dharanimuthusamy98@gmail.com

1. Isolation of Milk
Clotting Enzymes
DHARANI MUTHUSAMY
M.TECH DAIRY CHEMISTRY
COLLEGE OF FOOD AND DAIRY TECHNOLOGY
dharanimuthusamy98@gmail.com

2. Source of Enzymes
Animals BacteriaFungi Higher
plants
Chymosin (EC 3.4.23.4)
Porcine Pepsin
Bovine pepsin
Chicken pepsin
Endothia parasitica
Mucor pusillus var.
Lindt
Mucor miehei
Aspergillus oryzae
MTCC 5341
B. cereus ,
Bacillus subtilis,
Pseudomonads ,
Bacillus licheniformis,
Bacillus megaterium
papain & chymopapain,
Bromelain,
Cynzra cardunculus
, Solanum toruum,
ash gourd (Benincasa
cerifera)
, Cirsium aruense
1 2 3 4

3. Isolation of bovine chymosin
Extraction Of Enzyme From Abomasal Tissue
Buffalo calf abomasal tissues,excisedwithin12 h of death from calves1–2months old, were flushed
with distilled water and minced into small pieces then freeze-dried under vacuum and 100 g of the minced tissue
was homogenized in 2 l distilled water before straining through muslin. The remaining debris from the
homogenate was removed by centrifugation at 5000 g at 4 ºC for 10 min and re-extracted with 1 l distilled water.
Precipitation
Aluminium sulphate solution (0.33 M) was added with continuous stirring until the pH of the
solution reached 4.0. The pH was then immediately raised to 5.8 by addition of 0.5 M-disodium hydrogen
phosphate, which resulted in the formation of a gelatinous precipitate. The precipitate was removed by
centrifugation at 5000 g at 4 8C for 10 min
Dialysis
the supernatant was subjected to ultra filtration using a 100 kDa nominal molecular weightcut-off
hollowf ibre cartridge .The filtrate was again ultra filtered using a 10 kDa membrane (Millipore Corporation) to
concentrate the extract to a final volume of 100 ml. This resulted in the retention of chymosin within the
molecular weight range 10–100 kDa. The retentate was dialysed using a dialysis membrane of 10-kDa cut-off size
against 2 l of distilled water at 4 ºC.

4. What is Pepsin?
 Crude rennet extract contains active chymosin and an inactive precursor (prochymosin). Addition of acid
to the extract facilitates conversion of prochymosin to chymosin and allows the extract to reach
maximum activity.
 Chymosin has now been produced in Escherichia coli and Saccharomyces cereuisiae by recombinant DNA
techniques. Cheese making trials comparing recombinant chymosin with calf rennet have found no
significant differences between the two .
 the optimum pH for chymosin stability is between 5.3 and 6.3, and that the enzyme is moderately stable
at pH 2.0. As the pH is raised from 6.3 to neutrality the activity of the enzyme is destroyed at an
increasing rate. A region of instability near pH 3.5 was also noted.

5. a. pepsin is an aspartic protease, a family of gastric enzymes which catalyze the hydrolysis of peptides
chains in the first step of their digestion. There are several kinds of pepsin, namely pepsin A, B and C,
gastricin and chymosin .
b. All these enzymes are synthesized as a zymogen, an inactive form of the enzyme which is stable until the
active form is required. In the particular case of pepsin, it is called “pepsinogen”. Through activation of
pepsinogen in acid media active pepsin is obtained.
c. Despite a lot of microbial processes to obtain proteases are known, their origin is mainly animal. For
example, in adult pig stomachs, the predominant gastric protease is pepsin A, and there are small
amounts of pepsin B and gastricin

6. Method 1: Swine stomachs were used for the pepsinogen extraction. The stomachs were
rinsed with water and then fundus, easily recognizable by its reddish colour, was
separated. Fundus was frozen and subsequently grinded until a 3 mm particle size,
approximately. Then it was mixed with 5 parts of water and 3 of ice and stirred for 1
hour. pH was kept at around 7-7.5 and temperature about 1.5 ºC. Finally, the mixture
was centrifuged 5 min at 2800 g.The solid phase was discarded and the pepsinogen
solution (supernatant) was obtained.
Method 2 : coagulation of pepsinogen was achieved by lowering pH of the supernatant of
Method 1 to 4 with a HCl solution (3 N). Under these conditions, pepsinogen was
coagulated and was separated by centrifugation at 4000 g, being the supernatant
discarded. Afterwards, pepsinogen was diluted with a dilution ratio of 1:10 (substrate:
water), giving rise to another pepsinogen solution
Method 3: the concentrated pepsinogen obtained in Method 2 was lyophilized (Labconco
LyphLock6 lyophilizer) at 18ºC and 5 μmHg for 24 h. The final product (a brownish
dust) was diluted 1:10 in water
Porcine pepsin

