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Freezing of Dairy Products


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One of the best ways of shelf-life extension is freezing for all kind of food products and additives. Although freezing was applied to dairy product for preservation about 1930s, it was developed for general use after World War II. Freezing methods include plate contact, air blast, individual quick freezing systems, and cryogenic freezing. Frozen dairy-based products are generally divided into two groups. Some frozen dairy products are preferred for their long shelf life and they are thawed for being component for further processing. Other groups are frozen for developing some properties of them, such as structure, texture, aeration, and so on. In this chapter, the second category is focused and it covers ice cream, related frozen, aerated desserts, ice milk, and frozen yogurt. Frozen yogurt should be much like the commercial yogurt and should be characterized also by developed acidity from fermentation.
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Part III
Shelf Life of Dairy Products
Technological Options to Prolong Shelf Life
Advances in Dairy Products, First Edition.
Edited by Francesco Contò, Matteo A. Del Nobile, Michele Faccia, Angelo V. Zambrini, and Amalia Conte.
© 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.
One of the best ways of shelf‐life extension is freezing for all kind of food products and
additives. It provides significantly long‐term shelf life and easy to apply for every kind
of foods. Although new preservation techniques such as high‐pressure, infrared irra-
diation, pulsed electric field, and ultrasound, are increasingly popular, freezing method
is also a generally preferred method regarding its simplicity and conventionally
However, freezing also affects the physical properties of components because of the
conversion of water in it. This phenomenon occurs during energy removal of the prod-
uct from its initial temperature through below freezing temperature. General applica-
tion for freezing requires temperature decrease up to storage level around –18°C.
Cooling curves and phase diagrams give information about precooling, supercooling,
freezing, tempering, eutectic, ice nucleation, and glass transition points of the products.
All procedures and technologies obey and can be corrected due to explained phase
points of the products.
The other crucial point of freezing is microbiological and chemical concern. Freezing
inhibits or slows down reactions covering physicochemical and biochemical reactions
in food matrix but does not stops and remove them permanently. Thus, during storage,
some of the deteriorative activities continue.
Storage temperature, time, and thawing procedures are all quality factors for prevent-
ing quality loss of frozen products. Research indicates that although microbial activities
stop under –18°C, other reactions continue, such as enzymatic and nonenzymatic
changes, even though motion of molecules is inhibited due to freezing of water mole-
cules (Kennedy, 2000; Rahman, 2007; Sun, 2012) .
Freezing process means removal of both sensible and latent heat of the products.
During freezing, three phases—precooling/chilling, phase change, subcooling/temper-
ing—are observed. In precooling, only sensible heat is removed and the temperature of
the product is diminished to facilitate ice crystallization of free water. Then phase
change is observed and latent heat of fusion is removed. The phase change step is
Freezing of Dairy Products
Sebnem Tavman1 and Tuncay Yilmaz2
1 Ege University, Engineering Faculty, Food Engineering Department, Izmir, Turkey
2 Manisa Celal Bayar University Engineering Faculty, Food Engineering Department, Manisa, Turkey
Freezing 267
crucial due to formation of ice crystals so as to get high‐quality frozen foods. After
water is converted to ice, then subcooling starts. Having polymers such as fat, protein,
and carbohydrates makes food freezing more complicated, and the freezing curve is
different compared to pure substances.
During and after freezing, temperature has important effects on stability of frozen
foods. These effects can be grouped as follows: normal stability, which slows reactions;
neutral stability, which has no effect; and reversed stability, which eases reactions.
Concentration during freezing creates unfrozen intact water that might change such
properties as ionic strength, pH, water activity especially change in oxidation reduction
potential is crucial for shelf life. Oxygen removal from ice crystals also problematic and
it was noted that during freezing some damages occur, such as osmotic, solute induced,
and structural, regardless of aqueous system in food.
For slow cooling, crystallization occurs in the outer cells slowly, and if time is
sufficient, water moves due to osmotic pressure. Slow freezing is responsible for cell
shrinkage and membrane damage. Additionally, this water may not turn back after
thawing because of cell structure damage.
Membrane damage causes internal cell materials (potassium ion, galaktosidase, low
molecular solutes, amino acids, RMA, DNA) leakage, which results in cell death.
Osmotic dehydration is the other type of damage. The occurrence of repulsive forces
gives tendency to large anisotropic stresses in the membranes, concluding in deforma-
tion, phase separation, and formation of a nonlamellar phase. Furthermore, salt addi-
tion and lowered pH also play a role in the complex nature of freeze injury and cell
death. On the other hand, regardless of freezing speed, salt accumulation in unfrozen
water may kill the cell. To baffle or prohibit cell damage due to salt concentration, cryo-
protectants such as sugar are added to the water phase.
The quality of the frozen food can be improved by controlling freezing process, as
well as careful pre‐ and post‐freezing storage and preparation. Freezing rate is vital, of
course. In general, fast freezing is preferable to slower freezing. Especially for plant
material, freezing speed is important compared to other food components such as meat
and dairy products. Note, however, that some products will crack or be damaged if they
exposed directly to extremely low temperature for a long time.
Freeze cracking damage can be explained by volume expansion and contraction
expansion. Volume expansion occurs during ice formation, and the amount of empty
space in microstructure is the major factors. During rapid cooling, nonuniform
contraction causes internal stress. And due to fast freezing, crust formation occurs at
the surface, which is capable of cracking. It was found that size, moisture content,
density, elasticity modulus, Poisson’s ratio, and porosity are the major factors for freeze
cracking. Although very high freezing speed may totally damage the food product, no
single property can be expressed for cause of crack (Rahman, 2007; Sun, 2012).
In current applications, fast freezing is chosen for process cost and space. Also, fast
freezing is invariably lethal for live organisms such as yeast, depending on producing
intracellular ice crystals (Rahman, 2007). This lethality may be desired or not, depend-
ing on the type of food material. Care must be taken for foods such as culture‐added
ones. Freezing decreases the viable microbe population by 10% to 60%, which can be
increased gradually in storage period. Fragility of microorganisms differs considerably,
but actually population diminishes in freezing and then increases exponentially during
3.1.1 Freezing ofDairy Products
the thawing period. Although the maximum advised storage for preventing microbial
spoilage is between –9oC and –12oC, enzymes can cause deterioration.
In general, Gram‐negatives are more sensitive to frozen death than Gram‐positives.
Nonsporulating rods and sphericals are resistant, while Clostridium and Bacillus
remain stable by freezing. Stationary‐phase bacteria are more stable than growing‐
phase bacteria.
Water is everything in the freezing process and has different phases during and at the
end of freezing. Broadly, it divides into two groups, called free (freezable) water and
bound (unfreezable) water. Unfreezable water keeps its liquid phase at very low tem-
perature. The biggest problem and product damage comes from that unfreezable water
in frozen food matrix, and it is responsible for enzymatic deteriorative reactions during
storage. The concept of glassy state should be applied to ensure frozen food stability. In
the glassy state, molecular movement is reduced as much as possible. In general,
unfreezable water molecules are mobilized by solutes, and this immobilized amount
of water can be estimated for different foods by mathematical calculations and
Freezing Methods
If applied properly, freezing is the cheapest way of food preservation, compared to
canning and drying. The most important point is if the temperature of the material
begins at a temperature colder than room temperature or even close to freezing point,
better‐controlled crystallization occurs in the freezer. Different types of food systems
are available due to the variety of food products and production characteristics. Freezer
selection should match product requirements, reliable, sanitization requirements, and
economic factors.
Freezing methods include plate contact, air blast, individual quick freezing systems,
and cryogenic freezing. Heat transfer rates are about 5–2000 W/m2K for convective
cooling and 0.5 to 1.5 W/mK for conductive cooling. Some new freezing techniques or
combinations are being developed for their potential benefits, technical and economi-
cal advantages, and quality enhancements.
Freezing Dairy Products
Although freezing was applied to dairy product for preservation about 1930s, it was
developed for general use after World War II. Some factors were identified, relating to
the stability in storage of frozen dairy products. These factors are fat emulsion disrup-
tion, protein flocculation, and developing off‐flavor and bacteriologic problems in
defrost products. In common words there is no significant difference between fresh and
frozen milk samples including some properties such as pH, acidity, acid degree value,
peroxide value, protein sediment, apparent viscosity, or coliform counts. Furthermore,
it was detected that bacterial counts in frozen milk is lower than fresh one.
Frozen dairy‐based products are generally divided into two groups. Some frozen
dairy products are preferred for their long shelf life and they are thawed for being com-
ponent for further processing. Other groups are frozen for developing some properties
of them, such as structure, texture, aeration, and so on. In this chapter, the second cat-
egory is focused and it covers ice cream, related frozen, aerated desserts, ice milk, and
frozen yogurt.
Freezing 269
The composition of dairy products is unique compared to the other food materials such
as plant and meat. Especially properties and amount of fat content makes dairy product
controversy for freezing process. Freezing by slow cooling makes more damage to fat
globule and degredates the aroma of the end product compared to faster one. Moisture
content of dairy products vary around 87% to 91% involving significantly more physical
changes during freezing phenomenon. For milk freezing, it is not economical due to its
high water content and preconcentration required before freezing. If the freezing is applied
properly to the dairy products, their aroma will be comparable to their original state. If the
thawed product is pasteurized and homogenized for further processing for dairy or other
food products, all side effects of freezing and frozen storage can be recovered.
