recovery and purification of silk sericin from silk waste

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Recovery and Purification of Silk proteins from waste of silk industry.

OBJECTIVE:

To recover and purify silk protein sericin from waste silk from silk industry by

various processes and equipment.

ABSTRACT

Sericin is the second main component in cocoons, which are removed in the silk reeling

process of the raw silk industry and in the silk waste degumming of the spun silk industry.

The main amino acid of sericin, serine, exhibits a skin moistening and anti-wrinkle action,

which is interesting to use for film formation in this study. The extraction conditions of

sericin from two silk wastes, pieced cocoon and inferior knubbs were studied to find the

optimum extraction conditions. Boiling water extraction was considered based on the

response surface methodology (RSM) in order to identify the important factors for the

sericin extraction. The two factors considered were time and temperature. Both factors were

needed to be independent parameters in the predicted equation in order to improve the modelfit with R2 = 0.84. The components of extracted sericin were 18.24% serine, 9.83% aspartate,

and 5.51% glycine with a molecular weight of 132 kDa.

.

1Department of Biotechnology, RVCE, Bangalore.




INTRODUCTION

Silk has been of great scientific interest for centuries and a new insight about these polymers

have been surfacing with improved analytical methods and molecular biology tools. Silk is

composed of broad range of primarily protein based high molecular weight polymers.Silk is

produced by silkworm Bombyx mori which belongs to Bombycidae which is one of the

families that produce silk.Certain spiders belonging to Saturnidae family also produce silk.

Bombycidae produces a delicate twin thread of silk fibroin, which is coated by a protective

cover of sericin. Silk protein is a similar to collagen, elastin, keratin, fibroin, sporgin etc.

which is an essential constituent of cocoon filament.

Sericulture in India is an essential cottage industry. The post rearing operations are

fairly cost effective and silkworm rearing is still only considered as a side activity to main

farm activity. India is the second largest producer of silk in the world and has the distinction

of producing all the four varities of silk.Presently,India produces nearly 16,700mt silk and

reeled silk prices are in the range of Rs 900-1300/Kg,the pierced cocoons and waste silk

generated at the rearing are sold at Rs 80-100/kg,The waste contributes nearly 30 % of total

cocoon production. Waste silk can be classified as waste from the cocoon, rearing waste and

thread waste. Silk waste can be used as coarse yarn and spun silk, which can be incorporated

in natural rubber to achieve the physiochemical properties. It is also possible to utilize the silk

waste by extracting fibroin and sericin from silk polymer. This helps to make sericulture a

viable agro industry.

Structure of Silk

Silk is a continuous strand of two-filaments cemented together forming the cocoon of

silkworm, Bombyx mori.Silk filament is a double strand of fibroin, which is held together by

gummy substance called silk sericine or silk gum.Silk fibroin is the protein that forms the

filament of silkworm and gives its unique physical and chemical properties.Silk adapts

various secondary structures,including α-helix,β-sheet,and crossed β-sheet.

Fibroin is a glycol protein composed of two equimolar protein subunits of 370 and 25

kDa covalently linked by disulphide bonds.Fibroin filament is made of both crystalline and

amorphous domains.

The use of recovered sericin from silk reeling and degumming processes in the silk

industry will add more value to sericin. Moreover, the recovery of sericin will minimize

the BOD and COD values in the waste water. Since sericin is composed of 80% amino acids

that contain hydrophilic groups, such as serine, aspartate, and glycine, it can absorb moisture

very well. Sericin is also a good antioxidant. Therefore, sericin is used in many applications,





for example, in the medical field as an anticoagulation agent and as an anticancer agent. In

cosmetics, sericin is used in skin and hair products. Sericin that has a molecular weight of

more than 20 kDa can be used as medical biomaterials, degradable biomaterials, compound

polymers, functional bio membranes and hydrogels (Zhang, 2002; Lee et al., 2004; Kongdee

et al., 2005). Sericin is interesting to determine its use as a biopolymer film. Biopolymer

films are generally made from thin sheets of biomaterials such as proteins, carbohydrates, fats

and biomass. These films can be used for food coatings to prevent water and oxygen

permeation between food products and the atmosphere. In this research, the extraction

conditions in boiling water of sericin from silk waste were studied in order to predict the

equations for computing the amount of extracted sericin under a defined condition.