7. Activation
For each method, pepsinogen solution
activation was required in order to
obtain an active pepsin solution. This
was achieved by lowering the pH to 2
with HCl 3 N. After 10 minutes pH was
raised to 2.75 with NaHCO3 2 N.
Finally the solution was decanted for
6 hours and Solutions 1, 2 and 3 were
obtained.

8. Bovine pepsin
Preparation of bovine stomach homogenate (BSH)
Sections of frozen gastric mucosa of adult bovine were homogenized with about 5
volumes of 50mM sodium phosphate buffer, pH 7.0. Then the homogenate was filtered,
fractioned and frozen. Before using it, the fat was withdrawn and the homogenate
centrifuged at 15,000×g for 30min. Total protein concentration and enzymatic activity
was carried out in the supernatant.
Pepsinogen activation
The procedure for pepsinogen activation was made at 20 ◦C by slow addition of HCl
concentrate to the homogenate solution in order to diminish the pH until the solution of
homogenate reached a value of 2.5. It was allowed to rest for 30 min.A slight turbidity
was observed in all the cases due to a minimal precipitation of proteins. Then it was taken
to pH 6.4 with concentrate NaOH (3 M). After that, the solution was centrifuged at
1000×g during 5min.

9. Chicken pepsin
 The proteolitic enzyme pepsin (EC 3.4.23.1) was purified from chicken stomach by a
modification of the method of Bohak (1970): after homogenazing the raw material,
the zymogen was extracted with NaCl and NaHCO3, activated with 3N HCl and
precipitated with NaCl (28% final concentration).
 The precipitate was lyophilised; fractions of it were suspended in 0.02N HCl. The
solution was filtered through a column (2.6 x 80 cm) of Sephadex G-100 at an
elution rate of 8-10 ml x cm-2 x h-1. Two protein peaks were obtained, the first one
corresponding to the pepsin (2.0 mg/ml in pooled fractions).
 Chicken pepsin used as a clotting agent for the industrial production of pasteurized
white cheese.

10. FISH PEPSIN
Fish PGs have distinct characteristics from
mammalian PGs including higher pI
higher pH optimum and Michaelis
constant lower optimum temperature and
thermal stability than mammalian PGs.
Fish PGs (or pepsins) have been purified and
characterized in various types of fishes including
Arctic capelin , rainbow trout ,Atlantic cod ,bolti fish
,Antarctic rock cod , sea bream, African coelacanth ,
Mandarin fish ,smooth hound , orange–spotted
grouper , albacore tuna and European eel .

11. Arctic capelin rainbow trout Atlantic cod
European eel albacore tuna Mandarin fish

12. Isolation of Enzymes from Fungi
Endothia parasitica
EndothIa parasitica, ATCC 14729, is rinsed from a potato dextrose agar slant, under sterile
conditions and introduced to one liter of the aqueous inoculum medium in a 2.8-1. Fernbach flask
(the medium has been previously sterilized in an autoclave for 45 minutes at 121 C.).The medium
has a pH of about 6.8.
After cooling (28 C) of the inoculum is added to the vessel containing 2000 ml. of sterile growth
medium and fermentation is carried out at 28 C. while air is introduced at the rate of 0.5 volumes
of pair per volume of broth per minute and while the medium is agitated at 1700 r.p.m.
After 48 hours, it is found that sufficient enzyme is present to enable 0.5 ml. of broth to curdle 5.0
ml. of sweet milk within 3 minutes. The final pH is about 6.0-6.5. After filtering ,the filter cake is
washed with 100 ml. of deionized water. The combined washing water and filtrate are freeze dried
and the freeze dried solids assay about 128000. The freeze dried solids are then reconstituted in
deionized water to five times the original broth concentration and the solution is cooled to 1 C.