The freezing process affects dairy components such as fat and nonfat solid. Fat is the
most fragile to freezing phenomenon. Expanding water crystals cause mechanical dam-
age to fat globules and make them coalescence and accumulate. Addition of sugar or
homogenization before freezing can prohibit this side effect and make emulsion stable
even after thawing.
The large effect of freezing can be observed on milk protein. During freezing, casein
micelles lose their stability and precipitate, especially after thawing. It makes products
thicken and casein flocks precipitate on the bottom of the package. Although this pre-
cipitation is unwanted, casein removal process called cryo‐casein is applied by cooling to
–10°C. Then the produce is thawed to remove casein from concentrated milk serum.
Casein accumulation is reversible if adequate agitation is applied but becomes perma-
nent after long‐term storage. Reasons for casein flocculation are high‐degree concentra-
tion, excessive heating (more than 77°C), storing under –23°C and higher than –18°C
and long‐term storage. State of lactose and serum proteins are the other factors. Lactose
is responsible for about 55% of freezing point depression. Sun (2012) found that slow
freezing is better than fast freezing for protein stability due to lactose nucleation proce-
dure. When dissolved lactose limits the concentration of the salt in unfrozen phase and
ensures high viscosity. Dialysis of the milk product before freezing contributes protein
For aerated dairy desserts, components such as ice, air, and fat have different effects,
but their interaction is determinant for the end product structure. Quality and shelf life
of the product are affected by ice phase.
Dairy product freezing is quite problematic. First of all, milk is daily available all over
the world. Also, there are many challenges due to its unique composition, and frozen
dairy desserts are preferred to be eaten in frozen form, so there is no special problem
related to thawing as observed in other frozen foods.
Ice Cream
Ice cream is the most popular frozen dairy product. It is defined as sweetened product
composing of milk fat and milk nonsolid fat, and it is frozen by agitation to be aerated.
Composition of ice cream should be compatible with local legal requirements. Freezing
of ice cream divides into two basic categories: high shear application for extensive ice
crystallization and air incorporation, which is satisfied by rapid freezing. Rapid freezing
should promote small‐sized ice crystal due to consumer quality acceptance.
Ice industry is dominated by continuous freezers, which have scraped surface heat
exchangers. Rotating knife blades remove the ice layers and incorporate them into the
3.1.1 Freezing ofDairy Products
mixture while satisfying aeration. Mixing, cooling, and aeration processes take place
simultaneously. The mechanisms that lead to ice formation in an ice cream freezer are
quite complex. Ultimately, the product exiting the freezer contains numerous small ice
crystals. The dissolved sugar lactose, salts, and other components make initial freezing
temperature about –2.5°C. The ice crystals in ice cream at the exit of the freezer are
somewhat block shaped and vary in size from a few microns to over 100 mm. Very
small ice crystals gives ice cream its smooth, cool character. When crystals become
larger, the ice cream may be considered coarse. The dynamic whipping and freezing
allows fat network formation in the product. It was noted that ice cream is composed
of emulsion and foam. Recrystallization is the other concern about frozen products. It
happens after melting, growth, and ripening that occur after the initial ice crystal
phase has been developed. Although recrystallization occurs with no change in ice
phase volume, it leads to changes in the distribution of ice crystals within the system,
based on the thermodynamic difference in melting points between large crystals and
small ones.
Olson etal. (2003) studied properties of frozen dairy desserts processed by microflu-
idization of their mixes. Their study investigated sensory properties and meltdown
rates of ice creams having different composition processed either by homogenization or
microfluidization. They found that microfluidization produced nonfat and low‐fat ice
creams usually had a slower meltdown without affecting sensory properties.
Fermented Milk Product
Freezing process can be applied to fermented foods, including beneficial cultures such
as yogurt, while inhibiting spoiling activities. It is known that yogurt is a well‐estab-
lished dairy product and is protected by developed acidity (lactic acid) from fermenta-
tion of lactose by bacterial culture. Bifidobacteria has beneficial effects for human
intestine, including antagonistic effects on enteropathogens and suppression of liver
tumorigenesis. During acidification, casein micelles in the milk are destroyed and they
establish the typical acid gel.
Frozen yogurt, therefore, should be much like the commercial yogurt and should be
characterized also by developed acidity from fermentation. Because frozen yogurt is not
standardized in regulations, a wide range of products exists, due to citric acid content,
which acidity is not developed by bacterial culture.
In the frozen yogurt process, fermentation is the core phenomenon. For sweet mix,
the cream sugar, stabilizer, skim milk powder, and skim milk are combined and pasteur-
ized, homogenized, cooled, before mixing with yogurt . The completed frozen yogurt
mix is then aged and prepared for flavoring and freezing.
Yogurt acidification cultures are freeze‐resistant, such as strains of Lactobacillus aci-
dophilus, and can be stored frozen at –80°C. Many strains of the culture may also sur-
vive at –30°C. The basic concern about viability is storage temperature compared to
freezing speed. As an example, only 1 log cycle and 40%–70% fermentation activity lost
was observed at –80°C for 1 year for culture of dairy products such as yogurt. However,
fermentation activity was less than 10% when cultures were stored for 1 year at –30°C.
The fermentation activity of Streptococcus thermophilus was similarly reduced to 10%–
60% after 1 year of storage at –30°C. Protective solutes can be used to improve sur-
vival rates.
Freezing 271
Cheese is another group can be freezable. Properties of cheese such as meltability,
elasticity, oil formation, and cohesiveness are changed after freezing and thawing. Due
to the acceptability, although some types of cheeses such as cream cheese, unripened
cammeembert, and brick cheese are suitable for freezing, gouda and cheddar cheese do
have not good properties after freezing.
It is common knowledge that although high‐moisture cheeses have short storage life
at refrigerated temperatures, the shelf life of cheese during storage could be extended by
freezing. Mozzarella is prevalently consumed due to being a high‐moisture cheese used
as a pizza component, and studies have focused on this cheese type to investigate differ-
ent methodologies and analyzing the effect of different operating conditions on the
main characteristics of the cheese. Several authors have studied the changes in texture,
melted functional properties and proteolysis of frozen Mozzarella cheese (Bertola etal.,
1996b; Califano and Bevilacqua, 1999; Ribero etal., 2007).
Califona etal. (1999) studied changes in organic acid content of frozen low‐moisture
Mozzarella cheese. It was detected that there were no important difference between
fresh and frozen cheese for organic acid content even after freezing and stored at –20°C
for 14 to 21 days before thawing.
The initial freezing point of mozeralla cheese was measured by Ribero etal. (2007). In
their study, the influence of the water‐soluble solids on the freezing point depression of
unsalted Mozzarella cheese was analyzed. It was determined that both NaCl and other
soluble solids found in the aqueous phase have drastic effects on freezing point depres-
sion of mozzarella cheese.
Bertola etal. (1996a; 1996b) studied characteristics of frozen low‐moisture mozza-
rella. They investigated apparent viscosity, free oil formation, and meltability of cheeses
after freezing and frozen storage at –20°C. They determined that there was no quality
loss between frozen and unfrozen samples.
Van Hekken etal. (2005) studied the effect of frozen storage on the proteolytic and
rheological properties of soft goat milk cheese. They showed that the rheology and
proteolysis of soft cheese were not sensitive to freezing and long‐term frozen storage.
Soft cheeses had fragile textures that showed minimal change after freezing or over 28
days of aging at 4°C. Factors such as the formation and thawing of ice crystals in the
cheese medium and the limited proteolysis of the caseins caused only minimal impact
on cheese texture. It is advised that frozen storage of soft cheeses may be possible for
year‐round supply with minimal loss of textural quality.
Cheddar is the other investigated frozen cheese in literature. Researchers manufac-
tured different cheddar cheeses varied in several compositions and frozen them, then
thawed. They showed that the most acceptable body and texture in frozen and thawed
cheeses were normal fat, normal moisture cheeses and high fat, normal moisture
cheeses after 8 weeks of aging. The TCA‐soluble N increased significantly during fro-
zen storage and also after thawing (Kasprzak etal., 1994).
Other Dairy Products andStarter Cultures
In addition to ice cream, milk, yogurt, and cheese some other dairy products can be
frozen, such as cream and butter. Cream can be frozen in bulk containers for shelf‐life
increment, but after thawing, fat and serum separation is an extremely big concern. If
pasteurized and homogenized to restore emulsion, this kind of cream can be used as
3.1.1 Freezing ofDairy Products
a component in cream soups, recombined milk, butter, or ice cream. Sugar addition
or increasing nonfat solid content can prevent fat coalescence considerably, due to
lowering the freezing point. Freezing rate is not the determinant factor on fat
Although quality characteristics of butter are not sensitive to freezing conditions
regarding having lower water content up to 20%, freezing after production of butter is
more acceptable than freezing after chilling step. Butter is a water‐in‐oil emulsion, so its
behavior during freezing is very different from that of most food products, for which
water forms a continuous phase. Because the major concern is off‐flavor gain during
storage, frozen butter should be covered properly. Storage time, temperature, and pack-
age type affected butter flavor, oxidative stability index OSI, peroxide value PV, and free
fatty acid value FFV (Krause etal., 2008).
