CHARACTERISTICS AND USES
OF NOVEL AND CONVENTIONAL
PRESERVATIVES FOR FRUIT
DRINKS AND BEVERAGES
2
E. Mani-López, M.A. Ríos-Corripio, A.C. Lorenzo-Leal,
E. Palou, A. López-Malo
Department of Chemical and Food Engineering, University of the Americas
Puebla, Puebla, Mexico
2.1
Fruit Beverages
Fruit beverages are produced from raw materials or preserved
semifinished products (like filtered-clarified juices, sieved puree,
and concentrates, among others). Their consumption has increased
worldwide in the last years due to different factors. One of these factors is their nutrimental content being generally low in minerals, proteins, and fat while rich in vitamins (A, C, and B group), moisture,
fiber, antioxidants, and polyphenols; so they could, probably, help
to manage dietary deficiencies. These beverages are also important
because some of them, like fruit juices and nectars, are listed in the
healthy eating dietary recommendations (Akusu et al., 2016; Chueca
et al., 2016; Horváth-Kerkai and Stéger-Máté, 2012, Kalia and Parshad,
2015; Petruzzi et al., 2017).
A big portion of the world’s fruit production, especially orange,
apple, grapefruit, mandarin, lemon, pineapple, grape, pear, tomato,
pomegranate, and cranberry is processed into juices, followed by fruit
nectars from fruits such as mango, guava, peach, apricot, passion fruit,
papaya, soursop (guanabana), strawberry, banana, and tamarind
(Reyes-De-Corcuera et al., 2014).
Processing adds an economic value to different raw materials, such as
transforming fruits into other food products, such as juices, nectars, and
musts/pomaces that could be stored and sold, reducing waste and minimizing losses that could occur to the fresh fruit (Curil et al., 2017). Over
the years, there has been an increase in processing technology, product
Preservatives for the Beverage Industry. https://doi.org/10.1016/B978-0-12-816685-7.00002-1
© 2019 Elsevier Inc. All rights reserved.
31
32 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
formulation, equipment design, and production of fruit beverages, resulting in a great range of fruit drinks differing in composition, raw materials,
nutrient content, sensory quality, and packaging; because of the industry development with the propose to find differentiated products that
meet consumers demands. Generally, fruit drinks are classified per their
content and/or composition, being the main difference among them the
brand name (Horváth-Kerkai and Stéger-Máté, 2012).
2.1.1 Classification
Mainly fruit beverages could be categorized into two groups: fruit
juice and fruit drinks. The first one is defined as 100% juice with no
extra ingredients added, and the second one is a beverage made from
fruits and other ingredients (like sugar), this kind of drinks usually
have only 10% of fruit juice (Leschewski et al., 2016).
Related to fruit content, Horváth-Kerkai and Stéger-Máté (2012)
indicated that there are three categories of fruit drinks: juices and fruit
musts, fruit nectars, and soft drinks with fruit content. Fruit musts
and juices are obtained by a mechanical process and thus have the
taste, color, and aroma of the original fruit, they are consumed fresh
immediately after production or preserved by heat treatments (such
as pasteurization). Therefore, additives, different from fruit, sugars,
and acids are not allowed in fruit musts and juices. At the same time,
this category is divided into two subcategories: transparent or cloudy
juices. The first subcategory refers to filtered juices and the second
one to juices with colloids (like citrus juices) and probably fruit fibers.
Nectars, in the other hand, are made from fruit juices diluted with
sugar syrup or with sieved juices from single or blends of fruit juices
(Horváth-Kerkai and Stéger-Máté, 2012).
On the other hand, Fellows and Hampton (1992) classified fruit
beverages as follows: (1) juices, as its name mentions it is a fruit juice
without additives, (2) nectars that contain between 25% and 50% of
fruit solids and must be drank immediately after opening, (3) squashes
that contain 25% of fruit pulp with sugar syrup, usually having preservatives and are diluted in water, (4) cordials that are known to be crystal clear squashes, and (5) syrups that are clear concentrated juices
that have high sugar contents; these drinks are mainly preserved by
pasteurization, but they maintain their natural acidity and/or their
high contents of sugar (Reyes-De-Corcuera et al., 2014).
Another way to classify fruit beverages is related to their shelf life,
such as fruit drinks that must be drank immediately after opening
(these kind of beverages could not need any preservative if properly
processed and packaged) and those that could be stored between
uses, containing allowed preservatives (Fellows and Hampton, 1992).
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 33
2.1.2
Quality Factors
Fruit beverages have high contents of carbohydrates, mainly easily metabolized dextroses (such as fructose and/or glucose), vitamins,
water, and complex nitrogen sources. The content of organic nitrogen
is usually low and they are generally presented as free amino acids.
Vitamin content depends on the type of fruit and the processes to
which the product is submitted; however, most of these beverages are
fortified. Also, they could have high contents of organic acids that are
influenced by pH (Akusu et al., 2016; Chueca et al., 2016; Reyes-DeCorcuera et al., 2014).
Quality of fruit beverages is given by different sensory (flavor, color,
aroma, appearance, and texture), nutrimental (vitamins, minerals,
and dietary fiber contents), and antioxidant factors (α-carotene, β-­
carotene, β-cryptoxanthin, anthocyanins, and lycopene); being some
parameters of their quality: pH, soluble solid, soluble solid to titratable
acidity ratio (expressed as percentage of different acids to BX), color,
cloud, vitamin C, and pulp contents (Reyes-De-Corcuera et al., 2014).
The quality of fruit drinks also depends on the quality of their raw
materials. Fruit ripeness is a crucial characteristic, because before of
the optimal fruit ripe there are less sugar content and aromas, and
when overriped, they could have less color compounds and reduced
acidity (sometimes reflected in vitamin C content). For example,
acidic fruit juices have desirable high contents of sugar and distinctive
aromas when they are in their optimal ripe (Curil et al., 2017; HorváthKerkai and Stéger-Máté, 2012).
Flavor, one of the most important and complex aspects of fruit
drinks, depends on different properties like viscosity, nonvolatile and
volatile compounds, and pulp content; it could be affected by treatment temperature and time. However, in some cases (like in apple
juice), the changes on flavor caused by thermal treatments do not affect the acceptance of the product (Reyes-De-Corcuera et al., 2014).
Another important aspect with regards to fruit beverages is color,
which is visualized at first sight by consumers, and could be affected
by enzymatic action or by anthocyanins, lycopene, and/or polyphenols losses (Danişman et al., 2015; Reyes-De-Corcuera et al., 2014).
Finally, some fruits have high contents of insoluble plant components (such as protopectin, fibers, hemicellulose, cellulose, starch, and
lipids) and colloid compounds (pectin, polyphenols, and proteins)
that could cause turbidity and precipitation in their juices. Depending
on the finished fruit drinks, insoluble components and colloid compounds should be entirely or partially eliminated by clarification or
filtration, with the propose of improving sensory (color, taste, aroma,
and flavor) attributes (Horváth-Kerkai and Stéger-Máté, 2012; ReyesDe-Corcuera et al., 2014).
