Preparation Method of Fluorescent Green Silk Floss Sheet and Its Product

Abstract

The invention discloses a preparation method of a fluorescent green silk floss sheet. Select a transgenic Bombyx mori variety with homozygous genotype and high expression level of green fluorescent protein gene, raise it until it becomes a mature silkworm, mount it on a folding cluster for cocooning, collect the cocoons, quickly freeze them at -20 to -18 °C for more than 48 hours, open the cocoons, take the cocoon shells for degumming, rinsing, dehydration, drying, and beat them into silk floss sheets, then the fluorescent green silk floss sheets are obtained. The silk floss sheets prepared by the invention maintain fluorescent green and present bright green under natural light, and have the advantages of being fluffy, soft, of high quality, and environmentally friendly, and can be used for the production of subsequent textiles such as silk quilts and silk pillows.

Description

Technical Field

[0001] The present invention relates to the field of silk processing. More specifically, the present invention relates to a method for preparing a fluorescent green silk floss sheet and its product. Background Art

[0002] Green fluorescent protein is a pigment protein that can fluoresce in living cells without any substrates and cofactors, and is widely used in many fields such as biology, medicine, and photovoltaic power generation. The enhanced green fluorescent protein gene (EGFP) is integrated into the silkworm genome by transgenic methods and fused with the silk fibroin gene of the silkworm to obtain a silkworm variety that can spin fluorescent green silk. The cocoons spun by this variety are green, and the fluorescent green is located in the silk fibroin. It will not disappear with the removal of sericin during subsequent processing and can be used without subsequent dyeing, which is an energy-saving and environmentally friendly material.

[0003] Silkworm silk mainly consists of sericin on the outer layer and silk fibroin wrapped in the middle. During the process of preparing silk floss from cocoons, most of the sericin on the outer layer needs to be removed. The preparation of traditional silkworm silk floss usually requires a high-temperature cocoon boiling and degumming process. However, pigment proteins are easily denatured and lose their color under high-temperature and other conditions, so it is not applicable to the preparation of silk floss from fluorescent green cocoons of transgenic EGFP silkworms. There is also research and development on colored silk floss in China, but the cocoons used are all natural colored cocoons. The pigments of natural colored cocoons are located in the sericin on the outer layer of the silk, while the pigments of the fluorescent green cocoons spun by transgenic EGFP silkworms are located in the silk fibroin on the inner layer of the silk. There are essential differences between the two, so the process of producing colored silk floss from natural colored cocoons is also not applicable to the preparation of silk floss from fluorescent green cocoons of transgenic EGFP silkworms. There are reports on using alkaline protease for degumming of silk cocoons containing green fluorescent protein, but this method has the following disadvantages: (1) a large amount of alkaline protease needs to be used, resulting in high production costs; (2) the degummed silk cocoons obtained by this method only maintain fluorescent properties under ultraviolet light; (3) continuous degumming in an alkaline protease degumming solution at 50°C for 120 minutes is required to achieve a degumming rate of more than 25%, which takes a long time and requires a large pressure for the heat preservation equipment.

[0004] Therefore, there is an urgent need to develop a method for making silk floss from fluorescent green cocoons of transgenic EGFP silkworms that is low in cost and can present bright green under natural light, so as to provide technical support for the application of fluorescent green cocoons. Summary of the Invention

[0005] The present invention provides a method for preparing a fluorescent green silk floss sheet and its product. The prepared silk floss sheet maintains fluorescent green and presents bright green under natural light, and has the advantages of being fluffy, soft, of high quality, and environmentally friendly, and can be used for the production of subsequent textiles such as silk quilts and silk pillows.

[0006] To achieve these objects and other advantages according to the present invention, a method for preparing a fluorescent green silk floss sheet is provided. Select a transgenic Bombyx mori variety with homozygous genotype and high expression level of green fluorescent protein gene, raise it to mature silkworms, mount them on a folding cocooning frame, collect the cocoons, quick-freeze them at -20 to -18 °C for more than 48 hours, open the cocoons, take the cocoon shells for degumming, rinsing, dehydration, drying, and beating them into silk floss sheets, thus obtaining the fluorescent green silk floss sheets.

