QUALITY AND PROPERTIES EVALUATION OF WHITE SOFT CHEESE MADE BY FREE OR IMMOBILIZED COAGULANT ENZYMES DURING STORAGE

CONCLUSION
Results of this experiment showed that the calf rennet , bovine and
chicken pepsins were successfully immobilized on paraffin / hony waxes
(1 :1 mixture) cloth strips. Yield and chemical composition of cheese
manufactured by immobilized enzymes was slightly lower than that of
free enzymes. Generally, small differences among all treatments,
contained either free or immobilized coagulants, were found. The amount
of clotting enzymes retained in free enzyme cheeses were relatively twice
as much as that of immobilized enzyme cheeses. Sensory evaluation
revealed, moreover, that immobilized enzymes cheeses were relatively
lower organoleptically than those of free enzyme cheeses. This study
demonstrated the beneficial effect of using this technique of
immobilization for cheese making to reduce the high cost of cheese
Egypt. J. of Appl. Sci., 36 (7-8) 2021 39
production by reusing the immobilized enzymes sevral times in clotting
numerous batches of milk. In addition this technique will compensate the
shortage of animal rennet in the local market.

QUALITY AND PROPERTIES EVALUATION OF
WHITE SOFT CHEESE MADE BY FREE OR
IMMOBILIZED COAGULANT
ENZYMES DURING
STORAGE
Ahmed, M. A.
Dairy Technology Department, Animal Production Research Institute,
Agriculture Research Center, Dokki, Giza, Egypt
ABSTRACT
The present study aimed to isolate calf rennet, bovine, and chicken
pepsin enzymes from different sources, purified and immobilized on
paraffin and honey waxes mixture (1:1, w/w) to use in white soft cheese
making as well as to compare it with the corresponding ones of free
coagulant enzymes. The enzymes were extracted from the stomach of three
different sources (big bovine stomach tissues, chicken gizzard and calf
abomasum tissues). White soft Cheeses were made from standardized cow's
milk (%3.64 fat) using the former coagulants either free or immobilized.
Cheese was stored at room temperature for 1, 30, 60, and 90 days.
Results indicated that cheese made using free enzymes had higher
values of yield, moisture, and titratable acidity and lower values of protein
compared with that made using immobilized enzymes. Cheese made using
free coagulant enzymes had, also, higher values of cheese ripening indices
and total volatile fatty acids. On the other hand cheeses made with bovine
and chicken enzymes had higher initial cheese ripening indices than the calf
rennet cheeses, during the storage period. All cheeses showed gradual loss
of yield, moisture, and pH values during storage while, protein, fat and
titratable acidity were increased in both immobilized or free enzymes
cheeses. Gradual increase in cheese ripening indices and total volatile free
fatty acids (TVFFAs) contents were noticed in all treatments, during 90 days
of ripening. Retained enzymes were found lower in cheese curds made
using immobilized enzymes and higher in whey of cheese made using free
ones. Organoleptic evaluation revealed that cheeses of calf rennet, either
free or immobilized, were slightly prefered organoleptically, all over the
storage period, than the other cheeses contained bovine or chicken pepsins.
Calf rennet cheeses were characterized by clean flavor, firm and good body
and texture. Bovine pepsin cheeses exhibit slightly bitter taste and to
somewhat friable body and texture at the end of the storage period.
Generally, no obvious differences were observed between free coagulant
enzymes and immobilized ones, through the storage period and all the
resultant cheeses were accetptable .This study demonstrated the beneficial
effect of using the immobilization technique for cheese making to reduce the
Egypt. J. of Appl. Sci., 36 (7-8) 2021 29-44
cost of production , by reusing the immobilized enzymes several times ,
compensation the shortage of animal coagulants (especially in calf rennet)
and maintenance the quality of the resultant soft cheese.
INTRODUCTION
Using calf rennet for milk coagulation is the most procedure used in
cheese making. However, the worldwide increase in cheese production
coupled with the reduced supply and increasing prices of calf rennet , has led
to search for alternative milk clotting enzymes as an appropriate rennet
substitute (Anusha et al., 2014). A part from this some religious factors
(Islam and Judaism) and others related to veganism of some consumers
have limited their use (Shah et al., 2014). Rennet calve was and still the
most widely used in cheese manufacturing. It is milk-clotting enzyme
preparation, which is extracted from the calf’s fourth stomach of claves
before its weaning (Guiama et al., 2010). Milk clotting properties of the
rennet are derived from chymosin as the main enzyme component (EC 3.
