"Development of a sericin extraction process from sericin rich bivoltine silkworm hybrid cocoon shells".

Field of Invention

The invention relates to development of a sericin extraction process from sericin rich bivoltine silkworm hybrid cocoon shells.

Background of Invention (BRIEF ABSTRACT)

Silk fiber consists of two types of self-assembled proteins; fibroin and sericin that are produced in the silk gland of Bombyx mori L. Fibroin is a major component (72-81%) of silk fiber serving as the core, while the sericin is a minor component (19.0-28.0 %). The hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. Silk cocoons of existing bivoltine silkworm races consist of about 25 % of the shell weight as sericin. Silk cocoons are cooked in boiling water to obtain silk. During cooking gummy substance sericin is removed by boiling it in water, and the water containing sericin, a valuable resource material with the potential diverse biological applications is disposed off as waste.

In Japan, a silkworm race "Sericin Hope" contains about 98.5% of sericin (Yamamoto: Annual Report: 2002) as compared to 20-30% of sericin in the normal bivoltine silk cocoons. In Korea also Wenchow (2001) bred a silkworm, namely "Hashojam " which produces only sericin. As no such silkworm breed yielding higher sericin was available in India, it became imperative to identify a sericin rich silkworm bivoltine hybrid, whose cocoon shells contained the higher percentage of sericin.

EXISTING STATE-OF-ART:

National Status: The state -of-art processes for commercial exploitation of sericin is not available in India. However, at the different silkworm breeding laboratories of India during silkworm breeding experiments, sericin is estimated from the silk cocoons to understand the quantity of sericin content present in the newly developed silkworm breed / hybrid which is considered as one of the aspects for assessing the breeds. Further, Gulrajani (2005; 2006) identified the process for sericin extraction from the reeling waste water by following different methods and his work was limited only to lab scale. But no patenting on sericin has been reported by him (Gulrajani, A Bio-molecule of Value 2005, 2006).

EXTRACTION METHODS DEVELOPED BY GULRAJANI (2005, 2006):

1. Normal cocoon shells are boiled in water and by concentrating or precipitating followed by drying.

2. Degumming liquor by coagulation, filtration and concentration of the residue or by ultra filtration.

3. Classical Method: Involves multiple processes using several inorganic chemicals and solvents for several months.

4. Sericin extract is freeze-dried, freeze-thawed and spray-dried into sericin powder.

5. By coagulation and ultra filtration.

All the above state -of- art processes for sericin extraction methods used normal hybrid cocoon shells as the source material which contains only about 25% sericin.
In the light of above explanation the detailed comparative study of this present invention with those of the reported state of art process is not attempted, because the present invention process extracts sericin from the unique sericin rich silkworm hybrid which yields 95-98% sericin. Therefore, the comparison of this invention process with that of the reported state-of art processes meant for extracting sericin is unequal and does not serve the intended purpose of the patent questionnaire.

International Status:

1. The silkworm cocoons are extracted with the hydrochloric acid solution at a 1: (95-105) ratio upon boiling for 2-4 hours. Then the mixture is allowed to cool. Fibroin is obtained upon filtration, while sericin is obtained upon neutralizing the acidic solution (Borysivna Patent number: UA65934, 2004)

2. The product is rinsed with the water to neutral pH and dried by spraying. High-temperature water (preferably distilled water), preferably heated to 90-130°C is circulated to cut cocoon obtained by cutting one end part of cocoon and removing pupa and impurity and washing the cocoon, preferably at 10-100 L/kg/min liquid flow rate for 10-180 min by using the above high temperature water in an amount of 5-100 g based on 1 g cut cocoon. Thereby, sericin is extracted from the cut cocoon to extract the objective compound. It is preferable to use cocoon obtained from silkworm aseptically bred by using an artificial feed not containing a harmful substance such as heavy metal (Minamoto Toshiaki, Patent number: JP2001039999,2001).

3. To provide a sericin-separating and recovering device which can stably recover the high molecular weight sericin by gradually freezing a silk fiber- degummed solution, namely a sericin aqueous solution, obtained by subjecting silk fibers to a high temperature high pressure treatment using high temperature water, at about 0 °C over a long time and then thawing the frozen product (Yamazaki Masao, Patent number: JP2001302693,2001).

4. The sericin polypeptide whose mean molecular weight is 200-5000 is one directly extracted from husks, frison and waste silk and made up by using enzymatic hydrolysis process. The said product can be made into solution or powder; can be used as raw material for producing cosmetics. Sericin polypeptide contains 20.3 mol% of essential amino acids necessary for human body and 75.1 mol% of polar amino acid, so that it possesses excellent hygroscopicity, thermo-insulating property and nutritive value (Chen Wenxing, Patent number: CN1228438, 1999).

5. This promoter of collagen production includes sericin and a hydrolyzate thereof as an active ingredient. The sericin and the hydrolyzate thereof is obtained by partially hydrolyzing the sericin contained in a silkworm cocoon, or raw silk in an alkali solution or by a enzyme treatment to elute the sericin, regulating the pH of the elute so as to be 3-5 by an organic acid or an inorganic acid, or precipitating the sericin hydrolyzate by mixing the elute with a water-soluble organic solvent such as methanol, filtering and drying the resultant sericin to provide an easily water-soluble material having 10,000-20,000 average molecular weight and containing 20-40mol% sericin. The skin care product made by formulating 0.000l-10wt. % sericin or hydrolyzate
thereof in terms of a dried product (Yamada Hideyuki: Patent number: JP10226653,1998)

6. High-purity sericin (including its hydrolyzae) obtained by extracting cocoons or raw silk, is used as the active component to give an antioxidant. Sericin contained in cocoons or raw silk is partially hydrolyzed with an acid, alkali, or enzyme, then extracted, and treated as follows: the extract is mixed with a water-soluble org. solvent (e.g. methanol, ethanol, or dioxane) and the resultant presipitate is separated by filtration and dried to give a sericin powder; or the extract is subjected to ultrafiltration or filtration with a reverse osmosis membrane and dried to give a sericin powder (Yamada Hideyuki; Patent number JP10140154,1998).

7. This anticoagulant comprises a sulfonated silkworm product, preferably a sulfonated fibroin or a sulfonated sericin. The product is preferably obtained by adding concentrated sulfuric acid at 10-90wt.% concentration in an amount of 0.5-500 pts.wt. based on 1 pt.wt. extracted substance to the substance extracted from, e.g. silk yarn or contents of a silk gland of a silkworm such as fibroin or sericin and carrying out the sulfonation treatment at a temperature within the range of 20-100 deg.C for about several minutes to several hours (Tamada Yasushi: Patent number: JP9227402,1997)

8. This invention relates to cosmetic preparations, especially hair care preparations or hair care products, containing protein hydrolysates of silk, pashmina, cashmere wool, merino wool and/or mohair, mussel thread extracts, so-called bysuss threads, and sericin and/or sericin hydrolysates. The preparations are mild for skin and hair and lead to an improvement of hair structure and physical-optical hair properties (Kobayashi Hatsumi: Patent number: JP2007210902,2007)

USES OP SERICIN:

Sericin can be applied in many fields such as cosmetics, biomaterials and textile (Zhang, 2002). Sericin has wide application in medical, pharmaceutical and cosmetics due to its susceptibility to proteolytic enzymes and some properties like gelling ability, moisture retention and skin adhesion .

Environment - friendly biodegradable polymers can be produced by blending sericin with other resins (Annamaria et al., 1998).Sericin is used in preparation of art pigments and for surface protection of articles. Sericin- coated film is used on the surface of refrigeration equipment because of its anti-frosting action (Tanaka and Mizuno, 2001). Use of the coated sericin film is an effective anti-frosting method that can be widely applied to refrigerators, deep freezers and refrigerated trucks and ships. Moreover use of the coated film on roads and roof can prevent frost damage. Sericin protein can be coated on surfaces of various durable materials to enhance functionality (Li, 1996). The material coated with the sericin has excellent weather ability, good permeability and does not warp on drying. Sericin blends with water- soluble polymers, especially with polyvinyl alcohol (PVA). A blended hydrogel made of sericin and fibroin and PVA is said to have excellent moisture absorbing and desorbing properties and elasticity (Yoshii et al., 2000).