[0007] Preferably, the degumming solution used for degumming is a mixed aqueous solution of 0.08 wt.% sodium hydroxide + 1.0 wt.% sodium silicate nonahydrate + 0.5 wt.% Nekal, and the temperature of the degumming solution is 35 - 45 °C.

[0008] Preferably, the relative humidity during quick-freezing is controlled within 50%.

[0009] Preferably, during raising, the doors and windows are closed in the early instar stage, and the windows are opened for ventilation in the late instar stage, and the window screens are closed. The raising conditions are: 26 °C throughout the whole instar, and the relative humidity is 65 - 75%.

[0010] Preferably, the mulberry leaves fed at each instar during raising are older than those fed to conventional Bombyx mori at the same instar.

[0011] Preferably, the temperature in the cocooning frame is 25 °C, the relative humidity is 55 - 65%, and the mounting period is 6 days.

[0012] Preferably, the degumming includes the following steps:

[0013] Degumming: Open the cocoons, take the cocoon shells for vacuum penetration, evacuate to negative pressure, put them into a degumming solution 40 - 60 times their weight and soak for 15 - 25 s, introduce air until the vacuum degree is zero, and continue to soak for 20 - 40 min. Among them, the degumming solution is a mixed aqueous solution of 0.08 wt.% sodium hydroxide + 1.0 wt.% sodium silicate nonahydrate + 0.5 wt.% Nekal, and the temperature of the degumming solution is 35 - 45 °C;

[0014] Deep degumming: Take the degummed cocoon shell complex for rinsing, dehydration, and put it into a deep degumming solution 80 - 120 times its weight and soak for 30 - 50 min. Among them, the deep degumming solution is a 1 wt.% anhydrous sodium carbonate solution, and the temperature of the deep degumming solution is 35 - 45 °C.

[0015] Preferably, the temperature of the degumming solution is 40 °C, and the temperature of the deep degumming solution is 45 °C.

[0016] Preferably, after degumming, the obtained silk floss is rinsed clean, placed in a dehydrator, dehydrated at 500 rpm for 10 min, dried by blowing air at 35 °C, and finally placed in a cotton-beating machine to beat into silk floss sheets, thus obtaining the fluorescent green silk floss sheets.

[0017] The fluorescent green silk floss sheets obtained by the described preparation method.

[0018] The present invention has at least the following beneficial effects:

[0019] First, the color of the fluorescent green silk floss sheet prepared by the present invention exists in the fibroin and will not be lost with the removal of sericin. It is a good material for preparing high-quality colored silk floss products. The silk floss sheet prepared by the present invention is visible green under natural light and shows fluorescent green under ultraviolet light. This characteristic can be used as an anti-counterfeiting mark for the product.

[0020] Second, after the fluorescent green cocoons are quickly frozen for more than 48 hours, the silkworm pupae are frozen to death, while the fluorescent green color of the cocoon shells is not affected and even looks greener. Compared with the traditional method of drying cocoons to kill silkworm pupae, the present invention not only meets the requirements of inactivating transgenic living organisms but also can effectively maintain the color of the cocoon shells, and is time-saving and highly efficient.

[0021] Third, the present invention uses low-temperature (below 50 °C) degumming throughout the process, without the need for cocoon-cooking equipment, is easy to operate, causes little damage to fibroin, and through multiple tests and calculations, the degumming rate is above 25%, and the degumming effect is good.

[0022] Fourth, no special chemical additives are added throughout the process of the present invention. Sodium hydroxide, sodium silicate nonahydrate, Nekal, anhydrous sodium carbonate, etc. used for degumming are conventional drugs and are used in low amounts. They can be completely removed after multiple rinses. The prepared silk floss sheet has no chemical residue and is safe.

