Sericin Hydrolysate Research Articles

Functional modification of thermostable alkaline protease from Bacillus halodurans SE5 for efficient production of antioxidative and ACE-inhibitory peptides from sericin

In this work, the hydrolysis efficiency of recombinant protease_SE5 (r_SE5) on sericin was improved by protein engineering. The S166D variant, obtained from the first round of site-directed mutagenesis, exhibited a 1.4-fold increase in protease activity (602 U/mg) and sericin hydrolysis efficiency (123 μg peptide/mL). Further substitution of Ser166, located on a low-affinity calcium-binding loop, by Glu consequently improved sericin hydrolysis capability. The S166E variant showed remarkable protease activity (671 U/mg) and sericin hydrolysis efficiency (140 μg peptide/mL) with no changes in thermostability compared to r_SE5. Interestingly, the purified S166E efficiently hydrolyzed sericin, with a maximal peptide yield of 540 μg/mL within 60 min. The S166E-derived sericin peptide also exhibited higher ABTS radical scavenging activity (6470 μmol TEAC/L) and angiotensin-I-converting enzyme (ACE) inhibition activity (40%). Additionally, the identification of peptides by LC-MS/MS revealed that two smaller peptides (HHSGVNR and GWWWNSD) with a higher proportion of key amino acid residues were produced by S166E. These peptides were the potential bioactive peptides predicted by the BIOPEP database. Therefore, this study demonstrates the structure–function relevance between the low-affinity calcium-binding loop and hydrolysis of sericin by protease_SE5. S166E is a more powerful protease for producing value-added sericin hydrolysate, which can be used as an active ingredient for food and pharmaceutical applications.

Covalent binding of flavonoids with silk sericin hydrolysate: Anti-inflammatory, antioxidant, and physicochemical properties of flavonoid-sericin hydrolysate conjugates.

To construct food ingredients with improved bioactivities and physicochemical properties, two sericin hydrolysate-flavonoid conjugates, bearing quercetin and rutin covalently bound to sericin, were prepared under alkaline conditions. UV spectroscopy and SDS-PAGE confirmed that sericin hydrolysate and oxidized flavonoids formed conjugates, which were primarily the result of covalent interactions. Changes and differences in molecular weight distribution, secondary and tertiary structures, surface hydrophobicity, and surface morphology were observed. Anti-inflammatory activities were evaluated by basing on inhibitory activity against nitric oxide (NO) production and 15-lipoxygenase (15-LOX). The conjugates showed significantly improved (p<.05) anti-inflammatory and emulsifying properties than unmodified sericin hydrolysate. Meanwhile, the covalent interaction had a positive effect on the antioxidant activity of sericin hydrolysate. PRACTICAL APPLICATIONS: We had prepared flavonoid-sericin hydrolysate conjugates and evaluated the effect of covalent binding of flavonoids on the physicochemical, anti-inflammatory, antioxidant, and emulsifying properties of sericin hydrolysate, which is beneficial to broaden the range of applications of sericin and flavonoids in the food, cosmetics, and pharmaceutical industries.

Factors affecting sericin hydrolysis and application of sericin hydrolysate in sericin films.

Sericin is a natural protein and a by-product obtained from silk processing. To enhance the antioxidant properties of sericin, sericin hydrolysis was studied. The solvent effects (distilled water, citric acid and hydrochloric acid) and hydrolysis methods (heat treatment (water bath) and mild ultrasonic treatment at 20%, 40% or 60% amplitude) were investigated on the properties of sericin hydrolysate (SH). Furthermore, solvent effects (distilled water and 15% ethanol) were examined for the properties of the sericin films incorporated with selected SH. The SH samples from acid hydrolysis and the ultrasonic method had a darkened visual appearance. However, the degree of hydrolysis and antioxidant activity of SH increased with ultrasonic-assisted acid hydrolysis. The molecular weight (MW) of sericin was notably reduced. As expected, hydrochloric acid hydrolysis resulted in a lower MW for the SH than from citric acid. Thus, SH from hydrochloric acid and 20% amplitude in the ultrasonic method were selected to produce a sericin film. As revealed, using distilled water as a general solvent provided films with lower solubility and water vapor permeability but higher tensile strength. Furthermore, the addition of SH enhanced the antioxidant properties of its hydrolysate as a novel protein packaging film material for various applications.