34 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
2.1.3 Pathogenic and Spoilage Microorganisms of
Interest
Deterioration occurs in fruit beverages when there is a loss of nutrients, physicochemical changes, and/or microbial growth (Niir Project
Consultancy Board, 2012). Consumers usually reject foods that present changes in appearance, smell, or taste; this phenomenon is known
as food deterioration or spoilage. Spoilage primarily occurs because of
the proliferation of natural microbiota; in fruit beverages also because
they are in contact with air and environmental microorganisms during
handling (Chueca et al., 2016; Petruzzi et al., 2017; Aneja et al., 2014b).
Among the deteriorative microorganisms, yeasts are the most common group related to fruit drink deterioration; they can grow at low
pHs, high sugar contents, and low water activities, being fruit drinks
an ideal growing environment because of their high contents of carbohydrates, organic acids, and complex nitrogen sources (Chueca et al.,
2016). This group of microorganisms could cause turbidity, flocculation, pellicles, clumping, and production of CO2 and alcohol; mainly
due to metabolites of yeast activity in fruit drinks (Kregiel, 2015; Aneja
et al., 2014b).
Another important microbial group is the molds, which grow at almost the same conditions as yeast and could present mycelial mats
and musty or stale off-flavors in fruit beverages. Bacteria, contrary to
the previous groups, are more sensitive to low pHs, being lactic acid
bacteria (LAB) the most common bacteria group present in fruit
drinks, causing mainly off-flavors (Chueca et al., 2016; Kregiel, 2015;
Aneja et al., 2014b).
On the other hand, pathogenic microorganisms could also be
present in fruit drinks, generally because of contaminated raw materials (especially fruits). Lately, foodborne outbreaks related to fruit
beverages have increased; therefore, spoilage and pathogen bacteria
could be indicators of low quality in these kinds of drinks. Some of the
pathogenic and spoilage microorganism that have been found in selected fruit beverages are listed in Table 2.1 (Petruzzi et al., 2017; Aneja
et al., 2014b).
2.1.4 Main Preservation Methods
Among preservation methods that can be applied to fruit drinks,
thermal treatments are the main ones since they have provided undeniable results for a long time. These treatments are based on heating
the products until inactivation of spoilage and/or pathogenic microorganisms. Due to low acidity products (pH below to 4.5) vulnerability to microbial contamination and growth, thermal treatments and/
or adding of preservatives, became mandatory to produce stable shelf
life products (Petruzzi et al., 2017; Reyes-De-Corcuera et al., 2014).
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 35
Table 2.1 Pathogenic and Spoilage Microorganisms
Reported in Selected Fruit Beverages
Microorganism
Fruit Beverage
Reference
Apple and orange juice
Alberice et al., 2012; Bevilacqua et al., 2013a;
Pei et al., 2014
Khallaf-Allah et al., 2015
Aneja et al., 2014a
Somavat et al., 2013
Aneja et al., 2014a
Parker et al., 2010
Aguilar-Rosas et al., 2013
Aganovic et al., 2014
Aneja et al., 2014a
Chueca et al., 2016
Parker et al., 2010
Aganovic et al., 2014; Char et al., 2010
SPOILAGE BACTERIA
Alicyclobacillus
acidoterrestris
Acetobacter spp.
Bacillus coagulans
Bacillus subtilis
Clostridium sporogenes
Lactobacillus brevis
Lactobacillus plantarum
Leuconostoc spp.
Leuconostoc fallax
Listeria innocua
Orange nectar
Orange and carrot juice
Tomato juice
Orange and carrot juice
Papaya nectar
Apple juice
Tomato juice
Orange and carrot juice
Apple juice
Papaya nectar
Apple, orange, and tomato juice
PATHOGENIC BACTERIA
Bacillus cereus
Escherichia coli
Orange and carrot juice
Apple, prickly pear, mango, tomato,
orange, and carrot juice
Shigella flexneri
Listeria monocytogenes
Orange juice
Mango, pineapple, orange, and carrot
juice
Acerola, cashew, apple, and mango
nectar blend
Carrot, orange, and watermelon juice
Salmonella spp.
Salmonella Typhimurium
Staphylococcus aureus
Papaya, soursop, and guava nectar
Apple and orange juice
Carrot and orange juice
Aneja et al., 2014a
Ait-Ouazzou et al., 2013; Aganovic et al., 2014;
Aneja et al., 2014a; García-García et al., 2015;
Luis-Villaroya et al., 2015
Dewanti-Hariyadi, 2014
Backialakshmi et al., 2015; Firouzabadi et al.,
2014; Kamdem et al., 2010; Ngang et al., 2014
Da Silva et al., 2011
Danyluk et al., 2012; Sinchaipanit et al., 2013;
Dewanti-Hariyadi, 2014
Gabriel et al., 2015; Parker et al., 2010
Dewanti-Hariyadi, 2014; Park and Kang, 2013
Aneja et al., 2014a; Sinchaipanit et al., 2013
SPOILAGE YEAST AND MOLDS
Alternaria spp.
Cladosporium spp.
Colletotrichum spp.
Fusarium spp.
Geotrichum spp.
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Continued
36 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
Table 2.1 Pathogenic and Spoilage Microorganisms
Reported in Selected Fruit Beverages—cont’d
Microorganism
Fruit Beverage
Reference
Penicillium digitatum
Pichia spp.
Rhodotorula spp.
Saccharomyces cerevisiae
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Prickly pear, tomato, orange and carrot
juice
Apple and orange juice blend
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Aganovic et al., 2014; Aneja et al., 2014a;
García-García et al., 2015
Aganovic et al., 2014; Tyagi et al., 2014a
PATHOGENIC YEAST AND MOLDS
Aspergillus flavus
Aspergillus terreus
Aspergillus niger
Candida krusei
Candida parapsilosis
Curvularia
Penicillium islandicum
Orange and carrot juice
Orange and carrot juice
Orange, carrot and tomato juice
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Orange and carrot juice
Aneja et al., 2014a
Aneja et al., 2014a
Aganovic et al., 2014; Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Aneja et al., 2014a
Usually, fruit beverages are preserved by pasteurization, having the
target to reduce close to 5 log of the most resistant microorganism detected in the specific product. Pasteurization can be accomplished by
different techniques, such as, high temperature-long time (HTLT); this
thermal process uses temperatures between 90°C and 120°C for times
around 1–2 min, and it is based in outside heat generation, which is
transferred into food by mechanisms of convection or conduction.
Another technique is the one called high temperature-short time
(HTST), which ensures product safety, maintaining in some cases desirable bioactive compounds. This method uses temperatures ≥80°C
and times ≤30 s. Other variations are: mild temperature-long time
(MLTL) with temperatures below 80°C and times longer than 30 s, and
mild temperature-short time (MTST) also with temperatures below
80°C and periods of ≤30 s (Chueca et al., 2016; Petruzzi et al., 2017).
When fruit beverages are in contact with high temperatures,
even for short periods of time, there could be undesirable changes
in sensory and composition quality, decreasing fruit drinks benefits
to health. Sometimes, fruit drinks such as, blended fruit juices, are
pasteurized more than once because when juices and nectars are
extracted they are submitted to pasteurization, and then again when
they are blended (before packaging) causing even more damage in the
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 37
product qualities (Aganovic et al., 2016; Chueca et al., 2016; Petruzzi
et al., 2017).