[0023] Fifth, because the EGFP gene has strong hydrophilicity, the present invention is carried out under conditions with a slightly lower relative humidity than that in conventional silkworm rearing and cocoon mounting. The ratio of abnormal cocoons can be effectively reduced. The relative humidity during quick freezing is controlled within 50%, which is a relatively dry environment. The surface sericin is prone to denaturation, which is beneficial for degumming and also convenient for selling cocoons.

[0024] Other advantages, objectives, and features of the present invention will be partially reflected by the following description and partially understood by those skilled in the art through the research and practice of the present invention. Brief Description of the Drawings

[0025] Figure 1 It is a photo of the fluorescent green cocoons of the present invention;

[0026] Figure 2 It is a photo of the fluorescent green silk floss sheet of the present invention under white light;

[0027] Figure 3 It is a photo of the fluorescent green silk floss sheet of the present invention under ultraviolet light;

[0028] Figure 4 It is a photo of the cocoon shells of the present invention under different temperature treatments of quick freezing at -18 °C, normal temperature, and drying at 70 °C. Detailed Description of the Invention

[0029] The present invention will be further described in detail below with reference to the accompanying drawings, so that those skilled in the art can implement it according to the text of the specification.

[0030] It should be understood that terms such as "having", "comprising", and "including" used herein do not exclude the presence or addition of one or more other elements or their combinations.

[0031] It should be noted that in the following embodiments, the experimental methods are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial sources unless otherwise specified.

[0032] The present invention provides a method for preparing a fluorescent green silk floss sheet, comprising the following steps:

[0033] Rearing: Select a transgenic EGFP silkworm variety with homozygous genotype and high expression level of green fluorescent protein gene, rear it until it becomes a mature silkworm, mount it on a folding cluster for cocooning, collect the cocoons, and quickly freeze them. The contradiction between color retention and degumming has always been a difficult problem in the processing of natural colored silk floss. In the present invention, the color of the fluorescent green silk floss sheet exists in the fibroin and will not be lost with the removal of sericin, making it a good material for preparing high-quality colored silk products. Feed the silkworms with mulberry leaves that are a bit older than those used for rearing conventional silkworms at each instar, which can make the cocoon color greener. Since the EGFP gene has strong hydrophilicity, the present invention adopts conditions with a slightly lower relative humidity than that for rearing conventional silkworms and mounting them for cocooning, which can effectively reduce the ratio of irregular cocoons. After the fluorescent green cocoons are quickly frozen for more than 48 hours, the silkworm pupae are frozen to death, while the fluorescent green color of the cocoon shells is not affected (even looks greener). In this study, the fluorescent green cocoons were also placed at -18°C and quickly frozen for more than half a year, and the cocoon color remained bright, which is an important innovation point of the present invention. The relative humidity in the cold storage for quickly freezing the cocoons is controlled within 50%, which is a relatively dry environment. The surface sericin is prone to denaturation, which is beneficial for degumming and also convenient for selling the cocoons. As Figure 4 shown, the cocoon shells stored at -18°C for one month, the cocoon shells placed at room temperature, and the cocoon shells baked at 70°C for 2 hours are respectively presented. It can be seen that the quickly frozen and room-temperature cocoon shells are both bright in cocoon color, while the baked cocoon shells are yellowish.

[0034] Degumming of cocoon shells: Unwind the silkworm cocoons, take the cocoon shells for vacuum penetration, evacuate to negative pressure, immerse the cocoon shells in a degumming solution that is 40 - 60 times their weight for 15 - 25 s, introduce air until the vacuum degree is zero, and continue to immerse for 20 - 40 min. Among them, the degumming solution is a mixed aqueous solution of 0.08 wt.% sodium hydroxide + 1.0 wt.% sodium silicate nonahydrate + 0.5 wt.% Nekal, and the temperature of the degumming solution is 35 - 45 °C; Although the degumming conditions of high temperature and high alkalinity have good effects, they can damage the silk fibroin, and a large number of literature reports that green fluorescent protein will denature under conditions above 70 °C. The whole process of this invention uses low temperature (below 50 °C), does not require cocoon cooking equipment, is simple to operate, and has little damage to silk fibroin; and through multiple tests and calculations, the degumming rate is above 25%, and the degumming effect is good, which is an important innovation point of this invention.

