WO2023044687A1 - Method for improving strength of bombyx mori cocoon silk - Google Patents

Method for improving strength of bombyx mori cocoon silk Download PDF

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Publication number
WO2023044687A1
WO2023044687A1 PCT/CN2021/120018 CN2021120018W WO2023044687A1 WO 2023044687 A1 WO2023044687 A1 WO 2023044687A1 CN 2021120018 W CN2021120018 W CN 2021120018W WO 2023044687 A1 WO2023044687 A1 WO 2023044687A1
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Prior art keywords
silkworm
cocoon silk
improving
solution
graphene oxide
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PCT/CN2021/120018
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French (fr)
Chinese (zh)
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任玉莹
范雨萌
黄曼莹
屈建威
李兵
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苏州大学
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Priority to PCT/CN2021/120018 priority Critical patent/WO2023044687A1/en
Publication of WO2023044687A1 publication Critical patent/WO2023044687A1/en

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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/04—Silkworms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/90—Feeding-stuffs specially adapted for particular animals for insects, e.g. bees or silkworms

Definitions

  • the invention relates to a method for improving cocoon silk strength of silkworm, in particular to a method for improving silkworm cocoon silk strength by adding nanometer graphene oxide.
  • the silkworm ( Bombyx mori ) is an important economic insect, which has been domesticated and raised for more than 5700 years.
  • the silkworm is a complete metamorphosis of Lepidoptera, which goes through egg, larva, pupa and adult throughout its life. Its nutrient intake is mainly during the larva feeding on mulberry leaves.
  • Silkworms can usually be divided into 5 instars, namely 1st, 2nd, 3rd, 4th and 5th instars, and the mulberry consumption of the 5th instars accounts for more than 85% of the total age.
  • the silkworm After the silkworm feeds on the mulberry leaves, it digests and absorbs the nutrients in the mulberry leaves to provide energy for its own growth and development, and also provides raw materials for the accumulation of silk substances. Therefore, the absorption and utilization of nutrients in mulberry leaves directly affect the synthesis of silk protein in silkworms, which is related to the cocoon production of silkworms. From the third day of the fifth instar, the protein eaten is mainly used for the synthesis of silk protein.
  • nanomaterials have superior thermal, magnetic, light-sensitive characteristics and surface stability than conventional particles, so they are used in various aspects.
  • Graphene oxide (GO) is widely used as a reinforcing filler for biomaterials due to its excellent mechanical properties, low price, good biocompatibility, and high reactivity.
  • Graphene oxide has been used to fabricate a series of high-performance composite materials, such as GO-SF nanomembrane with 300 MPa ultimate stress and 145 GPa modulus, GO-SF fiber with high fracture stress and high tensile strength, initial modulus A high-strength GO-SF scaffold of 4.8 GPa, and a GO-RSF biomaterial system with high Young's modulus.
  • high-performance composite materials such as GO-SF nanomembrane with 300 MPa ultimate stress and 145 GPa modulus, GO-SF fiber with high fracture stress and high tensile strength, initial modulus A high-strength GO-SF scaffold of 4.8 GPa, and a GO-RSF biomaterial system with high Young's modulus.
  • the purpose of the invention is to provide a method for improving the cocoon silk strength of the silkworm, so as to promote the high-quality development of the silk industry.
  • the technical solution adopted in the present invention is: a method for improving the cocoon silk strength of silkworm, comprising the following steps: (1) mixing nano-graphene oxide, vinyl acetate, ⁇ -sitosterol with water to obtain Solution; (2) The solution prepared in step (1) is added to 100kg of mulberry leaves with a dose of 8-12L solution to spray mature leaves, and after the mulberry leaves are dried, feed silkworms in the middle and late stages of the fifth instar to improve the strength of silkworm cocoons.
  • a feed for improving cocoon silk strength of silkworm the preparation method of which comprises the following steps: (1) mixing nanometer graphene oxide, vinyl acetate, ⁇ -sitosterol with water to obtain a solution; (2) preparing The solution is sprayed on the mature leaves, and after the mulberry leaves are dried, the feed for improving the cocoon silk strength of the silkworm is obtained.
  • the present invention develops an additive; in step (1), the concentration of nano-graphene oxide in the solution is 1.5-2.5g/L; the concentration of vinyl acetate The concentration of ⁇ -sitosterol is 1-2 mg/L; the concentration of ⁇ -sitosterol is 1-2 mg/L.
  • the sheet thickness of the nano-graphene oxide is less than 1 nm, which is the nano-graphene oxide that conventionally feeds silkworms.
  • Mature leaves are existing conventional products, which refer to the leaves whose branches are counted from the top to the seventh position, and the insect-mouthed leaves, diseased leaves and dry leaves are removed during selection.
  • air drying is natural air drying.
  • the middle and late stages of the 5th age are common sense, generally the 4th day to the 5th day of the 5th age.
  • the feeding method of the present invention is the same as the technology of silkworm rearing and conventional mulberry leaf feeding.
