9/7/13 DT-5: Lesson 31.
ACCELERATED RIPENING
CHEESE TECHNOLOGY
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B. Tech. (Dairy Technology) ► DT-5 ► Resources ► Lesson 31. ACCELERATED RIPENING
Module 15. Accelerated ripening of cheese
Lesson 31
ACCELERATED RIPENING
31.1 Introduction
31.2 Accelerated Ripening of Cheese
31.3 Methods of Accelerated Ripening
31.3.1 Use of elevated temperatures of ripening
31.3.2 Addition of exogenous enzymes
[Link] Coagulant
[Link] Plasmin
[Link] Other proteinases
[Link] Exogenous Lipases
[Link] ß-Galactosidase (Lactase)
31.3.3 Use of adjunct cultures
31.3.4 Modification of starter bacteria/Starter cultures
[Link] Genetically engineered starters
[Link] Stimulation of starter cells
[Link] Modified starter cultures
[Link] Heat and freeze shock treated cells
[Link] Lysozyme treated cells
[Link] Mutant starter cultures
31.3.5 Use of high pressure treatment
31.1 Introduction
The ripening of cheese is a complex process of concerted biochemical changes, during
which a bland curd is converted into a mature cheese having the flavor, texture and aroma
characteristics of the intended variety. Ripening is an expensive and time-consuming
process, depending on the variety, e.g. Cheddar cheese is typically ripened for 6-9 months
while Parmesan is usually ripened for two years. Extended ripening period involves
increased cost due to refrigerated storage, space, labour apart from considerable loss in
weight and higher capital cost. Acceleration of cheese ripening can also be a means of
increasing the production of cheese in developing countries where investment in storage
facility can be a limiting factor.
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31.2 Accelerated Ripening of Cheese
Cheese ripening is essentially a process which involves metabolism of key constituents of
milk, i.e. lactose, proteins and fat. Most of the lactose is lost in whey but the residual
lactose is quickly metabolized to lactate by combined action of starter bacteria and native
bacteria of milk. This lactate serves as an important precursor compound for certain
reactions which occur during ripening. Citric acid fermentation also plays a role in cheese
ripening. Milk contains very low level of citrate, most of which is lost in whey. However,
the residual citrate in the curd is acted upon by citrate fermenting microorganisms like
Leuconostoc sp. to produce important flavor compounds like diacetyl, acetoin, etc.
Lipolysis of milk fat is vital for production of important flavor compounds. Milk fat is
hydrolysed during ripening by the action of enzymes. These enzymes may originate from
milk like lipase or they may come through coagulant like rennin or they may be produced
by cheese microflora. Lipolysis converts milk fat into free fatty acids and glycerol. Free
fatty acids, particularly short chain fatty acids, contribute directly to cheese flavor and they
also act as precursors for various catabolic reactions including esterification of
hydroxyacids to form lactones, formation of thioesters by reaction with sulphydryl
compounds and β-oxidation of fatty acids to alkan-2-ones. Other principal metabolism
during cheese ripening is proteolysis, wherein protein is broken down to peptides by the
action of residual coagulant and the principal indigenous proteinase in milk, plasmin. These
peptides are then hydrolyzed by enzymes derived by starter and non-starter microflora of
the cheese. The production of small peptides and amino acids is caused by the action of
microbial proteinases and peptidases respectively. The final products of proteolysis are
amino acids, the concentration of which depends on the cheese variety.
All these biochemical changes which occur during ripening are slow and take about 6
months to years, depending on the variety of cheese. For example, Cheddar cheese is
typically ripened for 6-9 months while Parmesan is usually ripened for two years. Such
extended period of ripening involves capital investment in form of product and utilities like
storage rooms, refrigeration etc. Therefore, various technological interventions are applied
to shorten ripening period. This process of reducing normal ripening period is called as
accelerated ripening.
31.3 Methods of Accelerated Ripening
Ripening is a slow and consequently, expensive process that is not fully predictable or
controllable. Therefore, there are economic and possibly technological incentives to
accelerate ripening. The principal methods by which this may be achieved are: an elevated
ripening temperature, exogenous enzymes, modified starters and adjunct cultures. At least
some of the methods used to accelerate ripening may also be used to modify flavor and in
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effect to create new varieties/ variants. Slow flavor development and low flavor intensity
are major problems with reduced-fat cheese and the methods used to accelerate ripening in
general are applicable to low-fat cheese also. Several promising developments have
emerged for rapid flavor development in Cheddar cheese and other varieties.