[0035] Deep degumming of cocoon shells: Take the cocoon shells for rinsing and dehydration, and immerse the cocoon shells in a deep degumming solution that is 80 - 120 times their weight for 30 - 50 min. Among them, the deep degumming solution is a 1 wt.% anhydrous sodium carbonate solution, and the temperature of the deep degumming solution is 35 - 45 °C, which is an important innovation point of this invention; No special chemical additives are added during the whole degumming process. Sodium hydroxide, Nekal, anhydrous sodium carbonate, etc. used for degumming are conventional drugs, and the dosage is low. They can be completely removed after multiple rinses, and the prepared silk floss sheets have no chemical agent residues and are safe.

[0036] Post-treatment: Take the cocoon shells for rinsing, dehydration, drying, and beating them into silk floss sheets, then fluorescent green silk floss sheets are obtained. The prepared silk floss sheets are visible green under natural light and show fluorescent green under ultraviolet light. This characteristic can be used as an anti-counterfeiting mark for this product.

[0037] <Example 1>

[0038] Rearing of silkworms: Rearing transgenic silkworm varieties with homozygous genotype and high expression level of green fluorescent protein gene in a closed silkworm rearing room; Keep the doors and windows closed from the 1st to 3rd instars, and open the windows for ventilation from the 4th to 5th instars, but the window screens need to be closed. The average rearing temperature throughout the whole instar is 26 °C and the relative humidity is 68%; For each instar, feed mulberry leaves that are a little older than those used for rearing conventional silkworms.

[0039] Treatment of fluorescent green cocoons: Raise the above-mentioned silkworms to mature silkworms, place them on a folding lattice for mounting, the temperature in the lattice is 25 °C and the relative humidity is 56%, and collect the cocoons six days after mounting. The collected cocoons are quickly frozen at -18 °C for 60 h, and the relative humidity in the quick-freezing cold storage is 49%.

[0040] Degumming of silkworm cocoons: Step i: Open the quick-frozen silkworm cocoons. Put the cocoon shells into a vacuum infiltration device, evacuate the air. After the vacuum reaches -0.09 MPa, open the water inlet valve to add degumming liquid at 40 °C. The dosage of the degumming liquid is based on the ratio of cocoon shells to liquid of 1:50. After adding enough, close the water inlet valve and soak for 20 s. Then open the air inlet valve to let air in. After the vacuum returns to zero, close the air inlet valve and continue to soak for 30 min. The composition of the degumming liquid is: a mixed aqueous solution of 0.08% sodium hydroxide + 1.0% sodium silicate nonahydrate + 0.5% Nekal BX.

[0041] Step ii: Take out the vacuum-infiltrated and soaked cocoon shell complex, rinse it with clear water for 30 min, and dehydrate it at 500 rpm for 5 min in a dehydrator; then put the dehydrated cocoon shells into a 1% anhydrous sodium carbonate solution at 45 °C according to the bath ratio of 1:100 for deep degumming for 50 min, and then rinse it with clear water 3 times.

[0042] Obtaining fluorescent green silk floss sheets: Put the rinsed clean silk floss in a dehydrator and dehydrate it at 500 rpm for 10 min. Then dry it by blowing air at 35 °C, and finally put it in a cotton-beating machine to beat it into silk floss sheets, thus obtaining fluorescent green silk floss sheets.

[0043] The photo of the fluorescent green cocoon in Example 1 is as Figure 1 shown. The photo of the prepared fluorescent green silk floss sheet sample under white light is as Figure 2 shown. It can show bright green under natural light, and the photo under ultraviolet light is as Figure 3 shown. It maintains fluorescent green. After multiple tests and calculations, the degumming rate is above 25%, and the degumming effect is good.

[0044] <Comparative Example 1>

[0045] Same as Example 1, except that the degumming of the silkworm cocoons only includes Step i and does not include Step ii. The obtained silk floss sheet has hard lumps.

