Claims:WE CLAIM:
1. Peptides of Sericin protein for adopting in cryoprotectent formulation; said peptides obtained by a method comprising acts of
a) adding an enzyme to Sericin protein solution in water;
b) adjusting pH of the solution to about 6.0 to about 6.5;
c) heating the solution at a temperature ranging from about 55° C to about 60° C for about 1.45hr to about 2.5hr ;
d) raising the temperature to about 90°C to deactivate the enzyme; and.
e) extracting the peptides for adopting in the cryoprotectant formulation.
2. The peptides of Sericin protein as claimed in claim 1, wherein the enzyme is selected from a group comprising papain (plant origin) - cysteine protease (EC 3.4.22.2) and Promod 24L (originated Bacillus subtilis).
3. The peptides of Sericin protein as claimed in claim 2, wherein the enzyme is Promod 24L (originated Bacillus subtilis).
4. The peptides of Sericin protein as claimed in claim 1, wherein the peptides are of molecular weight ranging from 75-20 kDa.
5. The peptides of Sericin protein as claimed in claim 1, wherein the Sericin is extracted from two groups comprising mulberry type - Bombyx mori and non-mulburry type comprising Antheraea mylitta and Samia cynthia ricini.
6. A cryoprotectant formulation comprising Tris(hydroxymethyl)aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of claim 1, ascorbic acid and antibiotics.
7. The formulation as claimed in claim 6, wherein the carbohydrate is selected from a group comprising glucose, fructose, and combination thereof.
8. The formulation as claimed in claim 6, wherein the antibiotic is selected from a group comprising 1,1'-[(1R,2R,3S,4R,5R,6S)-4-({5-Deoxy-2-O-[2-deoxy-2-(methylamino)-a-L-glucopyranosyl]-3-C-formyl-a-L-lyxofuranosyl}oxy)-2,5,6-trihydroxy-1,3-cyclohexanediyl]diguanidine, (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid and (2S,4R)-N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methylsulfanyloxan-2-yl]propyl]-1-methyl-4-propylpyrrolidine-2-carboxamide and combination thereof.
9. A method of preparation of cryoprotectant formulation comprising Tris(hydroxymethyl) aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of claim 1, ascorbic acid and antibiotics, said method comprising acts of
a) preparation of buffer solutions comprising acts of
(iii) dissolving Tris(hydroxymethyl)aminomethane, D- fructose and Citric acid in water to form a solution; and
(iv) adding glycerol to the solution, stirring and autoclaving to obtain buffer solution.
b) preparation of soylecithin solution by dissolving soylecithin in water; and
c) mixing buffer solution of step (a), soy lecithin solution of step (b) with sericin peptides of claim 1, ascorbic acid and antibiotic; stirring to obtain the formulation.
10. The method of preparation of cryoprotectant formulation as claimed in claim 9, wherein autoclaving is carried out at a temperature ranging from about 90°C to about 100°C and pressure ranging from about 4psi to about 6psi for a period ranging from about 3min to about 8min.
11. A method of preservation of parts of animal and plant origin, said method comprising acts of freezing the part with cryoprotectant formulation comprising Tris(hydroxymethyl)aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of claim 1, ascorbic acid and antibiotics.
12. The method as claimed in claim 11, wherein the parts of animal origin is selected from a group of semen, eggs, tissues and organs; and plant origin is selected from tissues and organs.
, Description:TECHNICAL FIELD
The present invention is in relation to cryopreservation. In particular the invention is in relation to a cryoprotectant formulation comprising peptide(s) obtained from Sericin for preserving semen, eggs, animal and plant tissues; and method of preparation of the formulation. The formulation helps in the maintenance of the quality of the semen and animal tissues. The invention is also in relation to Sericin peptides that can be used in the cryopreservation and method of obtaining the same.
BACKGROUND
Cryopreservation is a commonly adopted method for preserving biological materials including animal tissues, eggs, semen, cell organelles and organs. The method involves very low temperatures of about -80°C or below in said preservation. It is mainly adopted for biological materials wherein the preservation of genetic material is important. In the recent past, it is also being adopted in the preservation of organs, tissues.
However cryopreservation has specific challenges, for example in the cryopreservation technique commonly adopted in bovine industry for the preservation of semen, most of the spermatozoa do not survive due to the physiological changes and that affect quality of the sperms for fertilization. The physiological changes are observed due to the formation of ice crystals that hinder the motility of the sperm. The literature informs about some attempts to overcome the challenges associated with the cryopreservation through variation in cryoprotectant formulations.
There are two types of cryoprotectants, permeating cryoprotectants and non-permeating cryoprotectents. The most common cyroprotectant used for semen is glycerol and dimethyl sulphoxide; sucrose and other disaccharides are added to said solution to improve the effect of cryoprotectants.
The egg yolk is most extensively used in the cryoprotectants to improve the quality of the medium for the cryopreservation. However, egg yolk medium has its own limitations of being contaminated owing to its source, thus limiting the usage of the medium for possible contamination. Further, egg yolk formulation has very small shelf life and has to be freshly prepared as and when it is required, posing a tedious problem as the customization of the formulation may vary leading to ambiguous results. Hence an alternative to egg yolk is required to be explored and adopted to provide a medium for cryoprotectants.
The present invention aims to provide an alternative to the egg yolk that helps in the cryopreservation, a formulation for improved cryopreservation and the methods thereof.
STATEMENT OF INVENTION
Accordingly the present invention provides extraction of Sericin, peptides from Sericin protein by hydrolysis using protease enzymes, adoptable in cryoprotectant formulation, method of obtaining the Sericin peptides, a formulation comprising peptides of Sericin of present invention and method of preparation of the formulation.
BRIEF DESCRIPTION OF FIGURES
The features of the present invention can be understood in detail with the aid of appended figures. It is to be noted however, that the appended figures illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope for the invention.