In this regard, various approaches have been used to accelerate cheese ripening process
which include:
• Use of elevated temperatures of ripening
• Addition of exogenous enzymes
• Use of adjunct cultures
• Genetic modification of starter bacteria
• Use of high pressure treatment
31.3.1 Use of elevated temperatures of ripening
Cheese is ripened at uniform temperature and humidity, depending on the variety of
cheese. Ripening temperatures of some of the varieties of cheeses are as follows:
Emmental, 22-24°C (for part of ripening, i.e., the critical ‘hot room’ period); mold and
smear-ripened cheeses, 12-15°C; Dutch varieties, 12-14°C; and Cheddar, 6-8°C (the
ripening temperature for Cheddar is exceptionally low). The above temperatures are the
maximum in the profiles and are usually maintained for 4-6 weeks to induce the growth of
a desired secondary microflora. Thereafter, the cheese is transferred to a much lower
temperature (e.g. 4°C). Cheddar is an exception, since it is normally kept at 6-8°C
throughout the ripening process. It can be observed from the ripening temperatures of
most of the varieties that in no case it is more than 20-25°C as keeping cheese above this
temperature causes texture to be too soft and the cheese deforms readily. It may also
cause exudation of fat and excessive loss of moisture. Thus, the scope for accelerating the
ripening of most cheese varieties by increasing the ripening temperature is quite limited
except for Cheddar cheese which is ripened at low temperature of 6-8°C. This is again
limited to cheese which is made from good quality pasteurized milk and is
microbiologically safe. This approach is the simplest and cheapest method for accelerating
ripening as no additional costs are involved rather there may be savings due to reduced
refrigeration costs.
Several studies have been carried out to ripen Cheddar cheese at elevated temperatures.
For example, the duration of ripening can be reduced by 50% by increasing the ripening
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temperature from 6°C to 13°C, without adverse effects. The highest temperature that can
be used continuously is about 16°C, although 20°C could be used for a short period; 12-
14°C is probably optimal. Ripening can be accelerated or delayed by raising or reducing
the temperature at any stage during the process.
31.3.2 Addition of exogenous enzymes
Cheese ripening is essentially an enzymatic process and hence it should be possible to
accelerate ripening by augmenting the activity of key enzymes. However, addition of single
enzyme, which accelerates one particular reaction, is unlikely to produce a balanced flavor.
Hence, the need for addition of mixture of enzymes in proper ratio has been advocated by
several research workers. The addition of combinations of various fungal proteases and
lipases to Cheddar cheese has been reported to reduce ripening time by 50%. Lipase in
combination with proteinase gave good cheese flavor with low levels of bitterness. A
lipase/proteinase preparation derived from Aspergillus oryzae released C6-C10 fatty acids to
produce typical Cheddar cheese flavor. To achieve more intense and balanced flavor in
buffalo milk Cheddar cheese, use of mixture 0.001% lipase and 0.01% protease has been
advocated. A number of options are available, ranging from the quite conservative to the
more exotic.
[Link] Coagulant
The proteinases in the coagulant is principally responsible for primary proteolysis in most
cheese varieties and it might, therefore, be expected that ripening could be accelerated by
increasing the level or activity of rennet in the cheese curd. Although, it is suggested that
chymosin is the limiting proteolytic agent in the initial production of amino nitrogen in
cheese, several studies have shown that increasing the level of rennet in cheese curd
(achieved by various means) does not accelerate ripening and in fact probably causes
bitterness. Chymosin produces only relatively large oligopeptides which lack a typical
cheese-like flavor and may be bitter. Chymosin-produced peptides are hydrolysed by
bacterial (starter and non-starter) proteinases and peptidases and hence it would seem that
increased chymosin activity should be coupled with increased starter proteinase and
peptidase activities in order to accelerate ripening.
Chymosin has very little activity on ß-casein in cheese, probably because the principal
chymosin-susceptible bond in ß-casein, Leu192-Tyr193, is in the hydrophobic C-terminal
region of the molecule which appears to interact hydrophobically in cheese, rendering this
bond inaccessible. However, Cryphonectria parasitica proteinase preferentially hydrolyses
ß-casein in cheese (possibly because its preferred cleavage sites are in the hydrophilic N-
terminal region) without causing flavor defects. Rennet containing chymosin and
Cryphonectria parasitica proteinase might be useful for accelerating ripening.