Antioxidant Hydrolysate of Sericin from Bombyx mori Cocoons

Sericin was obtained from Bombyx mori cocoons in 28% yield with 92.06% purity. Its hydrolysate was produced by an enzymatic method. A fraction with molecular mass < 3 kDa was collected using ultrafiltration. The contents of 19 peptides with molecular masses in the range 600–3000 Da were determined using MALDI TOF/TOF. Their amino-acid sequences were found. The antioxidant activity of sericin hydrolysate (IC50) was 0.07 mg/mL according to absorption of ABTS radicals and 1.09 mg/mL according to absorption of OH radicals. These were 4.5 and 1.2 times greater, respectively, than that of sericin.

Accurate prediction for adsorption rate of peptides with high ACE-inhibitory activity from sericin hydrolysate on thiophene hypercross-linked polymer using CoMSIA in 3D-QSAR model

Efficient screening of angiotensin converting enzyme inhibitory (ACE-I) peptides from agricultural or edible sources attract increasing attention. However, their purification process from the complex natural system is still semi-empirical or even uncontrollable, which has seriously reduced their screening efficiency. Herein, inspired by the prediction of ACE-I activity, 3D-QSAR was proposed to predict the adsorption performance of peptides from sericin hydrolysate (SH) having high ACE-I activity on porous hypercross-linked polymers according to their molecular structures. Thiophene hypercross-linked polymer (T-HCP) possessing better screening capacity for ACE-I peptides was chosen as our research object in this work. The sequence and relative adsorption rate of 101 peptides in SH were analyzed by LC-MS and was used as a database to construct the relationship of peptide’s chemical structure and adsorption performance on T-HCP by Comparative molecular similarity indices analysis (CoMSIA) from 3D-QSAR. Optimum CoMSIA revealed that enhanced interaction of hydrophobicity and H-bond between T-HCP and the peptide was conducive to increase the adsorption performance of di- to hexa- peptides. Based on these relationships, the adsorption capability of 24 designed peptides with distinguished hydrophobic and H-bond fields was predicted on T-HCP by using optimum CoMSIA and the results of half of these were verified, which showed high consistency with their predicted adsorption rate. Interestingly, these peptides having higher adsorption capacities on T-HCP also possessed higher ACE-I activity. This can be attributed to the high concentration of aromatic surface with π-π interaction and weak-polar CSC group with H-bond interaction on T-HCP material, which is ideal for the selective adsorption of peptides with higher ACE-I activity from SH. This study provides important theoretical guidance for the industrial screening of bio-functional peptides from complex protein mixtures.

Enzymatic valorization process of yellow cocoon waste for production of antioxidative sericin and fibroin film

