• Journal of Microbiology and Biotechnology
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• v.23 no.12
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• pp.1779-1784
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• 2013

Although enzyme-hydrolyzed silk fibroin has been reported to enhance cognitive function before, it has been still unknown which peptides can improve memory. Here we report that amino acid sequences of three novel peptides were identified from fibroin hydrolysate. Fibroin hydrolysate was obtained by hydrolysis with protease after partial hydrolysis with 5M $CaCl_2$. Synthesized peptides derived from these sequences improved scopolamine-induced memory impairments in mice. We confirmed this hydrolysate had effects that improved learning and memory abilities by performing the Rey-Kim test. From this hydrolysate of silk fibroin, amino acid sequences of eight peptides were identified by LC-MS/MS. Three peptides (GAGAGTGSSGFGPY, GAGAGSGAGSGAGAGSGAGAGY, and SGAGSGAGAGSGAGAGSGA) were synthesized to investigate whether they could improve memory. Passive avoidance test and Morris water maze test were performed, and all peptides showed memory-enhancing abilities on scopolamine-induced memory impairments in mice. In this study, we identified three novel peptides that could improve memory, and that silk fibroin hydrolysate was a mixture of various active peptides that could enhance memory.

• KSBB Journal
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• v.13 no.1
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• pp.114-118
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• 1998

The effects of fibron solubilization conditions on molecular weight distribution of enzymatically-hydrolyzed silk peptides were investigated. The weight-averaged molecular weights of silk proteins prepared by solubilization with calcium chloride, ethylenediamine and sulfuric acid were 41600, 3308, and 1268 dalton, respectively. Silk peptides in the average molecular weight range of 600-1200 dalton were obtained by protease treatment from solubilized silk fibroin. After the acid hydrolysis of silk protein using hydrochloric acid for 24 hr, silk protein was hydrolyzed to peptides whose average molecular weight and free amino acid content were 145 dalton and 80%, respectively. It was possible to control molecular weight distribution of silk peptides by the combination of solubilization and hydrolysis methods. Among the various treatment methods, acid solubilization followed by protease treatment had an advantage of molecular weight control for the peptide production.

• Microbiology and Biotechnology Letters
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• v.34 no.2
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• pp.115-120
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• 2006

Enzymatic hydrolysis of silk fibers were investigated for the preparation of soluble silk peptides by ten food-grade proteases from Bacillus, Aspergilius, and plant sources. Silk fibers were dissolved for 1 hr in a 2:1 cosolvent (50% $CaCl_2$: ethanol) by heating at $90^{\circ}C$. The silk solution was filtered to remove Impurity particles and desalted for 50 hours by a dialysis process to remove the used cosolvent. When the silk hydrolysis was performed at $45^{\circ}C$ for 2 hours, most proteases from Bacillus and Aspergillus generated large amounts of insoluble aggregates. On the contrary, proteases from plant sources produced much less aggregates during prolonged incubations and also exhibited high hydrolysis activities. In regards of the solubility and broad molecular sizes of produced silk peptides, Bromelain was finally selected and applied for the enzymatic hydrolysis of silk fibers.

• Journal of Microbiology and Biotechnology
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• v.26 no.12
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• pp.2228-2228
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• 2016

• Microbiology and Biotechnology Letters
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• v.30 no.1
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• pp.39-45
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• 2002

It was found that the peptides originated from the hydrolysates of silk fibroin have in vitro immunostimulating effects in murine macrophage RAW264.7 cells. The stimulation effects on nitric oxide (NO) production resulted from treatments of acid or enzymatic hydrolysates were measured. The silk fibroin preparation isolated from cocoon was most efficiently digested by acid hydrolysis. Even though the sole treatment of acid hydrolysate stimulated the NO production in dose-dependent pattern, a part of its activity was found to be caused by the contaminated endotoxin, LPS. When each endotoxin-free hydrolysates obtained by filtering it through an ultrafiltration membrane of molecular weight (MW) cut-off 10,000 to eliminate LPS was used, the peptic hydrolysate with lowest degree of hydrolysis showed the highest activity. The fractions of peptic hydrolysate with MW ranges of 1,000∼10,000, 500∼1,000 and below 500 also showed a higher MW-higher activity correlation. From the analyses of amino acid composition of each hydrolysate, it was found that the contents of arginine, lysine, alanine and glycine residues affected the activity level of hydrolysate. The results of this study showed a possibility of utilizing fibroin as a source for immunostimulating (chemopreventive) functional peptides.

• International Journal of Industrial Entomology and Biomaterials
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• v.2 no.1
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• pp.7-13
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• 2001

Enzymatic hydrolysis of different forms of silk fibroin (soluble, gel and insoluble forms) by industrial and commercial enzyme preparations to obtain aqueous and powdered silk fibroin in relatively mild conditions was investigated. A mono-enzymatic hydrolysate systems were tested for hydrolysis of water-soluble form of fibroin as most productive form of protein substrate. Insoluble forms of substrate usually were hydrolyzed less effective. In some cases from soluble fibroin substrate gel was formed during hydrolysis process. This hindered intermixing and decreased rates of hydrolysis. Insoluble sediments were formed in enzymatic hydrolysates in other cases. These sediments and also sediment after chemical hydrolysis were purified and tested on amino acids content for comparison. Sediments formation in these conditions are considered as pure tyrosine isolation method. Obtained hydrolysates were characterized by gel-chromatography analysis and other standard biochemical methods. Possibility of application of enzymatic hydrolysis for preparation of silk fibroin hydrolysates is discussed.