Figure 1: shows ice recrystallization inhibition activity of Sericin peptides from Bombyx mori.
Figure 2: shows pictures of cryopreservation samples (a) Water ( Negative Control) (b) Casein ( Protein control), (c) AFP1 (Protein Positive control) (d) Composite sericin (e) P1 ( Papain hydrolysed Sericin with mol.wt>10KDa), (f) P2 ( Promod 24L hydrolysed sericin with mol.wt>10KDa), (g) P3 (Promod 215P hydrolysed sericin with mol.wt>10KDa), (h) P4 (Chymotrypsin hydrolysed sericin with mol.wt>10KDa.

Figure 3: shows hypothermia protection activity of Sericin peptides
Figure 4: shows Sericin peptides molecular weight ranges from 75-20 kDa after hydrolysis.
DETAILED DESCRIPTION OF INVENTION
It is to be understood that this description is not intended to limit the invention to the particular embodiments described in the detailed description, drawings, and claims herein. The disclosure covers all modifications and equivalents that fall within the ambit of the invention.
It may further be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by person skilled in the art.
The present invention is in relation to peptides of Sericin protein for adopting in cryoprotectent formulation; said peptides obtained by a method comprising acts of
a) adding an enzyme to Sericin protein solution in water;
b) adjusting pH of the solution to about 6.0 to about 6.5;
c) heating the solution at a temperature ranging from about 55° C to about 60° C for about 1.45hr to about 2.5hr ;
d) raising the temperature to about 90°C to deactivate the enzyme; and.
e) extracting the peptides for adopting in the cryoprotectant formulation.
In an embodiment of the invention, the enzyme is selected from a group comprising papain (plant origin) - cysteine protease (EC 3.4.22.2) and Promod 24L (originated Bacillus subtilis).
In another embodiment of the invention, the enzyme is Promod 24L (originated Bacillus subtilis).
In still another embodiment of the invention, the peptides are of molecular weight ranging from 75-20 kDa.
In still another embodiment of the invention, the Sericin is extracted from two groups comprising mulberry type - Bombyx mori and non-mulburry type comprising Antheraea mylitta and Samia cynthia ricini.
The present invention is in relation to a cryoprotectant formulation comprising Tris(hydroxymethyl)aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of present invention, ascorbic acid and antibiotics.
In still another embodiment of the invention, the carbohydrate is selected from a group comprising glucose, fructose, and combination thereof.
In still another embodiment of the invention, the antibiotic is selected from a group comprising 1,1'-[(1R,2R,3S,4R,5R,6S)-4-({5-Deoxy-2-O-[2-deoxy-2-(methylamino)-a-L-glucopyranosyl]-3-C-formyl-a-L-lyxofuranosyl}oxy)-2,5,6-trihydroxy-1,3-cyclohexanediyl]diguanidine, (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid and (2S,4R)-N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methylsulfanyloxan-2-yl]propyl]-1-methyl-4-propylpyrrolidine-2-carboxamide and combination thereof.