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[Link] Plasmin
Plasmin contributes to proteolysis in cheese, especially in high-cooked varieties in which
chymosin is extensively or totally inactivated. Plasmin is associated with the casein micelles
in milk, which can bind at least 10 times the amount of plasmin normally present, and is
totally and uniformly incorporated into cheese curd, thus overcoming one of the major
problems encountered with the use of exogenous proteinases to accelerate cheese ripening.
Addition of exogenous plasmin to cheese milk accelerates the ripening of cheese made
therefrom, without off-flavor development.
[Link] Other proteinases
The possibility of accelerating ripening through the use of exogenous (non-rennet)
proteinases has attracted considerable attention over the past 20 years. The principal
problems associated with this approach are ensuring uniform distribution of the enzyme in
the curd and the prohibition of exogenous enzymes in many countries. The earliest reports
on the use of exogenous enzymes to accelerate the ripening of Cheddar cheese appear to
be those of Kosikowski and collaborators who investigated various combinations of
commercially available acid and neutral proteinases, lipases, decarboxylases and ß-
galactosidase. Acid proteinases caused pronounced bitterness but the addition of certain
neutral proteinases and peptidases with the salt gave a marked increase in flavor after one
month at 20°C but an overripe, burnt flavor and free fluid were evident after one month at
32°C. Incorporation of the enzyme-treated cheese in processed cheese gave a marked
increase in Cheddar flavor at 10% addition and a very sharp flavor at 20%. Good quality
medium sharp Cheddar could be produced in 3 months at 10°C through the addition of
combinations of selected proteinases and lipases.
With the exception of rennet and plasmin (which adsorbs on casein micelles), the
incorporation and uniform distribution of exogenous proteinases throughout the cheese
matrix poses several problems:
Proteinases are usually water-soluble, and when added to cheese milk, most of the
enzyme is lost in the whey, which increases cost
Enzyme-contaminated whey must be heat-treated if the whey proteins are recovered
for use as functional proteins. The choice of enzyme is limited to those that are
inactivated at temperatures below those that cause thermal denaturation of whey
proteins
The amount of Neutrase that should be added to milk to ensure a sufficient level of
enzyme in the curd reduces the rennet coagulation time, yields a soft curd in which at
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least 20% of the ß-casein is hydrolyzed at pressing and reduces cheese yield.
[Link] Exogenous Lipases
Lipolysis is a major contributor, directly or indirectly, to flavor development in strong-
flavored cheeses, e.g. hard Italian, Blue varieties, Feta. Rennet paste or crude preparations
of pre-gastric esterase (PGE) are normally used in the production of Italian cheeses.
Rhizomucor miehei lipase may be used for Italian cheeses, although it is less effective than
PGE; lipases from Penicillium roqueforti and Penicillium candidum may also be
satisfactory. The ripening of blue cheese may be accelerated and quality improved by
added lipases. A blue cheese substitute for use as an ingredient for salad dressings and
cheese dips can be produced from fat-curd blends by treatment with fungal lipases and P.
roqueforti spores. Although Cheddar- and Dutch-type cheeses undergo little lipolysis
during ripening, it has been claimed that addition of PGE or gastric lipase improves the
flavor of Cheddar cheese, especially that made from pasteurized milk.
[Link] ß-Galactosidase (Lactase)
ß-galactosidase (lactase) hydrolyses lactose to glucose and galactose, results in stimulation
of lactococci and shortens the lag period of growth of lactococci. Lactose hydrolysed cow
and buffalo cheese milks have been reported to reduce manufacturing time, improve flavor
and accelerate ripening. It has been found that the lactase from Kluyveromyces lactis
available as Maxilact contains a proteinase, which is responsible for increased levels of
peptides, and free amino acids.
31.3.3 Use of adjunct cultures
Adjunct cultures are specifically selected strains, which are intentionally added to
accelerate ripening of full fat cheese and for flavor enhancement of low fat cheese.
Adjunct cultures reportedly decrease bitterness and contribute desirable flavor compounds.