Sericin plays a major role in the medical and pharmaceutical field. It has antioxidant action and suppresses in vitro lipid peroxidation and found to inhibit tyrosinase activity. It makes sericin the most valuable natural ingredient for food and cosmetics (Kato et al. 1998). It is also an antioxidant, antimicrobial agent for polluted air treatment (Sara Saravart et al, 2003). Sericin has been found to possess wound healing property and can be used as wound healing covering material in the form of film (Wu et al, 1996). Sericin also has adhesive property due to its chemical composition. Silk sericin membranes are good bandage material and the film has adequate flexibility and tensile strength. Due to its good biocompatibility and infection resistant nature, it is a novel wound coagulant material. It has the potential to find application in the development of contact lenses (Nakamara et al, 1996). Intake of sericin containing food relieves constipation, suppresses development of bowel cancer and accelerates the absorption of minerals (Siqin Zhaorigetsu, 2001; Sasaki Masahiro, 2007)

Sericin when given orally causes a decrease in the development of colonic aberrant crypt foci. The incidence and the number of colon tumours are suppressed by consumption of sericin. It is also used as germicidal, antifungal, antibacterial and anticoagulant (Arunee Kongdee et al, 2005)

In addition to the above mentioned medical and pharmaceutical uses of sericin, it has been used as component of cosmetics. Sericin alone or in combination with silk fibroin has been used in the skin, hair and nail cosmetics. Sericin when used in the form of lotion, cream and ointment shows increased skin elasticity, anti-wrinkle and anti-aging effects (Henne and Hoppe, 1986; Yamada et al., 19981 Ogawa and Yamada, 1999).Sericin powder in the form of sericin hydrolysate coated talc,mica,titantia,iron oxide and nylon have been used to formulate foundation cream and eyeliners (Yamada and Yuri, 1998). Sericin gels with pluronic and carbol act as moisturizer by repairing natural moisturing factor as well as trance epidermal water loss from the skin (Padamwar and Pawar, 2004).

LITERATURE SURVEY:

Mishra and Dash (1992) reported the utility of sericultural wastes and byproducts in Agriculture.

Hossain et al (1993) reported the evaluation of silkworm pupae meal as dietary protein source for catfish (Heteropneustes fossilis Bloch).A laboratory trial of 60 days evaluated silkworm pupae meal (SWP) as fish meal substitute in isonitrogenous diets for fingerling catfish (Heteropneustes fossilis) about 2.1 g at 0, 25, 50, 75 and 100 of dietary protein. Based on growth rate, feed conversion ratio, protein efficiency ratio and apparent net protein utilization, diets containing 50 and 75 SWP protein, respectively, produced the best growth (P0.05) while the control diet (0 SWP) produced lowest growth. Apparent protein digestibility (APD) for all diets was fairly high, ranging from 78.80 to 86.72 with control diet producing the lowest. In general, APD values increased with increasing SWP in diets. Compared with the control, fish fed on diets containing higher levels of SWP had lower carcass moisture and higher carcass protein and lipid contents. It was concluded that
SWP meal can be used as a substitute for fish meal in fish feed and at up to 75 of protein in catfish diets did not affect growth.

Ji, and Huang (1994) reported the multipurpose utilization of sericultural resources in China. The latest progress in the multipurpose utilization of sericultural byproducts in China are reviewed, focusing on silkworm faeces, pupae, cocoons, silkmoths and mulberries. From silkworm faeces are extracted pigments such as chlorophylls and carotenoids, and from the pupae an oil is isolated. Hormones and antibacterial peptides of the adults are used, and also the cellulose and fruit juice of the mulberry plants.
Taniguchi (1980 Studied on the simple compost of shoot rearing waste of silkworm, Bombyx mori. (V). Utilization effect of the so-called waste mulberry shoot compost. Magdi et al., (1989) reported the Versatile, fashionable handicrafts from silk waste. The bye-product of silkwaste is very cheap and easily available so techniques are developed for utilization of silkwaste for handicrafts. One such handicraft is greeting cards which helps to improve the economic standard of some people. Even garland, Flowers, wall hangings, carpets. Hankeys etc., are prepared out of silk waste.

Sharma and Madan (1994) reported the Potential of mulberry (Morus alba) biomass.A short review, describing the biology, silviculture and uses (including sericulture, as a protein rich substrate for mushroom growing, as fuel wood, as a source of various chemicals, as an edible fruit, for wood) of this important multipurpose tree. An integrated approach to utilization of the species is advocated, which includes use of the foliage for sericulture, the thin branches and sericulture waste for mushroom cultivation, the spent residues and raw leaves and stems for anaerobic digestion for biogas production, and the slurry from the biogas digesters as organic fertilizer.
Sengupta et al (2000) reported the Utilization of tasar silk waste through NMC/Core spun yarn. The main object of silk industry is to produce raw silk for the preparation of fabrics. To achieve this, different kinds of silk wastes are also produced as by-products which require careful handling to produce further useful product to bring end products at competitive level by lowering the cost of production. The present study focuses on the utilization of different wastes for the preparation of diversified products towards product development and diversification attitude to attract consumers.

Magdi et al, (1989) reported the Versatile, fashionable handicrafts from silk waste .The bye-product of silkwaste is very cheap and easily available so techniques are developed for utilization of silk waste for handicrafts. One such handicraft is greeting cards which helps to improve the economic standard of some people. Even garland, Flowers, wall hangings, carpets. Hankeys etc., are prepared out of silk waste.

Terada et al., (2002,) reported that the Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma s Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-0, human hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lines was accelerated in the presence of sericin. In comparison with bovine serum albumin (BSA), a widely used supplement in serum-free medium, sericin had an equivalent effect on the proliferation of the hybridomas and sericin additively stimulated the proliferation with BSA. Although heat easily denatures and inactivates most proteins, the activity of sericin was not affected by autoclaving.

Kongdee et al. (2005,) reported that the Modification of cellulose fiber with silk sericin . From ESEM and FTIR-ATR results, it was found that sericin coated onto cotton surfaces as a film. Increasing sericin content in the finishing solution increased the amount of coated sericin, and a greater depth of color in dyed samples and reduced free formaldehyde content in treated samples were observed. The sericin content in samples was found to have a negligible influence on tensile strength and crease recovery angle. With increasing sericin content, electrical resistivity of the samples dramatically decreased and water retention increased, indicating that sericin-treated fabrics may be comfortable to wear because of its maintenance of moisture balance with respect to human skin. Because cotton textile coated with sericin exhibited low formaldehyde content and no biocidal activity against Klebsiella pneumoniae and Staphylococcus aureus, the fabric may reduce skin irritation and disturbance of physiological skin flora arising from textile contact. Silk Anne Bellina skin care (1973) protein and amino acids provide intense moisture to the skin, leaving it "silky" soft without a heavy, greasy feel. Silk protein also known as silk peptide is from the center of the silk worm yarn. After special technical processing the center becomes silk protein.lt can be absorbed directly by the human skin. It can combine with the skin cell to revitalize the skin. It is the most advanced unique nutritive material for skin and hair products in the current
international market. It also can effectively control growth of melanin in the skin. Silk amino acids (silk proteins) are used to prevent dehydration and is mostly found in eye rejuvenation gels and creams.
Siqin zhaorigetu, et al. (2001) reported that the Silk Protein, Sericin, Suppresses Colon Tumorigenesis. This study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2-dimethylhydrazine (10 mg/kg body weight) for the initial 10 wk. The results suggest that dietary sericin suppresses the development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.

Radar (2003), a report on Genetically Engineered Crops. Building with Silk the gene for fibroin has been transferred from a silkworm to a goat, and is expressed as a component of its milk. Genes for the two spider silk proteins were transferred to cells cultured from cow udders. Those cells then made the proteins. Happily, the spider silk can be spun by forcing a solution of its two protein components through a tiny nozzle.The proteins self-assemble into spider silk strands. The same genes have since been transferred to live goats and when those goats are old enough to produce milk; it should be possible to make large quantities of spider silk very cheaply. Silk is an extraordinarily strong material, stronger than steel.

Soft drinks, cosmetics, candies, contact lenses, and other goods containing cocoon extract and promising health and beauty benefits are prepared. Biotechnological research is underway on putting silkworms to medicinal use. A soft drink christened Silk Water contains a protein found in silk known as glycine. Glycine is said to check the production of melanin in the skin, which causes tanning, freckles, and blotches. The fruit-flavored beverage is also spiked up with 18 amino acids extracted from silk by processing it into powder and dissolving this in water. The hands of workers who manually process silk are smooth despite long hours of exposure to water, the firm embarked on joint research with Hiroshima University and found sericin, another silk protein, to be responsible for the phenomenon. Other silk-related products on the market include candy containing silk fibers—offered as a health food— and shampoo and conditioner enriched with silk protein.