1.1 Sericin extraction and properties

1.11 Sericin extraction in boiling water

Two types of silk waste, pierced cocoons (the result of breeding moths that have emerged

from their cocoons to produce eggs required for the next crops) and inferior knubbs

(outer portion of the cocoon layer obtained in the first process of reeling cocoons), from

Bombyx mori (Thai Golden Silk, Multivoltine strain) were used in this study.These raw

materials were analyzed moisture and sericin contents following the standard method

(ISA, 1993). The sericin contents were determined based on the gravimetric method. Sericin

extraction in boiling water was studied with two important factors, which were temperature in

the range of 82-120°C (above 105°C using autoclave) and time in the range of 10-60 min.

The experimental design chosen for this study was the central composite design (CCD) and

then was performed by response surface methodology (RSM). This design was a full factorial

design with all combinations of the factors at two levels (high, +1, and low, -1). The centre

point (coded level 0), which was the midpoint between the high and low levels, was repeated

with five points. The range and levels of the independent variables with actual andcoded

levels were given in Table 1. The total number of test runs needed for this design was 13.

The sericin extraction was carried out by subjection

silk waste of 20 grams in boiling water at a liquid ratio of





1:30 with various temperatures and times. After extraction,

sericin solution was separated by hydraulic pressing at 2.5

MPa for 1 min. The silk waste was washed with hot water (100

ml) at 80°C and then pressed again for 1 min to separate the

extracted solution. The collected solution was measured for

volume and the % dry solid was determined by hot air oven

method at 105°C in order to calculate the weight of

total extracted soluble solid. Protein concentration in the

sericin solution was determined using the Lowry method

(Lowry et al., 1951). The percentage of sericin extraction

was calculated based on sericin content in the raw material at

different experimental designs. RSM was applied to analyze

the effect of independent variables on the response parameter

(% sericin extraction) by matching the response studied (Y)

with the code factors using the polynomial model associated

with the experimental design as defined in Equation 1.

(1)

where Y is the dependent variable, A0

is the constant coefficient, Ai

is the linear coefficient, Aii

is the quadratic coefficient, and Aij

is the two factors interaction coefficient.

2.1.2 Statistical analyses





Statgraphic 3.0 for Windows (Statistical Graphics

Corp.) was used for regression and ANOVA analysis.

Response surface graphs were obtained from the regression

equation in actual levels of variables, keeping the response

function on the Z axis with X and Y axes representing the

independent variables.

a yield of 77% for both.

For pierced cocoons,

Y = -948.66+17.27X1

+1.726X2

-0.0763X1

2

+0.0078X1X2

-0.0352X2

2

(4)

R

2

= 0.84, p-value 0.0097

Maximum condition at 115°C, 37 min, %Sericin extraction

= 77.14%

For inferior knubbs,

Z = -1344.24+25.73X1





+1.56X2

-0.1181X1

2

-0.0060X1X2

-0.0106X2

2

(5)

R

2

= 0.83, p-value = 0.0120

Maximum condition at 108°C, 43 min; %Sericin extraction

= 77.10%

As Y = %sericin extraction,

X1

= Temperature (°C), X2

= Time (min)

3.1.2 Properties of extracted sericin

SDS gel electrophoresis was used to determine the

molecular weight of sericin from pierced cocoons. It was

found that the extracted sericin had a molecular weight distribution above 132 kDa compared

with a standard marker

as shown in Figure 2. This result was different from sericin

extracted from Bombyx mori cocoons with saturated aqueous

salt solution, which had molecular weights of 150, 250, and





400 kDa (Takasu et al., 2002). It was implied that the extracted

sericin was partly degraded by boiling water at high temperatures. The amino acid

compositions of sericin were given for

18 kinds as shown in Table 2. The main amino acids in the

sericin extracted from the pierced cocoons by boiling water

were serine (32.74%), aspartic acid (17.64%), and

glycine (9.89%). This composition was similar to those

sericin extracted from the Bivoltine strain of different sources;