Starter cultures in dairy products are traditionally in liquid form. After the 1960s,
commercial requirements led to new solutions due to a need for proper transportation
and shelf life. Newly developed technologies such as freeze‐dried cultures are preferred
nowadays, and they can directly inoculate into the cheese vats, eliminating the need for
using buffers and bulk tanks. These cultures are called direct vat set (DVS) or direct‐to‐
vat inoculation (DVI) cultures. DVS cultures can be packed in an inert gas, and their
activity can be prolonged up to one year for frozen and two years for freeze‐dried
Bertola, N. C., etal. (1996a). Effect of Freezing Conditions on Functional Properties of Low
Moisture Mozzarella Cheese. Journal of Dairy Science 79 (2), 185–190.
Bertola, N. C., etal. (1996b). Textural Changes and Proteolysis of Low‐Moisture
Mozzarella Cheese Frozen under Various Conditions. LWT—Food Science and
Technology 29 (5‐6), 470–474.
Bylund, G. (2003). Dairy processing handbook. Lund, Sweden: Tetra Pak Processing
Systems AB.
Califano, A. N., Bevilacqua, A. E. (1999). Freezing low moisture Mozzarella cheese:
changes in organic acid content. Food Chemistry 64 (2), 193–198.
Hui, Y. H. (2004). Handbook of frozen foods. New York: Marcel Dekker.
Kasprzak, K., Wendorff, W. L., Chen, C. M. (1994). Freezing Qualities of Cheddar‐Type
Cheeses Containing Varied Percentages of Fat, Moisture, and Salt. Journal of Dairy
Science 77 (7), 1771–1782.
Kennedy, C. (2000). Managing frozen foods. Boca Raton, FL: CRC Press.
Krause, A. J., etal. (2008). The effect of refrigerated and frozen storage on butter flavor and
texture. Journal of Dairy Science 91 (2), 455–465.
Law, B. A., Tamime, A. Y. (2010). Technology of cheesemaking. Malden, MA: Blackwell.
Olson, D. W., White, C. H., Watson, C. E. (2003). Properties of frozen dairy desserts
processed by microfluidization of their mixes. Journal of Dairy Science 86 (4),
Rahman, S. (2007). Handbook of food preservation. Boca Raton, FL: CRC Press.
References 273
Ribero, G. G., Rubiolo, A. C., Zorrilla, S. E. (2007). Initial freezing point of Mozzarella
cheese. Journal of Food Engineering 81 (1), 157–161.
Sun, D.‐W. (2012). Handbook of frozen food processing and packaging. Boca Raton, FL:
Taylor & Francis
Van Hekken, D. L., Tunick, M. H. & Park, Y. W., 2005. Effect of frozen storage on the
proteolytic and rheological properties of soft caprine milk cheese. Journal of Dairy
Science 88 (6), 1966–1972.
Advances in Dairy Products, First Edition.
Edited by Francesco Contò, Matteo A. Del Nobile, Michele Faccia, Angelo V. Zambrini, and Amalia Conte.
© 2018 John Wiley & Sons Ltd. Published 2018 by John Wiley & Sons Ltd.
Dairy products are susceptible to physical, chemical and microbiological deterioration
throughout storage and distribution (Cha and Chinnan, 2004). In these products, con-
tamination can occur in the liquid milk, resulting in the inoculation of the whole prod-
uct, or after milk gelation, in this case, contaminating cells remain near the contact area.
The probability of consuming a contaminated cheese has even been estimated at 65.3%
for soft cheese made with raw milk (Cao‐Hoang etal., 2010). Depending on the type of
cheese, the physicochemical properties of the matrix, the origin of the contamination
(from raw materials, during the cheese making or ripening), and the contaminant spe-
cies, pathogenic bacteria can develop more or less toward the interior of the product.
During processing, the pathogenic bacteria risk is diminished by the pasteurization of
raw milk, the control of the length of maturation, and of the storage temperature of
cheese. This temperature—together with some intrinsic properties such as pH, water
activity, and the presence of antimicrobial compounds produced by starter culture—
constitute a hurdle system. However, colonization of cheese surface by microorganisms
constitutes a significant risk due to its high water content and favorable pH for micro-
bial growth (Conte etal., 2013). In the dairy industry, quality is traditionally measured
on the entire process of milk handling involving raw materials, product manufacturing,
packaging, storage, and distribution. Farmed animals represent a major reservoir of
pathogens that can be transferred to milk. The predominant human bacterial patho-
gens that can potentially be transferred to milk include mainly Listeria monocytogenes,
Salmonella spp., Staphylococcus aureus, and pathogenic Escherichia coli. Raw milk pro-
vides a potential growth medium for the development of these bacteria (Farrokh
etal., 2013).
Although pasteurization destroys potential pathogenic microorganisms, post‐pasteurization
processing can lead to the recontamination of dairy products. L. monocytogenes can cause
illnesses, extending from those with mild flu‐like symptoms to more serious. Between 1998
and 2008, at least 25% of reported outbreaks of listeriosis in the United States were of dairy
origin (Cartwright et al., 2013). Listeria can contaminate the dairy environment from
Antimicrobial Compounds Applied to Dairy Food
Luisa Angiolillo, Annalisa Lucera, Matteo A. Del Nobile and Amalia Conte
Department of Agricultural Sciences, Food and Environment, University of Foggia, Italy
Antimicrobial Agents ofPlant Origin 275
manure or improperly fermented silage and can be introduced in the human food supply
chain. S. aureus is a causative agent of bovine mastitis capable of producing thermostable
enterotoxins. Dairy products contain low levels of enterotoxigenic staphylococci; however,
temperature abuse above 10°C and poor starter culture activity during fermentation are fac-
tors involved in dairy‐related outbreaks of staphylococcal intoxication (Cretenet et al.,
2011). E. coli O157:H7 is a Shiga toxin‐producing Escherichia coli (STEC) serotype of high
virulence. The number of cases of severe disease caused by STEC in dairy products has
remained quite low, probably thanks to compliance with good hygienic practices at the
farm level.
Salmonella is found in the environment and in the gastrointestinal tract of farmed
and wild animals. A total of 108,614 confirmed cases of salmonellosis were reported in
the European Union in 2009 (EFSA, 2012). Dairy products along with meat and eggs are
the most common causes of foodborne Salmonella infection. Salmonellosis from con-
taminated milk and dairy products has been associated with inadequate pasteurization
and post process contamination. Control measures in the food dairy industry are
designed to prevent or minimize bacterial contamination, including the appearance or
growth of foodborne pathogens.
Good manufacturing practices, sanitation, and hygiene measurements for raw mate-
rial and food industry environment do not completely eliminate the occurrence of food-
borne outbreaks, however. Thus, research into increasing the role of controlling
pathogenic and spoilage microorganisms in food is essential. Strategies such as thermal
treatment (high or low), water activity (aw) reduction, nutrient restriction, acidifica-
tion/fermentation (low pH), altering redox potential (Eh), modified atmosphere pack-
aging, nonthermal physical treatments, or the use of antimicrobials have been
traditionally applied to enhance the shelf life of dairy products. Antimicrobial agents
also include chemical compounds added to control spoilage microorganisms.
The classification of antimicrobials is extremely difficult. However, depending on
origin, they can be divided into traditional (chemical substances) and novel substances,
called “naturals” (from animal, plant, and microbial sources). Details on each group are
reported below.
Antimicrobial Agents of Plant Origin
In recent years, antimicrobial properties of essential oils (EOs) have been documented,
and interest in these EOs persists. A wide range of natural antimicrobial agents derived
from essential oils has attracted much attention in the food and packaging industries as
a replacement for synthetic ones to protecting food products from microbial contami-
nation. The essential oils are aromatic oil liquids obtained mostly from plant material.
They exhibit antiviral, antibacterial, antimycotic, antitoxigenic, antiparasitic and insec-
ticidal properties, and their use is allowed in European Union (EU) in food (as flavoring,
either extracted from plant material or synthetically manufactured), perfumes and
pharmaceuticals (Burt, 2004). Several essential oils were used as antimicrobial agents to
prolong the shelf life of dairy products.
The potential antimicrobial effectiveness of rosemary essential oil (REO) on
Escherichia coli inoculated on Coalho cheese was evaluated. The E. coli strain EC16 was
used to inoculate two samples of commercial cheese, and then one sample was added
3.1.2 Antimicrobial Compounds Applied toDairy Food
with 20% (v/v) REO, and another sample was used without the addition of REO as a
control. Results showed that during seven days of refrigeration, the rosemary essential
oil has an inhibitory activity against the E. coli strain EC16 in vitro and in vivo (Ribeiro
etal., 2013). The antimicrobial effectiveness of basil oil and their principal constituents
(linalool or methylchavicol) was evaluated. Tsiraki and Savvaidis (2013) assessed the
effect of basil essential oil on the quality characteristics of whey cheese “Anthotyros.
