Scientific Bulletin. Series F. Biotechnologies, Vol. XXV, No.

1, 2021
ISSN 2285-1364, CD-ROM ISSN 2285-5521, ISSN Online 2285-1372, ISSN-L 2285-1364

MILK-CLOTTING ENZYMES OBTAINED FROM PLANTS

IN CHEESEMAKING - A REVIEW

Sorin NITU, Mihaela GEICU-CRISTEA, Florentina MATEI

University of Agronomic Sciences and Veterinary Medicine of Bucharest, 59 Mărăşti Blvd,

District 1, Bucharest, Romania

Corresponding author email: [email protected]

Abstract

The paper aimed to present a review of plant proteases used as milk coagulants in cheesemaking. Plant proteases have
been used as milk-clotting enzymes since ancient times. These milk-clotting enzymes are starting to become an
alternative to the calf rennet. Due to a high price of the calf rennet and a very limited availability, religious restrictions
or lacto-vegetarian diet, milk-clotting enzymes obtained from plants are the subject of extensive research. They are
present in a various plants and can be obtained from all plant parts: root, stem, leaves, flowers, fruits, seeds. Most
research has shown that plant milk-clotting enzymes belong to aspartic proteases but have been reported also enzymes
from serine proteases and cysteine proteases with this activity as well. Plant proteases with milk-clotting activity have
been researched in terms of coagulation activity and proteolytic activity. Most plant milk- clotting enzymes develop an
excessive proteolytic activity leading to lower yields of cheese, defects of texture and bitter flavours. The research will
continue in order to meet the increasing global demand for a good and diversified cheese production.

Key words: cheese, milk-clotting activity, plant proteases.