• Biomolecules & Therapeutics
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• v.19 no.2
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• pp.224-230
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• 2011

In order to develop silk peptide (SP) preparations possessing cognition-enhancing effect, several candidates were screened through in vitro assays, and their effectiveness was investigated in facilitated brain aging model rats. Incubation of brain acetyl-cholinesterase with SP-PN (1-1,000 ${\mu}g/ml$) led to inhibition of the enzyme activity up to 35%, in contrast to a negligible effect of SP-NN. The expression of choline acetyltransferase (ChAT) mRNA of neural stem cells expressing ChAT gene (F3.ChAT) was increased by 24-hour treatment with 10 and 100 ${\mu}g/ml$ SP-NN (1.35 and 2.20 folds) and SP-PN (2.40 and 1.34 folds). Four-week subcutaneous injections with D-galactose (150 mg/kg) increased activated hippocampal astrocytes to 1.7 folds (a marker of brain injury and aging), decreased acetylcholine concentration in cerebrospinal fluid by 45-50%, and thereby impaired learning and memory function in passive avoidance and water-maze performances. Oral treatment with SP preparations (50 or 300 mg/kg) for 5 weeks from 1 week prior to D-galactose injection exerted recovering activities on acetylcholine depletion and brain injury/aging as well as cognitive deficit induced by D-galactose. The results indicate that SP preparations restore cognitive functions of facilitated brain aging model rats by increasing the release of acetylcholine, in addition to neuroprotective activity.

• Korean Journal of Pharmacognosy
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• v.33 no.4 s.131
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• pp.312-318
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• 2002

Effect of the acid hydrolysate of silk fibroin on obesity was investigated in obese(057BL/KsJ-db/db) mice. After 8 weeks feeding of 1%(w/w) or 3%(w/w) fibroin hydrolysate, the extents of reduction in body weight were significantly higher than that of obese control. The weight reduction in female mice was higher than that in male mice. Plasma leptin in male mice increased up to 1.8-fold higher level than obese control by feeding hydrolysate. In case of female mice, however, it rather decreased with increased feeding concentration of hydrolysate. From the results of high glycine and serine contents of peptide fractions contained in fibroin hydrolysate, it was inferred that fibroin peptides might affect xylosyltransferase(XT) activity on chondroitin sulfate synthesis causing to change susceptibility of adipocytes to hormones such as insulin followed by the reduced leptin synthesis in female mice. The result of the higher lipolysis in hydrolysate-fed group than obese control indicated that the reduction in body weight was due to the increased lipolytic activities in male and female mice in common.

• Journal of Sericultural and Entomological Science
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• v.41 no.2
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• pp.108-115
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• 1999

An improvement of methods in fibroin hydrolysates preparat significantly enlarges their applications to practical use. Acidic hydrolysis by hydrohloric acid is one of the methods for silk fibroin depolymeriza6tion. A low yield of final product and long time of the process are the demerits of this method. Possibility of preparation of water-soluble silk hydrolysates with more yield and less expenses is investigated in this study. Such possibility is occurred with the increasing tratment temperature and simultaneously decreasing treatment time, concentration of hydrochoric acid respectively, the concentration of sodium hydroxide used for neutraliza6tion of hydolysates after hydrolysis. Colour is decreased in this case and a small amount of activated, too. Protection of hydrolysates against precipitation after neutraliza6tion, and separation and during concentrating process is the other merit of this method. Creamy-coloured insoluble silk powder is the remainder of hydrolyzed fibroin. This is the only the immobiliza6tion of enzymes and other physiological active substances. Fine particles of this powder can be used as additives for artificial diets and cultural media, as well as raw materials for polymer membranes, etc.

• Korean Journal of Pharmacognosy
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• v.33 no.1 s.128
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• pp.21-28
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• 2002

Antidiabetic effects of the acid hydrolysate of silk fibroin were investigated by oral administration to animal models for diabetes mellitus, Fibroin protein was extracted from cocoon and digested to peptides of low-molecular weight range (mainly below 3,000) and amino acids by acid hydrolysis, Feeding of the fibroin hydrolysate resulted in a significant recovering effect on reduction of body weight gain and a lowering effect on blood glucose gain in streptozotocin-induced diabetic Sprague Dawley rats (STZ rats) which were used as an insulin-dependent diabetic animal model. But the body weight and blood glucose level in C57BL/KsJ-db/db mice (db/db mice), an non-insulin-dependent diabetic animal model, were not changed significantly by the feeding, On the other hand, plasma leptin levels increased according to increased feeding amount of the hydrolysate in STZ rats and db/db mice in common, It was concluded from the results that the fibroin hydrolysate might stimulate the insulin secretion by recovering or activating pancreatic ${\beta}$ cells and result in the increased plasma leptin level. It was also deduced that the antidiabetic improvements in body weight and blood glucose gain in STZ were thought to be due to the increased insulin secretion, but in db/db mice of which the diabetic symptoms were caused by insulin resistance, the stimulated secretion of insulin was unlikely to be able to change body weight and blood glucose level significantly.