  • the inventiveness of the present invention lies in the limitation of the concentration of nano-graphene oxide and the use of vinyl acetate and ⁇ -sitosterol to improve the strength of silkworm cocoons without reducing the incidence of silkworm silkworms. This technical effect cannot be expected from the prior art .
  • the present invention has the following advantages: 1.
  • the present invention uses nano-graphene oxide as the main component additive to feed silkworms to increase cocoon silk strength by more than 30%, the effect is remarkable, the cocoon output is increased by more than 8%, and the quality of silkworm cocoons can be significantly improved; 2.
  • the method disclosed by the invention uses less additives, concentrates time for feeding silkworms, has wide sources of materials, and is low in cost; it does not need to change production rules, is convenient to operate, and is easy to popularize and apply.
  • Silkworm of the present invention is all existing products.
  • Silkworm species Suxiu ⁇ Chunfeng, bred and preserved in our laboratory, one silkworm (25,000 grains) was raised in each of the experimental area and the control area, and the specific feeding method was conventional.
  • Nano-graphene oxide Graphene oxide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of ⁇ 98% and a sheet thickness of less than or equal to 1 nm; ) Co., Ltd. products, all meet the requirements of conventional consumption.
  • Mature leaves are existing conventional products, which refer to leaves whose branches are counted from the top to less than 7. When selecting, insect-mouthed leaves, diseased leaves and dry leaves are removed.
  • the mulberry tree variety is Yu 711.
  • Cocoon silk strength is determined by the National Silk and Clothing Product Quality Supervision and Inspection Center. Cocoon silk strength is defined by absolute strength/cocoon silk fineness, which is a conventional method. The judgment of the disease is a conventional method for silkworm rearing.
  • Control example Add 10L of water to 100kg of mulberry leaves to spray mature leaves. After the mulberry leaves are dry, add 5-year-old silkworms. Give mulberry twice a day. The amount of mulberry is the same as the usual amount. nest.
  • Cocoon silk strength 4.06cn/D; output: 45kg; incidence number (head): 0 head.
  • Embodiment one kind of method that improves silkworm cocoon filament strength nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and ⁇ -sitosterol The content of sitosterol is 1.5 mg/L; according to the dose of 100kg mulberry leaves, add 10L solution to spray mature leaves, after the mulberry leaves are dried, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day, A total of 4 feedings were added, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
  • Cocoon silk strength 5.28cn/D, an increase of 30.05% compared with the control area; cocoon yield 49kg, an increase of 8.89% compared with the control area; number of diseased (heads): 0 head.
  • Embodiment 2 A kind of method of improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and ⁇ -sitosterol The content of sitosterol was 1.5 mg/L; add 10L solution dose to 100kg mulberry leaves to spray mature leaves, after the mulberry leaves were dried, add silkworms from the 1st day to the 3rd day of the 5th instar, twice a day for a total of Add food for 6 times, and the rest are fed with pure mulberry leaves. The amount of mulberry is the same as the usual amount, and the grid box is automatically loaded.
  • Cocoon silk strength 4.28cn/D, an increase of 5.42% compared with the control area; cocoon yield 46kg, an increase of 2.22% compared with the control area; number of diseased (heads): 0 head.
  • Embodiment three a kind of method that improves silkworm cocoon thread strength: nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and ⁇ -sitosterol The content of sitosterol is 1.5 mg/L; according to 100kg mulberry leaves, add 10L solution dose to spray mature leaves, after the mulberry leaves are dried, add silkworms from the 6th day to the 7th day of the 5th instar. Give mulberry twice, add food 4 times altogether, the amount of mulberry is the same as the usual amount.
  • Cocoon silk strength 4.45cn/D, an increase of 9.61% compared with the control area; cocoon yield 48kg, an increase of 6.67% compared with the control area; number of diseased (heads): 0.
  • Embodiment 4 Nano-graphene oxide is added into water to prepare a solution, wherein the concentration of nano-graphene oxide is 2g/L; add 10L solution dose to 100kg mulberry leaves to spray mature leaves, and add food to the 5th age after the mulberry leaves are dried. From the 4th day to the 5th day, silkworms were fed with mulberry twice a day, and the rest were fed with pure mulberry leaves. The amount of mulberry was the same as the usual amount.
  • Cocoon silk strength 3.95cn/D, an increase of -2.71% compared with the control area; cocoon yield 43kg, an increase of -4.44% compared with the control area; number of diseased (heads): 5.
  • Embodiment five vinyl acetate and ⁇ -sitosterol are added in water, prepare solution, and wherein the content of vinyl acetate and ⁇ -sitosterol is 1.5 mg/L; Add the dosage of 10L solution according to 100kg mulberry leaves and spray mature leaves, After the mulberry leaves are dried, feed silkworms on the 4th and 5th days of the 5th instar, feed mulberry twice a day, add food 4 times in total, and feed the rest with pure mulberry leaves. The stacking is automatically loaded.
  • Cocoon silk strength 3.86cn/D, number of cases (heads): 0 heads.