INTRODUCTION cheeses were arguments for the development of

this production: storage stability, relatively
Milk is a valuable food, but relatively easy transport and diversification of the human
perishable due to its contamination with diet. Worldwide, at present, over 35% of the
microorganisms, since milking. Also, milk amount of milk obtained on farms is
production is, in many regions of the globe, transformed into cheese (Costin, 2003).
seasonal, so there are no constant quantities Cheeses are some of the most complex and
during the year. Methods of preserving milk in dynamic foods. They are results of an applied
various forms and convenient conditions have biotechnology, each piece can be considered a
been discovered and perfectioned. Thus, at bioreactor in which numerous and complicated
present, the milk components are fractionated: reactions take place. Finally, is obtained a
the fat is transformed into butter, which can be product with specific sensory and nutritive
preserved by freezing, the dry matter in the characteristics. Cheeses are one of the most
milk is transformed into powdered milk with valuable sources of protein for the global diet
variable fat content, which does not require low and are an excellent source of nutrients such as
temperatures. Milk proteins can be separated by fats, minerals and vitamins. They can
precipitation or separation through membranes sometimes have a therapeutic role: patients
and preserved by drying in the form of protein with reduced gastrointestinal absorption and
concentrates. food allergies are currently treated with casein
A most common and historical method of hydrolysate (milk protein). Casein hydrolysate
preserving milk is processing in cheese, a more is a biologically active peptide, which plays an
complex process that involves concentrating important role in various physiological
protein along with a variable fraction of fat and disorders.
minerals, eliminating a significant amount of Cheeses are dairy products that have played a
water and lactose. The cheeses can be stored key role in human nutrition for centuries.
for a few weeks to several months. The Cheese making is essentially a dehydration
advantages resulting from the possibility to process, in which milk fat and casein are
transform the main components of milk into concentrated 6-10 times (Abebe & Emire,
66
2020). From ancient times the animal rennet is including milk-clotting proteases that coagulate
used in the manufacture of cheese. milk; belonging to the coagulation process, it
Milk coagulation is the main stage for cheese produces the separation into a solid part (curd)
production. Milk-clotting enzymes have been and a liquid part (whey). The presence of these
used in cheese making for thousands of years, enzymes is very important in the stomachs of
and they appear to be the oldest known young mammals for digesting the breast milk
application of enzymes; the earliest indication with which they are breastfed. The main active
of cheese making dates back to cave paintings enzyme in the rennet is chymosin or rennin (EC
around 5000 BC (Shah et al., 2013). 3.4.23.4, according with International Union of
Historically, most enzymatic preparations used Biochemistry Enzyme Commission IUB or
for cheese have been extracted from the EC), along with pepsin and lipase.
stomachs of ruminants, but coagulants from The natural calf rennet is extracted from the
microorganisms and plants have also been used inner mucosa of the fourth chamber of the
at very early dates (Bunty & Nabindra,. 2020). stomach of young, unweaned calves. The
The stomach of the ruminant, especially the rennet extracted from the old calves, fed with
calf, is the source of rennet. It contains grass or cereals, contains less chymosin or not
chymosin (EC 3.4.23.4) as the main enzymatic at all, but more pepsin. This rennet can only be
component and has been widely used in the used for some special types of milk and cheese.
manufacture of cheeses. Cheese production Each ruminant species produces a special type
increased significantly (3.5 times higher than in of rennet to digest the milk of its own species,
1961), but animal rennet production decreased so there is, for example, a goat's rennet for
due to the limited availability of herbivore milk-clotting of goat's milk or a lamb's rennet
stomachs (Nasr et al., 2017). for milk-clotting of sheep's milk.
The animal rennet is obtained on a commercial There are many alternative sources of milk-
scale from the stomachs of young ruminants clotting enzymes, from plants and
(calves, lambs, kids). A single calf produces microorganisms, that can replace animal rennet.
only 5 to 10 g of rennet. The enzyme helps to Fermentation produced chymosin (FPC), by
coagulate casein in milk. The property of the applying genetic engineering tools on the
enzyme to coagulate milk is important in terms microbial organism, is mainly used in the
of cheese quality and yield (Mahajan & manufacture of cheese in North America and
Chaudhari, 2014). Europe because it is cheaper and has a better
Research has long been focused on the quality than animal rennet. Enzymes produced
discovery of a milk-clotting enzyme that would by microorganisms are suitable as milk-clotting
satisfactorily replace animal rennet. Various substitutes but there has been a lot of interest in
factors, such as the high price of animal rennet, coagulation enzymes extracted from plants.
various religious restrictions (e.g. Islam and These enzymes are present in almost all types
Judaism), diet (lactovegetarians) or the inter- of plant tissues and it appears that all
diction on the use of recombinant calf rennet proteolytic enzymes have the ability to
(in France, Germany and the Netherlands) have coagulate milk under appropriate conditions.
encouraged the search for alternatives sources Almost all enzymes used to coagulate milk
of milk-clotting (Roseiro et al., 2003). Research belong to aspartic proteases but enzymes from
has been directed towards the discovery of other groups such as cysteine and serine
milk-clotting enzymes that would satisfactorily proteases are also used.
replace calf rennet in cheese making, including Plant extracts have been used since ancient
microbial, recombinant, and herbal enzymes times to coagulate milk to make cheese,
(Mahajan & Chaudhari, 2014). especially in Mediterranean countries, West
The most important substitute enzymes that Africa and Southern Europe. Thus, Homer
meet the requirements of cheese making suggested in the Iliad and the Odyssey that the
include microbial, recombinant enzymes and Greeks clotted milk with a fig extract (Ben
enzymes that have been isolated from plants. Amira et al., 2017).
Animal rennet is a complex of enzymes Milk-clotting plant proteases have become a
produced in the stomach of any mammal, subject of growing interest in cheese industry,