The present invention is also in relation to a method of preparation of cryoprotectant formulation comprising Tris(hydroxymethyl) aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of present invention, ascorbic acid and antibiotics, said method comprising acts of
a) preparation of buffer solutions comprising acts of
(i) dissolving Tris(hydroxymethyl)aminomethane, D- fructose and Citric acid in water to form a solution; and
(ii) adding glycerol to the solution, stirring and autoclaving to obtain buffer solution.
b) preparation of soylecithin solution by dissolving soylecithin in water; and
c) mixing buffer solution of step (a), soy lecithin solution of step (b) with sericin peptides of present invention, ascorbic acid and antibiotic; stirring to obtain the formulation.
In still another embodiment of the invention, autoclaving is carried out at a temperature ranging from about 90°C to about 100°C and pressure ranging from about 4psi to about 6psi for a period ranging from about 3min to about 8min.
The present invention is also in relation to a method of preservation of parts of animal and plant origin, said method comprising acts of freezing the part with cryoprotectant formulation comprising Tris(hydroxymethyl)aminomethane, carbohydrate, citric acid monohydrate, glycerol, Soy lecithin solution, Sericin peptide of present invention, ascorbic acid and antibiotics.
In yet another embodiment of the invention, the parts of animal origin is selected from a group of semen, eggs, tissues and organs; and plant origin is selected from tissues and organs.
An embodiment of the present invention provides Sericin peptides ranging from 75 to 20 kDa (figure 4), adoptable for usage in the cryopreservation. The specific peptides are obtained by the hydrolysis of Sericin protein from the species selected from two groups comprising mulberry type such as Bombyx mori, non-mulberry type comprising Antheraea mylitta and Samia cynthia ricini. In an embodiment of the present invention, Bombyx Mori species is used for exemplary purpose to obtain Sericin. In another embodiment of the invention, the Sericin peptides are obtained by hydrolysis of Sericin obtained from Bombyx Mori using four different enzymes; Papain, Promod 24L (bacterial origin), Promod 215P (fungal origin) and Chymotrypsin (Table 1).
It is observed that the peptides obtained by the hydrolysis, carried out at a pH ranging from about 5.0 to about 7.5 at a temperature ranging from about 55°C-65°C using the enzymes, Papain (plant origin) - cysteine protease (EC 3.4.22.2) and Promod 24L (originated Bacillus subtilis); can be adopted in the cryopreservation formulation.
TABLE 1: SERICIN PEPTIDES OBTAINED BY DIFFERENT ENZYMES.
Enzymes pH Range Temp for activation (°C)
for 2 hrs Temp for de-activation (°C)
for 40 min Termed as
Papain
(plant origin) 6.0-6.5 60-65 80-90 P1
Promod 24L
(bacterial origin) 5.0-7.5 60-65 80-90 P2
Promod 215P
(fungal origin) 5.0-7.5 55-60 80-90 P3
Chymotrypsin
(animal origin) 7.8 23-30 80-90 P4