  • Embodiment 6 Nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added to water to prepare a solution, wherein the concentration of nano-graphene oxide is 5g/L, and the content of vinyl acetate and ⁇ -sitosterol is 1.5 mg/L L; according to 100kg mulberry leaf adding the dose of 10L solution to spray mature leaves, after the mulberry leaves are dried, add food to the 5th instar 4th day and the 5th day silkworm, give mulberry twice a day, add food 4 times altogether, all the other adopt pure Mulberry leaves are fed, and the amount of mulberry is the same as the usual amount, and the box is automatically loaded.
  • Cocoon silk strength 5.22cn/D; number of cases (heads): 58.
  • Embodiment 7 A kind of method of improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 1.5g/L, vinyl acetate and ⁇ -sitosterol -The content of sitosterol is 1.5 mg/L; add 10L solution to 100kg mulberry leaves to spray the mature leaves, after the mulberry leaves are dry, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day , fed a total of 4 times, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
  • Cocoon silk strength 5.02cn/D; number of cases (heads): 0 heads.
  • Embodiment 8 A method for improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and ⁇ -sitosterol are added to water to prepare a solution, wherein the concentration of nano-graphene oxide is 2.5g/L, vinyl acetate and ⁇ -sitosterol -The content of sitosterol is 1.5 mg/L; add 10L solution to 100kg mulberry leaves to spray the mature leaves, after the mulberry leaves are dry, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day , fed a total of 4 times, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
  • Cocoon silk strength 5.25cn/D; number of cases (heads): 0 heads.
  • the present invention can increase the cocoon strength of silkworm cocoons efficiently by feeding a certain concentration of graphene oxide (2g/L), vinyl acetate, and ⁇ -sitosterol at a specific time (5 days and 4-5 days). It can also increase the output of silkworm cocoons and has broad application prospects.
  • the existing technology uses nano-graphene oxide to add feed, hoping to improve the strength of silkworm silk. Generally, the amount used is large, at least 1/1000, and as much as 15/1000. Only considering the silk strength, there is no research on the impact of silkworm disease, etc. Limit its practical production application.
  • the present invention only uses nano-graphene oxide, vinyl acetate, ⁇ -sitosterol and pure mulberry leaves without other additives.
  • nano-graphene oxide combined with vinyl acetate and ⁇ -sitosterol is sprayed on mulberry leaves, and then according to the conventional Feeding method Adding silkworms does not require other special feeding methods.
  • silkworms it was found that while the strength of silkworms was significantly improved, the number of diseased heads was not increased, which is a method that is beneficial to production and application.

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Abstract

Disclosed in the present invention is a method for improving the strength of bombyx mori cocoon silk, comprising: first preparing a solution from nano graphene oxide, vinyl acetate and β-sitosterol; spraying mature leaves with the solution according to the dosage that 8.0-12 L of the solution is added into 100 kg of mulberry leaves; and airing, and feeding 5-year bombyx mori for 4-5 days, such that the strength of the bombyx mori cocoon silk can be improved by more than 30%, and the yield can be increased by more than 8%. According to the biological function of the nano graphene oxide and the physiological features of bombyx mori, a prepared silkworm additive is wide in source and low in cost, the disclosed method is high in usability, convenient to operate, remarkable in effect and suitable for application and popularization in silkworm areas in China.

Description

一种提高家蚕茧丝强力的方法A method for improving silkworm cocoon silk strength 技术领域technical field
本发明涉及一种提高家蚕茧丝强力的方法，具体涉及一种通过纳米氧化石墨烯添食提高家蚕茧丝强力的方法。The invention relates to a method for improving cocoon silk strength of silkworm, in particular to a method for improving silkworm cocoon silk strength by adding nanometer graphene oxide.
背景技术Background technique
家蚕( Bombyx mori)是重要的经济昆虫，已经被驯化饲养5700余年。家蚕是完全变态的鳞翅目昆虫，一生经过卵、幼虫、蛹和成虫。其营养物质的摄取主要幼虫期间取食桑叶。家蚕通常可以分为5个龄期，分别为1龄、2龄、3龄、4龄和5龄，其中5龄期的食桑量占全龄的85%以上。 The silkworm ( Bombyx mori ) is an important economic insect, which has been domesticated and raised for more than 5700 years. The silkworm is a complete metamorphosis of Lepidoptera, which goes through egg, larva, pupa and adult throughout its life. Its nutrient intake is mainly during the larva feeding on mulberry leaves. Silkworms can usually be divided into 5 instars, namely 1st, 2nd, 3rd, 4th and 5th instars, and the mulberry consumption of the 5th instars accounts for more than 85% of the total age.
家蚕取食桑叶后，通过消化、吸收桑叶中的营养物质，为自身的生长发育提供能量，也为丝物质的积累提供了原料。所以对桑叶中营养物质的吸收和利用是直接影响家蚕丝蛋白的合成，关系到家蚕的蚕茧产量，5龄第3天开始，食取的蛋白主要用于丝蛋白的合成。After the silkworm feeds on the mulberry leaves, it digests and absorbs the nutrients in the mulberry leaves to provide energy for its own growth and development, and also provides raw materials for the accumulation of silk substances. Therefore, the absorption and utilization of nutrients in mulberry leaves directly affect the synthesis of silk protein in silkworms, which is related to the cocoon production of silkworms. From the third day of the fifth instar, the protein eaten is mainly used for the synthesis of silk protein.