In 1997 the National Institute of Sericultural and Entomological Science of the Ministry of Agriculture, Forestry, and Fisheries,Japan with the cooperation of major contact lens maker succeeded in developing the basic technology for applying silk protein to the production of soft contact lenses. According to the institute, contact lenses made with silk protein are friendlier to the eyes than those made with synthetic materials. By inserting the genes of substances beneficial to human health in silkworm chromosomes, it hopes to breed "cocoon factories." Sasaki, (2003) developed "MU-silk" finishing :Research has started in Japan into a treatment capable of covering the surface of various textiles with a film of natural silk. Dr H Ueyama and two other researchers have developed a new treatment, called "MU-silk", that can give silkiness to a variety of different textiles. This technology makes effective use of real silk and started with the discovery of the solvent
"MU" that dissolves silk easily without decomposing it. Tyler (2001) The subject line changed repeatedly: Heian tears" - "Silk and aristocratic tears" - "tear-drops" - "Heian tears: lit or fig?" - "Soggy silk" - "rivers of blood" - "Hemp" - "Paper" - "Blood and guts" - "silk stains" "Silk absorbs moisture, which makes it cool in the summer and warm in the winter. Silk is a natural protein fiber, like human hair, taken from the cocoon of the silkworm. The natural glue, sericin, secreted by silkworms and not totally removed during manufacturing of the silk, is a natural sizing which is brought out when washing in warm water. Most silk fabrics can be hand washed. A tightly woven silk will not "shrink". Silk garments, however, can shrink if the fabric has not been washed prior to garment making. A good shampoo works well on silk. It will remove oil and revitalize your silk. Do not use an alkaline shampoo or one which contains ingredients such as wax, petroleum, or their derivatives, as these products will leave a residue on your silk and may cause "oil" spots. If static or clinging is a problem with your silks, a good hair conditioner. Yu-Qing (2005)reported the Synthesis of silk sericin peptides-L-asparaginase bioconjugates and their characterization,The natural silk sericin, recovered from Bombyx mori silk waste by degumming and degrading, is a water-soluble peptide with different molecular masses, ranging from 20 to 60 kDa. It is composed of 15 sorts of amino acids, among which the polar amino acids with hydroxyl, carboxyl and amino groups such as aspartic acid, serine and lysine account for 72%. The covalent attachment of the silk sericin peptides to L-asparaginase (ASNase) produces silk sericin peptides-L-asparaginase (SS-ASNase) bioconjugates that are active, stable, have a lower immune response, and have extended half-lives in vitro in human serum.

Terada S.et al., (2002) in their report opined that Sericin, a protein derived from silkworms, accelerates the proliferation of several mammalian cell lines including a hybridoma s Sericin, a constituent of the silkworm cocoon, was added to the culture of four mammalian cell lines: murine hybridoma 2E3-0, human hepatoblastoma HepG2, human epithelial HeLa and human embryonal kidney 293 cells. The proliferation of all cell lines was accelerated in the presence of sericin. Since BSA is obtained from bovine serum and the risk of infections such as bovine spongiform encephalopathy cannot be eradicated, sericin derived from insects could be a preferable culture medium supplement for stimulating the roliferation of mammalian cells. Kongdee, et al., (2005) reported modification of cellulose fiber with silk sericin It was found that sericin coated onto cotton surfaces as a film. Increasing sericin content in the finishing solution increased the amount of coated sericin, and a greater depth of color in dyed samples and reduced free formaldehyde content in treated samples were observed. The sericin content in samples was found to have a negligible influence on tensile strength and crease recovery angle. With increasing sericin content, electrical resistivity of the samples dramatically decreased and water retention increased, indicating that sericin-treated fabrics may be comfortable to wear because of its maintenance of moisture balance with respect to human skin. Because cotton textile coated with sericin exhibited low formaldehyde content and no biocidal activity against Klebsiella pneumoniae and Staphylococcus aureus, the fabric may reduce skin irritation and disturbance of physiological skin flora arising from textile contact.
8md formulations are featured in Allure, American Spa and "O" Oprah magazines, md formulations Moisture Defense Antioxidant Treatment Masque providing intensive hydration for dry skin, excellent for sensitive and Rosacea skin types.May use as a "sleep facial mask" all night. Perfect for air travel to counteract dry cabin air. md formulations-
facial mask-dry skin is clear, completely unnoticeable on the skin. |

Gently, tap around Eyes, Neck, Back of your handslmd formulations- facial mask-dry skin benefits This "super-hydration" md formulations treatment instantly softens and rejuvenates while soothing even sensitive skin.Silk Anne Bellina skin care 1973 protein and amino acids provide intense moisture to the skin, leaving it "silky" soft without a heavy, greasy feel. Silk protein also known as silk peptide is from the center of
the silk worm yarn. Silk protein is a natural moisturizer and adjusts the moisture in the skin to maximum benefit. It can make the skin cutin and keep rich moisture to make the skin more elastic. It also can effectively mcontrol growth of melanin in the skin. So it has fantastically good results for skin beautification. Silk protein and amino acids leave skin silky smooth and help reduce pore size as they protect the skin from dehydration, penetrate the skin to leave a silky smooth feel. Silk amino acids (also called silk proteins and sericin) are obtained by the hydrolysis of silk - a natural protein fiber. The soft lustrous thread obtained from the silk cocoon woven by the silkworm is so converted and used in cosmetic preparations. It gives the skin a very soft and velvety feel when
used in a cosmetic product, and has some good water-binding properties and silk amino acids (silk proteins) also has a certain amount of mantioxidant action. Silk amino acids (silk proteins) are used to prevent dehydration and is mostly found in eye rejuvenation gels and creams.

Siqin zhaorigetu,et al., (2001) reported that the Supplemental Silk Protein, Sericin, Suppresses Colon Tumorigenesis in 1,2- Dimethylhydrazine-Treated Mice by Reducing Oxidative Stress and Cell ProliferationThis study was done to discover the underlying mechanism of the inhibitory effect of sericin against colon tumorigenesis. Mice were fed a diet with 30 g/kg sericin for 115 d, and given a weekly injection of 1,2- imethylhydrazine (10 mg/kg body weight) for the initial 10 wk. Dietary supplemental sericin caused a 62 reduction in the incidence of colonic adenoma (PD/0.05), but did not affect the incidence of colonic adenocarcinoma. Sericin intake significantly reduced the number of colon adenomas. The results suggest that dietary sericin suppresses the
development of colon tumors by reducing oxidative stress, cell proliferation, and nitric oxide production.

What are Silk Amino Acids, and why do they matter?
Silk amino acids (also called silk proteins or sericin) are obtained by the hydrolysis of silk - a natural protein fiber. The soft lustrous thread obtained from the silk cocoon woven by the silkworm is converted and used in Best Shot shampoo and conditioners. It gives your pets skin a very soft and velvety feel. Silk amino acids also have a certain amount of antioxidant action and used to prevent dehydration of the coat and skin.

Another advantage of protein enriched Best Shot! Silk amino acids (silk proteins) are used to prevent dehydration and are mostly found in eye rejuvenation gels and
creams.Adding silk amino acids to a skin care product imparts certain qualities to the product - both enhance the working of the product and the way in which the product feels.Silk amino acids are classed as non-irritating to the skin and eyes and are non- toxic.When our silk amino acids are analyzed they typically contain the following amino acids - Alanine, Arginine, Aspartic acid, Cystine, Glutamic acid, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Proline, Serine, Threonine, Tyrosine and Valine.Amino acids are naturally substantive compounds which arereadily absorbed onto negatively charged substrates - such as skin.

Michaille (1990) reported the.Cloning and characterization of the highly polymorphic Ser2 gene of Bombyx mori.Three alleles of the sericin (Ser) 2-encoding gene (Ser2), called L, C and mC, were isolated from a Bombyx mori genomic library, and two related ones, called mCL and Cv, were also characterized in B. mori European strains. The Ser2 gene gives rise to two middle silk gland mRNAs by differential splicing. The size of a short mRNA (3.1 kb) is constant, but the length of a longer one ranges from 5 to 6.4 kb depending on the Ser2 allele. These length variations probably result from unequal recombinations in a region which contains about 30 well conserved 45-bp repeats coding for a Ser-like peptide. Furthermore,the L allele (and probably the mCL one) contains a 4.4-kb retrotransposon, resembling the copia-like ones of Drosophila.