The adoptability of the peptides in cryopreservation are analysed by ice-recrystallization inhibition activity (Table 2).
TABLE 2: ICE-RECRYSTALLIZATION INHIBITION ACTIVITY OF SERICIN PEPTIDES
GROUPS CRYSTAL SIZE AS FUNCTION OF TEMPERATURE IN (µm)
Water 13.0 ± 2.6
(-25.4±0.9°C) 14.3 ± 2.5
( -16.8±1.3°C) 43.7 ± 2.5
(-11.6±0.7 °C) 68.7 ± 6.5
(-5.5±1.1 °C)
Casein 26.3 ± 3.1
(-23.0±1.4°C) 39 ± 4.0
( -17.3±0.9°C) 44.7 ± 5.9
(-12.4±0.7°C) 75.3 ± 5.5
( -6.6±0.6°C)
AFP1 6.3 ± 2.5
( -20.1±1.5°C) 7.0 ± 2.6
(-13.1±1.6°C) 7.7± 1.5
( -9.2±1.6°C) 8.3 ± 2.5
(-5.6±1.1°C)
Composite Sericin 14.3 ± 3.5
(-21.3±1.7°C) 18.7 ± 2.5
( -18.6±1.0°C) 19.3 ± 2.5
(-13.5±2.1°C) 22 ± 3.6
(-7.3±1.1°C)
P1
4.7 ± 1.5
(-22.7±1.1°C) 5.0 ± 2.6
(-17.2±1.5°C) 5.3 ± 2.5
(-12.8±1.3°C) 6.0 ± 3.6
( -6.3±0.9°C)
P2 12.0 ± 2.6
(-20.5±2.1°C) 14.0 ± 2.0
( -14.9±1.5°C) 14.7 ± 3.1
(-11.6±1.2°C) 15.7 ± 3.1
(-6.5±1.8°C)
P3 19.0 ± 2.0
(-22.7±2.1°C) 25.7 ± 3.5
(-14.1±1.2°C) 31.3 ± 4.0
(-11.2±1.4°C) 34.0 ± 5.6
(-7.5±0.9°C)
P4 25.7 ±1 3.5
(-22.5±0.7°C) 29.7 ± 3.1
(-16.5±1.7°C) 35.7 ± 6.5
(-12.1±1.7°C) 38.3 ± 6.5
(-7.4±1.2°C)

From the above data and graph (figures 1 and 2) it can be concluded that the Sericin hydrolysates P1, P2 from Bombyx mori with molecular wt>10KDa showed ice recrystallization inhibition activity as they either controlled the size of the crystals or did not allow the crystals grow at all and gave blunt edges to the ice as is clearly demonstrated in the figure 1 and figure 2.
Four fractions of Sericin hydrolysates are taken for the next phase of study, the Hypothermia protection activity in Lactobacillus bulgaricus.
Table 3: HYPOTHERMIA PROTECTION ACTIVITY OF SERICIN PEPTIDES
GROUP MEAN±SD For Survival Rates for Lactobacillus bulgaricus
Negative Control (Water) 27.51±2.94
Positive Control (5% Glycerol) 63.48±2.06
Casein ( Protein negative Control) 2.90±0.67
AFPI (Protein Positive Control) 67.96±1.37
Sericin from Bombyx Mori (Mol wt>10Kda)
Composite sericin 43.77±1.87
P1 76.60±2.67
P2 65.38±1.54
P3 51.53±0.64
P4 41.73±1.35

From the above Table -3 and graph (figure 3) it is seen that P1 and P2 peptides of Sericin showed hypothermia protection activity higher than that of positive control (5% glycerol) and protein positive control (AFP 1) informing that these two fractions possess cryo-preservation activity as the lactobacillus frozen in these fractions showed high degree of survivability.
Since only the fractions P1 and P2 obtained cleared Ice re-crystallization inhibition activity and Hypothermia protection activity, they are adopted to be used in the cryopreservation formulation. Preliminary examination was done with P1 and P2 fractions for post thaw cryopreservation studies. From the studies, P2 fraction is found to be more efficient than P1 fraction. Hence P2 fraction is adopted to be used in the cryopreservation formulation.
In another embodiment of the invention, a formulation for cryopreservation is developed and its activity as cryoprotectant, a semen extender is studied. The formulation comprise peptides from Sericin protein of present invention, soy lecithin, buffer, sugar, citric acid monohydrate, glycerol, ascorbic acid, benzyl penicillin and streptomycin in appropriate proportion as exemplified for a 100ml formulation given below.
Typically, a 100ml of the cryoprotectant formulation comprises
Part (i)-Tris Buffer solution
Common name Chemical name Quantity
Trizma base Tris(hydroxymethyl)aminomethane 2.42%
D-fructose D-levulose 1.0%
Citric Acid monohydrate - 1.36%
Glycerol Propane-1,2,3-triol 6.8%
The aforesaid materials are dissolved in distilled water to make upto 100 ml.
Part (ii) Soy lecithin solution
Common name Chemical name Quantity
Soy lecithin Soy Phoshatidyl Choline 2.0%

