密炼熔融被高分子所封锁形成气囊发泡体，当温度升高气囊越发膨胀，气囊内的芽孢杆菌漂浮在气体中，当芽孢杆菌接近气囊壁的高温，本能地向温度低的空间移动。请参考图2-4，从3D穿隧式摄像图可以窥视，气囊的体积介于200～250nm，芽孢杆菌体积介于8～25nm，因此气囊中的气体间接或直接保护芽孢杆菌在高温时维持体温的恒定，并且可通过物理性调节来减少温度对芽孢杆菌的影响甚至灭失。
该生物降解塑料母粒能够缩小与聚烯类塑料的比重差异，由于芽孢杆菌NK101与聚烯烃树脂塑料密炼熔融过程中，温度达到65℃～75℃时嗜氧扩繁生长，使聚烯类塑料产生微细气泡，此特性致使聚烯类塑料膨胀发泡，在同样的体积上，添加芽孢杆菌NK101的聚烯类塑料的比重为0.919～0.926，比不添加芽孢杆菌NK101的聚烯类塑料(比重为0.923～0.93)轻0.04。
经检测，由生物降解塑料母粒制成的塑料在堆肥、土壤掩埋的环境中(湿度、温度)153天有机固体物总降解量49.11％、阳光照射(光降解)或浸泡水中可缓速降解，排除以上的环境，由生物降解塑料母粒制成的塑料成品的保存期与无降分解塑料是一致的，保存期可达1年以上。
3、降解检测
生物降解塑料母粒可制造聚烯类成品，如膜、袋、吸塑品、硬质塑料，可依 照物性需求，调整聚烯烃材料使用量范围介于50～95％，生物降解塑料母粒使用量范围介于5～50％。本实施例中数值及数值范围百分比均为重量百分比。
根据三次实验，每次两年的验证表明(北京国家塑料制品质量监督检验中心认证，国塑检〔2013〕C0292)，聚烯烃材料添加生物降解塑料母粒，达到5％以上即具有诱蚀生物降解聚烯烃有机物的性能，添加比例与物性需求成反比。生物降解塑料母粒添加量越多，诱蚀生物降解性越强，物性的横向、纵向拉力与延伸力越差。根据实物测试，塑膜厚度0.008mm、母粒添加比例5％为基点，每增加0.001mm，添加比可增加1～1.2％，50％是最高的添加极限。
取成品聚烯烃材料含量54％，生物降解塑料母粒含量46％，制成0.035mm膜片试样，在153天对聚烯烃材料(碳酸钙为无机物)的聚烯烃降解率达到90.9％。参比材料为纤维素。
表1 试样基本特性
Figure PCTCN2021139199-appb-000002
表2 二氧化碳释放量和生物分解百分率
Figure PCTCN2021139199-appb-000003
表3 二氧化碳产生量及生物分解百分率
Figure PCTCN2021139199-appb-000004
Figure PCTCN2021139199-appb-000005
图5为2012E0568试样和参比材料纤维素降解过程中二氧化碳释放量曲线图。
本发明中生物降解塑料母粒诱蚀生物降解聚烯烃高分子，其主要作用原理是在聚烯烃类材料中植入经过深加工的芽孢杆菌NK101，此芽孢杆菌NK101具有承受高温的性能，在造粒、压出、射出、成膜或制袋的高温下成分不被破坏，当袋、膜使用后被埋入土壤中或堆肥内，芽孢杆菌NK101生长繁殖，产生的微生物酶诱引土著菌集结并进行分解，使袋、膜产生裂解成细片状(4～5cm)，经过生态化学螯合袋、膜分解成粉粒状(0.01～0.1mm)，受生态中的后生动物群吞食、消化，最终成为二氧化碳、水以及碳酸钙，还原为生态环境中的无害物。
添加生物降解塑料母粒的制品，通过FDA、RoHS、12类重金属、21类化学毒性检验、黄变4级、横纵向拉力的最高等级测试，取得了降解性能、安全与物性的优质评价。
添加生物降解塑料母粒的制品，铅、镉、汞、六价铬、多溴联苯(PBB)、多溴二苯醚(PBDE)的测试结果符合欧盟RoHS指令2002/96/EC的重订指令2011/65/EU附录Ⅱ的限值要求。
根据美国FDA法规要求，测定与食品接触而用于涂料的组分生物降解塑料母粒的聚乙烯的最大可萃取量，测试方法参考US FDA 21 CFR 177.1520 d(3)(ⅱ)&d(4)(ⅱ)，测试结果如下表：
常规模拟液 时间 温度 最大允许限值 样本检测值
正己烷 2hr 50℃ 53％(w/w) 3.7(w/w)
二甲苯 2hr 25℃ 75％(w/w) 5.7(w/w)
加入生物降解塑料母粒的包装袋，长度700±5mm，宽度510±5mm，缝线处为双边，100mm内单丝数不少于47条，经机械性能检测结果，物理性能如下表所示：
Figure PCTCN2021139199-appb-000006
牢固度：随机抽取5个包装嗲，2.5m±2mm高空自由落体后破包率≤4袋。
包装袋适用温度：≥100℃。
纸袋材料对水泥强度的影响：3天抗折强度比≥93％、3天抗压强度比≥95％.