The experiment was performed by adding the basil EO to the cheese samples to a final
concentration of 0.4% (v/w). The sensory evaluation and microbiological data showed
that the combined use of modified atmosphere packaging (MAP) and vacuum packag-
ing (VP) with added basil EO extended the shelf life of fresh Anthotyros by approxi-
mately 10 to 12 days for samples packaged in MAP and 6 days for those in VP as
compared to aerobic packaging. The principal constituents of basil oil (linalool or
methylchavicol) were mixed with low‐density polyethylene (LDPE) pellets to manufac-
turing antimicrobial films (AM). Suppakul et al. (2008) tested the inhibitory effect
against microbial growth on cheddar cheese wrapped with the antimicrobial films and
stored at 4°C. In addition, cheese samples inoculated with E. coli or Listeria innocua
were wrapped with the AM films, stored at refrigerated (4°C) or at abuse (12°C) tem-
peratures. In abuse temperature conditions, methylchavicol‐LDPE based film exhibited
a higher efficacy of inhibition than that of linalool‐LDPE based film. An antimicrobial
starch‐based film was realized by incorporating linalool, carvacrol, and thymol in the
coating of the film. Then, the inhibitory effect against S. aureus and A. niger of the
antimicrobial agents was tested on the surface of cheddar cheese (Kuorwel etal., 2011;
2012). Current studies suggest that linalool, carvacrol, or thymol, could be successfully
coated onto starch‐based films to produce packaging materials that exhibit activity
against S. aureus and A. niger. The inhibitory effect of the essential oils depended on the
concentration of the active agent in the film coating and was found to be in the order of
Ramadan etal. (2014) studied the antimicrobial effectiveness of black cumin seed oil
against some pathogenic bacteria (S. aureus, E. coli, L. monocytogenes and S. enteritidis)
inoculated in soft cheese during cold storage. For the cheese supplemented with essen-
tial oil (0.1% or 0.2%, v/w), the counts of the inoculated pathogens were significantly
reduced by about 1.3 log10 and 1.5 log10 CFU g‐1 after 21 days of storage.
A study was conducted by Belewu etal. (2012) to compare the effect of different levels
of eucalyptus oil (EO) and lemongrass oil (LO) on the shelf life of fresh West African
soft cheese. The experiment was performed by adding the essential oils at different
concentrations (75% EO + 25% LO and 50% EO + 50% LO) to the cheese samples;
moreover, cheese kept in the whey was used as control. The results showed that the
eucalyptus oil 75% plus 25% lemongrass had a positive impact on the nutritional, sen-
sory, and microbial values; whey did not significantly enhance the nutritional, sensory,
and microbial qualities of the soft cheese. The antibacterial activity of oregano and
thyme essential oils added at doses of 0.1 or 0.2 and 0.1 mL/100 g, respectively, to feta
cheese inoculated with E. coli O157:H7 or L. monocytogenes and stored under modified
atmosphere packaging was investigated. In feta cheese with oregano oil at the dose of
0.1 ml/100 g, the E. coli O157:H7 or L. monocytogenes, strains survived up to 22 or 18
days of storage, respectively, whereas at the dose of 0.2 mL/100 g up to 16 or 14 days of
storage, respectively. The antibacterial activity of thyme oil at 0.1 mL/100 g against
Antimicrobial Agents ofPlant Origin 277
either E. coli O157:H7 or L. monocytogenes was similar to that of oregano oil at 0.1
mL/100 g in feta cheese (Govaris etal., 2011).
The efficiency of plant essential oils as natural food preservatives in Fior di latte cheese
was assessed by Gammariello etal. (2008). The Fior di latte cheese samples were pack-
aged in polypropylene tubs using either brine or active brine. The use of natural com-
pounds such as lemon, sage, and thyme in Fior di latte cheese inhibited the growth of
microorganisms involved in spoilage. Conte etal. (2007) evaluated the effectiveness of
three different concentrations (500, 1000, 1500 ppm) of lemon extract on the microbial
quality decay kinetics during storage of mozzarella cheese. The active agent was added
to salt solutions with and without adding sodium alginate to obtain, respectively, an
active solution and an active gel. The results confirmed that the investigated substance
might exert an inhibitory effect on the growth of spoilage microorganisms such as coli-
forms and Pseudomonas spp. without affecting the functional dairy microbiota and the
sensorial characteristics of the product. The shelf life of Minas frescal cheese made with
oregano essential oil was estimated. Different concentrations of oregano essential oil
(0.0075% and 0.015%) were used in the samples, and the sample without active agents
was used as control. The researchers found that the count of Staphylococcus coagulase
positive, aerobic mesophilic, psychrotrophic, coliforms, and Salmonella were within the
limits established by applicable Brazilian legislation until to 28 days. Moreover, the sen-
sorial analysis of all samples did not show significant differences for the attributes
assessed (Santos etal., 2014). An alginate‐based edible coating loaded with essential oils
of rosemary and oregano was applied on ricotta cheese in order to evaluate the physical,
chemical, microbiological, and sensory characteristics. The authors found that there was
no difference between the samples in relation to sensory analysis, mass loss, texture, and
color (Tavares etal., 2014). Also, Asensio etal. (2014) evaluated the antimicrobial activity
of Argentinean oregano essential oils on flavored ricotta cheese. The results showed that
ricotta flavored with oregano essential oil improves their physicochemical characteris-
tics and lessens its microbiological count, especially ricotta supplemented with oregano
essential oil obtained from variety Cordobes.
Thyme and oregano essential oils were used against S. aureus both in vitro and on
fresh cheese. In vitro test, the antimicrobial power against the S. aureus of the EOs of
thyme and oregano has been proven; whereas, in vivo the results showed that the con-
centration of S. aureus remained almost unchanged for all types of cheeses produced
(Amatiste et al., 2014). The effects of some plant‐derived essential oils (cinnamon,
cumin, mint, dill, and caraway essential oils) on the shelf life of Labneh traditional fer-
mented milk were tested (Thabet etal., 2014; Wafaa etal., 2013). The results suggested
that the cinnamon oil at 0.3% extended the shelf life for up to 24 day at 6±1°C with
acceptable taste and flavor, and without any microbial spoilage. The dill and caraway
essential oils enhanced the cheese quality and the organoleptic properties and also
extended the shelf life to 28 days.
Also, Otaibi and Demerdash (2008) estimated improvement of the quality and shelf
life of concentrated yogurt (Labneh) by the addition of some essential oils. In particular,
thyme, marjoram, and sage were added at concentrations of 0.2, 0.5, and 1.0 ppm.
Results indicated that the Labneh containing 0.2 ppm thyme, marjoram, or sage oils was
the most acceptable after the control. An amount of 0.2 ppm thyme, marjoram, or sage
oils increased the shelf life of Labneh for up to 21days. Mohamed etal. (2013) found that
3.1.2 Antimicrobial Compounds Applied toDairy Food
among seven screened essential oils, caraway as well as dill seeds oil had the highest
antibacterial effect. The two selected essential oils were incorporated in cheese yogurt
manufactured from milk, which was previously contaminated by five strains of patho-
gens (L. monocytogenes, S. aureus, Bacillus cereus used as Gram‐positive bacteria and E.
coli and S. typhimurium used as Gram‐negative). The results highlighted that the cara-
way and dill seeds oils had the same ability to inhibit the growth of all pathogens and
were the most inhibiting to L. monocytogenes and S. typhimurium. The physicochemi-
cal and microbiological qualities of yogurt samples treated with anise volatile oil and its
oleoresin at varying concentrations (0.1 to 1.0 g/L) were assessed up to 20 days at 4±1°C.
The results revealed that incorporation of essential oil and oleoresin of anise at 1.0 g/L
concentration was moderately effective in controlling the growth of spoilage microor-
ganisms and also suggested that it had no adverse effect on the physicochemical proper-
ties of yogurt (Singh etal., 2011).
Alsawaf and Alnaemi (2011) carried out a study on antibacterial effect of Nigella
sativa (seed and oil) on the quality of soft white cheese. Nigella sativa seed (1% and 3%)
and oil (0.3% and 1%) were tested on some food poisoning and pathogenic bacteria as
well as on the total bacterial count. A decrease in the total bacterial count of S. aureus,
Brucella melitensis, and E. coli count in cheese samples treated with N. sativa seed and
oil was observed. Moreover, N. sativa oil was more effective as an antibacterial agent
than seed. Dried spearmint and its essential oils were used as preservatives in white
cheese. The authors found that the oil showed significant reduction in the total bacte-
rial count, proteolytic and psychrotrophic bacteria count, as compared with control
(Foda etal., 2009). An innovative Karish cheese was made by adding curcumin (0.3%
w/v) to obtain a new dairy product named Karishcum. The behavior of pathogenic bac-
teria in artificially contaminated cheese revealed that the addition of aqueous curcumin
extract achieved a reduction of bacterial counts, about one log cycle of S. typhimurium,
two log cycles of Pseudomonas aeruginosa and E. coli, respectively. S. aureus, B. cereus
and L. monocytogenes vanished at the end of the storage period (Hosny etal., 2011).
Moreover, Metwalli (2011) achieved a Karish cheese by mixing propolis (5%, 10%), gar-
lic, and ginger (0.5%), respectively, essential oils, in order to improve the quality of the
cheese. Total bacterial count, yeast, and mold decreased in all treated samples with
respect to the control cheese; lipolytic bacteria counts were affected by propolis more
than essential oils. The use of propolis and essential oils increased the shelf life of
Kareish cheese for 30 days while imparting good flavor.
Essential oils can be also used as antimicrobial volatile compounds incorporated in
sachets. Pires etal. (2009) and Han etal. (2014) studied the antimicrobial effectiveness
of sachets incorporating allyl‐isothiocyanate (AIT), rosemary, and thyme oils, respec-
tively, on the quality of mozzarella cheese. The sachet incorporated with AIT was tested
against growth of yeasts and molds, Staphylococcus spp. and psychrotrophic bacteria on
sliced mozzarella cheese. The authors found that yeasts and molds were the most sensi-
tive to the antimicrobial effects; while, psychrotrophic bacteria species were the most
resistant to the antimicrobial action. The efficacy of the volatiles of the oils (rosemary
and thyme) embedded in a sachet containing microcellular foam starch was tested to
reduce bacterial growth of L. monocytogenes inoculated in shredded mozzarella cheese.