AbstractBACKGROUNDCocoon waste, solid waste from the silk industry, is currently utilized as low‐cost spun silk yarn. In this study, the enzymatic valorization process of yellow cocoon waste to produce antioxidative sericin hydrolysate and fibroin film was demonstrated.RESULTSThe enzymatic process involving thermostable alkaline protease from Bacillus halodurans SE5 (protease_SE5) was superior to the conventional high temperature and high pressure and 0.5% Na2CO3 processes. Protease_SE5 produced sericin hydrolysate with high peptide concentration (0.335 mg mL–1) and provided degummed fibroin with a complete structure. Sericin hydrolysate from protease_SE5‐assisted hydrolysis showed remarkable radical scavenging activities on ABTS, DPPH and FRAP assays with 602, 3.90 and 24.8 μmol Trolox equivalents (TE) g−1 protein, respectively. After ultrafiltration and size‐exclusion chromatography, two active fractions (F1 and F2) were obtained from sericin hydrolysate with ABTS radical scavenging activity of 2120 and 3289 μmol TE g−1 protein, respectively. Identification of bioactive peptides by de novo sequencing was conducted, and seven candidate peptides were identified with high content of key antioxidative amino acids (His, Phe, Trp, Tyr and Arg) in the sequences. Moreover, bioactivities of several of the peptides were predicted by bioinformatics databases. Besides, the mechanical properties of enzyme‐derived fibroin film were also improved, with tensile strength and elongation at break of 62.1 MPa and 3.6% in the dry state, and 8.5 MPa and 140% in the wet state.CONCLUSIONThis enzymatic process could be an effective way for valorizing cocoon waste. The value‐added products from agricultural waste have promising applications in food, pharmaceutical and biomedical materials. © 2020 Society of Chemical Industry (SCI)

Silk sericin has significantly hypoglycaemic effect in type 2 diabetic mice via anti-oxidation and anti-inflammation

The sericin protein from silk-processing waste added to the normal diet at 0.8% (g%) level was administered orally to type 2 diabetic (T2D) mice to investigate its hypoglycaemic effects and mechanism. The oral protein is in the form of silk sericin hydrolysate, obtained from a boiling treatment of 0.025% calcium hydroxide solution. The protein significantly decreased fasting blood glucose, fasting plasma insulin, and glycosylated serum protein levels; improved oral glucose tolerance and insulin tolerance, and enhanced antioxidative activities. The protein could ameliorate the pathological damage in pancreatic β-cells and the liver tissue. It enhanced the expression of key proteins and enzymes, including insulin receptor, insulin receptor substrate, PI3K, phosphorylated-AKT, hepatic kinase, GLUT4, glycogen synthase, GSK3β, GLK, PFK1, PKM2, and AMPKα, which are related to insulin metabolism and glycolysis. The protein also reduced the expression of G6Pase, PCK, and ACC, which are related to gluconeogenesis and lipid metabolism in the liver, and decreased the expression of TNF-α, IL-6, P65, and IKKβ related to inflammation. In general, sericin could maintain normal glucose levels and regulate insulin secretion, insulin and lipid metabolism, and inhibition of inflammation. Therefore, sericin protein could be developed into a novel functional health food with significantly hypoglycaemic effect.

Characterization of thermostable alkaline protease from Bacillus halodurans SE5 and its application in degumming coupled with sericin hydrolysate production from yellow cocoon

Abstract Bacillus halodurans SE5 was newly isolated and grew well on a medium containing crude sericin extract from cocoon. Thermostable alkaline serine protease (protease_SE5) capable of decomposing sericin extract was purified to homogeneity from culture supernatant with a final yield of 25% and 2.01 × 104 U/mg. Among the six natural proteins tested, protease_SE5 showed the highest activity toward sericin. The sericin degumming, bio-bleaching coupled with sericin hydrolysate production from yellow cocoon by protease_SE5 and commercial Alcalase were demonstrated in the presence or absence of dithiothreitol (DTT). The addition of DTT enhanced the efficacy of both proteases. However, without DTT, the protease_SE5 had higher degumming ability and produced sericin hydrolysate 4-times higher than commercial enzyme based on the soluble protein concentration. SDS-PAGE and size exclusion chromatography analysis revealed that the maximal concentration of oligopeptides was observed with the hydrolysate prepared by protease_SE5 and showed higher antioxidant activity than those from Alcalase. The appreciable radical scavenging activities of the crude peptide (1.36 ± 0.07 mM) on ABTS, DPPH, and FRAP assay were 1568 ± 78, 3.6 ± 1.6, and 13.6 ± 0.4 μmol TE/L, respectively. Protease_SE5 has potential application for one step degumming and preparation of bioactive peptides from yellow cocoon.

