INTERNSHIP TRAINING REPORT ON PAPAIN PRODUCTION AND ANALYSIS OF VARIOUS

PARAMETERS

IN

SENTHIL PAPAIN AND FOOD PRODUCTS ( Pvt.) LTD,

SF NO.395/1/B-2
VEERAPANUR, PICHANUR.PO
COIMBATORE-641110

SUBMITTED BY

PRIYANKA.S
RAMA KRISHNAN.V
DEPARTMENT OF BIOTECHNOLOGY
KUMARAGURU COLLEGE OF TECHNOLOGY
COIMBATORE - 641049

UNDER THE GUIDANCE OF

Ms. LAKSHIMI ANAND C M. Sc . , Ph.D

HEAD OF QUALITY CONTROL
SENTHIL PAPAIN AND FOOD PRODUCTS (pvt.) LTD.

ACKNOWLEDGEMENT

We express our immense pleasure to express out deep gratitude to Mr.R.Vinoth kannan , General
mmanager, Senthil Papain and Food Products (P) Ltd., Coimbatore for providing us the great chance
to undergo training in this prestigious institution.

We also thank Ms. Lakshmi Anand C , Head of QC ,Senthil Papain and Food Products (P) Ltd.,
Coimbatore , for her unlisted encouragement which helped me in completing my internship training.

We also extend our thanks to Mr.Mannikannan , Senior Manager in R&D and Mr.Subramanian ,
Technical manager at Senthil Papain and Food Products (P) Ltd., for the support which made me
move forward with my internship training.

We also thank Mr.Vignesh , Senior executive at Senthil Papain and Food Products (P) Ltd., for
providing necessary informations and research opportunities on enzyme and food technology
during our training.

We also extend our thanks to all the Technical ( Ms. Soundarya , Ms. Tamilarasi , Mr. Kannan ,
Mr.Robin B Alex ) and Non-technical staffs of Senthil Papain and Food Products (P) Ltd., for
treating us like a family and for the timely support to move forward with training.

Lastly , we thank our family and friends for their encouragement and everlasting love which
helped us to finish this internship training.

1.INTRODUCTION
1.1 PAPAYA

The papaya is a fruit from plant Carica papaya, one of the 22 accepted species in the genus Carica
of the family Caricaceae. Papaya is native to Mexico and extends to South America and has
become naturalized throughout the Caribbean Islands, Florida and several countries of Africa.
Additional crops are grown in India, Australia, Malaysia, Indonesia, the Philippines, Thailand and
the U.S. state of Hawaii.

Papaya plants grow in three sexes: male, female, hermaphrodite. The male produces only pollen,
never fruit. The female will produce small, inedible fruits unless pollinated. The hermaphrodite
can self-pollinate since its flowers contain both male stamens and female ovaries. Almost all
commercial papaya orchards contain only hermaphrodites.

Kingdom: Plantae
Order: Brassicales
Family: Caricaceae
Genus: Carica
Species: C. papaya

India and Brazil are the major producers of papaya, together providing 57% of the world total of
12.4 million tons in 2013. Gaining in popularity among tropical fruits worldwide, papaya is now
ranked fourth in total tropical fruit production after bananas, oranges, and mango.

2. PAPAIN

Papain, also known as papaya proteinase I, is a cysteine protease (EC 3.4.22.2) enzyme present in
papaya (Carica papaya) and mountain papaya (Vasconcellea cundinamarcensis). Papain belongs
to a family of related proteins with a wide variety of activities, including endopeptidases,
aminopeptidases, dipeptidyl peptidases and enzymes with both exo- and endo-peptidase activity.

2.1 STRUCTURE OF PAPAIN

The papain precursor protein contains 345 amino acid residues and consists of a signal sequence
(1-18), a propeptide (19-133) and the mature peptide (134-345). The amino acid numbers are
based on the mature peptide. The protein is stabilised by three disulfide bridges. Its three-
dimensional structure consists of two distinct structural domains with a cleft between them. This
cleft contains the active site, which contains a catalytic diad that has been likened to the catalytic
triad of chymotrypsin. The catalytic diad is made up of the amino acids - cysteine-25 (from which
it gets its classification) and histidine-159. Aspartate-158.