At the same time, pasteurization can inactivate some enzymes
(peroxidase, polyphenoloxidase, pectin esterase, and polygalacturonase) that cause undesired changes. Such is the case of polyphenoloxidase, which is the one responsible for browning and degradation of
polyphenols and natural pigments in some fruit juices, causing losses
of the antioxidant activity and discoloration (Aganovic et al., 2016;
Petruzzi et al., 2017; Reyes-De-Corcuera et al., 2014).
Fruit drinks can also be preserved by aseptic packaging, being another method that utilizes high temperatures; this method consists in
sterilizing and processing the package and the fluid independently and
then, under aseptic conditions, hermetically seals them when brought
together. However, like pasteurization, these treatments could also
cause undesirable effects leading to quality and freshness reduction,
reflected in flavor, color, texture, appearance, nutrient, and pigment
losses. Therefore, some no-thermal methods such as ultrahigh pressures, electric pulses, UV, and/or ultrasound, have been proposed in
different studies (Aguilar-Rosas et al., 2013; Carbonell-Capella et al.,
2017; Horváth-Kerkai and Stéger-Máté, 2012; Petruzzi et al., 2017;
Pillai and Shayanfar, 2015; Shah et al., 2016).
On the other hand, chemical preservatives are also utilized for preserving fruit beverages, being the most common ones sodium benzoate and potassium sorbate. The type of chemical preservative to be
utilized in fruit drinks depends on the selected properties (physical
and chemical) of the beverage as well as of the preservative. Product
pH, vitamin content, packaging, and conditions of storage also may
influence the choice of the additive. However, consumers demand
for more natural, fruit products has increased over the years (Kregiel,
2015; Rupasinghe and Yu, 2012).
To minimize damages, as degradation of nutritional and fresh characteristics of fruit drinks, it is recommended refrigeration temperatures during storage and transportation (Aganovic et al., 2016; Chueca
et al., 2016; Petruzzi et al., 2017). The preservation of quality factors is
a key goal of the fruit beverage industry; therefore, the importance of
maintaining equilibrium between safety and nutritional quality of raw
materials (Petruzzi et al., 2017).
2.2 Preservatives
Foods and beverages often contain different types of food additives, among which preservatives play an important role (PetanovskaIlievska et al., 2017); these are included in one of the 26 major
additive categories that are utilized in foods (Kregiel, 2015). Before
the advent of preservatives, food was placed in containers such as
38 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
clay jars to preserve them from spoiling. Food storage can be traced
back to every ancient civilization such as Egyptian, Greek, Roman,
Sumerian, and Chinese (Anand and Sati, 2013). Preservatives are defined as substances that are added to products such as food or beverages to prevent, stop, and/or delay any food deterioration due to
microbial growth. An ideal food preservative remains effective until
the product is consumed (Gbonjubola and Josiah, 2012). The principal properties of preservatives include the following: retard or reduce the growth of undesirable microorganisms, fungi, and bacteria,
do not affect food texture or taste, be safe for human consumption,
and extend food shelf life (lengthen the time before a food product
begins to spoil). Shelf life is determined by rates of growth of spoilage microorganisms and chemical degradation of food components
(Akinwande et al., 2012).
Fruit juices are important commodities in the global market,
providing vast possibilities for new value-added products to meet
consumer demand for convenience, nutrition, and health. Fruit
juices are spoiled primarily due to proliferation of acid tolerant and
osmophilic microbiota. There is also risk of food-borne microbial
infections or intoxications, which may be associated with the consumption of fruit juices. In order to reduce the incidence of outbreaks, fruit juices are preserved by various techniques (Aneja et al.,
2014b). Traditionally, the stability of fruit drinks and beverage has
been achieved by thermal processing. However, thermal processing
tends to reduce the product quality and freshness; therefore, preservatives are a good option because these products display several advantages such as retention of sensory qualities and nutritional values
over traditional thermal processing (Rupasinghe and Yu, 2012). Food
preservatives may be classified as natural and conventional, which
play a very important role in the beverage industry, citric acid is a
good example of a naturally occurring preservative, sodium benzoate and potassium sorbate are representatives of the second type.
The choice of appropriate preservatives for fruit drinks and beverages should take into consideration specific product requirements,
the type of spoilage organisms associated with it, the product pH,
the intended shelf life, and the mode of application. The pH and nutritional parameters are among the most decisive factors in choosing
a preservative. In general, preservatives are only effective when the
initial microbial contamination level is low. Most microbiological
problems arise because of poor quality raw materials and poor process hygiene which lead to overcoming of the preservation system
applied during manufacture by the spoilage organisms (Riikka et al.,
2011). Good hygienic practices are essential to guarantee the quality
of the products (Sospedra et al., 2012).
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 39
2.2.1
Conventional Preservatives
Conventional preservatives are chemical substances that stop the
growth and activities of the microorganisms and help to preserve foods
for a longer time without affecting their natural characteristics; they
include antimicrobial agents and antioxidants (Fig. 2.1). Antimicrobial
agents are used to prevent or inhibit spoilage caused by molds, yeasts,
and bacteria (Dhaka et al., 2016). Some antimicrobial agents are benzoates, nitrites, calcium propionate, and sorbates. Antioxidants are
agents that are utilized to prevent the oxidation caused in the food
material. Selected antioxidants are butylated hydroxytoluene (BHT),
butylated hydroxyanisole (BHA), formaldehyde, and some organic
acids (Seetaramaiah et al., 2011). The majority of the preservatives
that are commonly utilized today are conventional rather than natural
(Anand and Sati, 2013) and have been utilized by the food industry for
decades. During processing and storage of fruit drinks and beverages,
they can suffer microbial contamination; thus, shelf life of beverages
can be extended by the addition of conventional preservatives that are
applied to improve their microbiological stability.
2.2.1.1 Sulfur Compounds and Benzoic and Sorbic Acids and
Their Salts
There are two types of packaged fruit drinks, those that are drank
straight after opening and those that are consumed in portions, so
Fig. 2.1 Conventional preservatives commonly utilized in fruit drinks and beverages.
40 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
they must be stored between drinking times. The former groups
should not require any preservative if they are properly processed and
packaged. However, the latter group must contain a certain amount of
permitted preservatives to have a long shelf life after opening. As soon
as the juice is expressed from the fruit, it starts to deteriorate, both as
a result of chemical activity (enzymatic action) and microbial spoilage. Chemical preservatives often supplement other types of preservation methods, to ensure an economical, safe, and flavorful product
for months or even years after preparation (Yadav et al., 2014). Some
research has revealed that the shelf life of fruit drinks without any preservative was <2 days because of their high sugar content and favorable acidity conditions for the growth of yeast and molds. Heating the
fruit drinks to 90°C increased their shelf life for some days but resulted
in losses of color and nutritional quality of the fruit drink. Hence,
conventional preservatives are commonly utilized. The constituents
of processed fruit drinks are mainly water, sugar, chemicals preservatives, colors, and fruit pulp (Shahnawaz et al., 2013). There are several
conventional preservatives that can be added to fruit drinks among
which benzoic and sorbic acids are widely utilized (Ruziye and Arzu,
2013). Processors need to check with local authorities or regulatory
agencies to find the maximum permitted levels. Preservatives typically
utilized on commercially available products contain bacteriostatic or
fungistatic compounds belonging to the group of the weak acids, such
as benzoic and sorbic acids. The effectiveness of these agents depends
on various factors including product pH, microbial load, and the intrinsic resistance of the microorganisms present after packaging.