The results showed that rosemary and thyme oil volatiles released from the sachet
restricted the growth of L. monocytogenes, resulting in a 2.5 log10 CFU/g reduction on
day 9 at 10°C compared to untreated samples.
Antimicrobial Agents ofMicrobial Origin 279
Antimicrobial Agents of Microbial Origin
Biopreservation can be defined as the extension of shelf life and food safety by the use
of natural or controlled microbiota and/or their antimicrobial compounds (Angiolillo
etal., 2014). Lactic acid bacteria (LAB) have antagonistic properties that make them
particularly useful as biopreservatives.
When LAB compete for nutrients, their metabolites often include active antimicrobi-
als such as lactic and acetic acid, hydrogen peroxide, and peptide bacteriocins. LAB
bacteriocins can be used for biopreservation and their combination with other pre-
servative techniques could be a way to control spoilage bacteria and other pathogens,
and inhibit the activities of a wide spectrum of organisms, including inherently resistant
Gram‐negative bacteria. Bacteriocins can be classified into five major classes:
Class I: Lantibiotics, which are small (2‐5 kDa), contain lanthionine (e.g., nisin)
Class II: unmodified, which are small (<5 kDa), also called Listeria active peptide (e.g.,
Pediocin PA1, and Leucocin A)
Class III: Large (>30 kDa), heat labile and are least characterized group (e.g., Helvetin
J and Enterolysin)
Class IV: complex, composed of protein plus one or more chemical compound (lipid
or carbohydrate) (e.g., Leuconosin S, and Lactococcin 27)
Class V: cyclic, head‐to‐tail cyclic backbone (e.g., Gassericin A, and AS‐48) (Cleveland
etal., 2001).
Most of the LAB bacteriocins identified so far are thermostable cationic molecules
that have up to 60 amino acid residues and hydrophobic patches. Electrostatic interac-
tions with negatively charged phosphate groups on target cell membranes are thought
to contribute to the initial binding, forming pores and killing the cells after causing
lethal damage and autolysin activation to digest the cellular wall. LAB bacteriocins,
have many attractive characteristics that make them suitable candidates for use as food
Protein nature, inactivation by proteolytic enzymes of gastrointestinal tract
Nontoxic to laboratory animals tested and generally nonimmunogenic
Inactive against eukaryotic cells
Generally thermo‐resistant (with antimicrobial activity after pasteurization and
Broad bactericidal activity affecting most of the Gram‐positive bacteria and some,
damaged, Gram‐negative
Genetic determinants generally located in plasmid, which facilitates genetic manipu-
lation to increase the variety of natural peptide analogues with desirable
The generally recognized as safe (GRAS) bacteriocin nisin produced by Lactococcus
lactis subsp. Lactis isolated from milk was the first antibacterial peptide described in
LAB active against Gram‐negative and Gram‐positive bacteria. Nisin is a peptide com-
posed of 34 amino acid residues, classified as a class‐Ia bacteriocin or lantibiotic
(Cleveland etal., 2001). To date, it is the only bacteriocin that has been approved by the
World Health Organization for use as a food preservative. Nisin has been shown to be
effective in the microbial control of a number of dairy products, and its use has been
3.1.2 Antimicrobial Compounds Applied toDairy Food
widely assessed in cheese manufacturing at low pH. The use of nisin producing and
nisin resistant starter cultures appears to be a viable means of incorporating and main-
taining this bacteriocin, through the cheese‐making process, to control foodborne
pathogenic and spoilage bacteria.
Pinto etal. (2011) used nisin at different concentration (0, 100 and 500 IU mL) against
S. aureus in Minas traditional serro cheese, that is a traditional semi‐hard Brazilian
cheese manufactured with raw milk. Nisin effectively reduced S. aureus count with a 1.2
and 2.0 log reduction cycles.
Nisin was added also to a chilled high‐fat milk pudding dessert, one of the most popu-
lar desserts in Japan, previously inoculated with spores of Bacillus thuringiensis, by
Oshima etal. (2014). They showed that nisin A inhibited spiked bacteria.
Nisin and other bacteriocines can be applied directly by incorporation in liquid sys-
tems (Gallo and Jagus, 2006) or in the case of food solid surfaces, using different tech-
niques like spraying, dipping, or brushing. According to Ture et al. (2011), direct
application of additives can have limited benefits because a loss of activity should be
present due to the interaction or reaction with other additives or components present
in the food matrix. Incorporation of antimicrobials in food interfaces by means of the
use of edible films where they are entrapped helps to decrease the rate of diffusion from
the surface to the bulk of the product (Kristo etal., 2008). For these reasons, Martins
etal. (2010) used an edible coatings made of galactomannans from Gleditsia triacanthos
with nisin against L. monocytogenes in ricotta cheese. Results showed that the cheese
coated with nisin‐added galactomannan film presented the best results in terms of
microbial growth delay. Sakacin C2 is a novel bacteriocin with a broad inhibitory spec-
trum secreted by Lactobacillus sakei with an antimicrobial activity against some food-
borne spoilage and pathogenic bacteria, including not only Gram‐positive but also
Gram‐negative bacteria such as E. coli and S. typhimurium. Yurong etal. (2012) studied
the effects of chemical composition including milk fat, the addition of emulsifiers and
preservatives, and homogenization on activity of sakacin C2 against E. coli in milk, lay-
ing the groundwork for the use of sakacin as a biopreservative in dairy products. They
found that milk fat in pasteurized and homogenized milk products (low‐fat milk and
whole milk) decreased the activity of sakacin C2 against E. coli ATCC 25922. Although
underutilized in the majority of cases, some enterocins (class II batteriocin) produced
by enterococci are among the most active bacteriocins in combating L. monocytogenes.
However, direct application of enterocins may result in decrease or the complete loss of
antimicrobial activity due to problems related to interaction with food components
(Chollet et al., 2008). Alternatively, the incorporation of live bacteriocin‐producing
strain(s), either through direct addition to the food or in an immobilized form on pack-
aging, may present a potential benefit in controlling L. monocytogenes in dairy products.
Coelho etal. (2014) discovered that bacteriocin activity was only detected in the whey
of fresh cheese inoculated with two Enterococcus strains, but all cheeses made with
bacteriocin‐producing strains inhibited L. monocytogenes growth in the agar diffusion
bioassay. To test the effect of in situ bacteriocin production against L. monocytogenes,
fresh cheese was made from pasteurized cows’ milk inoculated with bacteriocin‐pro-
ducing LAB and artificially contaminated with approximately 106 CFU/mL of L. mono-
cytogenes. All strains controlled the growth of L. monocytogenes, although some
Enterococcus were more effective in reducing the pathogen counts. After 7 days, this
reduction was of approximately 4 log units compared to the positive control. In addition
Antimicrobial Agents ofMicrobial Origin 281
to bacteriocins, LAB exert their antimicrobial activity by their fermentation activity and
with the production of other substances with antimicrobial action.
Angiolillo etal. (2013) stated that the addition of Lactobacillus rhamnosus in an edible
sodium alginate coating applied on the surface of Fior di latte cheese exerted an antimi-
crobial activity against Pseudomonas and Enterobacteriaceae. In another study,
Angiolillo etal. (2014) elaborated a sodium alginate coating containing Lactobacillus
reuteri in combination with glycerol applied on the surface of Fior di latte cheese in
order to extend its shelf life by means of in situ production of reuterin. They demon-
strated that the active coating with L. reuteri was effective in prolonging Fior di latte
microbial quality improving also its final taste.
Some strains of L. reuteri have been recognized for their ability to produce reuterin
(b‐hydroxypropionaldehyde; b‐HPA) during anaerobic metabolism of glycerol
(Rodríguez etal., 2003). Reuterin is an antimicrobial compound soluble in water, resist-
ant to heat and stable over a wide range of pH values, that inactivates Gram‐negative
and Gram‐positive bacteria (Vollenweider etal., 2003). Direct addition of reuterin to
control foodborne pathogens such Salmonella spp., Escherichia coli O157:H7, Listeria
monocytogenes and Staphylococcus aureus has been investigated in milk and dairy
products (Arqués etal., 2008). Natamycin (or pimaricin) is a polyene antifungal antibi-
otic produced by Streptomyces natalensis. It is used to control fungus growth in the
surface of most cheese and is not effective against bacteria or viruses. Natamycin has
been used for many years in a large number of countries throughout the world as an
authorized preservation treatment for cheeses. It is commonly employed in dairy‐based
food products to prevent yeast and mold contamination (El‐Diasty etal., 2008).
Moreira etal. (2007) incorporated natamycin in a cellulose‐based film and evaluated
the antimicrobial efficiency of natamycin‐incorporated film in the production process
of Gorgonzola cheese. They found that films with 2% and 4% natamycin presented sat-
isfactory results for fungus inhibition. Ture et al. (2011) studied the effect of wheat
gluten (WG) and methyl cellulose (MC) biopolymers containing natamycin on the
growth of A. niger and P. roquefortii on the surface of fresh kashar cheese during storage
at 10°C for 30 days. Wrapping of A. niger‐inoculated cheese with MC films containing
5–20 mg NA per 10 g resulted in approximately 2‐log reductions in spore count. Two
mg NA per 10 g included into WG films was sufficient to eliminate A. niger on the
surface of cheese.