4.234) that often called rennet, which is considered the best coagulant factor
due to high specificity in breaking the bond between Phenyl alanine 105
and Methionine 106 in kappa-casein (Ahmed et al, 2009).
Cheese is traditionally made by batch system and current practice is
by using clotting soluble enzymes. The normal methods have three negative
aspects :1- The high cost of calf rennet 2- Using large quantities of enzymes,
which remain in the clotting cheese 3- The appearance of bitter taste when
the enzyme has highly proteolytic activity. The global increase in cheese
production and its consumption with low providing of rennet and
consequent increase in its prices have led to the attempt to fix the former
negative aspects of the coagulant enzymes as well as reusing them several
times when manufacturing cheeses (Gary and Johri, 1993).
Using of calf rennet as a milk clotting enzyme in the manufacture of
cheese has been predominant in the industry for centuries. Lately, a
worldwide shortage of this enzyme has been predicted due to the increase in
production and consumption of cheeses besides the simultaneous decrease in
the general availability of suckling calves' stomachs. Consequently, a great
deal of interest has been generated in research for other effective and
competitive substitutes for calf rennet . However, only a few other animal
proteases - such as pig , chicken and bovine pepsins -and some microbial
rennet preparations have been found suitable as a rennin substitute and are
presently used in cheese making (Green, 1977 ) .
Enzymes immobilization or stabilization is one of the most important
geometric strategies and defined as any capable technique of allowing reuse
or continuous use of biocatalyst. Based on the nature of the reaction that will
lead to the validation, the methods can be classified as physical and
chemical, for being inverse and irreversible and according to another widely
used classification system, three types of immobilization can be
30 Egypt. J. of Appl. Sci., 36 (7-8) 2021
distinguished: 1- linkage to the vector 2- retention (capsulation) 3- cross
linkage (Esposito, 2015). One of the most important properties of
immobilized enzyme is the simplicity, effectiveness and the cost that effect
on the used method , since the insoluble immobilized enzymes will allow
the continuity in the enzymatic processes, control the output configuration,
easily of separated enzyme , stop rapidly of resulting product and reuse it
and improve the stability of the enzyme (Cao et al, 2003). Recently, the
immobilization of milk clotting enzymes has indicated a renewed interest in
several potential new applications for the removal of the immobilized
enzymes after the thrombosis has been completed. In addition, enzyme
proteolytic effectiveness can also be controlled during the whole process,
when ripening of the cheese, give it stability and protection of the enzymes
from the external influences (Gary and Johri, 1993). Using the associated
enzymes in the cheese industry was not commercially viable, and until 1969
the immobilized enzymes were not used. Several studies have observed that
most immobilizing methods can be used in bind ripening enzymes to milk.
Taylor and Richardson (1978) was able to manufacture cheese
continuously by binding the coagulant product of DOI of( the genus Mucor)
with glass balls. It was found about 70- 90% of the stabilized enzyme’s
activity doesn’t alter through cheese industry because of the ability of
enzymes to protect themselves (Holmes et al, 1977). The main of that
research is to install the rennet (from the fungal organism Mucor miehei),
which is widely used in the manufacture of cheeses , on paraffin wax and
honey wax to compare it with the properties of free rennet , in order to
determine the optimal conditions. The paraffin wax has been used as a
supporting material due to its economical and technically safe on the
industrial and laboratory scale , improving the stability of the rennet and
reuse it after being immobilized after coagulation.
Several applications were utilized this technique of enzymes
immobilization in the field of dairy products such as : immobilizing different
alkaline proteases on anion exchange resin with glutar ( Ohmiya etal.,
1978), immobilizing chymosin on paraffin wax (Shindo etal.,1984),
immobilizarion of pepsin on paraffin wax ( Savangikar and Joshi,1978) ,
immobilizarion of calf rennet and Mucor miehei rennet in manufacturing
Ras cheese (Saad,1986 ) , immobilizarion of free and microbial rennet in
Domiati cheese (Mansour etal, 2011) .
Therefore, the main objective of the present study was to extract,
purify and use of milk clotting enzymes of bovine, chicken pepsin and calf
rennet either in free form or immobilized on mixture of paraffin & honey
waxes ( 1: 1 ,w/w ) , to study its effect on chemical, ripening indicies and
sensory properties of the resultant white soft cheese , during 90 days of
storage period.