另一方面，纳米材料由于其独特的小尺寸效应、表面效应、量子尺寸效应及宏观量子隧道效应而具有优于常规粒子的热、磁、光敏感特性以及表面稳定性，从而应用于各个方面。氧化石墨烯（GO）由于其优异的机械性能、低廉的价格、良好的生物相容性和高反应活性被广泛用作生物材料的增强填料。氧化石墨烯已用于制造一系列高性能复合材料，如具有300 MPa极限应力、145 GPa模量的GO-SF纳米膜，具有高断裂应力和高抗拉强度的GO-SF纤维，初始模量为4.8 GPa的高强度GO-SF支架，以及具有高杨氏模量的GO-RSF生物材料系统。On the other hand, due to its unique small size effect, surface effect, quantum size effect and macroscopic quantum tunneling effect, nanomaterials have superior thermal, magnetic, light-sensitive characteristics and surface stability than conventional particles, so they are used in various aspects. Graphene oxide (GO) is widely used as a reinforcing filler for biomaterials due to its excellent mechanical properties, low price, good biocompatibility, and high reactivity. Graphene oxide has been used to fabricate a series of high-performance composite materials, such as GO-SF nanomembrane with 300 MPa ultimate stress and 145 GPa modulus, GO-SF fiber with high fracture stress and high tensile strength, initial modulus A high-strength GO-SF scaffold of 4.8 GPa, and a GO-RSF biomaterial system with high Young's modulus.
虽然石墨烯添食家蚕已有报道，但对提高茧丝性能的研究都是基于添食饲料，未见仅添食桑叶即可提高茧丝性能的报道。为了推动纳米氧化石墨烯应用于养蚕生产，迫切需要开发基于纳米氧化石墨烯的一种提高家蚕茧丝强力的方法。Although it has been reported that silkworms are fed with graphene, the research on improving the performance of cocoons is based on adding feed, and there is no report that the performance of cocoons can be improved only by adding mulberry leaves. In order to promote the application of nano-graphene oxide in silkworm production, it is urgent to develop a method based on nano-graphene oxide to improve silkworm cocoon silk strength.
技术问题technical problem
本发明的目的是提供一种提高家蚕茧丝强力的方法，以促进蚕丝产业的高质量发展。The purpose of the invention is to provide a method for improving the cocoon silk strength of the silkworm, so as to promote the high-quality development of the silk industry.
技术解决方案technical solution
为达到上述发明目的，本发明采用的技术方案是：一种提高家蚕茧丝强力的方法，包括以下步骤：（1）将纳米氧化石墨烯、乙酸乙烯酯、β－谷甾醇与水混合，得到溶液；（2）将步骤（1）制备的溶液按照100kg桑叶添加8～12L溶液剂量喷洒成熟叶，待桑叶晾干后添食家蚕5龄期中后期，实现家蚕茧丝强力的提高。In order to achieve the above-mentioned purpose of the invention, the technical solution adopted in the present invention is: a method for improving the cocoon silk strength of silkworm, comprising the following steps: (1) mixing nano-graphene oxide, vinyl acetate, β-sitosterol with water to obtain Solution; (2) The solution prepared in step (1) is added to 100kg of mulberry leaves with a dose of 8-12L solution to spray mature leaves, and after the mulberry leaves are dried, feed silkworms in the middle and late stages of the fifth instar to improve the strength of silkworm cocoons.
一种提高家蚕茧丝强力的饲料，其制备方法包括以下步骤：（1）将纳米氧化石墨烯、乙酸乙烯酯、β-谷甾醇与水混合，得到溶液；（2）将步骤（1）制备的溶液喷洒成熟叶，待桑叶晾干后，得到提高家蚕茧丝强力的饲料。A feed for improving cocoon silk strength of silkworm, the preparation method of which comprises the following steps: (1) mixing nanometer graphene oxide, vinyl acetate, β-sitosterol with water to obtain a solution; (2) preparing The solution is sprayed on the mature leaves, and after the mulberry leaves are dried, the feed for improving the cocoon silk strength of the silkworm is obtained.
本发明根据纳米氧化石墨烯的生物学功能，结合家蚕的生理特征，开发了添加剂；步骤（1）中，溶液中，纳米氧化石墨烯的浓度为1.5～2.5g/L；乙酸乙烯酯的浓度为1～2 mg/L；β-谷甾醇的浓度为1～2 mg/L。According to the biological function of nano-graphene oxide and the physiological characteristics of silkworm, the present invention develops an additive; in step (1), the concentration of nano-graphene oxide in the solution is 1.5-2.5g/L; the concentration of vinyl acetate The concentration of β-sitosterol is 1-2 mg/L; the concentration of β-sitosterol is 1-2 mg/L.