Fajardo etal. (2010) studied chitosan coatings containing natamycin on the physico-
chemical and microbial properties of semi‐hard Saloio cheese. They proved that nata-
mycin‐coated samples presented a decrease on molds/yeasts of 1.1 log10 CFU g‐1
compared to control after 27 days of storage. Dairy propionic bacteria are commercially
important in the production of the “eye” and typical flavors in Swiss‐type cheeses. They
also produce organic acids, leading to an effective “natural” antifungal ingredient that
could be used in dairy industry. Tawfok etal. (2004) added lyophilized P. thoenii P‐127
metabolites by 1.5% to Domiati cheese milk with a consequent prolongation of soft
cheese shelf life.
Natural antimicrobials are rarely used as single compounds; they are usually used in
combination with others to provide hurdles for the growth of microorganisms without
affecting sensorial and nutritional characteristics. Pires etal. (2008) used a combination
of nisin and natamycin incorporated into a cellulose‐based film and applied on mozza-
rella cheese against molds and yeasts, Staphylococcus sp. and psychrotrophic bacteria.
3.1.2 Antimicrobial Compounds Applied toDairy Food
By the ninth day of storage at 12° ± 2°C, the count of yeasts and molds on cheese covered
with the antimicrobial film decreased 2 log10 units compared with the count on cheese
with control film. In a study, Resa etal. (2014) evaluated the effectiveness of natamycin
and nisin supported in tapioca starch films against Saccharomyces cerevisiae and L.
innocua in a mixed culture present on the surface of a model system and of Port Salut
cheese was evaluated. It was observed that the preservatives incorporated in starch
films controlled growth of both microorganisms present together on the surface of the
cheese during storage.
Antimicrobial Agents of Animal Origin
Animals produce several antimicrobial compounds, as first line of defense, which are
also found in products of animal origin such as, milk and eggs (Straus and Hancock
2006). The potential of several antimicrobials derived from animal sources as food pre-
servatives is increasingly being reported. Some of the well‐characterized antimicrobials
of animal origin are described in this section.
Chitosan, a complex polysaccharide naturally present in the exoskeletons of crustaceans
and arthropods, alternatively, can be produced from some fungi (A. niger, Mucorrouxii,
P. notatum) (Tayel etal., 2010). It has gained considerable attention for commercial
applications in food (Leleu etal., 2011). Moreover, it has been found to be nontoxic,
biodegradable, biofunctional, and biocompatible; moreover, several researchers
reported that the chitosan has strong antimicrobial and antifungal activities (Hague
etal., 2005; Kim and Rajapakse, 2005). The biological activity of chitosan depends on its
molecular weight, deacetylation degree, chitosan derivatization, degree of substitution,
length and position of a substituent in glucosamine units of chitosan, pH of chitosan
solution, and, of course, the target organism. It is generally found that yeasts and molds
are the most sensitive group to chitosan, followed by Gram‐positive and Gram‐negative
bacteria (Aider, 2010). A study on the synergy of MAP and chitosan was conducted
successfully on stracciatella cheese (Gammariello etal., 2011). In this work, the addition
of different amounts of chitosan (0.010, 0.015, 0.020%) during cheese making, com-
bined with MAP prolonged stracciatella cheese shelf life (about 7 days), if compared
with the control sample (more or less 3 days). The results showed that the Pseudomonas
spp. growth was inhibited by combination of chitosan with MAP.
Del Nobile etal. (2009a) studied an integrated approach to prolong the shelf life of
Fior di latte cheese. The investigated strategy was based on the combination of chitosan
in the manufacture, either coating or active coating (lysozyme and ethylenediamine
tetraacetic acid disodium salt), combined with MAP. The authors reported that the
integrated approach developed allowed us to obtain a significant shelf life prolongation
to 5 days in comparison with the traditional packaging the latter showed a very short
shelf life limited to more or less 1 day. The use of chitosan and extracts of lemon and
sage was assessed during Fior di latte cheese making by Gammariello etal. (2010). The
active compounds were added to working milk at different concentrations. The results
highlighted that the presence of lemon extract and chitosan improved Fior di latte
Antimicrobial Agents ofAnimal Origin
cheese shelf life, whereas, the addition of sage extract negatively affected the sensory
properties. In particular, the chitosan at the concentration of 240 mg/Kg was effective
against coliforms and Pseudomonadaceae.
Coma etal. (2002) investigated the effect of edible film based on chitosan matrix and
tested her antimicrobial activity against L. monocytogenes. The chitosan film showed
100% of L. monocytogenes inhibition for at least 8 days. The latter results were validated
on Emmenthal cheese samples using L. innocua as model strain. The results showed
that the chitosan‐free samples exhibited 10 times higher colony forming units of bacte-
ria compared to chitosan‐coated ones. Besides, after 132 hours of storage, no colonies
were detected from chitosan‐treated samples. The chitosan was used also as antifungal
agent on Kariesch cheese, in this work the cheese was treated with 0.5% and 1.0% chi-
tosan solutions. The authors found that the chitosan‐treated cheese showed an improve-
ment of shelf life extended up to the 18th day of storage with respect to the control. The
results indicated that the application of chitosan on the Kareish cheeses improves the
mycological quality, in fact, a reduction of two logaritmic cycles in the molds and yeasts
counts was observed in cheese samples treated with chitosan 1% compared with the
control at the end of storage period (El‐Diasty etal., 2012). Di Pierro etal. (2011) con-
ducted a study extending the shelf life of ricotta cheese by using a chitosan/whey pro-
tein coating. The data proved that the viable numbers of mesophilic and psychrotrophic
microorganisms were significantly lower in coated ricotta cheese than in control sam-
ples; besides, the coating delayed the development of undesirable acidity, better main-
tained the texture, and did not seem to modify sensory characteristics. As reported by
Duan et al. (2007), the antimicrobial activities of chitosan‐lysozyme (CL) composite
films and coatings were tested against microorganisms inoculated (L. monocytogenes, E.
coli, P. fluorescens) onto the surface of mozzarella cheese. Three different package appli-
cations (CL film, CL lamination on film, and CL coating) were assessed. The authors
reported that the CL composite films and coatings showed the greatest antimicrobial—
in fact, they significantly reduced the growth of the microorganisms inoculated and
molds in mozzarella cheese, although they had a lesser antimicrobial effect on yeast.
Lysozyme is an enzyme that is naturally present in avian eggs and mammalian milk and
is generally recognized as safe (GRAS). It has been used both in the pharmaceutical and
food industries. The white lysozyme of hen eggs is commonly used as a preservative for
meat, meat products, fish, fish products, milk and dairy products, and fruits and vegeta-
bles (Cegielska‐Radziejewska etal., 2009). It is well known that lysozyme is bactericidal
against Gram‐positive microorganisms (Boland et al., 2003), whereas it is essentially
ineffective against Gram‐negative bacteria, owing to the presence of a lipopolysaccha-
ride layer in outer membrane. However, its effectiveness could be increased through the
use of some chelating agents (EDTA) as membrane disrupting agents.
Different concentrations of lysozyme (0.25, 0.50, and 1.00 mg mL–1) +50 mM of
Ethylene‐Diamine Tetraacetic Acid (EDTA), incorporated in a sodium alginic acid‐
based coating, were evaluated on Fior di latte cheese shelf life (Del Nobile etal.,
2009b). The same concentrations of active compound were used in brine to package
the controls. As reported by the authors, an increase in the shelf life (104%) was
recorded for the coated samples, respect to controls packaged in brine without active
3.1.2 Antimicrobial Compounds Applied toDairy Food
compounds. This shelf life increase was slightly lower than that recorded with sam-
ples packaged in the active brine (151%), as a result of a more pronounced microbial
proliferation. Conte etal. (2011) studied the effects of lysozyme/EDTA disodium salt
(Na2‐EDTA) combined with MAP on shelf life of burrata cheese. Three concentra-
tions of enzyme (150, 250, and 500 mg/kg) were added to the burrata samples. The
results confirmed that the combination of lysozyme/Na2‐EDTA and MAP prolonged
cheese shelf life; in particular, the enzyme at the highest concentration was effective
against Pseudomonas spp.
Doosh and Abdul‐Rahman (2014) examined the effect of hen egg white lysozyme
(250 and 300 mg/kg) as a natural antimicrobial to prolong the shelf life of soft cheese
made from buffalo milk. The results revealed that the enzyme gave better result at 300
mg/Kg concentration, contributing clearly to reduce the development of total count of
bacteria also the count of psychrophilic bacteria and yeast and mould. The antimicro-
bial activity of lysozyme combined with EDTA against spoilage microorganisms (coli-
forms and Pseudomonas spp.) of dairy products was confirmed by Sinigaglia et al.