67
due to their easy availability and simple based on the formation of an aggregate protein
purification processes. The use of plant network, which consists mainly of a certain
proteases in cheese production promotes the group of proteins known as caseins. This
greater acceptability by the vegetarians and network includes water, fat and other
may improve their nutritional intake (Ben constituents of milk. Biochemical processes are
Amira et al, 2017). quite different between cheese and fermented
The presentation of enzymatic and dairy products, as the production of cheeses
technological properties of milk-clotting plant involves the separation of whey by casein,
proteases, previously studied in literature, could while in fermented dairy products the entire
provide a clear vision on key elements for the composition of milk is included in the final
selection of appropriate plant rennet. product.
Evaluation of enzymatic activities is the main Milk coagulation (clotting) in cheese making is
step in the selection of an appropriate substitute of several types, depending on the main agent
of calf rennet which can be successfully used in involved in the biochemical process: enzymatic
cheese making. It is achieved by monitoring of coagulation, acid coagulation, mixed
Milk-Clotting Activity (MCA) and Proteolytic (enzymatic and acid) coagulation.
Activity (PA).
Milk-Clotting Activity (MCA) is the most Enzymatic coagulation of milk
important property of enzymes used in cheese
making. Mechanism: the biocatalytic action of
It is the ability of the enzyme to specifically coagulating enzymes on casein, leads to the
hydrolyse of κ-casein. It can be measured by formation of "clot" gel.
different methods, such as Soxhlet, Berridge Enzymatic coagulation of milk represents the
and the units used are Soxhlet, Berridge or modification of casein micelles by limited
Rennet Units and International Milk-Clotting hydrolysis of casein under the action of milk-
Units (IMCU). clotting enzymes, followed by a network
Proteolytic activity (PA) expresses the degree aggregation of micelles induced by the
of proteolysis of the enzyme. presence of calcium ions (Fox et al., 2004).
High proteolytic activity leads to excessive The first commercial rennet was prepared,
cheese maturation, with advanced hydrolysis of standardized and sold by Chr. Hansen A/S,
protein chains and formation of nonspecific Denmark in 1874, and was probably the first
bitter-tasting peptides. commercial enzyme of any kind, world-wide
The MCA/PA ratio is the one that best used (https://www.chr-hansen.com). Animal
characterizes a commercially coagulating rennet is, by definition, an extract of ruminant
enzyme: the higher it is, the better the enzyme abomasum, ideally containing mainly
coagulates the milk, without advanced chymosin, the enzyme that is specific for the
proteolysis during maturation. hydrolysis of κ-casein and the destabilization of
Considering that the researches regarding the casein.
use of plant proteases as a substitute for animal However, depending on the age of the calves
rennet, carried out so far in Romania, are in the from which it is extracted, the rennet may
incipient phase, of pioneering, the present contain pepsin, which is an acidic protease with
review aims to summarize the latest research a wider range of action on the casein substrate.
findings on plant proteases with milk-clotting Both chymosin and pepsin, and indeed all milk-
activity presenting enzyme chemistry, clotting enzymes used in cheese technology,
production and techno-functional properties. are classified as aspartic proteinases with
Enzyme Commission number (EC) 3.4.23.
MILK COAGULATION - THE MAIN Because there are now several different types
STAGE OF CHEESE MAKING and sources of milk-clotting enzymes on the
market, the International Dairy Federation
The general process of milk coagulation (IDF) officially defines that the name "rennet"
(clotting) into dairy products, such as cheeses is reserved for ruminant stomach enzyme
and fermented dairy products (yoghurt) is preparations, and other types of milk- clotting

68
enzymes (especially microbial ones) should be isoelectric point of the micellar casein (pH 4.6).
called 'coagulants'. For cheese technology, A porous network of weakly bound aggregates
rennets and coagulants are usefully classified, is formed. Moreover, the concentration of
according to their source, as animal, vegetal, proteins in the gel network will be increased
microbial and GMO sources. due to the active participation of denatured
Chymosin is the most important and active whey proteins in the formation of the structure.
milk-clotting enzyme, involved in cleaving the
Phe105-Met106 peptide bond from κ-CN. In the case of the mixed coagulation of milk
Coagulation of milk induced by chymosin can the mechanism undertakes a symbiosis between
be described in three phases: the two previous procedures.
1. During the primary phase, the enzymatic
hydrolysis of κ-CN takes place as follows: MILK-CLOTTING PLANT PROTEASES -
Chymosin A GENERAL DESCRIPTION, TYPES
AND SOURCES