2.2 PROPERTIES OF PAPAIN

Appearance: fine white powder

Alternate names: papaya peptidase 1

Source: Carica papayalatex

Storage: 2-8 C

Molecular weight : 23000 Da

pH: 4.8-5.5

Colour : white to off white

Specific gravity : 0.4- 0.45

TSS: 5%-6%

Optimum pH: 6.0 - 7.0.

Extinction coefficient: 25.

Isoelectric point: pH 9.6

Activators: Papain is activated by cysteine, sulfide, sulfite, etc. It is enhanced when heavy metal
binding agents such as EDTA are also present. Kirschenbaum (1971) indicated that N-
bromosuccinimide enhances the activity.

Inhibitors: Specific inhibitors are AEBSF, antipain, cystatin, E-64, leupeptin, PMSF, TLCK and
TPCK.

Stability: Papain as a crystalline suspension is stable at 5oC for 6-12 months. Stabilizing agents
are EDTA, cysteine and dimercaptopropanol.

2.3 USES OF PAPAIN

2.3.1 INDUSTRIAL USES OF PAPAIN

1.Papain breaks down tough meat fibres, and has been used for thousands of years to tenderise
meat eaten in its native South America. Meat tenderisers in powder form with papain as an active
component are widely sold.

2.Papain can be used to dissociate cells in the first step of cell culture preparations. A ten-minute
treatment of small tissue pieces (less than 1 mm cubed) will allow papain to begin cleaving the
extracellular matrix molecules holding the cells together. After ten minutes, the tissue should be
treated with a protease inhibitor solution to stop the protease action. Left untreated, papain
activity will lead to complete lysis of the cells. The tissue must then be triturated (passed quickly
up and down through a Pasteur pipette) to break up the pieces of tissue into a single cell
suspension.

3. Papain is also used as an ingredient in various enzymatic debriding preparations, notably

Accuzyme. These are used in the care of some chronic wounds to clean up dead tissue.

4.Papain is added to some toothpastes and mint sweets as a tooth whitener.

5.Papain is the main ingredient of Papacarie, a gel used for chemomechanical dental caries
removal. It does not require drilling and does not interfere in the bond strength of restorative
materials to dentin.

2.3.2 HEALTH BENEFITS OF PAPAIN

1. Boosts Digestion

The enzyme has protein-digestive properties and the University of Michigan supports its use as an
enzymatic support for the intestines, stomach and the pancreas.

2. Skin and Wound Care

Due to papains beneficial capacities, it is currently being studied for topical applications on
burns, irritations, and wounds. It has also been used for ulcers and bedsores.

3. Resistant to Fungus

Agricultural Research found that extracts from Carica papaya (papain) could statistically reduce
the fungal pathogen causing pawpaw fruit rot.

4. Immune System Support

According to Memorial Sloan-Kettering Cancer Center, papain may act as an immune support
system for cancer treatment, as proteolytic enzymes such as papain help to modulate leukocytes
in the immune response.

5. Redness Resistant

Studies confirm that the papain enzyme has powerful resistance to redness and may help reduce
joint and prostate irritation.

6. Antioxidant

Papain holds compounds that may aid in protecting the body from cellular damage caused by free
radicals. One study published in the Journal of Dairy Science found that papain can offer a
potential alternative to chemical additives for increased survival of probiotic bacteria in yogurt
due to its oxidation properties. Similarly, it is also a powerful agent commonly used in food
preservation, as it reduces bacterial infestations and spoilage due to oxidation.