(2008). Mozzarella cheeses containing lysozyme (0.25 mg mL‐1) and different amounts
of Na2‐EDTA (10, 20 and 50 mmol L‐1) were studied. The authors found that the
lysozyme and Na2‐EDTA significantly inhibited the growth of coliforms and
Pseudomonadaceae during the first 7 days of storage, whereas the lactic acid bacteria
were not affected. An alginate/lysozyme nanomultilayer coating in Coalho cheese was
evaluated (Medeiros et al., 2014). The authors found that an shelf life extension of
cheese was obtained—in particular, mesophilic and psychrotropic microbial counts and
the visual valuation of fungal contamination were also found to be lower on coated
cheese than on uncoated cheese. The lysozyme, incorporated in zein and zein–wax
composite films, was tested only or in combination with catechin and gallic acid against
L. monocytogenes inoculated on fresh Kashar cheese. The results confirmed the antimi-
crobial effect of the enzyme; all lysozyme containing films prevented the increase of L.
monocytogenes counts in Kashar cheese, but it was only the zein‐wax composite films
with sustained lysozyme‐release rates which caused a significant reduction (–0.4 deci-
mals) in initial microbial load. The mixture of catechin and gallic acid showed no con-
siderable antimicrobial effect in cheese (Ünalan etal., 2013). Lysozyme is also used to
prevent the late gas blowing of hard cheeses that is caused by the growth of Clostridium
tyrobutyricum in Gauda, Edam, Provolone and Emmentaler in United States (Currel
and Dam‐Mieras, 2014).
Lactoferrin is a multifunctional iron‐binding protein belonging to the transferrin family
and it is found on mucosal surfaces and in biological fluids, including milk, saliva and
seminal fluid, indicating that it may play a protective role in the innate immune response.
Lactoferrin and its peptides are a promising class of antimicrobial compounds in the
fight against pathogenic microorganisms, including S. aureus, E. coli, and L. monocy-
togenes (Dionysius and Milne, 1997), Streptococcus mutans and Vibrio cholerae (Farnaud
and Evans, 2003). The bactericidal function of bovine lactoferrin is partially the result
of the direct interaction between the positive charged regions with anionic molecules
present on the surface of some microorganisms, which causes an increase in the mem-
brane’s permeability, inflicting damage to the bacteria (Haversen, 2010).
Antimicrobial Agents ofAnimal Origin
The antibacterial activity of lactoferrin added at doses of 2% and 4% to Minas fres-
cal cheese inoculated with S. aureus was estimated. Data revealed that the lactoferrin
prevented the increase of the S. aureus population in the cheeses at the two concen-
trations tested, and the antimicrobial effect showed to be dose‐dependent.
Furthermore, initially the enzyme presented an action bacteriostatic, then a bacteri-
cidal action was observed after the 15th day of storage when lactoferrin was added at
a concentration of 2%, and after the 8th day at the concentration of 4% (Santana da
Silva etal., 2012). Another study on Minas frescal cheese‐added lactoferrin (2 and 4
mg/g) was conducted by Inay etal. (2012). Psychotropic population in the cheeses
added by lactoferrin did not differ from control cheese, demonstrating that no anti-
microbial activity occurred in the products. Quintieri etal. (2012) studied antimicro-
bial efficacy of pepsin‐digested bovine lactoferrin (LFH) on spoilage bacteria
contaminating traditional high‐moisture mozzarella cheese. These natural substances
were effective when tested in vitro against five potential spoilage bacteria contaminat-
ing cold stored mozzarella cheese. Only the fraction containing lactoferricins, were
effective against E. coli as the whole pepsin‐digested hydrolysate. The LFH tested on
cold‐stored commercial mozzarella cheese delayed significantly the growth of pseu-
domonads and coliforms in comparison with the untreated samples. The pepsin‐
digested bovine lactoferrin was used also to prevent blue discoloration of mozzarella
cheese caused by P. fluorescens (Caputo etal., 2015). The authors discovered that the
mozzarella cheese samples treated with LFH and inoculated with a selected P. fluore-
scens strain showed no pigmentation and changes in casein profiles up to 14 days of
cold storage. Furthermore from day 5, the count of P. fluorescens spoiling strain was
steadily ca. one log cycle lower than that of LFH‐free samples. A plasma coating func-
tionalized by bovine lactoferrin (LB) and lactoferricin (LfcinB) was studied to make
an active packaging useful to control cheese spoilage by Pseudomonas. The LfcinB
immobilized on plasma confirmed its higher antimicrobial activity with respect to
BLF, against strains belonging to three Pseudomonas species (Quintieri etal., 2013).
Shashikumar and Puranik (2011) used lattoferrin at different levels (10, 15 and 20
ppm) in Paneer, a traditional Indian milk. It was observed that, as the level of lactofer-
rin in the product increased, there was a significant decrease in the bacterial growth
when compared to the control.
Lactoperoxidase is a native enzyme in animal secretions such as saliva, tear‐fluid, and
milk. Lactoperoxidase catalyzes the oxidation of thiocyanate ion by peroxide into short‐
lived reactive oxidation products, such as hypothiocyanite anion (García‐Graells etal.,
2000). This compound, in turn, oxidizes enzymes and other proteins in the bacterial cell
membrane that have exposed sulfhydryl groups, which inhibit the growth of microor-
ganisms (Kussendrager and Van Hooijdonk 2000). Lactoperoxidase system has been
proposed as a biopreservative in milk and other products. Arqués etal. (2008) studied
the effect of lactoperoxidase combined with nisin and reuterin against L. monocytogenes
and S. aureus in enhancing shelf and safety of cuajada, a semi‐solid cheese manufac-
tured in Spain. The most pronounced decrease in pathogen counts was achieved by the
triple combination, which acted synergistically on the inactivation of L. monocytogenes
and S. aureus in cuajada over 12 days at 10°C.
3.1.2 Antimicrobial Compounds Applied toDairy Food
Antimicrobial Agents of Chemical Origin
In cheese the most common problem is the development of molds and yeasts. In addi-
tion to an economic problem, mold growth on cheese is a potential health hazard.
Certain molds produce toxic substances called mycotoxins, which may cause acute or
chronic disease conditions in humans. These molds are able to produce mycotoxins not
only in cheese but also on a variety of dairy products.
The number of chemical preservatives approved to counteract the microbial problem
in dairy foods is remarkably small. Good milk and dairy products chemical preserva-
tives must be nontoxic, easy removable, leave no detectable residues, and be inexpen-
sive and easy to apply. The efficiency of chemical preservatives depends primarily on
the concentration of preservative, the composition of food and the type of microorgan-
ism to be inhibited. Widely used chemical preservatives are benzoic and sorbic acids
and their salts (sodium benzoate and potassium sorbate) (Tfouni and Toledo, 2002),
calcium lactate, and calcium ascorbate. They are generally used to inhibit mold and
yeast growth and are also effective against many bacteria (Can etal., 2011). Benzoic acid
is manufactured from toluene, a petroleum byproduct. It is classified as a carboxylic
acid, weakly acidic, with a pH of 2.8. Both benzoic acid and sodium benzoate have
inhibitory effects on yeast growth. Due to the fact that benzoic acid is toxic, the amount
of benzoates that can be added to foods is carefully controlled. Codex Alimentarius, an
international treaty dictating food safety standard, limits the amount of benzoic acid to
0–5 mg/kg of the body weight (WHO, 1996). Sodium benzoate, which is the sodium salt
of benzoic acid, is preferred compared to benzoic acid since its solubility in water is
higher than that of benzoic acid (Pylypiw and Grether, 2000). Akpan etal. (2007) com-
pared the effect of sodium benzoate and potassium metabisulphates on soya beans
yogurt shelf life. They found that the addition of sodium benzoate at a concentration of
20 mg/mL combined with refrigeration storage, improved yogurt shelf life for 21 days
thus proving better than potassium metabisulphates. In another study, Gul and
Dervisoglu (2013) revealed that the use of sodium benzoate and potassium sorbate
improved the microbiological quality of Kashar cheese that is a typical semi‐hard
Turkish cheese, only if combined with milk pasteurization and hygienic production
conditions. Lucera etal. (2014) elaborated an active sodium alginate coating with differ-
ent chemical preservatives: potassium sorbate, sodium benzoate, calcium lactate, and
calcium ascorbate in order to choose the best chemical preservative for Fior di latte
cheese, that is a typical Mediterranean pasta filata cheese. Only potassium sorbate and
sodium benzoate showed good results, delaying Pseudomonas spp. and
Enterobacteriaceae growth without affecting sensory characteristics. Faccia etal. (2013)
studied the effect of calcium lactate added in the brine of Fior di latte cheese with the
final results that 1.2% (w/v) of calcium lactate exerted an antimicrobial action against
Pseudomonas and Enterobacteriaceae. Sorbic acid and its salts have less harmful effect
than benzoic acid since they are metabolized rapidly like some fatty acids (as butyric,
caproic acid) in human and animals (Koyuncu and Uylaser, 2009). The acceptable daily
intake (ADI) value of sorbic acid and its salts is 0–25 mg/kg body weight as established
by the JECFA (WHO, 1974). Sorbate preservatives are effective inhibitors of most com-
mon microorganism that can attack foods, without affecting sensory quality. Öksuztepe
etal. (2010) studied the addition of potassium sorbate on microbiological quality of
Cokelek, that is a typical Turkish dairy product. The addition of potassium sorbate at
0.1% w/v revealed a strong antimicrobial activity against molds and yeast. Potassium
sorbate proved to be effective also in improving yogurt microbial quality as demon-
strated by Rajapaksha etal. (2013), which have added potassium sorbate at 0.1% (w/v)
during the inoculation of milk with starter cultures, with a final result of no contamina-
tion by coliforms, E. coli, Salmonella, and molds. Mastromatteo etal. (2015) studied the
combination of MAP and an active sodium alginate coating with 3% of potassium sorb-
ate on Fior di latte shelf life, finding that the treatment reduced physiological changes
and microbial growth increasing the shelf life of 157%. The study of Azza and Ahmed
(2010) used in conjunction bio and chemical preservation to improve the microbiologi-
cal quality of soft cheese. Specifically, they used Bifidobacterium longum and potassium
sorbate during the production process of soft cheese with the final result of a strong
inhibition of the main deteriorative microorganism of cheese, preserving at the same
time its organoleptic quality. Another class of chemical substances used in dairy sector
are sucrose esters which have shown antimicrobial property against Gram‐positive bac-
teria and Gram‐negative bacteria as described in Table Wagh (2013) explored
the antimicrobial properties of synthesized lactose monolaurate against four Gram‐
positive and Gram‐negative bacteria in milk. The study confirmed the antimicrobial
activity of lactose monolaurate against the Gram‐positive bacteria but no activity was
found against Gram‐negative bacteria.