1) raw material with serine 31.99%, aspartic acid 15.74%,

and glycine 14.20% (Vaithamsat and Kitpreechavanich,

2008), 2) degumming solution with serine 27.30%, aspartic

acid 18.80%, and glycine 10.70% (Wu et al., 2007), and 3) hot

water-soluble sericin with serine 28.00%, aspartic acid 17.97%,

and glycine 16.29% (Zhang et al., 2004). The

extracted sericin was used to make biopolymer since it had

a high molecular weight (more than 20 kDa) and can absorb

moisture well. These properties can be used for film formation

MATERIALS AND METHODS

Equipments

Principle of operation: The rotary evaporator is a device for gently and efficiently

evaporating solvents from a mixture. It consists of a heated rotating vessel (usually a large

flask) which is maintained under a vacuum through a tube connecting it to a condenser. The

rotating flask is heated by partial emersion in a hot water bath. The flask's rotation provides

improved heat transfer to the contained liquid; the rotation also strongly reduces the

occurance of 'bumps' caused by superheating of the liquid. The solvent vapours leave the





flask by the connecting tube and are condensed in the condenser section. The condenser

section is arranged so that the condensed vapours drain into another flask where they are

collected. It is a very efficient way of rapidly removing large quantities of solvent.

Procedure

1. The solvent collection flask of the unit should always be emptied prior use to prevent





accidentally mixing of incompatible chemicals. SAFETY FIRST!

2. The flask with the solution is placed on the rotary evaporator. The use of a bump trap

prevents the solution from accidentally splashing into the condenser (and being

contaminated). It is highly advisable to start with a clean bump bulb in case something

bumps over after all! This would allow the experimenter to recover the solution or solid.

3. A metal or Keck clip is used to secure the flask and the bump trap. The green one

shown below fits 24/40 ground glass joints. Similar blue clips fit 19/22 joints and the

yellow ones fit 14/20 joints, which will most likely used in the lab. If you break the bump

trap, you will have to pay for it!

4. The dial on the motor is used for speed control of the flask rotation. A typical rotavap

uses a variable speed sparkless induction motor that spins at 0-220 rpm and provides

high constant torque. A good setting here is 7-8.

5. The aspirator vacuum is turned on. On most models, the vacuum on/off control is

managed by turning a stopcock at the top of the condenser (left side of the abovediagram). This stopcock is later also used to vent the setup after the solvent is removed.

6. The flask is lowered into the water bath (or the water bath is raised to immerse the

flask in the warm water. (On most models, a convenient handle (with height locking

mechanism) moves the entire condenser/motor/flask assembly up and down. Often the

tilt of the condenser assembly can also be adjusted. The water bath temperature should

not exceed the boiling point of the solvent!! For small amounts of common solvents the

bath heater is not needed.

7. The solvent should start collecting on the condenser and drip into the receiving flask.

Some solvents (such as diethyl ether or dichloromethane) are so volatile that they will

also evaporate from the receiving flask and be discharged down the drain. To prevent

this, a cooling bath on the receiver or (on some models) use a dry-ice condenser can be

used. In addition, an additional trap (with dry-ice or liquid nitrogen) can be placed

between the vacuum source and the condenser unit. This is particularly important of amembrane pump is used as vacuum source.





8. Once all the solvent evaporated (or whatever is desired at this point), the vacuum is

released,. The flask is raised out of the water bath and the spinning is discontinued.

9. The bump trap has to be cleaned and the receiving flask is emptied upon completion of

the evaporation

Vacuum Drier

Principle Of Operation:The term drying generally refers to the removal of moisture from

substance. Vacuum tray drying is a batch drying processing many cases it may be desirable to

dry materials on trays more rapidly than can be done by passing a stream of air over them and

yet to maintain the temperature lower than would correspond to the evaporation of water at

atmospheric pressure. Vacuum tray drier are used for materials that cannot be subjected tohigh temperature as much be reached in a compartment dryer such as pharmaceuticals. They

are also suitable for materials which are sensitive when contacted to air or any oxidizing

gases. If the liquid to be vaporized is a valuable solvent that can be easily collected in a

condenser. The Vacuum tray drier is suited for drying of materials where the material is to be

obtained in a powder form

Procedure:

1. The sample taken in petri plate Is placed in the tray.

2.The petri plate contains sample that has to be dried and brought to powder form.

3.The tray is placed inside the vacuum drying chamber and the door is closed and tightened.