本发明中，纳米氧化石墨烯的片层厚度小于1 nm，为常规添食家蚕的纳米氧化石墨烯。成熟叶为现有常规产品，指枝条从上统计7位以下的叶片，选取时去除虫口叶、病害叶和干枯叶。In the present invention, the sheet thickness of the nano-graphene oxide is less than 1 nm, which is the nano-graphene oxide that conventionally feeds silkworms. Mature leaves are existing conventional products, which refer to the leaves whose branches are counted from the top to the seventh position, and the insect-mouthed leaves, diseased leaves and dry leaves are removed during selection.
本发明中，晾干为自然晾干。5龄期中后期为常识，一般是5龄第4天-第5天。In the present invention, air drying is natural air drying. The middle and late stages of the 5th age are common sense, generally the 4th day to the 5th day of the 5th age.
本发明的添食方法与家蚕饲养的技术和常规桑叶喂养一样。本发明的创造性在于通过纳米氧化石墨烯浓度的限定以及乙酸乙烯酯、β-谷甾醇的使用，实现了家蚕茧丝强力的提高，且不降低家蚕发病率，此技术效果无法从现有技术预期。The feeding method of the present invention is the same as the technology of silkworm rearing and conventional mulberry leaf feeding. The inventiveness of the present invention lies in the limitation of the concentration of nano-graphene oxide and the use of vinyl acetate and β-sitosterol to improve the strength of silkworm cocoons without reducing the incidence of silkworm silkworms. This technical effect cannot be expected from the prior art .
有益效果Beneficial effect
由于上述技术方案运用，本发明具有下列优点：1．本发明利用纳米氧化石墨烯作为主要成分的添加剂喂食家蚕以提高茧丝强力30%以上，效果显著，蚕茧产量增加8%以上，可显著提高蚕茧的质量；2．本发明公开的方法中使用的添加剂用量少、添食家蚕时间集中、材料来源广泛、成本低；无需改变生产规律，操作方便，易于推广应用。Due to the application of the above technical solutions, the present invention has the following advantages: 1. The present invention uses nano-graphene oxide as the main component additive to feed silkworms to increase cocoon silk strength by more than 30%, the effect is remarkable, the cocoon output is increased by more than 8%, and the quality of silkworm cocoons can be significantly improved; 2. The method disclosed by the invention uses less additives, concentrates time for feeding silkworms, has wide sources of materials, and is low in cost; it does not need to change production rules, is convenient to operate, and is easy to popularize and apply.
本发明的实施方式Embodiments of the present invention
下面结合实施例对本发明作进一步描述：本发明的家蚕、添加剂等都是现有产品。家蚕品种：苏秀×春丰，由本实验室育成及保存，试验区和对照区各饲养1张（25000粒），具体饲养方法为常规方法。纳米氧化石墨烯：氧化石墨烯购自上海源叶生物科技有限公司，纯度≥98%，片层厚度小于等于1 nm；乙酸乙烯酯、β－谷甾醇和羧基纤维素为生工生物工程（上海）股份有限公司产品，都符合常规食用要求。成熟叶为现有常规产品，指枝条从上统计7位以下的叶片，选取时去除虫口叶、病害叶和干枯叶，桑树品种为育711。The present invention will be further described below in conjunction with embodiment: silkworm of the present invention, additive etc. are all existing products. Silkworm species: Suxiu×Chunfeng, bred and preserved in our laboratory, one silkworm (25,000 grains) was raised in each of the experimental area and the control area, and the specific feeding method was conventional. Nano-graphene oxide: Graphene oxide was purchased from Shanghai Yuanye Biotechnology Co., Ltd., with a purity of ≥98% and a sheet thickness of less than or equal to 1 nm; ) Co., Ltd. products, all meet the requirements of conventional consumption. Mature leaves are existing conventional products, which refer to leaves whose branches are counted from the top to less than 7. When selecting, insect-mouthed leaves, diseased leaves and dry leaves are removed. The mulberry tree variety is Yu 711.
茧丝强力测定委托国家丝绸及服装产品质量监督检验中心检测，茧丝强力采用绝对强力/茧丝纤度进行定义，具体为常规方法。发病判断为家蚕饲养常规方法。Cocoon silk strength is determined by the National Silk and Clothing Product Quality Supervision and Inspection Center. Cocoon silk strength is defined by absolute strength/cocoon silk fineness, which is a conventional method. The judgment of the disease is a conventional method for silkworm rearing.
对照例：按照100kg桑叶添加10L清水的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄家蚕，每天给桑两次，给桑量和平常使用量一样，采用方格蔟自动上蔟。Control example: Add 10L of water to 100kg of mulberry leaves to spray mature leaves. After the mulberry leaves are dry, add 5-year-old silkworms. Give mulberry twice a day. The amount of mulberry is the same as the usual amount. nest.
茧丝强力：4.06cn/D；产量：45kg；发病数（头）：0头。Cocoon silk strength: 4.06cn/D; output: 45kg; incidence number (head): 0 head.