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Table Antimicrobial Effects ofSugar Esters.
Sugar Esters Microorganism Effect Reference
Sucrose monolaurate E.coli 0157:H7 Strong inhibition with sodium
Xiao 2011
Sucrose and fructose
palmitate and laurate
Bacillus cereus and
E. coli K12
Inhibition of Bacillus with 1% w/v
of sucrose laurate, limited
inhibition against E. coli
Habulin 2008
Erythritol and xylitol
Gram‐positive and
Inhibition of xylitol Monolaurate
against Bacillus cereus
Piao 2006
Synthetic sugar esters Gram‐positive and
Sucrose and maltose laurate
inhibited Bacillus and E.coli
Ferrer 2005
Maltose laurate,
Maltotriose laurate and
Sucrose laurate
All esters suppressed the growth
at0.05% to 2% w/v
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... Despite the increasing popularity of new preservation techniques such as ultrasound, highpressure, infrared irradiation and pulsed electric field, freezing is still generally the preferred process for shelf-life extension of food products. It may be attributed to its inexpensiveness, simplicity and conventionality (Tavman and Yilmaz, 2018). Freezing is considered a viable solution for increasing the interest in more sustainable supply chains globally (Baldwin, 2009). ...
... Regular freezing has a longer phase transition time which forms larger ice crystals that can cause fat globule destabilization. Expanding ice crystals disrupt fat emulsion resulting in the coalescence of fat (Tavman and Yilmaz, 2018). Fat globules are enclosed by milk fat globule membranes (MFGMs) which serve as protection against external damage. ...
... Moisture, total solids, titratable acidity and pH were not significantly different among treatments. It was reported that there was no significant difference between fresh or refrigerated milk and frozen milk samples in terms of pH, titratable acidity, peroxide value and apparent viscosity (Tavman and Yilmaz, 2018). All values obtained were within the range of milk standards (Kailasapathy, 2016). ...
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In the Philippines, milk production is often inconsistent due to seasonality leading to either shortage or surplus of milk. Before processing, dairy farmers and cooperatives subject their collected raw milk to low temperatures to delay the deterioration of milk quality. This process is performed to prevent profit loss, reduce food waste and generate a sustainable supply chain. The study determined the effects of refrigeration, freezing and blast freezing on preserving the quality of raw cow's milk before processing. Chemical, foaming, microbial and sensory characteristics were determined to compare the effect of each treatment. Milk samples were refrigerated at 4 °C for 12 h (RM), frozen at-18 °C for 16 h (FM), and blast frozen at-40 °C for 10 h (BFM). Before conducting the analyses, FM and BFM were thawed at 4 °C for 22 and 16 h, respectively. Fat (4.07%) and protein (2.97%) significantly decreased in FM while moisture, total solids, titratable acidity and pH did not significantly differ among treatments. The foam value and foam volume were significantly higher in RM (88.00% and 86.33 mL) and BFM (91.58% and 89.75 mL), respectively. BFM had the lowest counts of aerobic microorganisms (5.16 log10 CFU/mL), coliforms (1.38 log10 CFU/mL), and Escherichia coli (< 1.00 log10 CFU/mL). Milk samples were pasteurized at 63 °C for 30 min before the conduct of the sensory analysis. Sensory characteristics did not significantly differ among treatments. From the results, blast freezing can be generally considered the most viable solution for storing raw cow's milk before processing.
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Salt-free labneh samples were prepared from buffalo's milk using 2μL/100ml milk of both dill (T1) and caraway (T2) essential oils. Chemical, anti-oxidant and organoleptic properties of samples stored for 28 days at 7°C±2 were investigated. Microbiological examinations were also periodically carried out until 14 days of storage. Data illustrated that adding essential oils had slight effect on titratible acidity contents; however their values were increased in all samples during storage. Dill and caraway essential oils encouraged the production of total volatile fatty acids (TVFAs) contents in treated samples which were also gradually increased during storage. Whereas their lowest values were recorded for control samples either fresh or during 28 days of cold storage. Acetaldehyde and diacetyl contents were reached to their maximum values in treated samples (T1 & T2) till 14 days of storage then they were decreased till the end of storage period. Tyrosine took the same trend of TVFAs, while tryptophan took the opposite trend. Obtained results showed that T2 samples had the highest levels of tyrosine among all periods. On the other side; presence of dill and caraway essential oils enhanced the anti-oxidant activity and increased the scavenging of free radicals in treated samples comparing with control ones. Starter culture did not pronouncedly affected by using essential oils; while, total bacterial viable counts were increased for all samples till they reached their maximum counts after 7 days. Yeasts & molds as well as coliform bacteria were not detected in all samples through storage. Using dill and caraway essentials oils improved the sensory properties of salt-free labneh samples either fresh or during storage; where their body & texture became soft with desirable spreadability properties. Flavor of treated samples were also more pronounced and more favored. Essential oils enhanced the taste and odor of labneh also created refreshing mouth feeling. So, dill or caraway essential oil could be recommended for using to enhance the salt- free labneh quality as they are natural, cheap, healthy and medicinal materials.
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The present study examined the effect of partially purified hen egg white Lysozyme as a natural antimicrobial to prolong the shelf life of soft cheese. The mentioned enzyme with enzymatic activity equal to 39 unit/mL was added in two concentration 250 and 300 mg/kg curd of soft cheese made from buffalo milk treatments T1 and T2, respectively. Treatments cheese and control cheese (C) were storage for 15 days at 6±1°C. The results revealed that there were a significant differences in chemical composition between control and treatments of Iraqi Soft Cheese (ISC) at zero time, also the enzyme contributed clearly to reduce the development of total count of bacteria also the count of psychrophilic bacteria and yeast and mold in treatments cheese during storage, treatment T2 gave better result, in prolonged the shelf life of ISC as a regard to bacterial count and also to the results of the chemical analysis for protein degradation expressed as the percentage of non-protein nitrogen (NPN) from the total nitrogen (TN) (% NPN/TN), as well as fat degradation expressed as acid degree value (ADV) during storage and retained good quality all the periods of storage (15 days) according to the broader for BDI method compared with control cheese (C), which became outside the broader of acceptance after 3 days of storage. These results reflected on sensory evaluation result for T1 and T2 treatments which get the acceptance and the highest score along the period of storage.
Concentrated yogurt (labneh) was produced by straining cow milk set yogurt in cloth bags. Three plants derived essential oils cinnamon, cumin and mint oils, were added to final concentrations of 0.3, 0.5 and 0.8% each to extend the shelf life of labneh. The chemical, microbiological and organoleptic properties of the labneh stored at 6±1ºC for up to 24 days were determined. Addition of plant derived essential oils affected the pH and total volatile fatty acid values of the prepared labneh, while total solids and fat values were only slightly affected. Total therapeutic bacterial count, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. Bulgaricus in the treated labneh increased and reached a maximum after 8 days of storage where after it decreased until the end of the storage period. Coliform and staphylococcus bacteria were not detected, while yeasts and moulds were detected at insignificant in some treated labneh. Labneh containing 0.3% cinnamon, cumin or mint oils were organoleptically the most acceptable and it had a good body and texture that was similar to the untreated one. From the results of this study, it can be concluded that 0.3% of cinnamon can be used in order to increase the shelf life of labneh for up to 24 days, with higher level of total volatile free fatty acid and therapeutic bacteria counts and low level of total viable, molds and yeast count.
Frozen foods make up one of the biggest sectors in the food industry. Their popularity with consumers is due primarily to the variety they offer and their ability to retain a high standard of quality. Thorough and authoritative, the Handbook of Frozen Food Processing and Packaging provides the latest information on the art and science of correctly handling and containing frozen foods. The book is divided into five parts for ease of accessibility and comprehension. Fundamentals of Freezing explains the basics of freezing. Facilities for the Cold Chain focuses on freezing-related equipment and facilities. Quality and Safety of Frozen Foods stresses the importance of quality, safety, and the nutritional values of frozen foods. Monitoring and Measuring Techniques for Quality and Safety describes the methods and techniques used to measure and maintain the quality and safety of frozen foods. The final part, Packaging of Frozen Foods discusses topics such as the various packaging materials used, a description of packaging machinery, and the future developments foreseen in frozen food packaging. Providing chapters written by authors with esteemed academic and professional credentials, the Handbook of Frozen Food Processing and Packaging is an essential resource for scientists in the frozen food industry.