Hamada et al. (1987) reported the Haemolymph control of sericin gene expression studied by organ transplantation. A factor that affects synthesis of sericin mRNAs of Bombyx mori was analyzed by organ transplantation and allatectomy. When silk glands of the third instar larvae were transplanted into the abdomen of fifth instar larvae, substantial amounts of sericin mRNAs were induced in the transplant. The induced sericin gene activity was suppressed upon re- transplantation into the abdomen of fourth instar larvae and induced again when the second hosts grew up to fifth instar larvae. An allatectomy performed on fourth instar larvae promoted production of these mRNAs, suggesting that the synthesis of sericin mRNA is regulated by the titer of juvenile hormone.

Matsunami, et al.(2003) reported the Expression pattern analysis of SGF-3/POU-M1 in relation to sericin-1 gene expression in the silk gland.Embryonic and larval expression patterns of the sericin-1 gene and its presumed transcription factor, SGF-3/POU-M1, in the silk gland were analyzed by in situ hybridization and immunohistochemistiy. The sericin-1 transcripts were first detected at embryonic stage 26 in an increasing gradient pattern in the middle and posterior part of the middle silk gland (MSG), while at the same stage the SGF-3/POU-M1 was already present in the entire anterior silk gland (ASG) and in the MSG but with a decreasing gradient pattern. The latter expression pattern was consistently maintained through all larval stages, while the sericin-1 expression was detected during the feeding stages but disappeared at the molting stages.

These observations suggest that, although the SGF- 3/POU-M1 was proposed to be a positive transcription factor for the sericin-1 gene, the protein might function in a negative manner on sericin-1 gene transcription. Alternatively, it is also possible that the sericin-1 gene might require another unidentified factor.

Morio Katsuno of the Silk Science Research Institute Dainihon Raw Silk Foundation states, "We have discovered that a protein called sericin contained in silk in large quantities inhibits the effects of enzymes that is responsible for skin blemishes and wrinkles. This protein also dissolves well in water, which helps to moisturize the skin. Another plus is that it has ultraviolet (UV) ray-blocking properties, which is good because the UVs can induce skin cancer. One reason for this could be that cocoons are designed to protect silkworms." Nature's unknown latent powers are truly remarkable.Toshio yamamoto (2002) bred the silkworm race'Sericin -hope'. In the process of breeding for sericin silkworm breed, the naked pupa was crossed with a normal breed KCS83 and a hybrid Nd x KCS83 was developed. This was back crossed with the normal breed and their progenies Nd/Nd, Nd/+ were sibmated in every generation ro develop homogyzygous sericin breed. The F1 hybrid is better than the parent for ease in rearing and more sericin production., Wenchow (2001) bred a
silkworm, " Hashojam" whish does not make cocoon, but produces sericin.Gulrajani., (2005) have reported the sericin is gelatinous and is made up of 18 types of amino acids. Sericin is highly hydrophyllic which absorbs water. It contains hydrophyllic amino acids such as asparitic acids, serine, threonine, argenine which absorbs water. Sericin is
classified as Sericin I, the outer layer which is highly soluble in water, the middle layer, sericin II is less soluble than sericin I and sericin III is very close to fibroin which is insoluble and difficult to separate. Sericin has many physiological functions such as anti-oxidation activity, tyrosine kinase activity, UVB protection and moisture holding
capacity. Sericin is used as a finished or coating material for man made or natural fibre, fabrics and articles.Material prepared with sericin are bio-degradable.Preparation of photographic plates with 2% sericin as compared to 8% gelatin is economical. It is more resistant to high temperature and therefore adapted to warm climates. The nagative film
after developing dries more readily (Pigorini, 1916).Sericin helps in hair ,growth. Dumas (in 1960) patented the hair growth by using purified sericin.Besides moisturing creams, puffs, masks with anti bacterial, anti fungal, deodorant effects are mild to skin, improve foaming ability in skin cleanses and efficiently removes soil, sebum from skin.

PATENT SURVEY

3 results were found in the worldwide database for sericin from cocoons in the title or abstract. One reference out of the three pertaining to the extraction of protein from cocoons was taken in to consideration.

A.T. Borysivna Patent number: UA65934 reported that the extraction of sericin from silkworm cocoons by boiling with hydrochloric acid solution at a ratio of 1: (95-105) for 2-4 hours, neutralizing the pH with water and dried by spraying.
21 results were found in the Worldwide database for sericin processing in the title or abstract

Method of processing sericin is as following:

YANG CHAOYOU Patent number: CN1786299 extracted sericin by using acid, alkali liquor, aldehyde group, and hydrogen peroxide which are cost effective and environment friendly.

ARISAKA YUKIE; Patent number: JP2006052515 fabricated an artificial flower out of cocoon without impairing sericin bu adding oil. OKAMOTO KENICHI; Patent number: JP2004353117 reported the extraction of the sericin from the cocoons by electrolysis of water. The liquid extract was dried by spraying. YAMADA EIJI; Patent number: JP 2004143649 reported a method of coating sericin to a fibrous material by using a water soluble dichlorotriazine compound and stabilizing the shape. HAGIWARA TOSHIO (JP); Patent number: W003038181 reported the coating of sericin to the fibre material using water-soluble dichlorotriazine compound, diamines and alkanolamines.MIYASHITA TORU; Patent number: JP11050397 developed a skin contact paper which absorbs the fats and sweat of skin without fading of makeup by suspending a wet paper into a liquid pulp comprising of wooden fibre, bast fibre and sericin, drying the paper and making it smooth with' the help of a hammer. It is hygroscopic and retains moisture. SHIGA YASUHIRO; Patent number: JP2000119966 developed a method of coating sericin to a synthetic fibre by treating the fibre in an aqueous solution containing glycine, sericin and chitosan.MINAMOTO TOSHIAKI; Patent number: JP2001039999 Sericin was extracted from the cut cocoons of the silkworm raised aseptically by using an artificial feed. The cocoons were heated in water at 90-130 °C at 10-100 L/kg/min liquid flow rate for 10-180 min. The extracted sericin was used in cosmetics, feed and for fiber processing. WAKABAYASHI SOUHEE; Patent number: JP4202855 developed a process to coat synthetic yarn with sericin powder using water soluble synthetic resin as a fixative .The coated material possess the vapour and antiseptic properties.
10 results found in the Worldwide database for: sericin and uses in the title or abstract.Two of them are listed below:NAKAGAWA MOMOKI; Patent number: JP2008069116 developed a water soluble cosmetic sheet using chitosan, fish collagen and sericin. The sheet dissolved in water is applied on the skin to make smooth. MASUNARI YOSHIYA; Patent number: JP2007175685 reported the extraction of sericin using sericin extractor in spring water and utilize the same for bathing.
Approximately 59 results found in the Worldwide database for: sericin product in the title or abstract. Some of the useful products are listed below:

SASAKI MASAHIRO;

Patent number: JP2007314574 reported that the slightly digestible agent in sericin extracted from the cocoons is used in controlling constipation.SASAKI MASAHIRO (JP); Patent number: US2007275875 reported that sericin is used in preventing colon cancer, as food additive and as health drink. KIM SUNG SOO; Patent number: KR20050015178 reported that the Jelly or beverage composition containing sericin extracted from silkworm cocoon as effective ingredient. Sericin 15% by weight extracted from the silk cocoons is mixed with agar 0.4 to 0.60% by weight to give a solution, which is added to drinking water 84.58% by weight and then sterilized at lOOdeg.C for 20min.