Addition of different Lactobacillus spp. (L. casei and L. plantarum) to Cheddar cheese
milk to a level of 105-106 cfu/ml increased the levels of free amino acids to attain highest
flavor scores. Augmentation of starter culture with L. casei had definite and positive
influence on the flavor; body and texture of buffalo milk Cheddar cheese. The flavor
development and biochemical changes in buffalo milk Cheddar cheese is faster when L.
casei is supplemented with cheese.
The principal adjuncts used in accelerated ripening of cheese are mesophilic lactobacilli and
thermophilic lactobacilli. Inoculation with mesophilic Lactobacillus adjuncts enhanced
flavor and accelerated proteolysis at the level of small peptides and amino acids.
Essentially the same volatiles were produced in all cheeses but at very different
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concentrations. Mesophilic lactobacilli modify proteolysis; in particular, they result in a
higher concentration of free amino acids and improve the sensoric quality. In contrast to
mesophilic lactobacilli, thermophilic lactobacilli die rapidly in cheese, lyse, and release their
intracellular enzymes. Consequently, cheeses made with thermophilic Lactobacillus spp.
as starters contain high concentrations of amino acids (the concentrations are particularly
high in Parmesan cheese). Although thermophilic lactobacilli will not grow in Cheddar
cheese, their inclusion as a starter adjunct markedly intensifies the flavor of Cheddar.
Adjuncts of thermophilic lactobacilli and Streptococcus thermophilus are available
commercially.
31.3.4 Modification of starter bacteria/Starter cultures
[Link] Genetically engineered starters
Food-grade controlled lysis of Lactococcus lactis for accelerated cheese ripening is an
important approach. An attractive approach to accelerate cheese ripening is to induce lysis
of Lc. lactis starter strains for facilitated release of intracellular enzymes involved in flavor
formation. Controlled expression of the lytic genes lytA and lytH, which encode the lysin
and the holin proteins of the lactococcal bacteriophage phi-US3, respectively, was
accomplished by application of a food-grade nisin-inducible expression system.
Simultaneous production of lysin and holin is essential to obtain efficient lysis and
concomitant release of intracellular enzymes as exemplified by complete release of the
debittering intracellular aminopeptidase N. Production of holin alone leads to partial lysis of
the host cells, whereas production of lysin alone does not cause significant lysis. Model
cheese experiments in which the inducible holin-lysin overproducing strain was used
showed a fourfold increase in release of l-Lactate dehydrogenase activity into the curd
relative to the control strain and the holin-overproducing strain, demonstrating the
suitability of the system for cheese applications.
[Link] Stimulation of starter cells
The growth of starter cells may be stimulated by the addition of enzymes or hydrolysed
starter cells to cheese milk. Ripening of Emmental type cheese has been accelerated by
using starters grown to high cell numbers in media supplemented with protein hydrolysates
and metalloproteinase from Micrococcus caseolyticus.
[Link] Modified starter cultures
Addition of modified/attenuated starter culture is to increase the number of starter cells
without causing detrimental effect on the acidification schedule during manufacture, so that
the cells contribute only to proteolysis and other changes during ripening. Modified starter
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cultures with attenuated acid producing abilities are added with normal starter cultures
during cheese manufacture. Selection of starter strains with enhanced autolytic properties
and increased peptidase activity would provide a more balanced flavor.
[Link] Heat and freeze shock treated cells
Mixed strain starters or L. helveticus culture subjected to various heat-shock treatments in
an attempt to reduce their acid producing ability but to enhance their rate of autolysis.
Some workers used heat-shock cultures to attain large number of a mixed-strain starter,
containing Lactococcus, Leuconostoc or L. helveticus strains which were cultivated at a
constant pH, followed by heating to 69°C/15 s. Flavor scores increased with increasing
numbers of heat-shocked cells reducing the ripening time to 50%. A combination of neutral
proteinase with heat shocked L. helveticus to a final level of 4 x 106 cfu/g curd also
accelerated the ripening of the cheese. Addition of heat-shocked lactobacilli increased
peptidolysis and produced good flavor in low-fat semi-hard cheese. Flavor acceleration
could be significantly improved by augmentation of starter culture with freeze-shocked L.
helveticus in buffalo Gouda cheese. The combination of liposome entrapped proteinase
and freeze-shocked lactobacilli resulted in development of more intense flavor without
bitterness in UF cheese.