κ-CASEINE (- Phe105 - Met106 -) Proteases are enzymes that perform

para-κ-CASEINE + CMP proteolysis, initiating protein catabolism by
hydrolysis of the peptide bonds between the
The hydrophilic CMP (Casein Macro Peptide) amino acids in the structure of the polypeptide
portion is released into the whey. This causes chain that make up the protein. Over time, they
the loss of a negatively charged group and the have evolved several times so that the same
decrease of steric stabilization. When approxi- reaction can be performed by different classes
mately 70% of k-CN is hydrolysed (Walstra et of proteases by completely different catalytic
al., 2006), the colloidal stability of the micelles mechanisms.
is low enough to begin the second phase: Proteases are found in all organisms, from
2. Spontaneous secondary aggregation phase - prokaryotes to eukaryotes and viruses. These
gel formation as molecular chains that connect enzymes are multifunctional, having many
through hydrophobic calcium bonds to form a physiological functions in plants and animals.
three-dimensional network, followed by a They are involved in various physiological
subsequent solidification. reactions from the simple digestion of proteins
3. In the third phase, the whey is expelled from in food to extremely well-regulated cascade
casein by syneresis (more cross-linking reactions such as blood clotting or apoptosis.
contraction). They also act in germination, biological aging,
Coagulation is improved by lowering the pH, inflammatory processes, etc.
increasing the calcium concentration and Proteases act either by completely breaking
temperature (without aggregation below 20°C). down of peptide into amino acids (unlimited
Syneresis is increased by increasing proteolysis) or by breaking down specific
temperature, pH and pressure applied, e.g. peptide bonds (limited proteolysis) depending
stirring (Walstra et al., 2006). on the amino acid sequence that makes up the
protein. The activity can be a destructive
Acid coagulation of milk change such as suppressing the function of a
Mechanism: selected cultures of lactic acid protein or digesting the protein to amino acids
bacteria ferment lactose from milk and turn it or it can be an activation of a function,
into lactic acid; by lowering the pH to the respectively a signal in a signalling pathway.
isoelectric pH of casein, it precipitates and Proteases are classified, according to the
forms a "lactic" gel. catalytic residue, in the following groups:
Upon acid coagulation of milk, the micellar serine proteases, threonine proteases, cysteine
casein is modified by the low pH of milk. This proteases, aspartic proteases, glutamic acid
causes the colloidal calcium phosphate (CCP) proteases and metalloproteases.
to dissociate from the micelles, the negative Plant proteases used as milk-clotting enzymes
charges in the casein micelles being were reported only in aspartic, cysteine and
neutralized, with aggregation occurring at the serine proteases.

69
According to the International Union of involved in several physiological processes,
Biochemistry Enzyme Commission (IUB or such as fruit development and ripening, nutrient
EC), http://www.enzyme-database.org, reserve, degradation of storage proteins in
proteolytic enzymes with milk-clotting activity germinated seeds, activation of proenzymes,
are part of EC 3 Hydrolases, EC 3.4 acting on and degradation of defective proteins. CPs
peptide bonds (peptidases). comprise a family of enzymes, which consist of
Depending on the groups of the active centre, papain and related plant proteases, such as
there are 3 subclasses of endopeptidases with chymopapain, ficin, caricaine, bromelain,
milk-clotting activity (Shah et al., 2014). actinidine and microsciadin (Rezanejad et al.,
2015).
Aspartic proteases (EC 3.4.23) (APs)
Aspartic proteases have two aspartic residues in Serine proteases (EC 3.4.21) (SPs)
their catalytic centre and are involved in protein Serine proteases possess a residue of serine in
degradation during plant development process, their active centre and show several
protein storage mechanisms, responses to stress biochemical and physiological characteristics.
and pathogens and plant senescence. They are In plants, they are widespread among
most active at acid pH and have preferential taxonomic groups, from trees to crops for
specificity for cleavage at peptide bonds vegetables and herbs. They are present in
between hydrophobic amino acid residues almost all components of plants, but most
responsible for catalytic activity. abundantly in fruits. Serine proteases from
Aspartic proteases with milk coagulation cucurbits, grains and trees are usually classified
activity have been identified in artichokes together.
(Cynara scolymus), armory (Silybum Serine plant proteases have been found and
marianum), rice core, Centaurea calcitrapa. extracted from latex, seeds, flowers, stems,
Regarding cardoon (Cynara cardunculus), leaves and roots.
flowers are traditionally used in the Shah et al. (2014) described a few serine
Mediterranean region in the manufacture of proteases developed and researched: neriifolin,
cheese. It produces cardosine and cyprosine, a type of chymotrypsin serine protease, has
aspartic proteases that have been found to been purified from the latex of Euphorbia
accumulate in mature flowers (petals and neriifolia; religiosin A, B and C were isolated
pistils) but not in leaves or seeds. Cardosin A is from latex extracted from Ficus; dubiumin was
an abundant aspartic protease from purified from Solanum dubium seeds;
C. cardunculus pistils (Feijoo-Siota & Villa, cucumisin from Cucumis melo and lettucine
2010). from Lactuca sativa were isolated and used as
coagulating enzymes.
Cysteine proteases (EC 3.4.22) (CPs) Research in recent decades has expanded the
list of plants or parts of plants that contain
Cysteine proteases or thiol proteases are some enzymes with milk coagulation properties.
of the largest groups of proteolytic enzymes Milk-clotting plant proteases were
involved in many processes in both prokaryotes characterized and named by their plant sources
and eukaryotes (e.g., bacteria, parasites, plants, (Table 1).
invertebrates and vertebrates).
The catalytic mechanism of these enzymes Table 1. Milk-clotting plant proteases characterized from
different plant sources
involves a cysteine group in the active site.
Protease Source References
Cysteine proteases have great potential for use
Bromelain Pineapple (Ananas comosus) Harrach et al., 1998
in the food, biotechnology, and pharmaceutical
Papain Papaya (Carica papaya) Mitchel et al., 1970
industries due to their property of being active Ficin Common fig (Ficus racemosa) Devaraj et al., 2008
in a wide range of temperature and pH. The Cardosin Thistle (Cynara cardunculus) Ordiales et.al., 2012
activity of all cysteine proteases depends on a Actinidin Kiwi (Actinidia chinensis) Katsaros et al., 2010
catalytic dyad consisting of cysteine and Cucumisin Melon (Cucumis melo) Uchikoba & Kaneda,
1996
histidine. In plants, they are widespread among
Lettucine Lettuce (Lactuca sativa) Lo Piero et al., 2002
different taxonomic groups and prove to be