Egypt. J. of Appl. Sci., 36 (7-8) 2021 31
MATERIALS AND METHODS
Materials:
Milk supply
Fresh cow's milk was obtained from the herd belonged to Sidis
Experimental Station, Animal Production Research Institute, Agriculture
Research Center, Egypt .
Ammonium sulphate, sodium acetate, and other chemicals were
purchased from Gomhoria company, Beni Suef Governorate, Egypt.
Salt , paraffin and honey waxes and ordinary white cotton cloth : tailored
to strips ( 5× 1.5 cm ) and sheets 24.5 × 17 cm were used for enzymes
immobilization from the local market .
Preparation of crude coagulant enzyme extracts :
1. Rennet calf extract : was prepared according to the method of Ahmed
et al. (2013), with some modification.
2. Bovine Pepsin extract: The big bovine stomach tissues were obtained
from butcher in Beni Sweef, The abomasum's were obtained from cow of
3 years age . It was prepared by the methods of Fahmi and Amer (1962)
and Fahmi et al., (1979).
3. Chicken pepsin extract: was prepared according to the method
mentioned by Ali and Salim,(1983).
All enzyme extracts were kept in the refrigerator at 5 ± 1°C until used.
Immobilization of bovine pepsin, chicken pepsin and calf rennet
enzymes:
Immobolization was done according to Savangikar and Joshi,
(1978) methods .
White soft cheese making using free and Immobilized enzymes.:
White soft cheese was made according to the method adopted by
Metwalli et al. (1982). Cow's milk (3.60 fat), was salted at a level of 8%,
heat treated at 73°C for 5 sec., cooled to about 42°C and divided into six
equal parts as follows :
First part (F1): contained free calf rennet (control)
Second part (F2): contained free bovine pepsin
Third part (F3): contained free chicken pepsin .
The other 3 parts were carried out as follows:
Fourth part (Z1): contained immobilized calf rennet.
Fifth part (Z2): contained immobilized bovine pepsin.
Six part (Z3): contained immobilized chicken pepsin.
Milk of all treatments were incubated at 40ºC, draining the whey,
cutting the curds into cubes and pickled in 10% salt solution using the own
drained whey of each treatment. Cheese samples were taken when fresh
and after 30, 60 and 90 days of storage for analysis.
32 Egypt. J. of Appl. Sci., 36 (7-8) 2021
Analytical methods:
 Titratable acidity, moisture, total solids, fat and total protein contents of
milk and cheese were determined according to the methods described by
Ling (1963).
 pH values of milk and cheese were measured using a combined calomel
glass electrode pH-meter model HANNA instruments H 18424.
 Cheeses Yield: was calculated as a weight of cheese divided by the
weight of milk and expressed as a percentage value (Nelson et al.,
2004).
 Proteolysis: was carried out by the determination of: (1) Water soluble
nitrogen; Non protein nitrogen contents in cheese , according to Kuchroo
and Fox (1982). Phosphotungstic acid (5%) soluble nitrogen was
determined according to Jarrett, et al. (1982).
 Tota volatile fatty acids in cheese was determined according to
( Kosikowski,1987). and expressed as ml 0.1 N NaoH/100 g cheese .
 Residual rennet determination: was determined according to Dulley
(1974) .
 Organoleptic properties:
Cheese samples were evaluated according to the method of Pappas et
al. (1996) when fresh and after 30, 60 and 90 days of storage at room
temperature. An overall score points out of (100 points) was given for
flavour (50 points), body & texture (35 points) , appearance (10points) and
colour (5 points) by a panel test consisted of twelve. persons, mainly from
staff members.
RESULTS AND DISCUSSION
Chemical composition of raw cows milk
The chemical composition and pH value of raw cows milk was
illustrated in Table (1).
Table 1. Chemical composition of cows milk used in white soft
cheese making.
Ingredients Parameters
Protein 3.45 %
Fat 3.60%
Total solids 12.30%
Non-fat solids 8.75%
Acidity 0.16%
PH value 6.6
Cheese Yield
-Casein fraction of milk protein is the dominant factor affecting curd
firmness, syneresis rate, moisture retention, and ultimately affecting
cheese quality and yield (Lawrence, 1993).