Although preservatives can be successfully utilized in beverage
formulations, they should never be considered infallible and there is
no substitute for severe quality and hygiene controls at every stage of
manufacture (Steen and Ashurst, 2006). Due to utilization of sugars
in fruit drinks, yeasts are of most immediate concern. Together with
molds and bacteria, they can bring about deterioration in flavor, producing taints, off-notes, and differences in mouth feel. Fruit drinks
provide an ideal growth substrate for many microorganisms, given
adequate supplies of the required nutrients. Apart from water, typical
microbial requirements include sources of carbon (carbohydrates), nitrogen (amino acids), phosphorus (phosphates), potassium, calcium
(mineral salts), and traces of other minerals, for example, sulfur, iron,
cobalt, and even vitamins. Also, when beverages include fruit pulp or
caramel (coloring), there will be a greater susceptibility to spoilage by
certain microorganisms (Steen and Ashurst, 2006). Conventional preservatives, which perform by a direct inhibiting action on the microorganisms themselves are not new. Table 2.2 lists the most commonly
conventional preservatives utilized by the beverage industry; these
preservatives are generally recognized as safe (GRAS).
Table 2.2 Summary of the Main Characteristics of Conventional
Preservatives Utilized in Fruit Beverages
Preservative
E-No. (Codex
Alimentarius)
Alternative Form
Used at Equivalent
Level
E-No.
Microorganisms
pH
Dose (ppm)
Sulfur dioxide (gas)
E220
Sodium sulfite
E221
pH < 4.0
<20
Sorbic acid
E200
Sodium sorbate
E201
Range 4.0–6.5
300
Benzoic acid
E210
Sodium benzoate
E211
Bacteria, molds, and
yeasts
Yeasts, molds, and
bacteria
Molds, yeasts, and
bacteria
Low pH values
(2.0–4.5)
150–200
42 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
Sulfur dioxide (SO2) and sulfites have been extensively used as antimicrobials for many centuries and are very effective preservatives.
Because of its ease of production, gaseous SO2 was one of the first
chemical compounds manufactured and utilized by humans (Steen
and Ashurst, 2006). The oldest reference on sulfur dioxide dates back
to Roman times when sulfur was burnt and the unfermented juice
exposed to the fumes to help for preservation of wine. Sulfites were
added to casks of lemon and lime juices in the 19th century to preserve
the fruit juice and help to prevent scurvy on ocean-going ships. Sulfur
dioxide is one of the most versatile agents utilized for food preservation and is well known for its microbiocidal effect on bacteria, molds,
and yeasts (Steen and Ashurst, 2006); bacteria are more sensitive to
SO2 than fungi. Sulfites have numerous functions apart from their antimicrobial activity, as they are also used as antioxidants, antibrowning agents, and color stabilizers.
The free forms of sulfites are more active than the bound forms
of sulfur dioxide. In fruit drinks, the sulfur compounds or their salts
have the dual mission of being antiseptic and antioxidant; for the first
function, a concentration of at least 0.1% is required but for the second one, 0.02% would be sufficient. The literature shows examples of
the use of sulfites in fruit drinks specifically for lemon, orange, passion
fruit (Passiflora sp.), and grape beverages.
The microbiocidal effect increases as the pH falls below 4.0; because
of this, SO2 is ideally suited for most soft drinks formulations (Steen and
Ashurst, 2006). Sulfites are reactive compounds and their association
with certain juice components and microbial metabolites (especially
carbonyls) reduces their antimicrobial efficacy (Jarvis, 2003).
Sulfites and metabisulfites of sodium or potassium are added to
fruit juices as potential sources of sulfur dioxide, which acts as an antimicrobial agent and also stabilizes ascorbic acid. Use of metabisulfite
providing up to 400 ppm of sulfur dioxide in orange juice has been reported. However, such high levels of sulfur dioxide are likely to impart
a characteristic pungent smell to fruit juices (Shahnawaz et al., 2013).
Benzoic acid occurs naturally in a number of fruits and vegetables and is also found in its free form in some resins, principally in
gum benzoin (from Styrax benzoia) and in coal tar. Sodium benzoate is a sodium salt, obtained by reacting benzoic acid with sodium
hydroxide, it is a conventional preservative that has been in use for
many years, which in very low concentration inhibits yeast, mold, and
bacteria. Although the benzoic acid precursor is more effective in its
antimicrobial function, benzoate is used because of its much higher
solubility (about 200 times more water soluble), so this salt is favored
as a preservative for practically any processed food, especially soft
drinks due to the use of high-fructose corn syrup (HFCS) as sweetener
(Onwordi et al., 2017). As early as 1909, the “harmlessness” of sodium
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 43
­ enzoate as a food preservative was extensively verified. The United
b
States Food and Drug Administration (FDA) has studied sodium benzoate extensively and found that it is safe when consumed in amounts
found in normal diets. For a long time, sodium benzoate has been
GRAS as a direct food additive. Sodium benzoate is a type of preservative commonly utilized in foods that have an acidic pH (Gbonjubola
and Josiah, 2012). It is the free or undissociated form of benzoic acid
that exhibits preservative action, and hence its use is only effective
when low pH values are encountered, ideally below pH 3.0, at which
the degree of dissociation of the acid is reduced to <10% (Mitchell,
1990). Sodium benzoate may be utilized in foods at levels not to exceed good manufacturing practices. Most national and international
legislations provide for the use of sodium benzoate for fruit drinks.
Sodium benzoate has been used as a preservative for various juice extracts during processing steps and in the finished products. Although
fruit juices often have a pH suitable for the antimicrobial action of
benzoates, it is necessary to ensure that the acidity of the medium is
sufficient. But, for products which are richer in proteins, part of the
benzoic acid is combined with these, thus its preservative action may
be reduced. Sodium benzoate is most suitable for use as an antimicrobial agent in beverages that naturally are in the pH range below 4.5, or
can be brought into this range by addition of a water-soluble acidulant.
The maximum accepted level of benzoic acid in beverages stipulated
by national and European legislation is 150 ppm (Kusi and Acquaah,
2014). Current usage results in a maximum level of 0.1% in foods.