实施例一 一种提高家蚕茧丝强力的方法：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为2g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天和第5天家蚕，每天给桑两次，共添食4次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment one kind of method that improves silkworm cocoon filament strength: nano-graphene oxide, vinyl acetate and β-sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and β-sitosterol The content of sitosterol is 1.5 mg/L; according to the dose of 100kg mulberry leaves, add 10L solution to spray mature leaves, after the mulberry leaves are dried, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day, A total of 4 feedings were added, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
茧丝强力：5.28cn/D，较对照区增加30.05%；蚕茧产量49kg，较对照区产量增产8.89%；发病数（头）：0头。Cocoon silk strength: 5.28cn/D, an increase of 30.05% compared with the control area; cocoon yield 49kg, an increase of 8.89% compared with the control area; number of diseased (heads): 0 head.
实施例二 一种提高家蚕茧丝强力的方法：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为2g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第1天至第3天家蚕，每天给桑两次，共添食6次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment 2 A kind of method of improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and β-sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and β-sitosterol The content of sitosterol was 1.5 mg/L; add 10L solution dose to 100kg mulberry leaves to spray mature leaves, after the mulberry leaves were dried, add silkworms from the 1st day to the 3rd day of the 5th instar, twice a day for a total of Add food for 6 times, and the rest are fed with pure mulberry leaves. The amount of mulberry is the same as the usual amount, and the grid box is automatically loaded.
茧丝强力：4.28cn/D，较对照区增加5.42%；蚕茧产量46kg，较对照区增产2.22%；发病数（头）：0头。Cocoon silk strength: 4.28cn/D, an increase of 5.42% compared with the control area; cocoon yield 46kg, an increase of 2.22% compared with the control area; number of diseased (heads): 0 head.
实施例三 一种提高家蚕茧丝强力的方法：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为2g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第6天至第7天家蚕，7天后已经上蔟，每天给桑两次，共添食4次，给桑量和平常使用量一样。Embodiment three a kind of method that improves silkworm cocoon thread strength: nano-graphene oxide, vinyl acetate and β-sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 2g/L, vinyl acetate and β-sitosterol The content of sitosterol is 1.5 mg/L; according to 100kg mulberry leaves, add 10L solution dose to spray mature leaves, after the mulberry leaves are dried, add silkworms from the 6th day to the 7th day of the 5th instar. Give mulberry twice, add food 4 times altogether, the amount of mulberry is the same as the usual amount.
茧丝强力：4.45cn/D，较对照区增加9.61%；蚕茧产量48kg，较对照区增产6.67%；发病数（头）：0头。Cocoon silk strength: 4.45cn/D, an increase of 9.61% compared with the control area; cocoon yield 48kg, an increase of 6.67% compared with the control area; number of diseased (heads): 0.
实施例四：纳米氧化石墨烯加入水中，配制溶液，其中纳米氧化石墨烯的浓度为2g/L；按照100kg桑叶添加10L溶液剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天至第5天家蚕，每天给桑两次，其余采用纯桑叶喂食，共添食4次，给桑量和平常使用量一样。Embodiment 4: Nano-graphene oxide is added into water to prepare a solution, wherein the concentration of nano-graphene oxide is 2g/L; add 10L solution dose to 100kg mulberry leaves to spray mature leaves, and add food to the 5th age after the mulberry leaves are dried. From the 4th day to the 5th day, silkworms were fed with mulberry twice a day, and the rest were fed with pure mulberry leaves. The amount of mulberry was the same as the usual amount.
茧丝强力：3.95cn/D，较对照区增加-2.71%；蚕茧产量43kg，较对照区产量增产-4.44%；发病数（头）：5头。Cocoon silk strength: 3.95cn/D, an increase of -2.71% compared with the control area; cocoon yield 43kg, an increase of -4.44% compared with the control area; number of diseased (heads): 5.
实施例五：乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天和第5天家蚕，每天给桑两次，共添食4次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment five: vinyl acetate and β-sitosterol are added in water, prepare solution, and wherein the content of vinyl acetate and β-sitosterol is 1.5 mg/L; Add the dosage of 10L solution according to 100kg mulberry leaves and spray mature leaves, After the mulberry leaves are dried, feed silkworms on the 4th and 5th days of the 5th instar, feed mulberry twice a day, add food 4 times in total, and feed the rest with pure mulberry leaves. The stacking is automatically loaded.
茧丝强力：3.86cn/D，发病数（头）：0头。Cocoon silk strength: 3.86cn/D, number of cases (heads): 0 heads.
实施例六：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为5g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天和第5天家蚕，每天给桑两次，共添食4次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment 6: Nano-graphene oxide, vinyl acetate and β-sitosterol are added to water to prepare a solution, wherein the concentration of nano-graphene oxide is 5g/L, and the content of vinyl acetate and β-sitosterol is 1.5 mg/L L; according to 100kg mulberry leaf adding the dose of 10L solution to spray mature leaves, after the mulberry leaves are dried, add food to the 5th instar 4th day and the 5th day silkworm, give mulberry twice a day, add food 4 times altogether, all the other adopt pure Mulberry leaves are fed, and the amount of mulberry is the same as the usual amount, and the box is automatically loaded.