- Data in Table (2) revealed , generally, that the yield of free and
immobilized calf rennet cheeses were found the highest during storage
Egypt. J. of Appl. Sci., 36 (7-8) 2021 33
period than the other treatments. Saad, (1986) found that the yield of
Ras cheeses manufactured by immobilized enzymes was slightly
lower than that of free enzymes .The yield of fresh cheese made using
free calf rennet (control) was %23.2 decreased to 19.6% after 90 days
of storage, while those made using immobilized calf rennet was
21.8.%, decreased to 19.1 % .
- Chicken pepsin cheeses recorded yield values slightly more than
bovine pepsin cheeses .
- Yield of all free enzymes of fresh cheeses were ranged between 21.4
to 23.2 % decreased to 18.3 to 19.6 after 90 days of ripening . The
corresponding values of immobilized enzymes in fresh cheeses were
19.1 to 21.8 % decreased to 17.4 . The main reason for decreasing the
yield in pepsin cheese treatments either contain free or immobolized
enzymes was the weak coagulation activity of these enzymes as well
as its high rate of proteolysis, during storage , which reflect on the
total solids contents and subsequently on the yield .
- These observations were supported by (Bruno et al., 2010) who said
that the speed of hydrolysis of caseins influences in yield, and slow
degradation of α s-and β-caseins is guarantee production of a firm
curd, which is occurs when chymosin is used. Most rennet substitutes
are more proteolytic than calf rennet and cause diminished yields .
Physico-chemical composition
Result present in Table (2) illustrate the Physico-chemical
composition of white soft cheese during storage .
- Moisture was found the lowest in calf rennet cheeses (F1and Z1) than in
the other treatments (F2 ,Z2 , F3 and Z3) . Moisture contents in free
enzymes of fresh cheeses ranged between 63.40 - 66.70 % decreased to
60.90 – 61.90 % after 90 days of storage period while the similar ones of
the immobilized enzymes were 62.90 -64.60 % in fresh cheeses
decreased to 60.50 – 61.0 % .
- protein contents were higher in calf rennet cheeses than the other
treatments.It increased gradually during storage and decreased in all
treatments , at the end of the storage period owing to the action of
proteolytic microorganisms , and the higher rate of increase in the cheese
ripening indices ( soluble and non-protein nitrogen) . protein contents
were 14.40 -15.50 in fresh cheeses contained free enzymes decreased to
14.70 – 15.50 % , at the end of the storage period whereas those of
immobilized cheeses were 14.70 -15.80 % in fresh cheeses deceased
latterly to 15.40 – 15.60 % .
- Fat contents of all treatments ( free or immobilized ) were found
approximately near during storage period with the irrespective of chicken
34 Egypt. J. of Appl. Sci., 36 (7-8) 2021
pepsin treatments which were slightly higher . It ranged between 19.20 –
20.30 % in free enzymes cheeses and 18.80 – 20.20 % in fresh
immobilized ones . Fat contents were decreased in all treatments during
storage as a result of the action of the lipolytic bacteria .
- Titratable acidity (TA) was found low in free or immobilized calf rennet
fresh cheeses ( F1 and Z1 treatments) than the rest treatments , along the
storage period , because most of rennet substitutes ( bovine and chicken
pepsins ) are more proteolytic than calf rennet and resulting in higher
amounts of organic acids and other minor proteins used by the lactic acid
bacteria .
- TA in fresh cheeses contained free enzymes (F1 , F2 and F3) ranged
between 0.18 -0.20 % increased to 1.22 – 1.32 % after 90 days of storage
, while the immobilized ones were 0.16 - 0.19 % in fresh cheeses
increased to 1.16 – 1.21 % .
- pH values of all treatments behaved reverse trend to TA all over the
storage period.
Table 2: Physico-chemical analysis of white soft cheese made by
using free or immobilized coagulant enzymes , during
storage period.