Soy lecithin is dissolved in hot distilled water to make upto 100 ml of distilled water.

iii) Cryoprotectant formulation
20 % of Tris Buffer is mixed with 80% of the soy lecithin solution (after centrifugation) then 1.14% parts of the existing solution is removed and replaced with the below mentioned ingredients as follows:
I. 0.25% Sericin peptide solution (10%)
II. 0.088% Vitamin C
III. 0.5% Streptomycin
IV. 0.3% Benzyl penicillin.
In an embodiment of the present invention the buffer is preferably Tris(hydroxymethyl)aminomethane, however other buffer like Piperazine-N,N'-bis(2-ethanesulfonic acid) may also be used.
In an embodiment of the invention the antibiotics are selected from a group comprising 1,1'-[(1R,2R,3S,4R,5R,6S)-4-({5-Deoxy-2-O-[2-deoxy-2-(methylamino)-a-L-glucopyranosyl]-3-C-formyl-a-L-lyxofuranosyl}oxy)-2,5,6-trihydroxy-1,3-cyclohexanediyl]diguanidine (commonly known as streptomycin or Ambistryn), (2S,5R,6R)-3,3-Dimethyl-7-oxo-6-(2-phenylacetamido)-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (commonly known as benzyl penicillin) and (2S,4R)-N-[(1R,2R)-2-hydroxy-1-[(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-methylsulfanyloxan-2-yl]propyl]-1-methyl-4-propylpyrrolidine-2-carboxamide (commonly known as lincomycin).
In an embodiment of the present invention, the compatibility of the formulation as cryoprotectant for the post thaw quality of semen of bovine is evaluated based on following parameters. The formulation of the present invention is also compared with the two formulations comprising egg yolk and soylecithin respectively.
a) Post Thaw Motility
b) Live and Dead
c) Sperm Morphology
d) Hypo-osmotic Swelling test
e) Acrosome Integrity Test
f) Incubation test
g) Microbial load
The formulation of the present invention is adoptable as a medium in the preservation of semen of animals like bovine, ram; eggs of animals and human beings; tissues and organs of animals, human beings and plants. However, for exemplary purpose the cryopreservation of semen is studied in the invention and the compatibility is assessed.
a) Post thaw motility test
The frozen semen straws were thawed at 37°C in a water bath for atleast 20 seconds. Then a drop of medium containing sperms is placed on a pre-warmed glass slide. The motility is checked under the phase contrast microscope at 20X objective.
TABLE -4: POST THAW MOTILITY TEST
Formulation comprising MOTILITY(%)
1 2 3
Egg Yolk 50 50 50
Silk Protein P2 50 40 50
Soy lecithin 0 0 0

Results: The criteria for a frozen sample to be considered a good sample, it needs to have to minimum 50% motility. As per the above table 4, the formulation of present invention behaves as a good cryoprotectant - as semen extender.
b) Live and dead
A drop of semen sample was placed on the slide to which two stains were added nigrosine and eosin and smeared on a slide and then air dried 15 minutes. Then the slide was viewed under 20X and live sperms were transparent where as the dead sperms were stained pink(Table 5).
TABLE -5: LIVE AND DEAD
GROUPS Mean ± SD
Egg Yolk 75.37 ± 3.21
Silk Protein P2 65.67 ± 3.06
Soy lecithin 0.00 ± 0.00