3. COMPANY 'S PROFILE

Being a agro-tech product the success of papain production lies to a great extent in agriculture.
Though technology is vitial as part of the backward integration programe the company undertakes
total cultivation of papaya under different forms of collective farming. Over the past several years
of agriculture, the farmers have found papaya cultivation a big boost in augmentig thier
agriculture income. In many areas where papaya has been introduced by the company it was
found more profitable than even Banana and Sugarcane. Presentaly the area under cultivation
exceeds 900 acres and more areas are added every year as per the over all corporate planning for
the papain industry.
3.1 LOCATION

Senthil papain and Food products (P) Ltd. started in 1995 meets the demand of quality papain
globally as a major part. The manufacturing plant in located very close to the papaya cultivation
area and no plantation is further than 100kms from the factory. They collect papaya from
Sathyamangalam , Theni, Ottanchattaram, Navakarai. It is situated just off the main Tamilnadu -
Kerala National Higway No. 47, admist the lush green surrounding where abudant ground water
and other necessary infrastructure are avaliable. The main city Coimbatore, it is well connected
with a good international Airport and Road Terminals. The temperate climate is most suitable for
the cultivation of this particular variety of papaya known as CO2, which yields the best latex for
papain processing.

3.2 PRODUCTS MANUFACTURED

Senthil Papain and Food Products Private Limited is the leading manufacturer of ultra refined
papain powder. There are other products like stabilized liquid papain, granulated papain, meat
tenderizer and pectin. Senthil Papain & Food Products Ltd is an agro industry manufacturing
papain powder from papaya latex. Senthil Papain develops papain powder and fruit pulp. The
papain powder is used in the manufacture of beer, beverages, medicines & drugs, meat industry
and refining textile & garment industry. It also supplies to industries such as cattle feeds,
cosmetics, detergents sewage water purification and tanneries. Senthil Papain also has an in-
house tissue developing centre, seedling nursery and cold storage.

3.3 FACILITIES AVAILABLE

3.3.1 PRODUCTION UNIT

The company is accompanied with a higher production capacity of 4 tons per month of the
desired product. The effluent from the process will be controlled by the effluent treatment plant
(ETP). The ETP treated slurry is also utilised as manure for their own plantations.

3.3.2 QUALITY CONTROL DEPARTMENT

Quality of a product ensures its value and market ability. The company has a separate quality
control department stringent quality of their enzyme products before delivering to the customers
globally.

3.3.3 RESEARCH AND DEVELOPMENT DEPARTMENT

This division is engaged in developing new products , methods and protocols to launch new
products to meet the increasing global demand. The laboratories are well equipped for upstream ,
downstream processes and for the analysis of the final product.

4. EXTRACTION OF PAPAIN ENZYME

4.1 COLLECTION OF LATEX

Papain is obtained by cutting the skin of the unripe but almost mature papaya and then collecting
and drying the latex which flows from the cuts. Tapping of the fruit should start early in the
morning and finish by mid-late morning (ie during periods of high humidity). At low humidity
the flow of latex is low. Two or three vertical cuts (except the first cut, see below) 1-2mm deep
are then made, meeting at the base of the fruit. The incisions are made using a stainless steel razor
blade set into a piece of rubber attached to a long stick. The blade should not protrude more than
about 2mm as cuts deeper than 2mm risk juices and starch from the fruit pulp mixing with the
latex which lowers the quality.

Fruits should be tapped at intervals of about 4-7 days and for the first tapping it is usually
sufficient to make only one cut. On subsequent tappings the two or three cuts are spaced between
earlier ones (as explained above). After about 4-6 minutes the flow of latex ceases. A dish is used
to collect the latex and the latex is then scraped into a polythene lined box with a close fitting lid;
such a box should be stored in the shade. The use of a close fitting lid and keeping the box in the
shade are both important because they reduce the reactions which cause the loss of enzyme
activity. Foreign matter such as dirt and insects in the latex should be avoided. Latex adhering to
the fruit should be carefully scraped off and transferred to the collecting box. However, dried
latex should not be mixed with fresh latex as this lowers the quality.