Treatments
storage
period
(days)
parameters (%)
Yield
%
Moisture Protein Fat pH Acidity %
F1
(control)
Fresh 23.2 63.4 15.6 19.3 6.45 0.18
30 21.8 62.2 15.8 20.5 6.28 0.26
60 20.4 61.8 16.0 21.2 5.78 0.93
90 19.6 60.9 15.5 21.6 5.21 1.22
Z1
Fresh 21.8 62.9 15.8 19.0 6.55 0.16
30 20.5 61.8 16.1 20.2 6.37 0.25
60 19.2 61.4 16.7 20.4 5.83 0.87
90 19.1 60.5 15.4 21.2 5.38 1.16
F2
Fresh 21.4 66.7 15.2 19.2 6.36 0.19
30 18.7 64.5 15.6 20.2 6.19 0.27
60 17.3 62.0 15.8 20.9 5.38 0.96
90 16.6 61.4 15.6 21.3 5.24 1.29
Z2
Fresh 19.1 65.2 14.8 18.8 6.44 0.18
30 18.4 63.1 15.2 20.0 6.27 0.26
60 16.8 61.6 15.9 20.2 5.56 0.89
90 16.1 61.2 15.6 21.0 5.29 1.18
F3
Fresh 21.9 65.2 14.4 19.2 6.41 0.20
30 20.4 63.0 14.7 20.3 6.19 0.28
60 19.1 62.8 15.5 21.0 5.49 0.99
90 18.3 61.9 15.4 21.4 5.32 1.32
Z3
Fresh 20.4 64.6 14.7 18.9 6.53 0.19
30 19.3 62.3 15.0 20.1 6.13 0.27
60 18.2 61.8 16.1 20.4 5.35 0.91
90 17.4 61.0 15.4 21.2 5.35 1.21
F1 : Free calf rennet,control Z1 : Immobilized calf Rennet .
F2 : Free bovine pepsin Z2 : Immobilized bovine pepsin .
F3 : Free chicken pepsin Z3 : Immobilized chicken pepsin .
Egypt. J. of Appl. Sci., 36 (7-8) 2021 35
Proteolysis :-
The proteolytic activity in cheese can be estimated by the release
of a small nitrogen-containing fractions (soluble nitrogen/total nitrogen
(SN/TN) , phospho tungstic acid - soluble nitrogen/total nitrogen (PTA -
SN/TN) and non-protein nitrogen /total nitrogen (NPN/ TN )) from the
protein (Visser, 1977).
- Results in (Table 3) showed an increase in the percentage of these
ripening indices ( SN/TN, PTA - SN/TN and NPN/ TN ) in the free
enzymes of calf rennet ( treatment, F1 ) , bovine and chicken pepsin(
treatments F2 and F3 ) , compared to the immobilized ones ( Z1 ,Z2
and Z3 ) , during the storage period .
- The initial values of these fractions in free calf rennet cheeses were
lower than that of free chicken pepsin and bovine pepsin cheeses ,
respectively. Values of SN / TN were 6.54 ,7.34 and 8.12 % in free
enzymes cheeses , while the same values were 5.34, 6.91 and 7.23 %
in the immobilized ones .
- At the end of the storage period these values were increased to 23.68,
24.62 and 25.48 % respectively and to 22.41, 23.88 and 23.96 %, in
the immobilized ones .The main reason for increasing these values in
bovine and chicken cheeses was the higher rate of proteolysis took
placed by pepsin enzymes than the calf rennet enzymes.
- Results revealed , moreover , that the trend in the changes in NPN/TN
and PTA-SN/TN in cheeses was found the same as in the SN / TN.
- Slight differences between values of free and immobilized enzymes
were observed and bovine pepsin recorded higher values than chicken
pepsin , along the storage period .
- Several studies were done to compare calf rennet with pepsin enzymes
in the field of cheese production e.g Gordin and Rosenthal (1978)
who used calf rennet and chicken pepsin in the manufacture of
Emmental-and Kashkaval cheeses .
Lipolysis:
- Data in Table (3) show, also, that the amount of TVFFA were
increased gradually in all cheeses either contained free or immobilized
enzymes, throughout the storage period.
- Cheeses contained free enzymes calf rennet, bovine and chicken
pepsin had higher values of TVFFA compared with the immobilized
ones , during storage .
- The TVFFA contents in free enzymes fresh cheeses were 6.8 ,7.1 and
7.4 while it were 5.4, 6.4 and 6.6 in immobilized ones , respectively.
36 Egypt. J. of Appl. Sci., 36 (7-8) 2021
These values increased to 18.3 , 19.6 and 20.1 and 15.7, 16.2 and 16.8
, at the end of the storage period , respectively.
- Also, data cleared that the TVFFA were slightly higher in cheese made
with bovine pepsin than the other treatments.
Table 3: Changes in total , soluble nitrogen and total volatile fatty
acids (TVFFA) contents of white soft cheese made with free
or immobilized coagulants , during storage period.