For a frozen semen sample to be considered as good it needs to have atleast 50% live sperms. As per the above table the criteria is satisfied with the formulation of present invention. The sample is tested after freezing the sample for 48 hours.
c) SPERM MORPHOLOGY
The frozen semen straws are thawed at 37°C in a water bath for 20 seconds. An eosin-nigrosin stain is commonly used as a morphology stain. A drop of eosin, four drops of nigrosine and a small drop of semen are placed on a clean, grease free slide. The sperm is mixed with eosin and then immediately with nigrosin stain. The mixture is taken on the edge of a slide and pulled across the top of another slide leaving a smear allowed it to dry in air. 200 spermatozoa are counted under oil immersion at a magnification of 1000X in different areas of smear and classify them as normal, head abnormal, mid piece abnormal and tail abnormal sperms (Table 6).
TABLE -6: SPERM MORPHOLOGY
GROUPS Mean ± SD
Egg Yolk 5.33 ± 0.58
Silk Protein P2 4.67 ± 0.58
Soy Lecithin 60.67 ± 2.52

For a frozen semen sample to be considered a good sample it should not contain more than 20% abnormal sperms. The above table shows that the Sericin based extender does not increase the incidence of abnormal sperms thereby can be considered as a good cryopreservation medium.
d) HYPO-OSMOTIC SWELLING TEST( HOST)
The frozen semen straw was thawed at 37°C in a water bath for 20 seconds and then to 0.2ml of semen 0.8ml of distilled water was added (1: 4 ratio) and kept in the water bath for 5 mins and then one drop of sample was placed on a clean pre-warmed glass slide and the slide was viewed under the microscope at 40X objective.
TABLE -7: HYPO-OSMOTIC SWELLING TEST( HOST)
GROUPS Mean ± SD
Egg Yolk 68.67 ± 0.58
Silk Protein P2 66.00 ± 3.00
Soy Lecithin 0.000 ± 0.00

A healthy plasma membrane is very important for the health of sperm, hypo-osmotic swelling test is performed to check the health of the sperms in the semen. For a frozen semen sample to be considered a good sample it must atleast have 50% of sperms with an intact plasma membrane. The formulation of the present invention helps in maintaining the health of the sperm and the same is reflected from the table 7.
e) ACROSOME INTEGRITY TEST
The frozen semen straws are thawed at 37°C in a water bath for 20 seconds. One drop of the sample is placed on to the slide and a smear is made which is allowed to air dry. Once the sample has dried the slide is immersed in a coulter jar containing giemsa overnight. Next day the slide is removed from the stain and washed under tap water to remove the stain and kept for drying. Then the sample is viewed under 100X (oil immersion objective) of the phase contrast microscopy.
TABLE -8: ACROSOME INTEGRITY TEST
GROUPS Mean ± SD
Egg Yolk 87.00 ± 6.66
Silk Protein P2 80.00 ± 1.73
Soy Lecithin 0.000 ± 0.00

An intact acrosome is very important for the fertilizing affectivity of the sperm as the acrosome contains all the important enzymes required during fertilization. A good frozen semen sample should contain atleast 65% intact acrosome. Sericin based semen extender of the present invention shows that the intact acrosome is 80% (Table 8).
f) INCUBATION TEST
The frozen straws were thawed at 37°C in a water bath for 20 seconds. A drop of the sample was placed on a pre-warmed glass slide and motility was checked after every half hour for 90 minutes (Table 9).
TABLE 9: INCUBATION TEST

Egg Yolk
Silk protein P2
Soy lecithin
0h 30m 60m 90m 0h 30m 60m 90m 0h 30m 60m 90m
50 40 30 20 50 40 30 10 0 0 0 0
50 40 30 10 40 30 10 0 0 0 0 0
50 40 30 20 50 40 30 20 0 0 0 0

The sperms should be alive for atleast 48 hours inside the female tract in order to ensure higher chances of conception. Therefore this test is very important as the sperms need to maintain atleast 10% motility by the end of 90 minutes. For the frozen semen sample to be considered good it need to have atleast 50% motility at 0 mins and should have 10% motility by the end of 90 minutes. It is shown in the sperms frozen in the sericin based extender are able to maintain 10-20% by the end of 90 minutes.
f) MICROBIAL LOAD
The straws are thawed at 37°C in a water bath for 20 seconds and the content of the straw is spread on a petriplate containing nutrient agar medium and placed in an incubator at 37°C. The plates are incubated for 24 hours and then the number of colonies formed are counted (Table 10).
Table 10: MICROBIAL LOAD
Col CFU/ml
Egg Yolk Silk protein P2 Soy lecithin
Nil Nil 2500
Nil 1000 Nil