[0046] <Comparative Example 2>

[0047] Same as Example 1, except that the degumming of the silkworm cocoons only includes Step ii and does not include Step i. There are too many hard lumps after degumming, and it is difficult to pull them into silk floss sheets.

[0048] <Comparative Example 3>

[0049] Same as Example 1, except for the formula of the degumming liquid in Step i of the degumming of the silkworm cocoons. The composition of the degumming liquid is: a mixed aqueous solution of 0.1% sodium hydroxide + 1.0% sodium silicate nonahydrate + 0.5% industrial soap. Using this degumming liquid for degumming, the color and luster of the silk floss are maintained well, but it is not fluffy enough.

[0050] The number of devices and the processing scale described herein are used to simplify the description of the present invention. Applications, modifications, and variations of the present invention will be apparent to those skilled in the art.

[0051] Although the embodiments of the present invention have been disclosed as above, it is not limited to the applications listed in the specification and the embodiments. It can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily made. Therefore, without departing from the general concept defined by the claims and the equivalent scope, the present invention is not limited to specific details and the illustrated examples described herein.

Claims

1. A method for preparing a fluorescent green silk floss sheet, characterized in that, Select a transgenic silkworm variety with homozygous genotype and high expression level of green fluorescent protein gene, rear it until it becomes a mature silkworm, place it on a folding cluster for cocooning, collect the cocoons, quickly freeze them at -20~-18°C for more than 48 h, cut open the cocoons, take the cocoon shells for degumming, rinsing, dehydration, drying, and beat them into silk floss sheets, then fluorescent green silk floss sheets are obtained; Degumming includes the following steps: Degumming: Cut open the cocoons, take the cocoon shells for vacuum infiltration, evacuate to negative pressure, immerse them in a degumming solution with a weight 40~60 times that of the cocoon shells for 15~25 s, introduce air until the vacuum degree is zero, and continue to immerse for 20~40 min. Among them, the degumming solution is a mixed aqueous solution of 0.08 wt.% sodium hydroxide + 1.0 wt.% sodium silicate nonahydrate + 0.5 wt.% Nekal, and the temperature of the degumming solution is 35~45°C; Deep degumming: Take the degummed cocoon shell complex for rinsing and dehydration, immerse it in a deep degumming solution with a weight 80~120 times that of the cocoon shell complex for 30~50 min. Among them, the deep degumming solution is a 1 wt.% anhydrous sodium carbonate solution, and the temperature of the deep degumming solution is 35~45°C.

2. The preparation method of the fluorescent green silk floss sheet according to claim 1, characterized in that, The relative humidity during quick freezing is controlled within 50%.

3. The preparation method of the fluorescent green silk floss sheet according to claim 1, characterized in that, During rearing, close the doors and windows in the early instar stage, open the windows for ventilation and close the window screens in the late instar stage. The rearing conditions are: 26°C throughout the whole age, and the relative humidity is 65~75%.

4. The preparation method of the fluorescent green silk floss sheet according to claim 3, characterized in that, When rearing, the mulberry leaves fed in each instar are older than those fed to conventional silkworms of the same age.

5. The preparation method of the fluorescent green silk floss sheet according to claim 1, characterized in that, The temperature in the cluster is 25°C, the relative humidity is 55~65%, and the cocooning period is 6 days.

6. The preparation method of the fluorescent green silk floss sheet according to claim 1, characterized in that, The temperature of the degumming solution is 40°C, and the temperature of the deep degumming solution is 45°C.

7. The preparation method of the fluorescent green silk floss sheet according to claim 1, characterized in that, After the degummed silk floss is rinsed clean, place it in a dehydrator and dehydrate it at 500 rpm for 10 min, dry it with hot air at 35°C, and finally place it in a cotton beater to beat it into silk floss sheets, then fluorescent green silk floss sheets are obtained.

8. The fluorescent green silk floss sheet obtained by the preparation method according to any one of claims 1-7.

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