包装袋防潮性能：3天抗压强度比≥90％。
单丝拉力检验结果：单丝拉力合格率≥80％。
生物降解塑料母粒与聚乙烯作为原材料制备的包装袋，采用GB13735-92标准进行产品检验的结果如下表所示：
Figure PCTCN2021139199-appb-000007
生物降解塑料母粒有别于粮食性材料(以淀粉等天然物质为基础的生物降解塑料目前主要包括以下几种产品：聚乳酸(PLA)、聚羟基烷酸酯(PHA)、淀粉塑料、生物工程塑料、生物通用塑料：聚烯烃和聚氯乙烯等)，以诱蚀生物降解聚烯高分子为机制，在生产工艺、设备不需要做任何变动、增加或修改即可生产生物降解塑料制品，平价的成本具有强大的市场竞争力。
本文中应用了具体个例对发明构思进行了详细阐述，以上实施例的说明只是用于帮助理解本发明的核心思想。应当指出，对于本技术领域的普通技术人员来说，在不脱离该发明构思的前提下，所做的任何显而易见的修改、等同替换或其他改进，均应包含在本发明的保护范围之内。

Claims (10)

  1. 芽孢杆菌NK101，菌株名称为枯草芽孢杆菌SK01(Bacillus subtilis SK01)，保藏于中国典型培养物保藏中心，保藏编号CCTCC NO:M2020812。
  2. 权利要求1所述芽孢杆菌在降解塑料方面的应用，其特征在于，塑料中芽孢杆菌的添加量为塑料总重的0.05～0.2％。
  3. 一种芽孢杆菌微粒胶囊，其特征在于，由权利要求1所述的芽孢杆菌包覆在衣膜中构成，所述衣膜材料为乳酸或壳聚糖，和甲基硅油。
  4. 根据权利要求3所述的芽孢杆菌微粒胶囊，其特征在于，以干燥固体形态重量百分比计，各成分用量为：芽孢杆菌49～53％，乳酸或壳聚糖45～47％，甲基硅油1～4％。
  5. 权利要求3或4所述芽孢杆菌微粒胶囊的制备方法，其特征在于，是将芽孢杆菌NK101和衣膜材料经混合、干燥、研磨、过筛、精磨后添加甲基硅油混炼，形成衣膜包覆芽孢杆菌的微粒胶囊。
  6. 一种生物降解塑料母粒，其特征在于，由权利要求3或4所述的芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌经密炼造粒制成。
  7. 根据权利要求6所述的生物降解塑料母粒，其特征在于，以干燥固体形态重量百分比计，各成分用量为：芽孢杆菌微粒胶囊0.3～1％，聚烯烃12～16％、碳酸钙80～87％，分散剂2％，硬脂酸锌1％。
  8. 权利要求6或7所述生物降解塑料母粒的制备方法，其特征在于，包括以下步骤：
    (1)将芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌搅拌混匀，得混合物；
    (2)混合物经密炼机进行蜜炼熔合后，螺杆挤出机造粒；
    (3)送风冷却，过筛，得产品。
  9. 根据权利要求8所述的制备方法，其特征在于，步骤(2)中熔合温度为170～202℃，时间25～35分钟；螺杆挤出机造粒的温度190～202℃，时间2～5分钟。
  10. 一种生物降解塑料，其特征在于，由权利要求6或7所述的生物降解塑料母粒和聚烯烃材料制成，生物降解塑料母粒的添加量为生物降解塑料总重量的5～50％。
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