WOO JI HYUNG (KR); Patent number: KR8101586 reported that the sericin 1 % along with maltose, and ammonium nitrate were fermented at 30- 35°C, pH 7.0-7.4 is used as a degumming agent. HAN CHUNG SEOP (KR); Patent number: KR20020096607 developed a cosmetic composition comprising of 0.5 to 10.0% by weight of silk protein dissolved in neutral salt and hydrolysed with protease such as actinase or neutrase. This composition is used in the manufacture of soap, skin lotion, cream, pearl-off pack or bathing agent. KIMURA YOSHIHARU; Patent number: JP2006183231 developed the coating of sericin to a core fibre by dissolving sericin in an aqueous solution of 2 % by weight of sodium hydroxide and dipping the core fibre and drying. MAKITA MANABU Patent number: JP2006149510 reported that the hydrolysed silk is used as a head ornamental product because of its skin care property preventing inflammation of the skin and retaining the healthy skin. TERAMOTO HIDETOSHI; Patent number: JP2006111667 developed sericin hydrogel having a mol. wt. of >=50 kDa from silk gland, cocoon layer or cocoon using alcohol having excellent moldability.MEESILPA
PRATEEP; Patent number: JP2005171218 reported that the sericin extracted from the
That silk cocoons in water in the ratio of 1 : 20 by- weight, heated at about 110-120°C. under a pressure of <=14 lb/inch2for about 25-30 min. and used in the manufacture of soaps by the addition of 2.00-20.00 % by wt of sodium hydroxide or potassium hydroxide, and 1.00-30.00 %wt of glycerol. The product is safe, when used for smoothing and softening the skin and does not cause irritation. SHAO ZHENGZHONG (CN); Patent number: CN1583836 reported that a spongy porous silk protein was developed at a low temperature in a frozen phase without using neither pore generating agent nor an inorganic solvent. ZHANG YUQING (CN); Patent number: CN 1477141 a water soluble sericin was developed by treating the cocoons in water or alkaline solution with high-temp and high-pressure, at pH 8-12.

NOGATA AKIHIRO; Patent number: JP2003239174 reported'the development of functional fibre product by coating sericin to the synthetic fibres which gives a comfort to human bodies due to high water-absorbing and releasing property, is not toxic to the human bodies.OSADA KATSUE; Patent number: JP2004018648. reported that sericin was ectracted from silk cocoons using 0.5-10 mmol/L of sodium carbonate and 0.5-10 mmol/L of sodium hydrogencarbonate and is used as an antioxidant. YAMAZAKI MASAO; Patent number: JP2001302693 reported the extraction of sericin at 0 ° C for a long time and thawing at high temperature and high pressure. SASAKI MASAHIRO; Patent number: JP2000312568 reported that the sericin was extracted in water using organic solvent such as methanol or dioxane. Thereafter, passing the extract is passed through an ultrafiltration membrane or reverse osmosis membrane and drying the solution. The sericin is used in beverages and health foods. SASAKI MASAHIRO;
Patent number: JP2000256210 reported that the sericin is obtained from the cocoons or raw silk by using organic solvent, such as methanol, ethanol and dioxane, to precipitate the sericin, then filtering and drying the precipitate to form sericin powder. The sericins are preferably administered at a daily dose of 1-1,000 mg per kg of the body weight of the patient suffering from colorectal cancer. CHEN WENXING (CN); Patent number: CN1228438 reported the extraction of Sericin polypeptide having molecular weight of 200-5000 from husks, frison and waste silk by using enzymatic hydrolysis process and is used as raw material in cosmetics.KIRIKAWA MAYUMI; Patent number:

JP2000044598 reported that the production of silk protein fine powder from grinding the scouring silk. SHIOMI HIDEKAZU; Patent number: JP11276876 reported that an aqueous solution of sericin was prepared from silk cocoons in water by maintaining a pH of 3-4 using organic or inorganic flocculating agent. The solution was filtered and dried to obtain sericin. The sericin possesses moisture retaining and cosmetically whitening effects, and further, suppresses the oxidation of oils/fats in an emulsion through anti-oxidation action to maintain the oils/fats in a good condition for a long time. OGAWA ATSUKO; Patentnumber: JP11193210 reported that the sericin hydrolyzate is obtained by partially hydrolyzing and eluting sericin contained in cocoon or raw silk using an alkali aqueous solution or by en2ymatic treatment. The yeast degradation product is obtained by decomposition of yeast due to autodigestion or proteinase or hydrolysis of yeast due to an acid. It is used in production of collagen, prevention of skin wrinkles and moisture-retention.YAMAt>A HIDEYUKI; Publication number: JP10226653 reported that the sericin was extracted by partially hydrolyzing the sericin from cocoon, or raw silk using an alkali solution or by a enzyme treatment in a pH of 3-5 by using an organic acid or an inorganic acid. The sericin was also extracted by mixing the cocoons in a water-soluble organic solvent such as methanol, filtering and drying the resultant sericin to obtain an easily water-soluble sericin having molecular weight of 10,000-20,000 with 20- 40 %mol sericin. YAMADA HIDEYUKI; Patent number: JP10140154 reported that the sericin from cocoons were hydrolysed using acid, alkali or enzyme and the extract was treated with the organic solvent such as methanol, ethanol, or dioxane. The precipitate was separated by Alteration and dried to obtain sfcricin powder.TEZUKA HIROMU; Patent number: JP9241131 reported that the hair tonic was obtained by adding one or more kinds of materials selected from galenicals such as Symphytum officinale extract, Chlorella pyrenoidosa extract and Ginkgo biloba leaf extract, lipase-inhibiting agents such as Spindus mukurossi extract, Cinchona succirubra extract and Glycyrrhiza extract, lipid peroxide-inhibiting agents such as Arnica montana extract and Paeonia lactiflora extract, and compounds such as photosensitive element 103 and tyrosine to the water-swollen product of sodium montmorillonite among natural or synthetic smectites.

A composite clathrate obtained by adding amino peptides obtained by the enzymatic decomposition or acidic or basic hydrolysis of silk-originated fibroin or sericin to the water- swollen product of aluminum magnesium silicate or hectorite may further be added to the above system .TAM AD A YASUSHI; Publication number: JP9227402 developed an anticoagulant from sericin. Concentrated sulfuric acid at 10-90 % by wt. was added to 0.5-500 pts. by wt.of sericin and by sulfonation at a temperature of 20-100°C for several hours anticoagulant was developed. NOMURA MASAKAZU; Patent number: JP8060547 reported the development of skin caring fiber product. The natural fibre was immersed in an aqueous solution containing sericin which in turn contains 20-40% by wt .of serine, an amino acid which gives 0.05-5.% wt of sericin protein to the fiber and possess a skin caring property.HATAKEYAMA HYOE; Patent number: JP8012738 developed a polyurethane compound prepared by treating sericin with polyisocynate and used in the films, molding and in fiber. VELO LUIGI MARIA (IT); Patent number: EP0608706 reported that the sericin was recovered from degumming of raw silk yarn, from silk fabric and from waste silk by ultra Alteration method.
Approximately 108 results found in the Worldwide database for: sericin protein in the title or abstract. The relevant ones are given below:

NAKAGAWA MOMOKI Patent number: JP2008069116 reported that the sheet cosmetic is composed of a water-soluble sheet formed from a composition containing one or more kinds of natural polymers selected from chitosan, fish collagen and sericin and used to smoothen the skin by dissolving the water-soluble sheet in water and applying the water- soluble polymer to the skin. KOBAYASHI HATSUMI; Patent number: JP2007210902 developed method for extracting polymeric silk protein from silkworm. The liquid silk was extracted from the matured silkworm. The liquid silk was heated in water to dissolve the silk at a temperature of 20-30°C. Sericin and fibroin having different solubility were separated from water. The silk protein was dried on a flat plate and stored as a film.