When handling fresh latex, care should be taken to ensure that it does not come into contact with
skin as it will cause burning. Neither should it come into contact with heavy metals such as iron,
copper or brass as this causes discolouration and loss of activity. Pots, knives and spoons should
not be used unless they are made from plastic or stainless steel. Fresh latex does not keep well
and should be dried to below 5% moisture (when it will have a dry and crumbly texture) as soon
as possible. After two or three months the fruits are ripe and should be removed from the tree.
The ripe fruits are edible but have very little sale value because of their scarred appearance.
However, the skin of the green ripe papaya does contain about 10% pectin (dry weight) and the
fruits could be processed to extract this.

4.2 PRODUCTION OF PAPAIN

Latex from different papaya cultivations are gathered together and stored in cold room at the
factory until the required amount is collected. The production of papain enzyme from latex
follows the following steps,

1. HOMOGENISATION

The collected latex is then homogenised. The process turns liquids into a state consisting of
extremely small particles distributed uniformly throughout the other liquid.

2. CENTRIFUGATION

The mixture is then seived at pore size 4mm to remove extraneous materials and then
centrifuged at 11,000 rpm.

3. FILTRATION

Then the mixture is filtered using plate and frame filter press with membrane pore size of
10mm and the filter cake is used as manure. The permeate is used for further processes.

4. ULTRAFILTRATION
The liquid from the last process is subjected to ultra filtration using membrane of pore
size 24kdm. Most of the micro organisms are removed and the liquid gets concentrated
in this step.

5. SPRAY DRYING

In this process the liquid is converted to a powder by removing the moisture

component from the liquid solution. The solution is sprayed through a nozzle into a
chamber where simultaneously hot air of 150 C being blown into it. The final powder is
then collected separately in a sterile area.

6. BLENDING

The produced powder is blended to get a homogeneous material with uniform particle
size distribution , texture and other required attributes.

6. MICROBIOLOGY

6.1 AEROBIC PLATE COUNT

6.1.1 FOOD AND DRUG ADMINISTRATION (FDA)

AIM

To estimate the number of viable aerobic bacteria present in the URPP by Food and drug
admistration.

MATERIALS REQUIRED

Cotton
Spatula
Micropipettes
Measuring cylinder
Test tubes
Conical flash
Weighing balance
Autoclave
Incubator

MEDIA REQUIRED

FDA media
0.85% saline for dilution
MEDIA PREPARATION

0.85% Saline water - 0.85g sodium chloride dissolved in 100 ml distilled water and pour
10ml per tube.

FDA MEDIA ( butter field's phosphate buffer )

Table

The above ingredients are dissolved water and pH is adjusted.

The media , saline , petriplate , spatula , micropipette and tips are properly packed and
autoclaved at 121 C for 20 minutes.

After autoclaving everything is taken into the inoculation room. The media which is to
be used in inoculation is to be kept at 45 C temperature in water bath.

PROCEDURE

1g of sample is added into 10 ml of autoclaved. This gives a 10 times dilution(1/10).

After thorough mix , 1 ml of the sample will be taken for plating.

Uniform mixing and exact volume transfer are very important. To get a correct count.

1ml of diluted sample is poured into the sterile petriplate and then 15 ml of FDA agar is
added and mixed evenly before the medium will get safety in the petri plate.

Then incubated at 37 C for 48 hours.

The result will be recovered after 48 hours of the incubation.

The colony count in the each plate will give the number of microorganism's present in
the sample.

CALCULATION

Colony forming units = No. Of colony * dilution factor / weight of the sample

RESULT

The presence of aerobic microorganisms in the sample should be less than 100.
6.1.2 INDIAN PHARMACOPIEA METHOD (IP)

AIM

To estimate the number of viable aerobic bacteria present in the URPP by IP method.

MATERIALS REQUIRED

Cotton
Spatula
Micropipettes
Measuring cylinder
Test tubes
Conical flash
Weighing balance
Autoclave
Incubator

MEDIA REQUIRED

IP media - soya bean caesin agar

0.85% saline for dilution

MEDIA PREPARATION

0.85% Saline water - 0.85g sodium chloride dissolved in 100 ml distilled water and pour
10ml per tube.