茧丝强力：5.22cn/D；发病数（头）：58头。Cocoon silk strength: 5.22cn/D; number of cases (heads): 58.
实施例七 一种提高家蚕茧丝强力的方法：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为1.5g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天和第5天家蚕，每天给桑两次，共添食4次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment 7 A kind of method of improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and β-sitosterol are added in water, prepare solution, wherein the concentration of nano-graphene oxide is 1.5g/L, vinyl acetate and β-sitosterol -The content of sitosterol is 1.5 mg/L; add 10L solution to 100kg mulberry leaves to spray the mature leaves, after the mulberry leaves are dry, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day , fed a total of 4 times, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
茧丝强力：5.02cn/D；发病数（头）：0头。Cocoon silk strength: 5.02cn/D; number of cases (heads): 0 heads.
实施例八 一种提高家蚕茧丝强力的方法：纳米氧化石墨烯、乙酸乙烯酯和β-谷甾醇加入水中，配制溶液，其中纳米氧化石墨烯的浓度为2.5g/L，乙酸乙烯酯和β-谷甾醇的含量均为1.5 mg/L；按照100kg桑叶添加10L溶液的剂量进行喷洒成熟叶，待桑叶晾干后添食5龄第4天和第5天家蚕，每天给桑两次，共添食4次，其余采用纯桑叶喂食，给桑量和平常使用量一样，采用方格蔟自动上蔟。Embodiment 8 A method for improving silkworm cocoon silk strength: nano-graphene oxide, vinyl acetate and β-sitosterol are added to water to prepare a solution, wherein the concentration of nano-graphene oxide is 2.5g/L, vinyl acetate and β-sitosterol -The content of sitosterol is 1.5 mg/L; add 10L solution to 100kg mulberry leaves to spray the mature leaves, after the mulberry leaves are dry, add silkworms on the 4th and 5th day of the 5th instar, and give mulberry twice a day , fed a total of 4 times, and the rest were fed with pure mulberry leaves. The amount of mulberry given was the same as the usual amount, and the grid box was automatically loaded.
茧丝强力：5.25cn/D；发病数（头）：0头。Cocoon silk strength: 5.25cn/D; number of cases (heads): 0 heads.
应用实例表明，本发明在特定时间（5龄4-5天），添食一定浓度的氧化石墨烯（2g/L）和乙酸乙烯酯、β-谷甾醇，可以高效提高家蚕茧丝的强力，还能增加蚕茧的产量，具有广阔的应用前景。现有技术利用纳米氧化石墨烯加入饲料，希望提高家蚕蚕丝强力，一般使用量大，最少为千分之一，多的达到千分之十五，仅考虑蚕丝强力，没有研究家蚕发病等影响，限制其实际生产应用。本发明仅采用纳米氧化石墨烯、乙酸乙烯酯、β-谷甾醇与纯桑叶，无需其他添加剂，首次将纳米氧化石墨烯结合乙酸乙烯酯、β-谷甾醇喷洒在桑叶上，再根据常规喂养方法添食家蚕，无需其他特殊饲养方法，预料不到的发现显著提升蚕丝强力的同时，未增加发病头数，为一种利于生产应用的方法。Application examples show that the present invention can increase the cocoon strength of silkworm cocoons efficiently by feeding a certain concentration of graphene oxide (2g/L), vinyl acetate, and β-sitosterol at a specific time (5 days and 4-5 days). It can also increase the output of silkworm cocoons and has broad application prospects. The existing technology uses nano-graphene oxide to add feed, hoping to improve the strength of silkworm silk. Generally, the amount used is large, at least 1/1000, and as much as 15/1000. Only considering the silk strength, there is no research on the impact of silkworm disease, etc. Limit its practical production application. The present invention only uses nano-graphene oxide, vinyl acetate, β-sitosterol and pure mulberry leaves without other additives. For the first time, nano-graphene oxide combined with vinyl acetate and β-sitosterol is sprayed on mulberry leaves, and then according to the conventional Feeding method Adding silkworms does not require other special feeding methods. Unexpectedly, it was found that while the strength of silkworms was significantly improved, the number of diseased heads was not increased, which is a method that is beneficial to production and application.

Claims (10)

  1. 一种提高家蚕茧丝强力的方法，其特征在于，包括以下步骤：A method for improving the cocoon silk strength of silkworm, is characterized in that, comprises the following steps:
    （1）将纳米氧化石墨烯、乙酸乙烯酯、β-谷甾醇与水混合，得到溶液；(1) Mix nano-graphene oxide, vinyl acetate, β-sitosterol with water to obtain a solution;
    （2）将步骤（1）制备的溶液喷洒成熟叶，待桑叶晾干后添食家蚕5龄期中后期，实现家蚕茧丝强力的提高。(2) The solution prepared in step (1) is sprayed on mature leaves, and after the mulberry leaves are dried, it is fed to the middle and late stages of the fifth instar silkworm, so as to improve the cocoon silk strength of the silkworm.