70
ISOLATION AND EVALUATION OF source must be considered in all the stages of
ENZYMATIC ACTIVITY OF MILK- choosing the source.
CLOTTING PLANT PROTEASES Certain intracellular enzymes are used
commercially without isolation and
Obtaining enzymatic preparations - purification, but most commercial enzymes are
theoretical considerations regarding the produced extracellularly by microorganisms or
extraction of enzymes plants or must be released from cells in solution
and subsequently processed (Figure 1).
In order to study the structural and / or bioche- Solid/liquid separation is generally required for
mical properties of an enzyme, the source that the initial separation of cell mass, removal of
best meets the isolation requirements and that cell debris after cell rupture, and collection of
meets, as far as possible, the following condi- precipitates. This can be done by filtration,
tions must be chosen: maximum catalytic centrifugation or aqueous biphasic partition.
activity - the enzyme present should not be In general, filtration or partitioning of aqueous
degraded or inactivated; maximum possible biphasic systems is used to remove unwanted
purity - it must not contain other large enzymes cells or cell debris while centrifugation is the
or molecules; maximum possible yield - resul- preferred method for collecting the required
ting from the percentage of activity recovered solid material.
compared to the activity of the original extract,
if the purification of the enzyme is also
followed.
In the strategy of choosing a biological material
for the extraction of an enzyme usable for
different purposes or for its study is necessary
to consider several factors such as: enzyme
abundance, because for any study, experiment
or application it is necessary to obtain total
protein extracts with high concentrations of the
enzyme of interest, biological sources rich in
the desired enzyme must be chosen;
availability and cost price: the biological
source must be accessible both geographically
and economically; intracellular localization: of
an enzyme is essential to know in order to
establish the most convenient method of
extraction; source characterization: the chosen
source must be perfectly characterized; when
the chosen source are plants, it is necessary to
know them genus, species and variety. It is
often necessary to know the area, the climate in Figure 1. Flow diagram of enzyme preparation
which the plant source developed as well as the (Chaplin & Bucke, 1990)
harvesting period; comparative studies. Some
enzymes have been studied in some species or Evaluation of milk-clotting plant proteases -
in different tissues of some species. In such an important step in validation as a rennet
cases it is very important to study the substitute
respective enzymes in different other species or
tissues in order to evaluate the evolution of the Experimental research to identify plant
enzyme, its properties compared to similar proteases with milk-clotting activity can be
ones, primary, tertiary structure, different conducted in a variety of ways but must
isoenzymes, etc. The stability of the enzyme consider the principles described in the
and the possible difficulties in handling the previous paragraph.