Fruit drinks normally require concentrations of 0.05%–0.10% sodium
benzoate in the finished products. Benzoic acid is also utilized in fruit
juices and other acid products for its antimicrobial activity, for example, in passion fruit juice; it was found that the sample containing benzoic acid showed better preservation. Since the two chemicals exert
either one or both of synergistic or concerted actions, it is possible that
benzoic acid makes a significant contribution to protection of ascorbic
acid. Other important factor in preserving with sodium benzoate is the
addition of the antimicrobial as early as possible in the food processing operations. The early addition of sodium benzoate will prevent the
microorganisms from forming enzymes, which may continue to cause
deterioration even though the microorganism growth will be inhibited
at a later stage in processing. Benzoic acid is often used in conjunction
with sulfur dioxide where it is claimed that the “joint” performance is
better, owing to a synergistic effect (Mitchell, 1990). One of the most
important considerations in preserving with sodium benzoate is the
maintenance of absolute hygiene and sanitation. It should be clearly
understood that although preservatives such as sodium benzoate
serve a very useful purpose in foods, they could not take the place of
cleanliness in food processing. Products that have already spoiled will
44 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
not benefit from the use of sodium benzoate as a preservative. The
most problematic spoilage organisms of soft drinks are notoriously
resistant to preservatives. Although an excellent activity is recognized
for benzoates, it should not be forgotten that their activity depends to
a large extent on the conditions in which they are utilized. In practice, the most advantageous way of applying benzoates is to add them
initially to the packaging, before introducing the content, using dissolved, because in this way all the product is impregnated with this
salt. The benzoic acid is then liberated by adding, shortly before the
final closure, the necessary amount of acid, which must also be previously dissolved. In the case of very acidic juices, it is still interesting to
use benzoate in dissolved form and also throw it slowly and with agitation to prevent the precipitation of benzoic immediately; since can
be so large that it is deposited without fulfilling its mandate. Recently,
sodium benzoate has been replaced in the majority of fruit drinks and
beverages by potassium sorbate (Saltmarsh, 2015).
Sorbic acid is a safe, nontoxic, and effective food preservative; its
salts are collectively known as sorbates; the salts of sorbic acid include
sodium sorbate, potassium sorbate, and calcium sorbate. These salts
are usually preferred for food applications. This is because they are
more soluble in water but the active form is the acid, with a pKa of
4.76; it is about as acidic as acetic acid. Over the last 40 years, sorbates
have been tested and extensively utilized for preserving a variety of
food products throughout the world (Mendonca, 1992), due to their
antimicrobial properties, preserved freshness, and keeping the original flavor of foods. Their antimicrobial effects and safety of concentrations used in foods have been well established (Lueck, 1980; Sofos and
Busta, 1981). Sorbic acid is a natural compound that is also made synthetically. First isolated from berries in the 1800s, it became commercially available for use as a food preservative in the 1940s and 1950s.
Sorbic acid controls and prevents the growth of yeast, mold, and bacteria (Mukta and Anu, 2013). A mixture of preservatives is usually
more effective than a single preservative due to their synergistic action
(Wind and Restaino, 1995). Sorbic acid may be used in a mixture with
other preservatives, for example, with sulfur dioxide; in synergy will
play a greater role. The optimal pH for the antimicrobial activity is below pH 6.5; sorbates show reduced activity with increases in pH; however, the upper limit of effectiveness is considerably higher than for
benzoic acid, at around pH 6.0–6.5. It is the undissociated form that inhibits microbial growth. Sorbates are generally used at concentrations
of 0.025%–0.100%. Sorbates have been specifically applied in different
types of fruit beverages, such as apple, orange, grape, lemon, pineapple, and pomegranate. Sorbic acid is on the FDA list of GRAS substances. The Select Committee on GRAS Substances Opinion asserted
in 1975 that sorbic acid poses no hazard to health when ­consumed at
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 45
the typical levels found in food, according to the FDA. It is an unsaturated fatty acid that can participate in normal metabolism function,
can be oxidized to water and carbon dioxide, and does not accumulate
in the human body.
2.2.1.2
Mode of Action
The mechanism of action of SO2 is not fully understood. One suggestion is that the undissociated sulfurous acid or molecular SO2 is
responsible for the antimicrobial activity. Its greater effectiveness at
low pH tends to support this. Ingram and Vas (1950) suggested lowering of pH of certain foods by addition of acid as a means of obtaining
greater preservation with SO2. It has been suggested that the antimicrobial action is due to the strong reducing power that allows these
compounds to reduce oxygen tension to a point below which aerobic
organisms can grow or by direct action on some enzymatic systems
(Jay et al., 2005).
Ought and Were (2005) mentioned that SO2 antimicrobial efficacy
is mainly based on the molecular form that freely diffuses through cell
membranes and decreases intracellular pH. Sulfites are also highly reactive, inactivating various macromolecules.
As mentioned before, the effectiveness of sodium benzoate as a
preservative increases with decreasing pH (increasing acidity). This is
because the ratio of undissociated (i.e., free) benzoic acid to ionized
benzoic acid increases as the pH decreases. It is generally accepted
that the undissociated benzoic acid is the active antimicrobial agent.
Although no definite theory has been yet proposed to explain its antimicrobial effect, it is believed to be related to the high lipid solubility
of the undissociated benzoic acid which allows it to accumulate on
the cell membranes or on various structures and surfaces of the bacterial cell, effectively inhibiting its cellular activity. Benzoic acid would
uncouple substrate transport and oxidative phosphorylation from the
election transport system by making the cytoplasmic membrane freely
permeable to protons (Lou et al., 2007). Olutimayin et al. (2001) reported that benzoates interfere with the utilization of acetate required
for the function of energy-rich compounds, which results in blockage
of cell metabolism. Although several studies have been performed on
the antimicrobial activity of sodium benzoate, it is difficult to obtain
substantial evidence on relative activities of sodium benzoate against
specific members of microbial species. Also, these studies mentioned
that benzoic acid and sodium benzoate have been reported to be
less active against Gram-negative bacteria. Actual field application
trials are recommended for assurance of satisfactory antimicrobial
activity against the microbial species in question. Among other factors, the lack of sensitivity to the antimicrobial agent by bacteria may
be due to inability of the agent to diffuse into the cell, and cellular
46 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
i­ mpermeability which leads to a reduced concentration of the antimicrobial compound available at the target site so that the cell may avoid
injury (Denyer and Russell, 2004).
Sofos et al. (1986) proposed that sorbic acid and its salts inhibit various bacteria, including spore formers, at various stages of their life cycle (germination, outgrowth, and cell division). This multiple action of
sorbates may be responsible for their broad effectiveness compared to
other antimicrobial agents. Inhibition of bacterial growth by sorbates
may result from alteration of cell membranes, inhibition of transport
systems and key enzymes, creation of a proton flux into the cell, or
more than one of these actions. In reduced pH environments, spore
formers are inhibited by both increased hydrogen ion concentration
and the presence of sorbate molecules. Recent evidence has suggested that sorbates inhibit spore germination by acting on postbinding stages of the process (e.g., the connecting reactions). Inhibition of
the connecting reactions by sorbates may be through some interaction
of these compounds with the spore membranes or through inhibition
of enzymes. Hesse et al. (2002) mentioned that the antimicrobial activity of sorbic acid in aqueous solution is pH dependent, with the
maximum effect occurring at low pH, thus favoring the undissociated state of the acid; because they are uncharged, undissociated acid
molecules are lipophilic and will penetrate plasma membranes and
thus enter cells. Theoretically, the higher-pH environment of the cell
cytosol (ca. pH 7.8 in Aspergillus niger) promotes the rapid dissociation of acid molecules into charged protons and anions, which cannot
subsequently diffuse back across the plasma membrane. Intracellular
acidification of the cell cytosol resulting from the accumulation of protons inhibits key metabolic activities involved in glycolysis and hence
inhibits adenosine triphosphate (ATP) yields.