Silk Sericin Hydrolysate is a Potential Candidate as a Serum-Substitute in the Culture of Chinese Hamster Ovary and Henrietta Lacks Cells.

The silk sericin hydrolysate (SSH) from the waste of silk processing as a substitute of fetal bovine serum (FBS) was used for the culture of Chinese hamster ovary (CHO) cells and Henrietta Lacks (Hela) strain of human cervical cancer cells. The survival ratio of these cells cultured in SSH media were similar to or higher than those in FBS media. Especially after the serum was replaced by low concentration of SSH at 15.0 μg/ml for 5 d, the proliferation of both cells was also similar to or higher than that of FBS group; the percentages of CHO and Hela cells in S-phase were 28.9 and 28.0%, respectively. The former is nearly two times that of FBS group, the latter is also higher than the control group. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that among the differentially expressed genes, the relative expression of CXCL12 gene of CHO cells in SSH group increased, was three times that of serum group, and the relative expression of LCN2 gene of Hela cells increased 2.8 times, indicating that these related genes were activated to promote cell growth and proliferation. These results fully illustrated the hydrolysated sericin has a potential use as serum substitutes in cell culture.

Amino acid composition and anti‐polyphenol oxidase of peptide fractions from sericin hydrolysate

SummarySericin hydrolysate (SH), prepared from enzymatic hydrolysis of sericin, was investigated for its antipolyphenol oxidase (PPO) properties. SH decreased PPO activity from both purified mushroom PPO and extracts from apple and eggplant, and retarded browning in fresh‐cut apple and eggplant. SH was a competitive inhibitor using catechol as a substrate. SH exhibited copper ion‐chelating power and reducing power abilities. Fractionation of SH using size exclusion chromatography resulted in four fractions, designated as F1, F2, F3 and F4, with PPO inhibition of 35.75%, 3.89%, 24.52% and 14.75%, respectively. Ser and Asp were major amino acids found in F1. Amino acid sequences in F1, as investigated by LC‐MS/MS using de novo sequencing, contained a high ratio of amino acids with chelating ability. Moreover, amino acids with reducing power ability and with antityrosinase ability were also identified in the sequences.

High-Throughput and Rapid Screening of Novel ACE Inhibitory Peptides from Sericin Source and Inhibition Mechanism by Using in Silico and in Vitro Prescriptions.

Several novel peptides with high ACE-I inhibitory activity were successfully screened from sericin hydrolysate (SH) by coupling in silico and in vitro approaches for the first time. Most screening processes for ACE-I inhibitory peptides were achieved through high-throughput in silico simulation followed by in vitro verification. QSAR model based predicted results indicated that the ACE-I inhibitory activity of these SH peptides and six chosen peptides exhibited moderate high ACE-I inhibitory activities (log IC50 values: 1.63-2.34). Moreover, two tripeptides among the chosen six peptides were selected for ACE-I inhibition mechanism analysis which based on Lineweaver-Burk plots indicated that they behave as competitive ACE-I inhibitors. The C-terminal residues of short-chain peptides that contain more H-bond acceptor groups could easily form hydrogen bonds with ACE-I and have higher ACE-I inhibitory activity. Overall, sericin protein as a strong ACE-I inhibition source could be deemed a promising agent for antihypertension applications.

Hypocholesterolemic effect of sericin-derived oligopeptides in high-cholesterol fed rats.