4.The heater is switched on and the vapor line is closed by immersing the coil in ice,so that

the vapors coming out is condensed.

5.The vacuum pump is switched on, required pressure should be set .

6.The temperature is maintained at or below 50.

7.When the sample is dried,the tray is taken out and the dried sample is scraped out into powder form.

Agarose gel electrophoresis

Electrophoresis is a technique used to separate and sometimes purify macromolecules -

especially proteins and nucleic acids - that differ in size, charge or conformation. As such, it

is one of the most widely-used techniques in biochemistry and molecular biology.

When charged molecules are placed in an electric field, they migrate toward either the positive or negative pole according to their charge. In contrast to proteins, which can have





either a net positive or net negative charge, nucleic acids have a consistent negative charge

imparted by their phosphate backbone, and migrate toward the anode.

Proteins and nucleic acids are electrophoresed within a matrix or "gel". Most commonly, the

gel is cast in the shape of a thin slab, with wells for loading the sample. The gel is immersed

within an electrophoresis buffer that provides ions to carry a current and some type of buffer to maintain the pH at a relatively constant value

The gel itself is composed of either agarose or polyacrylamide, each of which have attributes

suitable to particular tasks:

Agarose is a polysaccharide extracted from seaweed. It is typically used at concentrations of

0.5 to 2%. The higher the agarose concentration the "stiffer" the gel. Agarose gels are

extremely easy to prepare: you simply mix agarose powder with buffer solution, melt it by

heating, and pour the gel. It is also non-toxic.

Agarose gels have a large range of separation, but relatively low resolving power. By varying

the concentration of agarose, fragments of DNA from about 200 to 50,000 bp can be

separated using standard electrophoretic techniques.

Polyacrylamide is a cross-linked polymer of acrylamide. The length of the polymer chains is

dictated by the concentration of acrylamide used, which is typically between 3.5 and 20%.

Polyacrylamide gels are significantly more annoying to prepare than agarose gels. Becauseoxygen inhibits the polymerization process, they must be poured between glass plates (or

cylinders).

Acrylamide is a potent neurotoxin and should be handled with care!Wear disposable gloves

when handling solutions of acrylamide, and a mask when weighing out powder.

Polyacrylamide is considered to be non-toxic, but polyacrylamide gels should also be handled

with gloves due to the possible presence of free acrylamide.

Polyacrylamide gels have a rather small range of separation, but very high resolving

power. In the case of DNA, polyacrylamide is used for separating fragments of less than

about 500 bp. However, under appropriate conditions, fragments of DNA differing is length

by a single base pair are easily resolved. In contrast to agarose, polyacrylamide gels are used

extensively for separating and characterizing mixtures of proteins.

Protein estimation by Lowry’s method

Objective: To determine the concentration of proteins by Lowry’s method.

Reagents required:

1. BSA stock solution (0.2mg/ml),





2. Analytical reagents:

(a) 50 ml of 2% sodium carbonate mixed with 50 ml of 0.1 N NaOH solution (0.4 gm in 100

ml

distilled water.)

(b) 10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37% sodium potassium

tartarate

solution.

(c)Prepare analytical reagents by mixing 1 ml of (b) with 50 ml of (a)

3. Folin - Ciocalteau reagent solution (1N) Dilute commercial reagent (2N) with an equal

volume of

water on the day of use (2 ml of commercial reagent + 2 ml distilled water)

Principle:

The phenolic group of tyrosine and tryptophan residues ( amino acid) in a protein will

produce a blue purple color complex , with maximum absorption in the region of 660 nmwavelength, with Folin- Ciocalteau reagent which consists of sodium tungstate molybdate

and phosphate.

Thus the intensity of color depends on the amount of these aromatic amino acids present and

will thus vary for diff erent proteins.

Most proteins estimation techniques use Bovine Serum Albumin (BSA) universally as astandard protein, because of its low cost, high purity and ready availability.

The method is sensitive down to about 10 µg/ml and is probably the most widely used protein

assay despite its being only a relative method , subject to interference from Tris buff er,

EDTA, non ionic and cationic detergents, carbohydrate, lipids and some salts. The incubation

time is very critical for a reproducible assay. The reaction is also dependent on pH and a

working range of pH 9 to 10.5 is essential.