13. During the cooling period ,nitrogen is flushed over the solutions and solids to aid in the
exclusion of oxygen. Acetone, 25 C., is then added slowly, with stirring, to the enzyme solution until
an amount equivalent to twice the original volume has been added. Toward the end of the addition of
acetone, the precipitated enzyme becomes completely congealed.
The acetone-water supernatant layer is decanted from the vessel leaving behind the
viscous, congealed precipitate. The precipitate is then suspended in an equal volume of water and is
rapidly freeze dried; these solids contain 85% of the activity. A 15% (w./v.) aqueous solution of the
purified freeze dried solids is prepared, cooled to 1 C. and a volume of acetone, cooled to 25 C., is
added slowly, with stirring, until precipitate just begins.
The precipitate is allowed to settle and the supernatant is decanted and to this is added
slowly, with stirring, a volume of cold (-25 C.) acetone equivalent to 0.75 volume of the solution
originally taken. The precipitate which forms is allowed to settle, the supernatant layer is decanted
off, excess acetone is removed. The freeze dried solids contain 40% of the original activity and
assay about 1245,000. This precipitate is dissolved in an equal volume of Water and is dialyzed
against cold tap water for 5 days.The non-dialyzables are then freeze dried. The solid product has
an activity assay value of 1:l00,000

14. Mucor pusillus var. Lindt
and Mucor miehei
Mucor pusillus is grown on wheat bran for 72 hours
at 30°c. Wheat bran (2 kg) is mixed well with
tapwater (1.4 liters), and autoclaved at 115 ° for 20
minutes and inoculated with the spore suspension.
Inocula are prepared earlier by culturing on wheat
bran in 200-ml Erlenmeyer flasks (7 flasks) for 7
days at 30 °, and then suspending sporangiospores
in sterilized water. The inoculated bran is extracted
with tap water (10 liters) for 20 hours at room
temperature and filtered. A 6 liter filtrate contains
about 800 units/ml of milk-clotting activity (enzyme
yield: 2400 units/g of wheat bran).
One liter of the above extract is blended
well and precipitated with 3 liters of
ethanol at 14 °. The precipitate is
separated by centrifugation and dried
at 5 °.

15. Bacterial
proteases
1. Bacteria are reported, wild bacteria with a high milk-clotting activity (MCA) in submerged
fermentation show a great potential, due to shorter fermentation period, larger capacity of
extracellular secretion and higher material utilization ratio.
2. Some bacteria, such as Bacillus subtilis, Bacillus licheniformis and Enterococcus faecalis, have
been suggested as potential rennet substitutes
3. Most of the microorganisms mentioned were screened from soil, natto, milk or glutinous rice
wine samples.
Method of production
The bacteria were cultured in the medium and the bacterial strains were identified and
enriched in the beef broth. Later solid state fermentation(SSF) is carried out. bacteria
inoculums prepared above were inoculated into 100ml of a medium composed of sucrose in a
250-ml flask, and then were incubated at 37 ◦C, pH 7.0 with shaking at 150rpm for 21h,then
centrifuged to remove the bacterial cells, and precipitation with the addition of cold 95%
ethanol from the culture broth, followed by dialysis through a membrane with 10kDa cut-off.

16. Isolation of Enzymes from Plants
Accurately weight 5g of grinded powder papaya was dissolve in accurately measured
20ml distilled water, the water was added in ratio 1:5 and Water papaya mixture was
filtered by using filter paper.
Grinded pretreated crude enzyme was mixed with 40mM cysteine at a ratio of
3:1(w/v) and the suspension was adjusted to pH 5.6 using 6M HCL and then stirred for
15 min at 40c [15]. The mixture was filtered and pH of the filtrate was adjusted 9.0
using 6M NaOH. The insoluble material was removed by centrifugation at 9000xg for
30min at 4oc. The supernatant was precipitated with (NH4)2SO4 at 45% saturation.
The salt –enriched solution was incubated at 40c for 30min.
The precipitation was collected by centrifugation as above, and dissolved using
20mM cysteine. The solution was kept at 4 c before adding sodium chloride (10% w/v).
The mixture was slowly stirred for 30min before separating the papain by
centrifugation. The enzyme was dissolved in water and stored at 4 C,then followed by
the purification by sephadex G-75 for industrial use.
Papain