PICANO ROGER (CH); US2007041925 developed Skin and hair care preparation containing a combination of protein hydrolyzates.Sericin was mixed with pashmina, cashmere, wool, merino wool to prepare hair care products.SUGIMOTO KENICHI Patent number: JP2007204376 developed swelling control composition for hair. A composition for controlling the swelling of the hair comprised of sericin having olecular weight of 5,000-20,000 and vegetable protein having molecular weight of 200- ,000.MIN SIJIA WU (CN) ; Patent number: CN 1985791 Prepared skin care silk protein gel cream. The silk protein cream was produced by homogenizing the water soluble sericin at 0-90°.C with 70-98 %;water and used in producing high quality natural skin care cosmetics. SUGIMOTO KENICHI; Patent number: JP2007015936 developed HAIR COSMETIC. The hair cosmetic comprises a dibasic acid diester compound and sericin having molecular weight of 5,000-20,000, which prevents the elution of protein inside the hair,controls roughness and dryness of the hair even under a dry environment. JING LIN ZHANG (CN); Patent number: CN 1896265 developed natural-silk nutrient and silk polypeptide by enzyme method. Silk nutrient was prepared by using enzymes to increase the nutritive value. TSUBOUCHI KOZO; Patent number: KR20040081059 developed method of functional polypeptides originating from silk protein and use thereof in the field of medicament, cosmetics, foods and the like for skin care. An aqueous solution of protein in neutral salt was prepared and treated with peptide bond cleaving agent for producing functional polypeptide composition originating from silk protein .JU WAN SEOK; Patent number: KR20040073153 developed compositions for preventing or treating brain neuronal diseases and improving recognition function, (a) a pharmaceutical^ effective amount of silk peptide produced by decomposition of silk protein; and (b) pharmaceutically acceptable carriers, wherein the silk protein is silk sericin or silk fibroin; the decomposition of silk protein is carried out by using calcium solution such as calcium chloride, acid hydrolysis, protein decomposing enzyme such as thermoase, flavourzyme, sumizyme, protamex and protin and a combination thereof; and the average molecular weight of the silk peptide . is 300 to 100,000 dalton.KIM SUNG SOO; Patent number: KR20050015177 developed facacil pack composition comprising sericin protein extracted from cocoon for whitening skin.Sericin powder extracted from cocoons by freeze drying method was mixed with grape seed powder, apricot seed powder, job's tear powder, potato flour, green tea powder, chestnut skin powder, pine mushroom powder, lemon juice, vitamin C and le mon flavor for the preparation of facial pack.CHO BONG RIM (KR); Patent number: KR20030085738 developed hair tint composition containing silk and wool proteins. A hair tint composition comprising of 0.5 to 5.0% by weight of sericin from silk cocoons and wool protein by removing wax from silkworm cocoon using acid and an alkali.HAN CHUNG SEOP (KR); Patent number: KR20020096607 developed cosmetic composition containing silk protein. Soluble sericin was dissolved in neutral salt and hydrolysed with protease such as actinase or neutrase after removing the salt. The composition is formulated into toilet soap, skin lotion, cream, pearl-off pack or bathing agent. TANG YI (CN); Patentnumber: CN 1800473 developed Active sericin silk non-woven water jetting cloth process. In this process the non-woven cloth was coated with sericn and used for face pack, sanitary towel (pad), and wound paste. TAMURA TOSHIKI; Patent number: JP2006137739 developed method for producing proteins using silkworm middle silk gland-specific gene expression system.

Transgenic silkworms comprising GFP whose expression is regulated by the sericin gene promoter were produced. TSUBOTA YUKIKO; Patent number: JP2006117607 developed SKIN CARE PREPARATION. Skin care unit was prepared by using hydrolysed sericin along with antiinflammatory agent. ISHIKAWA OSAMU; Publication number: JP2005289889 developed DISINFECTANT FORMULATED WITH SILK PROTEIN. This invention is related to the disinfectant formulated with silk protein, especially sericin. It prevents drying of skin and chapped hand. MEESILPA PRATEEP; Patent number: JP2005171218 developed SILK SOLID SOAP AND METHOD FOR PRODUCING THE SAME. Thai silk or cocoons in a ratio of 1 :20 in weight in water were heated at 110-120°C at <=14 lb/inch2for about 25-30 min. The silk cleansing product contains 0.01-20.00 wt% of the sericin protein, 2.00-20.00 wt% of sodium hydroxide or potassium hydroxide, and 1.00-30.00 wt% of glycerol (for glycerinated soap) or 20.00-75.00 wt% of a vegetable or animal fat (for hard soap).SHAO ZHENGZHONG (CN); Patent number: CN 1583836 developed porous sericin sponge material. Preparation of sponge-like porous materials of silk protein is at low temperature to have frozen phase. No cross-linker nor pore-generating agent nor surfactant nor organic solvent with strong toxicity is used. TSUBOUCHI KOZO; Patent number: JP2004300142 developed.

METHOD FOR PRODUCING FUNCTIONAL POLYPEPTIDE ORIGINATING FROM SILK PROTEIN, AND ITS USE.

Sericin was extracted using neutral salt solution and treating with peptide bond-cleavihg material to cleave the peptide bonds between specific amino acid residues in the silk rotein. YAMAZAKI MASAO; Publication number: JP2004218122 developed METHOD FOR RECOVERING SILK PROTEIN. Sericin solution is dripped on and spread over a outer surface of circumferential hot drum and heated to dryness, and a filmy solid formed on the hot drum outer circumferential surface is peeled off with a scraper and recovered, into powdery sericin. SASAKI MASAHIRO;Patent number: JP2003252744 developed

MULTIFUNCTIONAL PROTECTIVE AGENT AGAINST ULTRAVIOLET
HAZARD.

The sericin possess the protection action against the skin from harmful ultraviolet rays but also having therapeutic activities for the damaged skin. YAMAMOTO TOSHIO; Patent number: JP2001245550 developed

BREED OF SILKWORM CAPABLE OF PRODUCING SERICIN
IN LARGE AMOUNT.
Silkworm mutant breed producing large quantity of sericin was developed. The breed can be identified from other breeds of the silkworm by detecting restriction fragments length
polymorphism(RFLP) of a silkworm chromosome DNA by using a silkworm cDNA derived from a gene carried by each of 28 pairs of silkworm chromosomes as a probe. NAKAMURA KAZUO; Patent number: JP2001106794 developed SERICIN-

CONTAINING POLYMERIC

HYDROUS GEL AND METHOD FOR PRODUCING THE SAME.

polymeric hydrous gel is produced by cross-linking and insolubilizing a blended material of the sericin with polyvinyl alcohol. SUZUKI NATSUMI; Patent number: JP2000191446 developed HAIR COSMETIC. Hair cosmetic comprises of sericin 0.5-5 % by wt.; sugar alcohol0.5-10 % by wt; a neutral amino acid, glycine, 0.05-10 by wt.%. This cosmetic is widely used in, solutions, foams, sprays.YAMADA HIDEYUKI; Patent
number: JP2000184868 developed
WATER FOR FOOD AND DRINK.
A sericin powder is obtained by treating a cocoon of a domesticated silkworm removed from a chrysalis in distilled water at 80 °C for 1 h, then concentrating the resultant eluate under a reduced pressure, removing a flocculant by filtration, adding ethanol thereto, separating the deposited substance by filtration, drying the separated substance and
providing the sericin powder. Furthermore, a sericin hydrolyzate powder is prepared by treating the cocoon of the domesticated silkworm after removing the chrysalis with an aqueous sodium hydrogen carbonate at 0.1 mol/L concentration at 80 °C for 1 h, eluting a sericin hydrolyzate and affording the sericin hydrolyzate powder in the same manner as that described above. This water for a food and drink is obtained by adding the resultant sericin powder and/or sericin hydrolyzate powder to a commercially available mineral water, heating the resultant mixture at 100 deg.C, completely dissolving the sericin powder and/or sericin hydrolyzate therein while carrying out the sterilization, adding the mineral water and filtering the obtained mixture. YOSHII FUMIO; Pa,tent number: JP2000169736 developed

HYDROGEL COMPOSITION CONTAINING SILK PROTEIN.

A hydrogel composition having rubberlike elasticity and long-lasting water retention characteristics by blending a water-soluble polymer with a silk protein and exposing the blend in an aqueous solution to a radiation to cause crosslinking. TSUBOUCHI KOZO; Patent number: JP11092564 developed METHOD FOR OBTAINING HIGH-MOLECULAR-WEIGHT SERICIN BY EXTRACTION. 1 g of cocoon , raw silk thread is immersed in 20-60 ml of an aq. urea soln. having a concn. of 2-8 mol/1 and sericin extracted at 100-130 deg.C for several min to several hr, pref. for 10 min or lower. After the completion of extraction, sericin having an average mol.wt. of 50,000 or higher is separated and recovered from the extractant e.g. by molecular- sieve chromatography, by using an ultrafiltration film, or by frational precipitation with an org. solvent. HIKODA TOYOHIKO; Patent number: JP5071003 developed FINE HAIR.The fine hair was obtained by imparting (A) protein fibers 7-1 lOmum in thickness and <=3mm in mean
length (e.g. scoured wild silk, wool, sericin-set silk) with (B) a separation improver such as silica gel, alumina sol or tannin. HIRABAYASHI KIYOSHI; Patent number: JP1256351 developed

FOOD USING SILK PROTEIN AND PRODUCTION THEREOF.