  2. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，将步骤（1）制备的溶液按照100kg桑叶添加8～12L溶液剂量喷洒成熟叶。The method for improving silkworm cocoon silk strength according to claim 1, characterized in that the solution prepared in step (1) is added to 100 kg of mulberry leaves and sprayed with 8-12 L of solution to mature leaves.
  3. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，溶液中，纳米氧化石墨烯的浓度为1.5～2.5g/L。The method for improving silkworm cocoon silk strength according to claim 1, characterized in that, in the solution, the concentration of nano-graphene oxide is 1.5-2.5 g/L.
  4. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，溶液中，乙酸乙烯酯的浓度为1～2 mg/L，β-谷甾醇的浓度为1～2 mg/L。The method for improving silkworm cocoon silk strength according to claim 1, characterized in that, in the solution, the concentration of vinyl acetate is 1-2 mg/L, and the concentration of β-sitosterol is 1-2 mg/L.
  5. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，晾干为自然晾干。The method for improving silkworm cocoon silk strength according to claim 1, wherein drying is natural drying.
  6. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，5龄期中后期是5龄第4天至第5天。The method for improving cocoon silk strength of silkworm silkworm according to claim 1, characterized in that, the middle and late stages of the 5th instar are the 4th day to the 5th day of the 5th instar.
  7. 根据权利要求1所述提高家蚕茧丝强力的方法，其特征在于，每天添食两次。The method for improving silkworm cocoon silk strength according to claim 1, characterized in that adding food twice a day.
  8. 一种提高家蚕茧丝强力的饲料，其特征在于，所述提高家蚕茧丝强力的饲料的制备方法包括以下步骤：A feed for improving cocoon silk strength of silkworm, characterized in that the preparation method of the feed for improving silkworm cocoon silk strength comprises the following steps:
    （1）将纳米氧化石墨烯、乙酸乙烯酯、β-谷甾醇与水混合，得到溶液；(1) Mix nano-graphene oxide, vinyl acetate, β-sitosterol with water to obtain a solution;
    （2）将步骤（1）制备的溶液喷洒成熟叶，待桑叶晾干后，得到提高家蚕茧丝强力的饲料。(2) Spray the solution prepared in the step (1) on the mature leaves, and after the mulberry leaves are dried, the feed for improving the cocoon silk strength of the silkworm is obtained.
  9. 权利要求8所述提高家蚕茧丝强力的饲料在提高家蚕茧丝强力中的应用。The application of the feed for improving cocoon silk strength of silkworm described in claim 8 in improving silkworm cocoon silk strength.
  10. 纳米氧化石墨烯、乙酸乙烯酯与β-谷甾醇在提高家蚕茧丝强力中的应用。Application of nano-graphene oxide, vinyl acetate and β-sitosterol in improving cocoon silk strength of silkworm.
PCT/CN2021/120018 2021-09-23 2021-09-23 Method for improving strength of bombyx mori cocoon silk WO2023044687A1 (en)

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Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1140666A (en) * 1965-04-02 1969-01-22 Ajinomoto Kk Artificial silkworm feed
CN1608489A (en) * 2003-10-17 2005-04-27 华岛编织股份有限公司 Fodder for silkworm and produced silk fed with the fodder and silk products using the silk
CN104886008A (en) * 2015-04-29 2015-09-09 东华大学 Graphene oxide-feeding silkworm rearing method for preparing high-strength silk and product of silk
CN107048100A (en) * 2017-05-10 2017-08-18 福鼎朝辉水产开发有限公司 A kind of silkworm feed additive and preparation method for improving mulberry silk intensity and electric conductivity
CN108077197A (en) * 2018-02-07 2018-05-29 四川红旗丝绸有限公司 A kind of method of young silkworm farm breeds
CN108354092A (en) * 2018-03-09 2018-08-03 中科国思生物科技研究（广州）有限公司 A kind of silkworm containing nano material adds food feed and preparation method
CN109864042A (en) * 2019-03-26 2019-06-11 青岛理工大学 A kind of nano-graphene preparing high thermal conductivity silk freshens food and educates silkworm method and its product

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1140666A (en) * 1965-04-02 1969-01-22 Ajinomoto Kk Artificial silkworm feed
CN1608489A (en) * 2003-10-17 2005-04-27 华岛编织股份有限公司 Fodder for silkworm and produced silk fed with the fodder and silk products using the silk
CN104886008A (en) * 2015-04-29 2015-09-09 东华大学 Graphene oxide-feeding silkworm rearing method for preparing high-strength silk and product of silk
CN107048100A (en) * 2017-05-10 2017-08-18 福鼎朝辉水产开发有限公司 A kind of silkworm feed additive and preparation method for improving mulberry silk intensity and electric conductivity
CN108077197A (en) * 2018-02-07 2018-05-29 四川红旗丝绸有限公司 A kind of method of young silkworm farm breeds
CN108354092A (en) * 2018-03-09 2018-08-03 中科国思生物科技研究（广州）有限公司 A kind of silkworm containing nano material adds food feed and preparation method
CN109864042A (en) * 2019-03-26 2019-06-11 青岛理工大学 A kind of nano-graphene preparing high thermal conductivity silk freshens food and educates silkworm method and its product

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