Treatments
Ripening
period
(days)
Parameters (%)
SN / TN NPN/TN PTA-SN/TN TVFFA
F1
(control)
Fresh 6.54 5.22 4.23 6.8
30 18.73 7.48 5.77 9.4
60 19.84 10.62 8.88 16.2
90 23.68 13.55 11.32 18.3
Z1
Fresh 5.34 4.36 4.01 5.4
30 17.28 6.92 5.32 8.2
60 18.77 9.64 7.96 14.3
90 22.41 12.11 10.42 15.7
F2
Fresh 8.12 6.72 4.76 7.4
30 20.44 8.33 6.13 10.3
60 21.86 11.13 9.27 18.6
90 25.48 14.04 12.53 20.1
Z2
Fresh 7.23 5.37 4.41 6.6
30 19.04 7.24 5.73 9.1
60 20.38 9.83 8.52 15.5
90 23.96 12.98 11.72 16.8
F3
Fresh 7.34 6.40 4.43 7.1
30 19.88 7.74 5.96 9.8
60 20.59 10.65 8.99 16.4
90 24.62 13.27 12.04 19.6
Z3
Fresh 6.91 5.12 4.28 6.5
30 18.68 6.82 5.74 8.7
60 19.87 9.54 8.28 14.9
90 23.88 12.38 11.47 16.2
See abbreviations under Table (2) .
Retention of free or immobilized coagulant enzymes in cheese curds
and whey :
- Coagulant enzymes plays important role in the ripening process of
the cheese by promoting hydrolysis of casein into large peptides. The
concentration of the coagulant enzymes in cheese depends on some
technological factors such as pre-treatment of milk, cooking
temperature, variety of cheese, type of coagulant, pH value of the
curd at cutting , pH at whey drainage, and moisture level of the final
cheese (Hurley et al., 1999).
- Various techniques have been used to eliminate the activity of
residual coagulants in cheese in order to study its role in cheese
ripening (Fox and McSweeney, 1996).
Egypt. J. of Appl. Sci., 36 (7-8) 2021 37
- Results in Table (4) showed the residual coagulant enzymes
retention in the curd and whey of the resultant cheeses contained
free or immobilized coagulant enzymes and expressed as
coagulation time (CT) per seconds. Results indicated that the rate of
retention of enzymes in the curd (CT) was proportional with the
amount of free or immobilized coagulant enzymes remained in the
curd or whey.
- Coagulation time were found higher in cheese curds made using
immobilized enzymes compared with those made using free ones .
Also it was lower in cheese curd made using free or immobilized
bovine pepsin than both chicken pepsin or calf rennet.
- The average values of CT for free calf rennet, bovine and chicken
pepsin cheeses were: 563, 490, and 523 sec., respectively. While the
average values of CT for immobilized ones were 832, 734 and 787
sec., respectively.
- The CT was higher in calf rennet whey than the other two treatments
cheeses. The average values of CT in the whey of free calf rennet,
bovine and chicken pepsin were: 287, 235 and 251 sec., while in
immobilized ones were 458, 384 and 373 sec. respectively.
Table (4): Effect of free or immobilized coagulant enzymes on the
residual coagulants remained in curd and whey , after
drainage stage.
Treatments Enzyme state
Coagulation time (sec.)
Whey curd
Calf rennet
Free (F1) 287 563
Immobilized (Z1) 458 832
Bovine pepsin
Free (F2) 235 490
Immobilized (Z2) 373 734
Chicken pepsin
Free (F3) 251 523
Immobilized (Z3) 384 787
See abbreviations under Table (2) .
Sensory evaluation of cheese
- Sensory evaluation of white soft cheese is a valuable criterion for
determining cheese quality and acceptability. The body, texture and
flavour are affected considerably by many factors such as type of
milk, pre-treatment of milk, season of milk production, addition of
starter to milk, type of coagulant used, percentage of salt in both the
cheese curd and the pickling solution , microflora of the cheese milk,
additives to the cheese milk to accelerate the cheese ripening and
storage temperature during ripening (Pappas et al., 1996). Chymosin
is the major enzyme of calf rennet, and it has been widely used in the
38 Egypt. J. of Appl. Sci., 36 (7-8) 2021
dairy industry to produce a stable curd with good flavour due to its
high specificity for the Phenyl alanine105-Methionine106 bond of kcasein
(Rao et al., 1998).