71
In addition, most of the articles written on this Table 2. Milk-clotting activity, protease activity and ratio
topic in the literature, outline the same of milk-clotting to protease activity of tested plants
(Dahot et al., 1990)
experimental work procedure:
1) Screening on a wider range of plants with Part of
MCA¹ PA² Ratio of
coagulant activity can be achieved, based on Name of plants plant
tested
(units/ml) (units/ml) MCA/PA
similarity, plants from the same family with a
Aloe L. sp. Stem 190 7 27.14
plant already proven with milk-clotting
Euphorbia Whole
activity, on the same plant can be investigated hista Plant
360 9 40.00
and compared also different parts of the plant Cereus
Stem 160 6 26.67
triangularis
(root, stem, leaves, flowers). Oseni & Euphorbia
Stem 600 5 120.00
Ekperigin (2013) compared the milk-clotting caducifolia
Euphorbia
activity of different parts of Sodom Apple nivulia
Stem 760 7 108.57
(Calotropis procera) (Figure 2). Opuntia
Stem with
role of 120 5 24.00
phylloclades
leaves
Ficus
Leaves 380 7 54.29
bengalensis
Ficus carica Leaves 1200 9 133.33

Ficus elastica Leaves 490 6 81.67

Calotropis
Flowers 170 6 28.33
procera
Calotropis
Leaves 390 9 43,33
procera
Leaves,
Carica papaya 1580 8 197.50
dried latex
Legend:
1 - The unit of milk-clotting activity was defined as the amount of
enzyme which clotted 1.0 ml of milk in one minute at 50°C.
2 - One unit of the protease activity was defined as the amount of
enzyme that liberated 1 μg of tyrosine under the standard assay
conditions.

Figure 2. Milk- clotting and proteolytic activity of Table 3. Ratio of milk-clotting activity/proteolytic
Calotropis procera plant (Oseni & Ekperigin, 2013) activity of some plant proteases and other coagulants
(Da Silva et al., 2013)
2) Extraction of milk-clotting enzyme from Name of milk- MCA¹ PA² Ratio of
plants, based on flow diagram described clotting enzyme (U/mg) (U/mg) MCA/PA
(Figure 1). Chymosin 269 0.08 3363
3) Protein content determination: the protein
concentration of each enzyme solution should Rennet from Mucor 438 0.16 2738
be determined by different methods in order to
Papain 208 0.51 408
report a standardized enzymatic activities per
protein unit. Protease of Ginger 314 0.19 1653
4) Preparation of a standard working substrate Protease of
(liquid milk from different species, with known 760 7 109
Euphorbia nivulia
fat and protein content, milk reconstituted from Protease of Quixaba 917 0.16 5731
powdered milk, corrected with calcium Legend:
chloride). 1 - One milk coagulating unit per millilitre (U ml–1) is defined as 400
t–1; the amount of enzyme that coagulates the milk in one minute has
5) Defining and applying a working method for 400 milk coagulating units. The variable t is the time required for the
determining the MCA e.g. procedure described first clots of milk to form
2 - One unit of activity is equivalent to a change in optical density of
by IDF (1992) (Da Silva et al., 2013). 0.01 nm per minutes at 440 nm.
6) Proteolytic activity determination by
different chemical and biochemical methods. Within the frame of research for a suitable
7) Ratio of milk-clotting to protease activity substitute for calf chymosin, which is strongly
can accurately indicate which plant proteases recommended enzyme, combining a strong
should be developed as a milk-clotting clotting activity with a low general proteolytic
enzymes as seen in Table 2 (Dahot et al., activity (Panayotov et al., 2014), should be
1990). realized comparative studies of coagulant
72
activities and MCA/PA ratios of plant proteases stronger proteolysis and lower production with
with chymosin and microbial rennet as seen in milk-clotting plant proteases (Everett & Auty,
Table 3 (Da Silva et al., 2013). 2008).