Microbial load is also very important parameter, as very high microbial load might affect the sperms and the viability and the motility of the sperms will not be good. The microbial load in a sterile sample should not exceed 5000 CFU/ml. It is seen from the above table that Sericin based extender does not exceed the limit.
From all the above 7 tests it can be finally concluded that the Sericin based formulation fulfils all the criteria for it to be successful a semen extender and it could be used as a replacement for egg yolk.
EXPERIMENTAL:
EXPERIMENT 1: EXTRACTION OF SERICIN
100g of cocoon (Bombyx Mori) is taken and soaked in 1800ml of water containing 1.25g of Potassium carbonate for 1.5hr. Then the cooker is heated to 60-70°C for 1hr. Then cocoons are squeezed and washed, then kept for drying in the hot air oven. Sericin obtained is 1350ml which is concentrated to 432ml (pH= 8.06) and used.
EXPERIMENT 2: HYDROLYSIS OF SERICIN
100ml (5%) Sericin is taken and hydrolysed with 0.1425g of PROMOD 24L enzyme. Before hydrolysis the pH of the solution is adjusted to 6.5 (pH adjusted with citric acid). This solution is then heated to 60°C for two hours to activate the enzyme and for the hydrolysis to take place. Then this solution is heated to 90°C to deactivate the enzyme.
EXPERIMENT 3: PREPARATION OF CRYOPROTECTENT FORMULATION

Step i: Preparation of TRIS Buffer
2.42g of Tris(hydroxymethyl)aminomethane TRIZMA Base, 1.0g D- fructose and 1.36g citric acid are dissolved in 100ml of water. Then 6.8g glycerol is added and stirred for some time in a magnetic stirrer and then this solution is autoclaved at 100°C at 5 psi for 15 mins.
Step ii: Dissolution of Soy lecithin
100ml of dimeneralised water is heated to 60°C and 2g soy lecithin is dissolved by adding small amounts of water with constant stirring till all the globules dissolve and form a homogeneous opaque solution. Then this soy lecithin solution is centrifuged for 10 mins at 7000 rpm to remove the centrifugate and use the solution.

Step iii: Final Formulation Preparation
20ml of Tris Buffer is mixed with 80ml of the soy lecithin solution (after centrifugation) then 1.14g of the existing solution is removed and replaced with remaining ingredients as follows: 0.25 g (%w/w) of Sericin peptide is added along with 88 mg(%w/w) of vitamin C and mixed well and to that 0.5g (%w/w) Ambistryn and 0.3g (%w/w) of Benzyl penicillin is added and mixed well till it completely dissolves to obtain the formulation.
A specific example of 100ml formulation comprises the constituents as follows-
SPECIFIC COMPOSITION: For 1X 100ml of semen extender
i) Tris Buffer solution
Common name Chemical name Quantity
Trizma base Tris(hydroxymethyl)aminomethane 2.42%
D-fructose D-levulose 1.0%
Citric Acid monohydrate - 1.36%
Glycerol Propane-1,2,3-triol 6.8%
The ingredients are dissolved in distilled water and made upto 100 ml
ii) Soy lecithin solution Preparation
Common name Chemical name Quantity
Soy Lecithin solution Preparation
Soy lecithin Soy Phoshatidyl Choline 2.0%
Soy lecithin is dissolved in hot distilled water and made upto 100 ml.

iii) Final Formulation Preparation
20 % of Tris Buffer is mixed with 80% of the soy lecithin solution (after centrifugation) then 1.14% parts of the existing solution is removed and replaced with remaining ingredients as follows:0.25% Sericin peptide solution (10%) of present invention, 0.088% Vitamin C, 0.5% Streptomycin and 0.3% Benzyl penicillin
The present invention is advantageous as the contamination is reduced and hence involves very small amount of antibiotics. The formulation of present invention can be prepared, stored for about twelve months and used as and when it is required saving the time and effort.
Thus the present invention provides unique peptides from Sericin protein which can be adopted in the cryoprotectant formulation and cryoprotectant formulation thereof and methods of preparation by cost effectively. The products of the present invention can be easily adopted as cryoprotectant for semen, tissues and other organs of animals and human beings.