Procedure





Diff erent aliquotes of BSA solutions were prepared by mixing stock BSA solution (1 mg/ ml)

– 0, 0.2, 0.4,0.6, 0.8 and 1 ml

The final volume in each of the test tubes was made upto 1ml by adding distilled water.

5ml of reagent C was added. Solutions were mixed well.

This solution was incubated at room temperature for 10 minutes.

0.5ml of reagent Folin Ciocalteau solution was added to each tube and incubated in dark for 30 minutes.

Absorbance was taken at 660 nm.

The absorbance against protein concentration graph was plotted to get a standard calibration

curve.

From the absorbance values of unknown samples , concentration was determined from the

standard calibration curve.

What is SDS-PAGE?

SDS (Sodium dodecyl sulphate) is a detergent used to denature proteins and give them a

negative charge

PAGE: Polyacrylamide Gel Electrophoresis

It is a technique to separate proteins by their molecular weight





Polyacrylamide gel

l





How can we interpret our SDS-PAGE results

Percent commonality is a measure of the similarity between the protein composition of 2

samples

How could this help us solve a question of “identity” in the case of different types of

seafood?

Introduction of Spectrometric Analyses





The study how the chemical compound interacts with different wavelengths in a given region

of electromagnetic radiation is called spectroscopy or spectrochemical analysis.

The collection of measurements signals (absorbance) of the compound as a function of

electromagnetic radiation is called a spectrum

REVIVAL OF LITERATURE

Silk thread produced by the domesticated silkworm, Bombyx mori; is composed of two kinds

of protein: a fibrous protein (fibroin) which forms the thread core, and a gum-like protein

(sericin) that surrounds the fibroin fibers to join them together. To obtain shiny aspect, softhandle, and elegant drape associated with silk fibers serisin is removed by boiling aqueous

solutions containing soap, alkali, synthetic detergents, or organic acids, or recently by

proteolytic enzymes (1) and discarded in the silk processing wastewater. Lately, there is a

growing interest in the recovery of sericin from silk industry waste water not only to reduce

the environmental impact of silk manufacturing (2) but also to utilize it as a valuable resource

for many industries including cosmetics, pharmaceutical, biomedical, and food (3-6).

For decades, much biochemical research has focused on the use of fibroin in biotechnological

materials and biomedical applications (7, 8). Sericin, which has excellent moisture absorption

and release properties, is antimicrobial (9), UV resistant and has pharmacological functions

such as anticoagulation (10), anti-cancer and antioxidant activities (11, 12) with inhibitory

action of tyrosinase (13), can find applications in functional biomaterials and textiles as well.

It can impart useful and unusual properties to polymer gels, membranes, foams, fibers, and

other composite materials (2). It can be used to produce cryopreservatives, anticoagulants,

and biocompatible materials. With its unique properties, sericin can be used in the surface

modifications of fibers and fabrics. In fact, it was used as a coating material for cellulose

fibers and the treated textiles exhibited a decrease in free formaldehyde content, resistance to

electricity, skin irritation and allergic reactions with increased water retention and only a

negligible decrease in the textile tensile strength (14). Sericin was also impregnated into

polyester fabric to overcome polyester hydrophobicity and to improve UV absorption

properties of the sericin treated fabrics (15).

Sericin, proposed as a candidate material for antimicrobial finishing of the textile by

incorporating its powder form into synthetic fibers during process, may offer some additional

advantages (16). It is a water-soluble natural protein constituting 25%-30% of a versatile





material like silk. The protein can be cross-linked, copolymerized, and blended with other

macromolecular materials to produce materials with improved properties (2, 17).

Considerable scientific research has demonstrated that improved functional property is

achievable for carrier-based textile finishing applications by manipulation of the carrier

particle size and morphology (18). Manufacturing of carrier-based dry powder formulations

can be achieved by various methods including milling, freeze-drying and spray drying (19).

The advantages of the spray drying process include the negligible possibility of degradation

of heat-sensitive molecules and rapid production of dry powders from the solution in a one-

step process with controlled particle characteristics (20-24); the disadvantages were reported

to be the difficulty of obtaining a narrow size distribution and the tendency to agglomerate

(19). Optimization and control of the powder dispersion and deposition properties could be an

important phenomenon in the development of dry powder sericin microspheres, which may

be attached to fabrics to impart unique properties.