TECHNOLOGICAL CONSIDERATIONS Grana and Provolone pasta filata cheeses with

OF MILK-CLOTTING PLANT milk-clotting plant proteases
PROTEASES USED IN DIFFERENT TYPE Grana and Provolone cheeses made with
OF CHEESES cardoon extract have a softer texture and a loss
of shape. These differences are explained by
At this moment, cheeses obtained with milk- the activation of enzymes at stretching
clotting plant proteases are normally produced temperatures (80-85°C) used to make these
on an artisanal scale, in small dairies. cheeses. In contrast, chymosin is inactivated by
Roseiro et al. (2003) summarized and described these temperatures. An interesting example is
a few technological considerations about this Gran Kinara, a cheese produced in North of
issue, as presented below. Italy with Grana Padano technology, but
Edam and Cheddar semi-hard cheeses with replacing the animal rennet with vegetable
vegetable proteolytic enzymes rennet. The use of vegetable rennet extracted
After 8 months of maturation, Cheddar cheese from the flowers of Cynara cardunculus, the
made with Ficus carica extract did not show common wild thistle that grows spontaneously
any difference between the control cheese also on the mountains, has allowed to offer a
produced with animal rennet; no difference rare, sought after alternative to traditional
compared to Cheddar cheese obtained with animal rennet, which can contribute decisively
Withania coagulans extract, except that the to the "zero lactose" of the Gran Kinara and to
vegetable extract had a slightly light texture. provide original and pleasing organoleptic
Cheddar cheese obtained with Streblus asper characteristics (Sousa, 1998).
extract is known to have bitter and acidic
aromas and a delicate texture while cheeses CONCLUSIONS
with Carica papaya leaf extracts also have
flavour defects such as bitterness. The use of Proteases from plants are used in milk
cardoon extract has been suggested to produce coagulation and cheese-making process
new types of soft cheeses and for improving the especially in Mediterranean countries, Middle
texture of low-fat Cheddar cheese. Edam East, West Africa and Southern Europe. They
cheese coagulated with cardoon had a softer currently do not have an industrial use because
texture and the whitish colour of the whey of high bitterness developed post-coagulation
suggested a higher degree of proteolysis and a and lower cheese yields. However, some
lower yield (Rao & Matur, 1979). researches and developments were done in the
understanding of their action and the control of
Camembert and Roquefort mold cheeses with the various parameters that influence cheese
vegetable proteolytic enzymes production.
Camembert cheese obtained with vegetable The most important aspects to consider in the
coagulant was matured before the one made study of this kind of plant proteases are type of
with animal rennet. There was also reported a coagulant, how often the plant source is found
slightly astringent taste in cheese made with in the spontaneous flora, enzymatic activities
cardoon at the beginning of ripening, which described by milk-clotting activity (MCA),
disappeared towards the end of ripening and proteolytic activity (PA) and MCA/PA ratio, as
which can be explained by the presence of a determinant parameter, concentration of plant
tannins and other substances in flowers. There proteases in the plant source.
was a higher proteolytic activity in Camembert The selection of a suitable plant protease with
sheep's milk cheese and, although the resulting milk-clotting activity must be based on the best
cheese was pleasant to the consumer, it had a MCA/PA ratio, the use of a low coagulant
softer texture, bitterness and a loss of yield. dose, the optimisation of various coagulation
The main differences in Roquefort cheese were

73
parameters keeping under control of ripening clotting agent, Food Sci. Technol, Campinas, 33(3):
step. 494‒499.
Devaraj, K. B., Gowda, L. R., Prakash, V. (2008). An
Plant proteases with milk-clotting activity have unusual thermostable aspartic protease from the latex
been developed to produce cheeses similar to of Ficus racemosa (L.). Phytochem 69:647–655.
those made with commercial rennet. Everett, D.W., & Auty, M.A.E. (2008). Cheese structure
The continuous improvements made to counter and current methods of analysis, International Dairy
their proteolytic activity should be based on a Journal 18(7):759‒773.
Feijoo-Siota, L., Villa, T. G. (2010). Native and
good selection of the type of milk for specific Biotechnologically Engineered Plant Proteases with
cheese variety made with these coagulants, the Industrial Applications, Food Bioprocess Technol.
use of different lactic bacteria cultures which DOI 10.1007/s11947-010-0431-4.
can inhibit the proteolysis. Fox, P. F., McSweeney, P. L., Cogan, T. M. and Guinee,
Good results have been observed for certain T. P. (2004). Cheese: Chemistry, Physics and
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