- Sensory evaluation revealed that cheeses of calf rennet, either free or
immobilized, were slightly prefered organoleptically, all over the
storage period, than the other cheeses contained bovine or chicken
pepsin .Calf rennet cheeses were characterized by clean flavor , firm
and good body and texture. Mansour et al. (2011) observed that
Domiati cheese made with free microbial rennet gained higher scores
than that using immobilized microbial rennet.
- Bovine pepsin cheeses exhibit slightly bitter taste and to somewhat
friable body and texture at the end of the storage period , owing ,
presumably, to the higher rate of proteolysis occurred by the pepsin
enzymes .
- Chicken pepsin cheeses were found slightly better than bovine pepsin
cheeses, during 90 days of storage.
- No obvious differences were observed between free coagulant
enzymes and immobilized ones, through the storage period. These
observations were in line with Shindo et al. (1984), who found no
great differences were observed between the quality of cheese
produced with immobilized enzyme and the cheese produced by the
free enzyme.
- Generally, all the resultant cheeses were acceptable with the
exception of bovine pepsin cheeses which were slightly inferior . It is
known that sensory properties of flavour, colour and texture are key
factors for final product acceptance (Abdul-Rahman, 2013).
CONCLUSION
Results of this experiment showed that the calf rennet , bovine and
chicken pepsins were successfully immobilized on paraffin / hony waxes
(1 :1 mixture) cloth strips. Yield and chemical composition of cheese
manufactured by immobilized enzymes was slightly lower than that of
free enzymes. Generally, small differences among all treatments,
contained either free or immobilized coagulants, were found. The amount
of clotting enzymes retained in free enzyme cheeses were relatively twice
as much as that of immobilized enzyme cheeses. Sensory evaluation
revealed, moreover, that immobilized enzymes cheeses were relatively
lower organoleptically than those of free enzyme cheeses. This study
demonstrated the beneficial effect of using this technique of
immobilization for cheese making to reduce the high cost of cheese
Egypt. J. of Appl. Sci., 36 (7-8) 2021 39
production by reusing the immobilized enzymes sevral times in clotting
numerous batches of milk. In addition this technique will compensate the
shortage of animal rennet in the local market.
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تقييم جودة وصفات الجبن الابيض الطري المصنع بواسطة الأنزيمات المجبنة
الحرة والمثبتة أثناء التخزين
محمد علي أحمد
مرکز البحوث الز ا رعية معيد بحوث الانتاج الحيواني قسم بحوث تکنولوجيا الالبان
أجريت ىذه الد ا رسة بغرض استخلاص کل من منفحة العجول الرضيعة وانزيم الببسين
البقري وببسين الدجاج باستخدام محاليل الاستخلاص المناسبة لکل أنزيم . وتم ترسيب
02-02 , %02- الأنزيمات باستخدام کبريتات الأمونيوم بنسب إشباع ت ا روحت بين 02
02-02 % عمى التوالي, وتم تثبيتيم عمى أشرطة قماش من القطن بواسطة خميط من , %
شمع الب ا رفين وشمع عسل النحل. وقد درست إمکانية استخدام ىذه الانزيمات المثبتة في تصنيع
الجبن الابيض الطري وذلک لتقميل کمية الانزيمات المجبنة ) من الببسين ( التي تتبقى في
الجبن والتي قد تؤدي الى ظيور المذاق المر , ومقارنتيا بمثيمتيا من الانزيمات الحرةعمى النحو
التالي :
1. تم الاستخلاص والتنقية ثم التجييز من ترکيز وتجفيف لمثلاثة انواع من الأنزيمات المجبنة
منفحة العجول الرضيعة والببسين البقري وببسين الدواجن وحفظت لحين الاستعمال.
0. تم تجييز اشرطة من الشاش ووضعت فوق لوح زجاجي وتم خمط الانزيمات المجبنة مع
الشمع المسال ) خميط من شمع الب ا رفين وشمع العسل 1:1 ( بنسبة ) 1 أنزيم : 4 شمع(
م وتترک عمى درجة ح ا ررة الغرفة º ثم الصب فوق اشرطة القماش عمى درجة ح ا ررة 02
) الأصمي لو ) 0,0 pH ليتصمب الشمع, واستخداميما في تجبين المبن عمى ال
0. تم تصنيع عدد 0 معاملات من الجبن الابيض الطري بطريقة متولى وأخرون ) 1020 م( مع
بعض التعديلات حيث تصنع معاممتان من کل نوع من الانزيمات المجبنة التي تم
تجييزىا احداىما بالأنزيم المثبت بخميط من شمع الب ا رفين وشمع العسل عمى أشرطة القماش
والمعاممة الاخرى بالأنزيم الحر, مع عمل ثلاث مکر ا رت لکل معاممة .