The aim of this study was to examine the physical characteristics--including particle size

distribution, shape and moisture content as well as agglomeration degree of the sericin

powders produced by a lab-scale spray dryer under various conditions. Since these

characteristics are governed by formulation and process variables, the significance of these

variables, as well as interactions between them, were examined using a factorial experimental

design and linear regression analysis. The sericin feed concentration was chosen asformulation variable, and the solution feed rate and drying temperature chosen as process

variables. This information will be useful for the optimization of the spray drying

manufacturing of sericin powders with different dispersion and deposition properties.

Degumming process

- Silk degumming with dried papaya’s latex

Raw silk was degummed by using degumming liquor with various concentrations of dried

latex of papaya fruit. Degumming liquor was comprised papaya’s dried latex ranges 0%, 1%,

2%, 3%, and 4 % owf respectively. The liquor ratio of raw silk to degumming liquor was 1 :

25 (g : ml).

Specimens were degummed at the temperature ranges 55, 65, 75 and 85 and time 10, 20, 30

and 40 minutes, then rinsed with cool and hot water and dried at 60 OC

- Silk degumming with soap and Sodium carbonate

Specimens were subjected to degum in a boiled alkaline solution containing 12 % owf soap

and 6 %owf sodium carbonate for 60 minutes at a liquor ratio of 1 : 30 (g : ml). Degummed

silks were rinsed with cool and hot water and dried at 60 OCStaining test with direct dyes





Hirus Supra Red 3BL 140% (C.I. Direct Red 80) was used to evaluate the remaining of

sericin after degumming. The degummed silk samples were dyed in an dye liquor comprised

Hirus Supra Red 3BL 140% 1 g/L with a liquor ratio of 1 : 200 (degummed silk : dye liquor)

at 100 OC for 2 minutes. The color strength of dyed samples (K/S values) was measured by a

spectrophotometer (Color Quest XE) under illuminant D65 with a 10O standard observer.

Color strength was calculated from the reflectance values using Kubelka-Munk equation as

follows R2)R1(S/K2−= (1)

Where R is the decimal fraction of the reflection of the dye fabric, K is the absorption

coefficient and S is scattering coefficient.

2.5 Fiber surface morphology analysis

Scanning electron microscope (SEM) was used to study surface morphology of degummed

silk. The samples were coated with gold by sputtering at room temperature. Scanning

electron micrographs of fibers were taken by scanning electron microscope (JSM-5410LV).

The instrument was operated at 15 kv.

SUMMARY

Sericin is the second main component in cocoons, which are removed in the silk reeling

process of the raw silk industry and in the silk waste degumming of the spun silk industry.

The main amino acid of sericin, serine, exhibits a skin moistening and anti-wrinkle action,

which is interesting to use for film formation in this study. The extraction conditions of

sericin from two silk wastes, pieced cocoon and inferior knubbs were studied to find the

optimum extraction conditions. Boiling water extraction was considered based on the

response surface methodology (RSM) in order to identify the important factors for the

sericin extraction. The two factors considered were time and temperature. Both factors were

needed to be independent parameters in the predicted equation in order to improve the model

fit with R2 = 0.84. The components of extracted sericin were 18.24% serine, 9.83% aspartate,

and 5.51% glycine with a molecular weight of 132 kDa.

Silk sericin microparticles were prepared by precipitation in organic solvent (methanol,

ethanol and iso-propanol). Effect of organic solvent on the molecular characteristics of silk

sericin microparticle were investigated by ATR FT-IR microspectroscopy. The results

suggested that the conformation of the microparticle were changing from random coil to β-

sheet and β-turn after the treatment. After being treated with methanol, ethanol and iso-





propanol, the β-sheet and β-turn proportion of microparticles were increased. The appearance

of β-sheet and β-turn conformation may be associated with the packing of molecular chains.

REFERENCES

This work was supported by the Kasetsart UniversityResearch Development Institute

(KURDI). The authors wish to thank Shinano Kenshi (Thailand) Co., Ltd., for supportingthe

raw materials, the Faculty of Ago-Industry, Faculty ofScience, and KAPI, Kasetsart

University, for the degummingprocess and instrument analyses.

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recovery and purification of silk sericin from silk waste

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