Sericin aqueous solution is mixed with aqueous solution of juice, soup, nutritional drink.
KIRIKAWA MAYUMI; Patent number: JP2000044598 developed SILK PROTEIN FINE POWDER AND ITS PRODUCTION. The silk protein fine powder consists
of 5-30 wt.% of sericin with an average molecular weight of 7,000- 300,000 and 95-70 %wt. of silk fibroin with an average particle size of <=30 &mu m and the maximum particle size of <=60 &mu m. This silk protein fine powder is obtained by scouring silk followed by drying and then grinding the silk-scoured product. TSUBOUCHI KOZO; Patent number: JP11092564 developed METHOD FOR OBTAINING HIGH- MOLECULAR-WEIGHT SERICIN BY EXTRACTION. 1 g of cocoons were immersed in 20-60 ml of an aq. urea soln. having a concn. of 2-8 mol/1 and extracted at 100-130 °.C for several min to several hr, pref. for 10 min or lower. After the completion of extraction, sericin having an average mol.wt. of 50,000 or higher is separated and recovered from the extractant e.g. by molecular-sieve chromatography, by using' an ultrafiltration film, or by frational precipitation with an org. solvent.
2 results found in the Worldwide database for: sericin in cosmetics in the title or abstract.

TSUBOUCHI KOZO; Patent number: KR20040081059 developed

PRODUCTION METHOD OF FUNCTIONAL POLYPEPTIDES

ORIGINATING FROM SILK PROTEIN AND USE THEREOF IN THE FIELD

OF MEDICAMENT, COSMETICS, FOODS AND THE LIKE FOR SKIN
CARE.
An aqueous solution of sericin from cocoons of Antheraea yamamai were extracted in neutral salt and treated with peptide bond- cleaving agent; and cleaving peptide bonds between specific amino acid residues of silk protein. KAWAHARA YUTAKA; Patent number: JP2003311213 developed METHOD FOR SEPARATING SERICIN ANDFIBROIN IN SILK. The method for separating the sericin and the fibroin comprises of irradiating silk with UV rays in a humidifying atmosphere and grinding the silk with a mill, etc., then sieving the powder with a meshed screen or vibration screen.

DRAWBACKS IN EXISTING STATE-OF ART & HOW THE DRAWBRACKS HAVE

BEEN OVERCOME BY YOUR INVENTION:
Two reported state-of- art sericin extraction processes in India:
I. The process for sericin extraction from the normal silkworm hybrid cocoon shells.
II. The process for sericin extraction from the reeling waste water by Gulrajani (2005, 2006).

I.DRAWBACKS IN THE PROCESS OF SERICIN EXTRACTION FROM THE

NORMAL SILKWORM HYBRID COCOON SHELLS:

The normal hybrid cocoon shells are boiled in water using sodium carbonate and soap solution for the extraction of sericin. The process of boiling of cocoons and evaporation of the water for its condensation to
obtain sericin is cumbersome. This process is lengthy and time consuming. In view of the above draw backs the above sericin processes are not commercially viable. This process is limited to the different silkworm breeding laboratories of India, where sericin is estimated from the normal silkworm cocoon shells to understand the quantity of sericin present in the newly developed silkworm breed / hybrid which is considered as one of the aspects for assessing the breeds / hybrids. But it is not commercially exploited so far.

II.DRAWBACKS IN THE PROCESS OF SERICIN EXTRACTION FROM REELING

WASTE WATER:

Silk reelers of different parts of India reeled the cocoons by Multi- End Silk Reeling machines, Cottage Basin and Charka to obtain the silk yarn. During the cocoon cooking, sericin is dissolved in the water and water is disposed of as waste. This silk reeling waste water is used for sericin extraction.

The main draw back lies in the non-availability of the silk reeling waste water from single silk reeling unit. Of the above three techniques, the most widely used ones are the charka and cottage basin where the required and viable silk reeling waste water is not available in any single reeling unit, and it needs to be collected from several reeling units which is time consuming and cost escalating. Multi-end silk reeling unit is expensive and are limited in number, and as such the availability of source material is also limited. Besides, the recovery of sericin is only about 20-25%.

II. DRAWBACKS IN THE PROCESS OF SERICIN EXTRACTION FROM THE

NORMAL SILKWORM HYBRID COCOON SHELLS:

The normal hybrid cocoon shells are boiled in water using sodium carbonate and soap solution for the extraction of sericin. The process of boiling of cocoons and evaporation of the water for its condensation to obtain sericin is cumbersome. The draw back lies in lengthy and time consuming process with low recovery of sericin (20-25%). In view of the above draw backs the above sericin processes are not commercially viable.

SOLUTION

In the above state-of- the art sericin extraction processes the source material used is normal silkworm hybrid cocoon shells. The sericin content in these cocoons constitutes about 20-25% of the weight of the cocoon shells. In the present invention process, a special sericin rich silkworm hybrid N x CSR26 has been identified and selected as an exclusive source material whose cocoon shells contain 95-98% sericin as against 25 % sericin in the normal silkworm hybrid cocoon shells. Moreover, this invention process involves direct method of sericin extraction from the sericin rich hybrid cocoon shells for the best possible commercial exploitation. Whereas the available state of the art processes involve indirect method of sericin extraction which is not commercially feasible because the source material is from the reeling waste water, where collection and processing involves long time and cumbersome.

In this invention, generation of adequate quantity of source material for sericin extraction process can be done in one place; the problem of collection and transportation of the source material as in the state of the art processes is totally avoided. Moreover, only one inorganic salt in low concentration viz., sodium carbonate is used, and the process time is kept at the minimum and the recovery of the end product sericin is 95- 98%. In the state-of-the art process, inorganic salt and soap in large
quantity are used involving longer process time with low recovery of sericin i.e., 20-25%.
Objects of Invention the main object of this invention is to develop a process for the extraction of sericin.

Other object is to develop a process which is pollution free.

Another object is to develop a user friendly process having low cost ingredients.
Yet another object is to develop a process wherein minimum equipment and infrastructures and chemicals are being used.
Further object is to develop a process having maximum productivity in optimal processing time.

Other object is to develop a process having maximum utility of mulberry sericin rich bivoltine silkworm hybrid 'NXCSR 26' cocoon shells.

Statement of Invention

This invention relates to a sericin extraction process from sericin rich bivoltine silkworm hybrid cocoon shells of NXCSR 26 comprising the steps of, stifling sericin rich hybrid cocoons at temperature of 100 to 110°C for 4-8 hours, removing the pupae from cocoons and using sericin rich empty shells, extraction using 0.05 to 1.5% liquor solution of sodium carbonate, preferably 0.2 to 0.7% sodium carbonate, wherein the liquor solution is heated at 90 to 100°C, 60 to 80g hybrid cocoon shells are added to the solution and boiled for 25-35 minutes, cooling the contents at room temperature and filtering through cheese cloth, neutralizing by addition of 0.05 to 0.15 N hydrochloric acid, 4-6 ml, dehydrating the neutralized filtrate by spray drying at 100-130°C at the rate of 4-6 litres in 50-70 mins wherein sericin is obtained as fluffy off white powder.
Brief description of accompanying drawings
Fig 1. Shows Attributes of sericin.
Fig 2. Figure showing sericin rich silkworm bivoltine hybrid cocoons. Fig 3. Shows SDS-Polyacrylamide gel electrophoresis of sericin along with the markers. Lane 1: Sericin; Lane 2: Protein marker.
Detailed description of Invention
In the present work, eleven polyvoltine and eight bivoltine silkworm breeds were crossed with sericin rich novel bivoltine silkworm breed and 38 silkworm hybrids prepared, evaluated for identification of sericin rich hybrids. Among the 22 polyvoltine hybrids the sericin content ranged between 85.71 (N x M26) to 90.01 % (N x NDV6). In the 18 bivoltine hybrids, it ranged between 94.16 (N x CSR6) and 96.21 % (N x CSR26). Of these, two hybrids viz., N x NDV6 and N x CSR26 were short listed based on higher sericin content and evaluation index values. Finally N x CSR26 was selected based on higher sericin content and better rearing performance.
This N x CSR26 was used as source material for sericin extraction. During the process of sericin extraction, 0.05 to 1.5% of sodium carbonate, 0.05 to 1.5% of sodium chloride and 0.05 to 1.5% of sodium bicarbonate were used to extract maximum quantity of extraction of sericin from sericin rich hybrid cocoon shells using minimum quantity of inorganic salts. Finally 0.2 - 0.7% of sodium carbonate was selected as the final concentration of the sodium carbonate solution for effective and maximum quantity of sericin extraction from the sericin rich bivoltine hybrid cocoon shells. Using 0.2 to 0.7% concentration of sodium carbonate solution, it is possible to extract 95-98% sericin from the sericin rich hybrid cocoon shells.