17. Bromelain

18. Novel Milk-Clotting Enzyme from
Brassica napus Seeds
Preparation and Extraction of The Crude Enzyme:
The rape dry seeds were crushed and mixed with dist. water at 9 °C with
continuous shaking over a period of 12 hours. The resulted extract was then
centrifuged at 3000 r.p.m for 15 minutes, and the supernatant was collected,
dialyzed against dist. water and then used as the crude enzyme preparation.
The seeds were chosen as the target plant tissue to be studied as they contain
high amounts of storage protein and hence a high percentage of proteolytic activity.
Shows a high specificity of action in only the acidic range.
Controls the unwanted proteolytic activity during ripening process.
Raphanus sativus (radish), Eruca sativa (arugula), Brassica alba (white mustard)

19. Proteolytic enzymes from other plants
Solanum coagulans Calotropis procera Helianthus annuus
Lactuca sativa E. psoraleoides Cynara scolymus

20. ACTION OF
MILK- CLOTTING
ENZYMES

21. Enzymatic Coagulation of Milk

22. Action of different Proteolytic Enzymes
Trypsin or ficin Seeds
Bacteria
Fungi
Advanced coagulation
high values of FC and amino- N
coagulum is transient
No coagulation of milk- inhibited by acids
bitter-tasting curds
high proteolytic activity – radish seeds
decreased rennet clotting time
Firm gel formation
High milk clotting activity
Safe producers
capacity to secrete large amount
low optimum pH, making gels denser
crystalline mucor rennin enzyme is similar to
that of calf rennin.
weaker in alkaline conditions
Ca ions –increases proteolytic activity of mucor
rennin
Mucor pusillus resembles pepsin, rennin.

23. Reference
E. Jurado a et al. (2012)Pepsin extraction process from swine wastes E. Jurado a et al. 20th International
Congress of Chemical and Process Engineering CHISA 2012 25 – 29 August 2012, Prague, Czech Republic
L Paez De Leon et al.(1995):Purification and assay of chicken pepsin, Cátedra de Salud Pública, Facultad de Ciencias
Veterinarias, Universidad Central de Venezuela, Maracay, Venezuela. 1995;46(4):237-41
Joseph L. Sardinas et al. Milk-curdling Enzyme elaborated By Endothia Parasitica: Apr. 21, 1964, Ser. No.
361,532 3 Claims. (C. 99-116)
Shieh, C.-J., Phan Thi, L.-A., & Shih, I.-L. (2009). Milk-clotting enzymes produced by culture of Bacillus subtilis
natto. Biochemical Engineering Journal, 43(1), 85–91.
Arima, K., Yu, J., & Iwasaki, S. (1970). [30] Milk-clotting enzyme from Mucor pusillus var. Lindt.
Proteolytic Enzymes, 446–459.
Natalia Imelio et al (2012)Pepsin extraction from bovine stomach using aqueous two phase systems: Molecular
mechanism and influence of homogenate mass and phase volume ratio. Journal of Chromatography B, 873 (2008)
133–138,2012.
Yidnekachew Welde and Abebe Worku Identification and extraction of papain enzyme from papaya leaf in
adigrat towen, northern Ethiopia :JMPS 2018; 6(3): 127-130 © 2018 JMPS

24. Rodney J. BrownC. A. ErnstromMark E. Johnson: Milk-Clotting Enzymes and Cheese
Chemistry,Part I—Milk-Clotting Enzymes, Fundamentals of Dairy Chemistry pp 609-654
Choudhery, A. K. and Mikolajcik, E. M. 1969. Rennin-like activity in milk of Bacillus cereus. J. Dairy Sci. 52, 896–896.
Gordin, S. and Rosenthal, I.1978. Efficacy of chicken pepsin as a milk clotting enzyme. J. Food Prot. 41, 684–
688.
Mohamed Mohey Eldin Elmaza et al.(2012)Screening Local Egyptian Seeds for Milk-Clotting Activity
and Physicochemical Characterization of Brassica Napus Seed Extract. J. Agric. Food. Tech., 2(2) 28-34,
Green, M. L. and Morant, S. V. 1981. Mechanism of aggregation of casein micelles in rennet-treated milk. J. Dairy
Res. 48, 57–63.
Brown, R. J. 1981. The mechanism of milk clotting. Proc. 2nd Bienn. Marschall Int. Cheese Conf. pp. 107–
112.

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