2.2.1.3 Quality Factors
The main aim of food processing is to provide well-being to humans through a safe and nutritionally adequate diet while meeting
their expectations of taste, aroma, and appearance. The food industry has as challenge, more long-lasting products by sacrificing to a
minimum their nutritional characteristics and sensory attributes. The
impulse of the fruit drinks industry, like the rest of the food sectors,
is known to seek improvement and competiveness within the market in order to offer a safe and quality product. The beverage industry
has used antimicrobials to prevent quality losses, meaning that the
sensory properties of the product are not affected during processing, procurement, storage, and consumption. In fruit drinks when
preservatives are added, the physical, chemical, organoleptic, and
nutritional characteristics must be maintained without the additive
having an adverse effect. Sarkar et al. (2014) showed that flavonoids
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 47
can be preserved by the use of sodium benzoate and the juices can be
consumed with same effectiveness as the fresh ones, even after refrigerated storage. The chemical preservatives utilized by the beverage industry are already very well established and there are few or no quality
losses that could cause in beverages. As mentioned above, sorbates
and benzoates are the main antimicrobials used by the beverage industry to preserve the quality of their products; however, they must
be handled with some care to avoid that the presence of them could
affect the sensory properties of the final product, an example of them
is sodium benzoate that at low pH values, may impart a slight tang in
taste attributable to the undissociated benzoic acid. If this effect is undesirable, it may be overcome by using other approved preservatives
is in conjunction with sodium benzoate to lower the concentration
of sodium benzoate below the taste threshold. For sorbic acid, one of
its disadvantages is that it affects the taste and pH of foods; however,
research has shown that the main conventional preservatives used in
beverages do not impart strange flavors and maintain the chemical
and organoleptic stability of the final product.
Some disadvantages associated with the presence of sulfur dioxide present in beverages are that some people can detect it as an unpleasant back note or taint and that it can provoke allergic reactions.
Asthma sufferers tend to be susceptible to the effect of gaseous SO2,
small traces of which can promote an asthmatic attack, and foods containing sulfites can have an associated risk of gas liberation when they
are swallowed (Steen and Ashurst, 2006).
Another important parameter that must be taken into account to
maintain the quality of the product when using preservatives is that
benzoic and sorbic acids and their salts are permitted food additives
by international laws for processing in restrictive amounts, but their
content must be declared and must not exceed the established limits
by legislation (Gomaa et al., 2013).
Lately, benzoate use has aroused some controversy around the
world; some studies suggested that benzoate decomposes to form
benzene a known carcinogen, especially in beverages that also contain ascorbic acid and/or citric acid; however, in most beverages
which contain it, benzene levels are below those considered harmful
for consumption. Heat, light, and shelf life can affect the rate at which
benzene is formed. Given this information, regulatory agencies in various countries have requested more information on the concentration
of benzene present and its potential toxicity for humans. For example,
the UK Agency reviewed the study originally published by Peter Piper
of the University of Sheffield. After reviewing this study, they found
that the relevance of exposure to humans was not clear. However,
many of the major soft drink and beverage companies have decided
to look for alternatives to ingredients that would allow them to “clean”
48 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
their labels by means of natural ingredients, while some directives on
the presence of benzene in beverages have companies try to limit the
irrational use of sodium benzoate.
The use of preservatives is important to maintain the stability of
fruit drinks and to be able to continue offering the consumer products
that make life easier. However, it is important to evolve. Current consumer demands for convenience and taste but also requiring products
with more “green,” “natural,” or “clean” labels, are encouraging the
search for suitable alternatives.
2.2.2 Novel Preservatives
Natural preservatives are considered as novel preservatives due to
being recently proposed to be utilized in fruit drinks and beverages.
Most of them are under research and very few are satisfying the desirable characteristics of a food preservative. A novel preservative for
fruit drinks and beverages must be effective against to a broad spectrum of microorganisms, stable at low pH and heat treatment, with
good aqueous solubility, from natural origin, cheap, easy to handle,
and without negative effect on sensory and physicochemical properties. Nevertheless, the ideal novel preservative does not exist, in spite
of good and interesting proposals reported. Naturalness is considering
an important attribute for consumers, thus “natural” foods are judged
as safe or healthy. On the other side, preservatives are necessary to
extend shelf life and ensure food safety, thus natural preservatives are
an alternative to be explored.
2.2.2.1 Essential Oils
An essential oil (EO) is a complex mixture of aromatic and volatile compounds obtained from raw material of plants such as flowers,
leaves, fruits, woods, seeds, barks, roots, and fruit peels, by steam or
dry distillation or pressing. EOs are a blend of compounds, and may
contain approximately 20–80 individual components (ICs) at different
concentrations. Most common EOs studied for fruit drinks and beverages applications are obtained from citrus, cinnamon, wild mint, lemongrass, eucalyptus, clove, black pepper, oregano, and thyme. Also ICs
from EOs had been tested to preserve fruit drinks and beverages such
as citral, eugenol, cinnamaldehyde, carvacrol, and thymol, among
others.
EOs have shown antimicrobial activity against spoilage yeasts
such as Saccharomyces cerevisiae, Torulaspora desbrueckii,
Zygosaccharomyces bailii, Yarrowia lipolytica, Pichia membranifaciens, and Dekkera anomala (Araújo et al., 2003; Belletti et al.,
2007). Eucalyptus EO (4.5 mg/mL) inactivated 2.5 log CFU/mL of
Saccharomyces cerevisiae during 8 days at room temperature in a 1:1
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 49
fruit juice mixture (apple:orange); when thermal treatment of 70°C
for 90 s was applied to the same juice mixture only a half of the concentration of EO was required to obtain the same inactivation level
at the same storage conditions (Tyagi et al., 2014b). The effect of suspended particles in the antimicrobial activity of EOs was evaluated by
Tserennadmid et al. (2011); they assessed the inhibitory effect of the
sage, juniper, lemon, and marjoram EOs against Geotrichum candidum,
Pichia anomala, Saccharomyces cerevisiae, and Schizosaccharomyces
pombe in cloudy and clear apple juice; minimum inhibitory concentrations (MICs) were between 1 and 4 μL/mL for every tested EO and
for both juices; however, higher MICs were required for cloudy apple
juice. Probably, the adherence of EOs to juice suspended particles resulted in a reduced antimicrobial activity. Spores of deteriorative bacteria such as Acinetobacter acidoterrestris were inhibited with lemon
EO (0.08%, 0.12%, or 0.16%) in lemon juice concentrate over 11 days in
refrigerated storage (Maldonado et al., 2013).
EOs have been assessed for their antimicrobial activity in fruit
juices also against pathogenic bacteria. EOs of Mentha arvensis L.
(0.625 μL/mL) and Mentha piperita L. (1.25 μL/mL) inactivated 5.0
log CFU/mL of Escherichia coli UFPEDA 224, Listeria monocytogenes
ATCC 7644, and Salmonella Enteritidis UFPEDA 414 in cashew, guava,
and pineapple juices after 1 h at 4°C (De Sousa Guedes et al., 2016). For
mango juice, different concentrations of EOs and contact time were
required to inactivate target bacteria at the same temperature (4°C).