The beneficial effect of cholesterol-lowering proteins and/or peptides derived from various dietary sources is continuously reported. A non-dietary protein from silk cocoon, sericin, has also demonstrated cholesterol-lowering activity. A sericin hydrolysate prepared by enzymatic hydrolysis was also expected to posses this effect. The present study was aimed at investigating the cholesterol-lowering effect of sericin peptides, so called "sericin-derived oligopeptides" (SDO) both in vivo and in vitro. The results showed that SDO at all three doses tested (10mgkg-1day-1, 50mgkg-1day-1, and 200mgkg-1day-1) suppressed serum total and non-HDL cholesterol levels in rats fed a high-cholesterol diet. Triglyceride and HDL-cholesterol levels were not significantly changed among all groups. The fecal contents of bile acids and cholesterol did not differ among high-cholesterol fed rats. SDO dose-dependently reduced cholesterol solubility in lipid micelles, and inhibited cholesterol uptake in monolayer Caco-2 cells. SDO also effectively bound to all three types of bile salts including taurocholate, deoxytaurocholate, and glycodeoxycholate. Direct interaction with bile acids of SDO may disrupt micellar cholesterol solubility, and subsequently reduce the absorption of dietary cholesterol in intestines. Taking all data together, SDO or sericin peptides exhibit a beneficial effect on blood cholesterol levels and could be potentially used as a health-promoting dietary supplement or nutraceutical product.

누에 실샘 가수분해물의 제조 및 특성 규명

Silk protein has been explored to be used for biomedical applications for several decades. However, it has not been used in this field cause to their irreversible crystallization after dissolving in water. The existing methods of silk protein hydrolysis using silkworm cocoon were used with harmful solvents and through a very complicated process. Therefore, we have developed novel methods for the production of water-soluble hydrolysate using silkworm gland. We manufactured two types of silkworm gland-derived hydrolysate (water-soluble SGH, SSGH; total SGH, TSGH) and compared the characteristics with commercial cocoon-derived sericin hydrolysate (CSH). The molecular weight of SGH ranged from 7 to 50 kDa (SSGH) and 5 to 15 kDa (TSGH) within glycine, alanine, and aspartic acid as a main amino acid composition. In contrast, CSH ranged from 15 to 50 kDa within serine and aspartic acid. The results of FTIR implied that SGH was more soluble form than CSH, as shown by the decrease in the -sheet structure at on amide I peak. In comparison with 10% fetal bovine serum, 0.1% (1 mg/ml) SSGH had equivalent effect on the proliferation of human dermal fibroblasts and mesenchymal stem cells. All results of the SSGH made by novel manufacturing process indicate the SSGH is more preferable as a culture medium supplement than cocoon-derived sericin.

Inhibitory effect of sericin on polyphenol oxidase and its application as edible coating

SummarySericin, a water‐soluble globular protein derived from silk industry wastewater, was investigated for food industrial applications. The results proved that sericin retarded polyphenol oxidase (PPO) activity. However, the degree of inhibition varied depending on the enzyme origins, the types of substrate, sericin content and sericin molecular size. Using catechol as a substrate, under the conditions studied, sericin lowered purified mushroom PPO and apple extract PPO activity by 40% and mango extract PPO by 75%. Kinetic studies on purified mushroom PPO indicated that the type of inhibition of sericin was dependent on the substrates used. Inhibitory effects of sericin increased as the sericin content increased. The reduction in sericin molecular size by enzymatic hydrolysis produced sericin hydrolysate with ability to decrease PPO activity approximately three times greater than that of sericin. Fresh‐cut Red Delicious apples coated with sericin showed significant reduction in weight loss and improvement in the colour and texture.

Enzymatic production of bioactive peptides from sericin recovered from silk industry wastewater

Bioactive peptides were produced from sericin, a by-product from silk industry wastewater, by using Protease P. The optimal hydrolysis conditions (pH of 8.40, temperature of 43.97 °C and the enzyme/substrate ratio of 3:100) were estimated by using response surface methodology with a maximum degree of hydrolysis of 14.43% and the minimum IC 50 value for the ferrous ion-chelating activity of 0.128 mg/mL. The freeze-dried sericin hydrolysate (SH), produced by using Protease P and under the above optimal conditions, contained 82% of crude protein, 13% of moisture and 5% of ash. The physiochemical analysis shows that the amino acids with hydroxyl groups (Ser and Thr) of SH accounted for 30% or so, and SH was a mixture of hydrophilic peptides mostly distributed among 250–4000 Da. SH exhibited excellent antioxidant activities in two measurements, including the ferrous ion-chelating activity and the reducing power. Moreover, SH also exhibited a notable tyrosinase-inhibitory effect in a dose-dependent manner. This study firstly showed an effective bioprocess for the production of sericin bioactive peptides, which may be used as valuable ingredients in the food, cosmetic and medicine industries.