The extracted sericin was neutralized by the addition of 0.05-0.15 N hydrochloric acid, and the neutralized filtrate is dehydrated by spray- drying method and sericin as a fluffy off-white powder is obtained. The sericin powder was subjected to SDS-PAGE electrophoresis to confirm the molecular weights of the protein fractions. Our sericin product also showed three protein fractions having molecular weights of 150, 250 and 400 kDa respectively. This observation is in conformity with the earlier works of Takasu, Yamada and Tsubouchi (2001) and Yu Qing Zhang et al., (2005). The physical properties of the extracted sericin is a light fluffy off white powder, odourless having a bulk density of 0.0869g / ml. The water regaining property is 7.15g / g protein and pH is 7.0 at 1% solution.

PREPARATION OF THE SOURCE MATERIAL FOR THIS INVENTION PROCESS:

Background:

China, Japan, Korea and Thailand have developed several methods for sericin extraction from silk cocoons for commercial exploitation. Information in respect of the extraction of sericin from cocoons for commercial exploitation is limited in India.
Identification of sericin rich bivoltine hybrid as source material for this invention process:
Initially 38 crosses were prepared by crossing with eleven polyvoltine and eight bivoltine pure breeds. After short listing based on high sericin content and better rearing performance, one hybrid namely N x CSR26 with 95-98% sericin was identified. This special sericin rich bivoltine silkworm hybrid is found to contain more sericin (95-98%) and less fibroin as compared with any other sericin rich silkworm hybrids. This hybrid became a source material for sericin extraction.
The sericin rich hybrid cocoons were stifled at a temperature of 100 to 110° C for 4-8 hours. The pupae are removed from the cocoons and the sericin rich empty shells are the source material for this invention process.
The cocoon characteristics of sericin rich hybrid is given in table 1.
TABLE: 1. COCOON CHARACTETRESTICS OF THE NEW SERICIN RICH
HYBRID (N X CSR26)
Hybrid Cocoon Shell Shell Sericin
SL. weight weight percentage percentage /
No (g) (g) (%) cocoon
~ N x CSR26 2.073±0.051 0.0794±0.014 3.83±0.282 98.5 ± 0.05
BENCH MARK LEVEL OF SERICIN EXTRACTION PROCESS

3. Laboratory equipment's
❖ Hot water bath
❖ Electronic balance
❖ Spray dryer
❖ PH meter Oven
❖ Measuring cylinder-1000 ml
❖ Round bottom flask

PROCESS DESCRIPTION

Step 1. Preparation of liquor solution: 2-7g of sodium carbonate is dissolved in 950- 1050 ml water to obtain a liquor solution of about 0.2 - 0.7% concentration of the solution.
Step II. The liquor solution is heated in a hot water bath. At 90 -100° C temperature, about 60-80g sericin rich silkworm hybrid cocoon shells are added to the solution and boiled for about 25-35 minutes.
Step III. The contents are cooled at room temperature and filtered through cheese-cloth. The pH of the filtrate is neutralized by the addition of 0.05 to 0.15 N hydrochloric acid.
Step IV. The neutralized filtrate is dehydrated at a temperature of 100- 130 ° C by spray-drying method at the rate of 4-6 litres in 50- 70 minutes and sericin as a fluffy off-white powder is obtained.
About 95-98 g of sericin is obtained by processing l00g of
sericin rich bivoltine silkworm hybrid 'N x CSR26' cocoon shells.
The viability lies in the fact that this invention process is least time
consuming, with high out put of the desired product.

FLOW CHART

BENCH MARK LEVEL OF SERICIN EXTRACTION PROCESS


THE PROPERTIES OF SERICIN EXTRACTED BY THIS PATENT PROCESS:

SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS OF SOLUBLE SILK PROTEINS:

The sericin / protein were extracted from mulberry silk cocoons using 0.2 to 0.7 % sodium carbonate in water. The different protein compositions were studied in a SDS- polyacrylamide gel electrophoresis. Three major protein fractions having molecular weights approximately of 400, 250 and 150 kDa were represented as three bands (Fig.4). This observation is in conformity with the earlier published results of Takasu, Yamada and Tsubouchi (2001) and Yu Qing Zhang et al., (2005).

Sodium dodecyl sulphate -Polyacrylamide gel Electrophoresis (SDA- PAGE) was carried out according to method of Laemmli (1951) using 10% cross linked gel as the resolving gel and 2.5% cross linked stacking gel . The resolving gel contained 30% acrylamide, 0.8% N, N methylene- bisacrylamide, 20% TEMED, 10% SDS and 1.5 M Tris-HCl buffer pH 8.8.
The Stacking gel contained 30% aciylamide, 0.8% N,N methylene- bisacrylamide, 20% TEMED, 10% SDS and 0.5 M Tris-HCl buffer pH 6.8. The various components were mixed in proportions to get the appropriate cross linking. Polymerization was initiated by adding ammonium per sulphate. Glycine (0.192M) buffer pH 8.3 containing 0.1%SDS was used as the reservoir buffer. The sample buffer comprised of 1.5M Tris, 10ml of glycerol and 50mg Bromophenol blue made up to 100ml in distilled water. Sample were mixed with sample buffer and placed in a boiling water bath for 20 min. Cooled and loaded onto the gel. Standard protein marker (Fermentas) also runs each time along with sample. Electrophoresis was carried out at a constant voltage of 100 volts till the dye band moved to the bottom of the gel (approximately for two and half hours). The gel was then stained with coomassie brilliant blue R-250 dye made in a solvent system consisting of methanol, glacial acetic acid and water (45:5:50 v/v). The gels were destained with methanol, acetic acid and water 50: 7: 43 v/v. Electrophoresis of sericin on SDS-PAGE. Sericin protein was stained by Periodic acid -Schiffs stain and the molecular weight markers were stained by Coomassie blue.

NOVEL FEATURES OF THE INVENTION:
Single step processing technique
Time saving technique

Maximum recovery of sericin from the sericin rich cocoons of bivoltine silkworm hybrid 'N x CSR26'.
Total avoidance of corrosive chemicals
User-friendly
Higher purity and multiple utility of the end product.
95-98% sericin was extracted from the sericin rich hybrid cocoons shells The ratio of source material to the end product is very high in this extraction process. In the sericin rich hybrid N x CSR26, 60-80g of cocoon shells converted to 57-78g of end product.

ADVANTAGES OVER OTHER KNOWN ALTERNATIVES INCLUDING

IMPORTED ALTERNATIVES:

Maximum utilization of cocoons of sericin rich bivoltine silkworm hybrid N x CSR26
Maximum sericin recovery at the lab scale.
Short processing technique
Gross reduction of production time and man hours.
Minimum use of chemical and corrosive solvents.
Pollution-free process.
Absence of harmful effects of residuary chemical in the end product.

We Claim

1. A sericin extraction process from sericin rich bivoltine silkworm hybrid cocoon shells of NXCSR 26 comprising the steps of:-

i) stifling sericin rich hybrid cocoons at temperature of 100 to 110°C for 4-8 hours

ii) removing the pupae from cocoons and using sericin rich empty shells

iii) extraction using 0.05 to 1.5% liquor solution of sodium carbonate, preferably 0.2 to 0.7% sodium carbonate, wherein the liquor solution is heated at 90 to 100°C hybrid cocoon shells are added to the solution and boiled for 25-35 minutes

iv) cooling the contents at room temperature and filtering through cheese cloth

v) neutralizing by addition of 0.05 to 0.15 N hydrochloric acid, 4-6 ml

vi) dehydrating the neutralized filtrate by spray drying at 100-130°C at the rate of 4-6 litres in 50-70 mins wherein sericin is obtained as fluffy off white powder.

2. A sericin extraction process as claimed in claim 1, wherein one inorganic salt sodium carbonate is used in low concentration.

3. A sericin extraction process as claimed in claim 1, wherein process time is kept at minimum.

4. A sericin extraction process as claimed in claim 1, wherein 60-80 gms silkworm hybrid cocoon shells are used.

5. A sericin extraction process from sericin rich bivoltine silkworm hybrid cocoon shells of NXCSR 26 as described and illustrated herein