IP MEDIA- soya bean caesin agar

In 100 ml of distilled water added 4g of soya bean caesin digest agar.

The above ingredients are dissolved water and pH is adjusted.

The media , saline , petriplate , spatula , micropipette and tips are properly packed and
autoclaved at 121 C for 20 minutes.

After autoclaving everything is taken into the inoculation room. The media which is to
be used in inoculation is to be kept at 45 C temperature in water bath.

PROCEDURE

1g of sample URPP is added into 10 ml of autoclaved. This gives a 10 times

dilution(1/10).
After thorough mix , 1 ml of the sample will be taken for plating.

Uniform mixing and exact volume transfer are very important. To get a correct count.

1ml of diluted sample is poured into the sterile petriplate and then 15 ml of IP agar is
added and mixed evenly before the medium will get safety in the petri plate.

Then incubated at 37 C for 48 hours.

The result will be recovered after 48 hours of the incubation.

The colony count in the each plate will give the number of microorganism's present in
the sample.

CALCULATION

Colony forming units = No. Of colony * dilution factor / weight of the sample

RESULT

The presence of aerobic microorganisms in the sample should be less than 1000

6.2 YEAST AND MOULD TEST

AIM

To check whether yeast and mould organisms present in the papain sample.

MATERIALS REQUIRED

Cotton
Spatula
Micropipette
Glass wares
Weighing balance
Autoclave
Vortex

BUFFER PREPARATION

Saline solution - 1.36g of sodium chloride is dissolved in 100 ml water.

MEDIA PREPARATION (PDA AGAR)

TABLE

ANTIBIOTICS

0.02g of Chloramphenicol and streptomycin added in 10 ml distilled water. From this 2ml
antibiotic solution is transferred to 100 ml of PDA.

The media , saline , petriplate , spatula , micropipette and tips are properly packed and
autoclaved at 121 C for 20 minutes.

After autoclaving everything is taken into the inoculation room. The media which is to
be used in inoculation is to be kept at 45 C temperature in water bath.

PROCEDURE

1g of sample URPP is added into 10 ml of autoclaved. This gives a 10 times

dilution(1/10).

After thorough mix , 1 ml of the sample will be taken for plating.

Uniform mixing and exact volume transfer are very important. To get a correct count.

1ml of diluted sample is poured into the sterile petriplate and then 15 ml of FDA agar is
added and mixed evenly before the medium will get safety in the petri plate.

Then incubated at room temperature for 5 days.

Count the yeast colonies on the third day and the mould colonies on the fifth day.

CALCULATION

Colony forming units = No. Of colony * dilution factor / weight of the sample

RESULT

The yeast and mould should be absent.

6.3 PATHOGEN TEST

AIM

To identify the pathogen microorganisms present in the URPP.

PRINCIPLE

Counting the number of colonies of microorganisms utilizing the carbon and other
sources present in the media.

MATERIALS REQUIRED

Conical flask
Weighing balance
Laminar air flow
Sample

REAGENT REQUIRED

Toddlewitt broth - 8.325g in 225ml distilled water

Soya bean caesin digestion media - 6.75g in 225 ml distilled water.

Macconkey agar

Blood agar

PROCEDURE

10g of sample URPP is added into the Toddlewitt and incubated at 37C for 48 hours.

Then it is subcultured in macconkey agar and blood agar plate and then incubated at 37
C for 48 hours.

The sub culture is observed.

The same procedure and soya bean caesin digestion media. If the sample contaminated
with microorganisms , there growth will occur.

OBSERVATION

TABLE

RESULT

The pathogen should be absent.

7. CONCLUSION
Papain has various applications in industries such as Food processing , Laboratory,
Veterinary and Medicine. This company's R&D department clearly insists on the
development of new product.

In review this internship has been an excellent and rewarding experience. I have been
able to meet and network with so many people that I am sure will be able to help me
with opportunities in the future. All the techniques was explained and learned from
production, quality control, and research departments