Sericin separation from silk degumming wastewater

This study was conducted to the recovery of sericin protein produced during silk degumming process. Sericin waste solution from conventional degumming process in Thailand contained high BOD (4840 mg/L), COD (8870 mg/L) and nitrogen content (0.11%). This indicated protein contamination and high cost for wastewater treatment. To reduce the treatment costs as well as to recover valuable sericin protein, membrane filtration and enzymatic hydrolysis of recovered sericin were studied. The results showed the quality improvement of wastewater (BOD, 158 mg/L; COD, 260 mg/L) as well as that an amount of sericin protein with molecular weight 2427–9863 Da were recovered after membrane filtration process. The recovered sericin was further enzymatically hydrolyzed to obtain sericin hydrolysate (average molecular weight 1046–2795 Da), which is mostly suitable for further cosmetics application.

A Sericin-Derived Peptide Protects Sf9 Insect Cells from Death Caused by Acute Serum Deprivation

Sericin is the silk protein enveloping fibroin fibers in cocoons. Sericin hydrolysate protects cultured Sf9 insect cells from death caused by serum deprivation; the activity depends on the repeats of 38 amino acids. A partial peptide from the 38 residues, SGGSSTYGYS, inhibited serum-deprivation death as well. Cell viabilities in the presence of 10% (v/v) foetal calf serum, no additives and 1 mM: SGGSSTYGYS were 96, 12 and 31% on the third day after inoculation, respectively. Aromatic residues seemed to be important because SGGSSTWGWS had the same activity as SGGSSTYGYS but SGGSSTAGAS had no activity.

The silk protein, sericin, protects against cell death caused by acute serum deprivation in insect cell culture.

Sericin is the silk protein that covers fibroin fibers and functions as a 'glue' in the cocoons of silkworms, and its most abundant component, Ser1, contains repeats of Ser- and Thr-rich 38 amino acid residues. The viability of Sf9 insect cells was 20, 57 and 49% on the fifth day and 41, 91 and 70% on the ninth day after serum deprivation in the presence of no additives, 3000 microg sericin hydrolysate and 350 microg SerD (the peptide containing the two repetitive units) ml(-1), respectively. Thus, the sericin samples were useful in preventing cell death and promoting cellular growth after acute serum deprivation.

Cryoprotective effect of the serine-rich repetitive sequence in silk protein sericin.

The silk proteins, fibroin and sericin, are produced in the silk gland of Bombyx mori, and hydrophilic sericin envelops fibroin with successive sticky layers in the formation of a cocoon. To study the biological functions of sericin, we focused on the serine-rich sericin peptide consisting of 38 amino acids, which is a highly conserved and internally repetitive sequence of a sericin protein. The corresponding gene was chemically synthesized, and the PCR-amplified gene was ligated to oligomerize sericin peptide and fused at the amino terminus to a His-tagged and proteolytic cleavage sequence in an inducible expression vector. When the dimers of sericin peptides were overexpressed in Escherichia coli, the transformants showed a prominent increase in cell viability after freezing in medium. Further, the purified dimeric sericin peptide from E. coli was found to be effective in protecting lactate dehydrogenase from denaturation caused by freeze-thaw. Both of these protective effects against freezing stress in cells and proteins were also observed with sericin hydrolysate. These results indicate that this unique sericin peptide, like sericin, has a high cryoprotective activity and will be valuable as a new biomaterial for industrial use.