The acid-producing ability of lactic acid bacteria can be markedly reduced by a sub-lethal
heat treatment while only slightly reducing enzyme activities. Heating at 59 or 69°C for 15
s was optimal for mixed mesophilic and thermophilic lactobacilli cultures, respectively.
Most (90%) of the heat-shocked cells added to cheese milk at a level of 2% (v/v) were
entrapped in the curd but entrapment efficiency decreased at higher levels. Proteolysis in
Swedish cheese was increased and quality improved by addition of the heat-shocked cells
to the cheese milk, L. helveticus being the most effective. The extent of proteolysis
increased pro rata the level of heat-shocked L. helveticus cells added but not for the
mesophilic culture. Bitterness was not observed in any of the cheeses. Heat-shocked
(67°C/10 s) L. helveticus cells accelerated amino nitrogen formation and enhanced flavor
development in Swedish hard cheese; when added alone, Neutrase accelerated proteolysis
but it caused bitterness which was eliminated when both heat-shocked L. helveticus cells
and Neutrase were added to the curd.
[Link] Lysozyme treated cells
Addition of lysozyme-sensitised cells to cheese milk at a level of equivalent to 1010 cells/g
of cheese indicated that intracellular dipeptidases were released and as a result,
concentration of free amino acids significantly increased. However, there was no effect on
the rate of flavor development. Economically, the use of lysozyme treated cells may not be
viable for large-scale cheese manufacture owing to the cost of the enzyme. Addition of
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lysozyme encapsulated in a dextran matrix to cheese milk at renneting, which would be
released at Cheddar cheese pH (5.2-5.4) leading to the lysis of the cells with release of
intracellular peptidases has also been suggested.
[Link] Mutant starter cultures
Because the rate of acid development is a critical factor in cheese manufacture, the amount
of normal starter cannot be increased without producing an atypical cheese. This has led to
the use of lactose negative mutants, which do not affect the rate of acid development but
provide additional enzymes. Like attenuated cells, these mutant cells serve as packets of
enzymes but are much easier to prepare and would appear to be the logical choice when it
is desired to increase the number of cells without a concomitant increase in the rate of acid
production. Lac- mutants of starter strains have been reported to provide packets of
uniformly distributed proteinases/peptidases, enhancing the production of peptides and free
amino acids without interfering with acid production during manufacture. Cheddar cheese
containing 1011cfu/ml of Lc. lactis subsp. cremoris (Lac Prt-) cells received highest flavor
scores. Some workers have showed advancement in ripening of 4-12 weeks after 6
months storage in Cheddar cheese with mutant starter.
It was also recommended to use the Prt starters to reduce bitterness in cheese. It was
claimed that the rate of proteolysis in Cheddar cheese made using Prt- starters was similar
to that in control cheese. Lac- Lactococcus strains with high exopeptidase activity are
commercially available as cheese additives. When inoculated into the cheese milk at
0.002%, individually or as a cocktail, the Lac- cultures enhanced cheese flavor over that of
the control. Assessment of proteolysis showed only minor differences between the cheeses
with respect to primary and secondary proteolysis but all adjunct-treated cheeses contained
higher levels of amino acids than the controls throughout ripening.
31.3.5 Use of high pressure treatment
Using high pressure treatment (HPT) is one of the technological approaches for
accelerated cheese ripening. HPT has emerged as a food processing technology primarily
due to increasing interest in novel methods for preservation of foods. Applying high
pressure to food products modifies interactions between individual components, influences
rates of enzymatic reactions and can inactivate microorganism.
In HPT, an increase in pressure tends to result in a decrease in volume, which enhances
chemical reactions, phase transitions and changes in molecular configurations. Irrespective
of the size and geometry, the pressure is instantaneously and uniformly distributed
throughout the food. The increased pressure affects the environment of bacterial cell and
many biochemical reactions in cells. HPT can cause conformational change in protein but
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small macromolecules like amino acids, vitamins etc. are not affected.
The application of HPT to cheese results in an increase in moisture content and pH and
cause changes to the cheese matrix and lysis of cells, which contribute to ripening. HPT of
cheese affects the pattern of proteolysis during ripening, the effect of which is dependent
on the type of cheese, magnitude, duration and temperature of pressure treatment. The use
of HPT appears to have advantages in both cheese manufacture and ripening. However,
additional research is required to define the operating conditions of HP treatment to
provide positive effects in cheese making and ripening.
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