Gelatin, Caesin, Serratopeptidase, Pepsin & Collagen

Objective

At the end of this lecture, student will be able to:

• Discuss the source, method of production, constituents, tests, uses of Gelatin Caesin, Serratopeptidase, Pepsin & its products and Collagen

Discuss the source, method of production, constituents, tests, uses of Gelatin

Gelatin

Source

• Protein derivative

• Evaporating the aqueous extract

• Skins, tendons, bones

• Domestic animals – Ox, Bos Taurus, the Sheep, Ovies aries

Family

• Bovidae

Gelatin – Method of Preparation

Pretreatment

• Skin and tendon

– Liming

– 15-20 or 40 days in dilute milk of lime

– Dissolves fleshy matter, removes chondroproteins, saponifies fat

• From bones

– Ossein, 12-18 % fat and 50 % mineral

– Defattened with organic solvent

– Treated with hydrochloric acid to remove minerals (Decalcification)

Gelatin- Preparation from pretreated material

• Heated with water in open pans at 85oC

• Direct heating and high temperature – avoided

• Converts insoluble collagen into soluble gelatin

• Clear fluid is run off and evaporated under pressure

• Run into metal trays and allowed to set into jelly

• Placed in trays with wire netting bottom

• Passed through a series of drying rooms at temperatures, increasing by about 10oC each time from 30oC to 60oC

• At high temperature – loses its jellying capacity

• High moisture content – flexible

Gelatin – Description

• Thin sheets / Shreds / Powder

• Colourless or pale yellow

• Odourless

• Tasteless

• Hard and brittle

• Insoluble in cold water, dissolves on heating, forms jelly on cooling

• Bloom strength

• Gelatin A and Gelatin B

Gelatin A – acid treated precursor product (Isoelectric pH-9)

Gelatin B – alkali treated precursor product (Isoelectric pH-4.7)

Chemical Constituents

– Glutin

– Free from chondrin, sulphur and tryptophan

– Major amino acid – Lysine

Chemical Test

• Evolves ammonia when heated with soda lime

• Precipitated by tri nitro phenol, tannic acid

• Not precipitated by alum, lead acetate or acids (Chondrin)

• Millons test: white precipitate, on warming – brick red colour

• Biuret test: violet colour (NaOH + Cu2SO4)

• With picric acid- yellow precipitate

Gelatin – Uses

• Preparation of capsules, paste, pastiliies, suppositories and gelatin sponge

• Absorbable gelatin sponge – haemostat

• Specially purified and pyrogen free gelatin – vehicle for IV injection

• Bacteriological culture medium

• Nutrient

• Preparation of commercial food products

Gelatin Products

• Absorbable gelatin sponge

• Absorbable gelatin films

Absorbable gelatin sponge:

• Sterile, absorbable, water insoluble, nearly white, porous matrix

• Whisking a warm solution of gelatin to a foam of uniform porosity and dried

• Cut into pieces, sterilised by dry heat, (150OC for 1 hour)

• Though insoluble in water, absorbed in body fluids and takes up not less than 30 times its weight of water

Absorbable gelatin films:

• Sterile, light amber obtained from specially prepared gelatin formaldehyde solution by drying, followed by sterilisation

• In the form of saline soaked sheets in surgical repairs of defects in membranes like pleura, duramater

• Serves as mechanical protective, temporary supportive and replacement matrix

Discuss the source, method of production, constituents, tests, uses of Caesin

Caesin

Source:

• Isolated protein from cow milk Bos taurus (Bovidae)

• Solids of cows milk: 80% total proteins

Extraction and separation:

• Cow milk is the commercial source of caesin

• At pH 4.6 acesin gets precipitated (Milk pH is 6.6)

Separation:

• Cream separated from milk

• Skim milk used for caesin separation

Precipitation:

Lactic acid caesin:

• Skim milk is pasteurised at 72C from 15 secs followed by cooling to room temperature

• Then inoculated with suitable Strain of lactic acid producing bacteria known as starters

• Milk is incubated without agitation for 14-16 hrs

• Lactose gets converted into lactic acid

• Caesin gets coagulated

• Others methods are mineral acid precipitation, Coagulation using enzymes (no change in pH, Chymosin (calf rennet) from calf stomach is used, during incubation enzyme specifically celaves one of the bond in K-Caesin, leading to breakdown of protein chain, this breakdown destabilises the caesin miscelles, hence coagulation happens

Cooking, washing and dewatering:

• After coagulation it is heated indirectly to 50-55C

• Dewatered with blow of hot air

• Then moisture content is controlled and packed

Physical properties:

Colour: White yellowish Taste: Characteristic Odour: Characteristic

Solubility: Acid acaesin isoluble at pH 4.7, fully soluble is alkaline medium, caesin insoluble at pH 7.1

Shelf life: 12 months

Chemical nature:

Consists of proline peptides and no disulfide linkages

Tests:

Biuret test, ninhydrin test, xanthoprotein test

Uses:

• Choice of material in paints until the advent of acrylic paints

• Caesin based glue in artworks and in aircrafts

• Cheese making

• Plastic and fibres production

Discuss the source, method of production, constituents, tests, uses of  Serratopeptidase

Serratopeptidase

• Proteolytic enzyme derived from the bacteria belonging to Genus Serratia present in the gut of silk worm

• Originally discovered from Serratia E15 species, now produced by fermentation biotechnology

• Very effective bacterial enzyme and better than trypsin and chymotrypsin

Uses:

• in inflammation

• Sputum liquefaction

• Enhancement of antibiotic effects due to removal of inflammatory barrier

Discuss the source, method of production, constituents, tests, uses of Pepsin

Pepsin

Synonym: Puerzyme

Source:

• Principal proteolytic enzyme of vertebrate gastric juice

• Inactive precursor form is pepsinogen produced in stomach mucosa

• Hormonal secretion of gastrin and scretin stimulate the release of pepsinogen into stomach

• Pepsinogen mixes with HCl in stomach and gets converted into pepsin

• Belongs to the group of Acid proteases

• Pepsin proteins:  Pepsin A, Pepsin B (parapepsin 1), Pepsin C (Gastricsin), Pepsin D (unphosphorylated form of pepsin A)

• Pepsin A is predominant gastric protease

• Pepsin A digest not less than 3000times its weight of coagulated egg albumin, strength can be manipulated with lactose

• Commercially prepared from glandular layer of fresh hog stomach

Physical properties:

Colour: White or light buff amorphous powder

Odour: Faint and meaty

Taste: Slightly acidic, saline

Solubility: soluble in water producig opalascent solution, insoluble in alcohol, ether and chloroform

Isoelectric pH: less than 1.0

Chemical nature:

• Monomeric with Beta protein with high percentage of acidic portions

• Porcine pepsin has 4 basic residues and 42 acidic residues and si O-phosphorylated at S68

• For the protein to be active, one of the two asparate residues in the catalytic region has to be protoated and the other deprotonated, happens between pH 1 and 5

Uses:

• Dehairing in leather industries

• To provide whiping quality of soy protein and gelatin in food manufacturing

• In food chemistry digestability of proteins assessed by pepsin

• Viable mammary epithelial cells are sub cultured using pepsin

Storage: well closed containers at cool place, away from sunight

Summary

• Gelatin is a protein derivative obtained from aqueous hydrolysis of the bones, skin and tendons of various domestic animals

• Quality of gelatin depends on the temperature and pressure used

• Gelatin A and gelatin B depends on the raw material used

• Major amino acid is lysine and free from chondrin, sulphur and tryptophan

• Gelatin is used in the preparation of capsule shells, in bacteriological culture medium etc