م ( ناتج کل معاممة عمي حده º 02- 4. تم أخذ عينات من الجبن المخزن في الشرش ) 02
عمى فت ا رت 1 يوم , شير , شيرين , وثلاث شيور . حيث أوضحت نتائج تحميل الجبن
المتحصل عمييا ما يمى :
أن التصافي في الجبن المصنع بالأنزيمات المثبتة کا ن أقل قميلا منو في تمک المصنعة 
بالأنزيمات الحرة, وانخفض تدريجيا مع تقدم مدة التخزين فى جميع المعاملات. وتلاحظ
ارتفاع التصافي مع منفحة العجول الرضيعة ثم ببسين الدواجن وأخي ا ر الببسين البقري عمى
التوالي.
کانت نسبة الرطوبة والحموضة اعمى نسبيا فى حين کانت نسبة البروتين منخفضة نسبيا 
فى الجبن المصنع بالأنزيمات الحرة عن تمک المصنعة بالأنزيمات المثبتة. حدث نقص
خلال فترة التسوية بينما لوحظ زيادة في کل من pH لکل من الرطوبة والتصافي وکذلک ال
الحموضة ونسبة البروتين والدىن في الجبن الناتج مع زيادة فترة التخزين .
Egypt. J. of Appl. Sci., 36 (7-8) 2021 43
ولقد أوضحت نتائج قياسات ثوابت التسوية لمجبن أنو لا توجد فروق کبيرة , حيث تبين أن 
الانزيمات المثبتة عمى ش ا رئط القماش تحمل البروتين وتحرر نتروجين ذائب بالماء ونتروجين
لا بروتيني ونتروجين ذائب في حامض الفوسفوتنجستک وذلک بدرجة أقل من الصورة الحرة
في حالة الثلاث انواع من الأنزيمات المجبنة , وکانت اکبر مع الببسين البقري عن ببسين
الدواجن ثم عن منفحة العجول الرضيعة عمى التوالي. کذلک الاحماض الدىنية الطيارة
سمکت نفس الاتجاه .
وجد ان نسبة الانزيمات المتبقية فى خثره وشرش جبن الانزيمات الحره کانت اعمى من 
نظيرتيا المحتويو عمى الانزيمات المثبتة طوال مده التخزين .
اظيرت نتائج التحکيم الحسى ان عينات الجبن المصنعة بالأنزيمات المثبتة حصمت عمى 
درجات متقاربة مع تمک المصنعة بالأنزيمات الحرة. وکانت درجات التحکيم مرتفعة نسبيا
مع منفحة العجول الرضيعة ثم ببسين الدواجن والببسين البقري عمى التوالي.
بعد نجاح استعمال منفحة العجول الرضيعة والببسين البقري وببسين الدواجن المثبتة عمى 
الشمع في صناعة الجبن الابيض الطري , أظيرت النتائج ان ترکيب الجبن المنتج ونتائج
التقييم الحسي مقاربة لمجبن المنتج باستعمال منفحة العجول الرضيعة , وىذا دليل عمى
کفاءة الانزيمات المثبتة في صناعة الجبن الطري و إمکانية إحلاليا محل منفحة العجول
الرضيعة.
اظيرت ىذه الد ا رسو امکانيو استخدام ىذه التقنيو الحديثو فى تحميل الانزيمات المجبنو 
بنجاح فى صناعو الجبن لانيا تخفض تکمفو الانتاج وکذلک امکانيو استخداميا م ا رت عديده
Savangikar and ( فى صناعو الجبن اى اکثر من 12 مره فى تجبن المبن ( 1978
مع عدم تاثيرىا عمى جوده الجبن بالاضافو الى Shindo et al., (1984) and Joshi
تعويضيا النقص فى کميات المنفحو الحيوانيو فى السوق المحمى
44 Egypt. J. of Appl. Sci., 36 (7-8) 2021