Escherichia coli and Salmonella Enteritidis were inactivated after 1 h
when 1.25 or 2.5 μL/mL were applied for both EOs, respectively; while
for Listeria monocytogenes 5 μL/mL of both EOs were required and
4 or 24 h of contact were necessary for Mentha arvensis and Mentha
piperita, respectively (De Sousa Guedes et al., 2016). Similar results
were reported by Leite et al. (2016) for Cymbopogon citratus EO added
to pineapple juice; they required 1.25 μL/mL for inactivated 5 log
CFU/mL of Escherichia coli UFPEDA 224 and Listeria monocytogenes
ATCC 7644 and 15 min of exposure, while for S. Enteritidis UFPE 414
the same concentration of EO inactivated it after 1 h of exposure at 4°C.
EOs should not change their antimicrobial activity when juice
or fruit beverage pH changed; despite low pH could increase EOs’
­hydrophobicity. This has been proved by Yuste and Fung (2002) in
apple juice; they studied the antimicrobial activity of cinnamon EO
(0.1%, 0.2%, or 0.3%) in apple juice at two pHs (3.7 or 5.0) and two storage temperatures (5°C or 20°C) inoculated with Listeria ­monocytogenes
Scott A. They evaluated the injured cells recovered, in broth heart infusion agar, after 1 h in apple juice with cinnamon EO, and they found
that the most important factor in antimicrobial activity was temperature; besides room temperature favored bacterial inactivation independently of juice’s pH. Synergy between antimicrobials suggest that
50 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
using two or more preservatives could enhance fruit beverages safety,
such as cinnamon EO (0.3%) plus nisin (≤200 ppm) for apple juice
stored at 5°C or 20°C inoculated with Salmonella Typhimurium and
Escherichia coli O157:H7 (Yuste and Fung, 2004).
EOs applications also have been assessed against native microbiota of fruit beverages (mesophilic bacteria, LAB, and mold and yeasts)
and successful reductions were obtained without modifications of
physicochemical properties. Table 2.3 presents selected studies of EOs
applications in specific juices and beverages.
Due to great impact on the sensory properties of food, EOs can be
used in combination with other technologies such as thermal treatment, ultrasound, or high-intensity pulsed electric field (HIPEF) for enhance shelf life or safety of fruit drinks and beverages. Sánchez-Rubio
et al. (2016) evaluated cinnamon leaf EO (0.02 mg/mL) combined with
ultrasound (24 kHz; 105 μm; 33.31 W/mL) and heat treatment (50°C);
30 min of treatment reduced significantly Saccharomyces cerevisiae in
orange juice (2.5 log CFU/mL) and pomegranate juice (2.8 log CFU/
mL), this treatment extended the shelf life of juices up to 28 days at
5°C. In orange juice, ultrasound (20 kHz, 0.4 W/mL, 15 min, <30°C)
Table 2.3 Selected Studies With Regards to
Antimicrobial Activity of Essential Oils (EOs) in Fruit
Juices’ Against Native Microbiota
Fruit Juice
Essential Oil
Microbial
Target
Tomato juice
0.5% of oregano or
thyme EOs
Lactic acid
bacteria
Pineapple
juice
0.2 μL/mL of
Cinnamomun tamala
leaves EO
4500–9000 mg/L of
clove EO
Mesophilic and
fungi counts
0.2 μL/mL of black
pepper EO
Mesophilic and
fungi counts
Watermelon
juice
Orange juice
Mesophilic count
Main Results
References
Extended the shelf life
of the juice at least
2 weeks
Inactivation of ≈0.3 log
CFU/mL after 28 days
at 10°C
9000 mg/mL decreased
mesophilic counts in
6–8 log cycles after
7 days at 37°C
Reductions <1 log
cycle after 28 days
at 4°C
Mohácsi-Farkas et al.,
2002
Kapoor et al., 2008
Siddiqua et al., 2014
Kapoor et al., 2014
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 51
plus 250 μL/mL of orange EO enhanced the juice shelf life (Khandpur
and Gogate, 2016). HIPEF was combined with EOs in order to inactivate different pathogenic bacteria; successful studies inactivated
(≈5 log cycles) of Salmonella Enteritidis and Escherichia coli O157:H7
in strawberry, apple, and pear juices were achieved with 0.5% or 0.1% of
cinnamon bark EO and HIPEF (35 kV/cm, 4-μs pulse length, T ≤ 40°C)
(Mosqueda-Melgar et al., 2008a). For tomato juice, the combination of
0.1% of cinnamon bark EO and HIPEF (35 kV/cm for 1000 μs at 100 Hz,
4-μs pulse length) was enough to inactivate ≈5 log cycles of Salmonella
Enteritidis (Mosqueda-Melgar et al., 2008b). Mild acid juices like melon
or watermelon required 0.2% of cinnamon bark EO and HIPEF (35 kV/
cm for 1709 μs at 193 Hz and 4-μs pulse duration) to reduce >5 log cycles
of Salmonella Enteritidis, Escherichia coli O157:H7, and Listeria monocytogenes (Mosqueda-Melgar et al., 2008c). High hydrostatic pressure
(HHP) treatment at 300 MPa (20 min) plus 200 μL/L of Citrus sinensis
or Citrus reticulate EO inactivated ≈3.5 or ≈2.5 log cycles of Escherichia
coli O157:H7 VTEC in orange or apple juices, respectively (Espina et al.,
2013). Under the same conditions (+)-limonene inactivated >5 log cycles in orange and apple juices. In addition, remained antimicrobial
activity was observed during the refrigerated stored of juices and up
to 3 log cycle reductions were achieved. Freeze-thaw treatment (FTT)
(−23°C/24 or 48 h; thawing at 7°C for 4 h) combined with lemongrass
(0.1–1 μL/mL), cinnamon leaf (2 μL/mL), or basil (2 μL/mL) EOs decreased Escherichia coli O157:H7 and Salmonella Enteritidis counts in
strawberry juice stored at 7°C (Duan and Zhao, 2009).
ICs of EOs had been also investigated in fruit juices and beverages.
The most studied ICs are cinnamaldehyde, carvacrol, eugenol, thymol, and geraniol (De Souza et al., 2016). ICs of EOs such as citral,
β-pinene, and linalool had been assessed in citrus-based beverages
inoculated with Saccharomyces cerevisiae (104.4 CFU/mL) and heat
treated (55 °C, 15 min); however, lower stability of beverages were observed during storage (60 days) at 28 °C (Belletti et al., 2010). In addition, authors evaluated mixtures of citral, β-pinene, and linalool but
poorly improvements of beverage stability were observed. In apple
soft drink, 40 ppm of (E)-2-hexenal and thermal treatment (55 °C for
10 min) were enough to inhibit the growth of Saccharomyces cerevisiae
[104 CFU/bottle (500 mL)] after 60 days of storage at 25°C (Belletti et al.,
2007). Citral (200 ppm) and mild treatment at 54°C (8 min) inactivated
4 log CFU/mL of Escherichia coli O157:H7 in apple juice; in this study,
the combination of both treatments had a synergistic effect, reducing
up to six times the time required to inactivate 90% of the population,
while citral reduced <0.5 log CFU/mL and thermal treatment inactivated <1 log CFU/mL if they were applied individually (Espina et al.,
2010). Synergistic effect of citral plus thermal treatment was maintained also if low concentrations of citral were added even at ­levels
52 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
of 5 or 20 ppm; Authors supposed that heat caused partial damage in
the cytoplasm and outer membrane allowing citral to easily reach the
interior of the cells and inactivate them.
EOs or ICs have been emulsified prior to be added to fruit beverages
in order to minimize sensory changes and enhance their antimicrobial
properties. Antimicrobial activity can be improved because solubility
of EOs or ICs in aqueous foods increased if they are emulsified; it is
well known that EOs or ICs have poor solubility in water. In addition, a
decrease in droplet size (nano-emulsions) in many cases can increase
antimicrobial activity. Table 2.4 shows selected studies that evaluated
the application of ICs of EOs emulsified in fruit beverages.
Interesting inhibition or inactivation results have been reported
by emulsifying or nano-emulsifying ICs of EOs; however, further sensory studies and cost-related analysis have to be performed in order to
evaluate their practical application.
Table 2.4 Selected Studies With Regards to the
Antimicrobial Activity of Individual Components (IC)
of Essential Oils (EOs) Emulsified or Nano-Emulsified
in Fruit Juices
Fruit
Beverage
Orange or
pear juices
Orange juice
Clarified
watermelon
juice
EO or IC Emulsified
Mixture of terpenes extracted
from Melaleuca alternifolia emulsified in palm oil and emulsifying
agents
Eugenol nano-emulsified in
sesame oil and Tween 80
trans-cinnamaldehyde
n­ ano-emulsified with Tween 20
Microbial
Target
Lactobacillus
delbrueckii
Staphylococcus
aureus NCIM
2672
Staphylococcus
aureus ATCC
12692
Main Results
Reference
Inactivation of 3 log
CFU/mL after 2 days
and up to 16 days of
storage at 32°C
0.3% of eugenol
decreased ≈3.2 log
CFU/mL after 24 h
and maintained up to
72 h at 4°C
The addition of 0.8%
of nano-emulsion
reduced the growth
of bacteria after 48 h
at 37°C
Donsì et al.,
2011
Ghosh et al.,
2014
Jo et al., 2015
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 53
Table 2.4 Selected Studies With Regards to the
Antimicrobial Activity of Individual Components (IC)
of Essential Oils (EOs) Emulsified or Nano-Emulsified
in Fruit Juices—cont’d
Fruit
Beverage
2.2.2.2
EO or IC Emulsified
Microbial
Target
Carrot juice
Isoeugenol emulsified in
β-lactoglobulin
Escherichia coli
K12
Carrot, apple,
and orange
juices
Carvacrol emulsified in capsul
(modified maize starch)
E. coli ATCC
35218
Main Results
Reference
Minimal biocidal
concentration in
carrot juice at 25 °C
was 155 mg/L and
348 mg/L at 6°C,
after 24 h (initial
inoculums 108 CFU/
mL)
At 1.5 μL/mL of
emulsion 4 log CFU/
mL were reduced for
apple and orange
juices and 3 log
CFU/mL for carrot
juice, after 4 days of
storage, counts were
maintained at these
levels up to 15 days
at 4°C
Nielsen et al.,
2016
Bacteriocins
Nisin is the most studied bacteriocin for fruit juices due to its antimicrobial activity against deteriorative thermoacidophilic bacteria
Alicyclobacillus acidoterrestris (Ruiz et al., 2013) as well as against beer
and wine spoilage LAB (Martínez-Viedma et al., 2008). In concentrated orange juice, the addition of 10 IU/mL of nisin decreased 29%
the D value for Alicyclobacillus acidoterrestris compared to thermal
treatment at 92°C alone (Peña et al., 2009). In apple juice, 10 IU/mL of
nisin reduced ≈3.2 log CFU/mL of cells or spores of Alicyclobacillus acidoterrestris after 29 days of storage at 30°C, while the same concentration of nisin and storage conditions did not affect the viability of cells
Char et al.,
2016
54 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
of Propionibacterium cyclohexanicum NCIMB 13575 in orange juice
(Walker and Phillips, 2008). Authors attributed the nisin resistance of
Propionibacterium cyclohexanicum to an adaptive mechanism; and
suggested further investigations to clear this mechanism. In kiwi juice,
100 IU/mL of nisin inactivated 3 log CFU/mL of Alicyclobacillus contaminans DSM 17975 or C-ZJB-12-33 after 4 days at 45°C, and maintained undetectable counts up to 14 days at the same temperature;
besides, nisin reduced the thermal resistance of Alicyclobacillus spp.
in the same food matrix (Jiangbo et al., 2016). Nisin loaded in chitosan
carageenan nano-capsules prolonged the shelf life of tomato juice up
to 6 months when compared with nisin alone at the same concentration (0.5 mg/mL), which only gave 1 day of stability at room temperature (Chopran et al., 2014).
Other bacteriocins such as enterocin AS-48 from Enterococcus
faecalis A-48-32 inhibited or inactivated LAB responsible for spoilage and ropy appearance of apple juice. The addition of 2.5 or 5 μg/
mL of enterocin AS-48 to fresh apple juice reduced ≈5 log CFU/mL of
exo-polysaccharide-producing lactobacilli (Lactobacillus collinoides
5, Lactobacillus diolivorans 29, and Pediococcus parvulus) or the 3hydroxypropionialdehyde-producing strains (Lactobacillus collinoides
9 and Lactobacillus collinoides 10) when apple juice was stored at 22°C
(Martínez-Viedma et al., 2008). This bacteriocin could be utilized to extend the shelf life of fresh apple juice prior to the fermentation process
to produce apple cider. When the effect of temperature was evaluated
on the effectiveness of bacteriocins in apple juice, as expected low temperature (4°C) enhanced antimicrobial activity and increased ≈1.8 log
CFU/mL reduction of P. parvulus 48 after 24 h of exposition compared
with room temperature (22°C). Furthermore, shelf life of apple juice was
extended up to 30 days (at 4°C or 22°C) when 0.613 AU/mL of enterocin AS-48 and HIPEF (35.5 kV/cm, 1000 μs treatment time, and 150 Hz
in bipolar mode) were applied to juice (Martínez-Viedma et al., 2010).
Bificin C6165 (produced by Bifidobacterium animalis subsp. animalis
CICC6165) at 40 μg/mL inactivated 4 log CFU/mL of Alicyclobacillus acidoterrestris DSM 3922 in commercial juices (apple, orange, peach, and
grape fruit) after 24 h of storage (45°C) and maintained these levels after
10 days; furthermore, endospores were reduced (>5 log/mL) in grape,
orange, and peach juices at concentration of 80 μg/mL of bacteriocin
(Pei et al., 2014). Paracin C is a bacteriocin produced by Lactobacillus
paracasei CICC 20241 and displayed antimicrobial activity against
Alicyclobacillus acidoterrestris DSM 3922; 30 μg/mL of paracin C inactivated 92.2% of target bacteria after 24 h in apple juice (Pei et al., 2017).
2.2.2.3 Enzymes
Lysozyme, lactoferrin, and lactoperoxidase system are frequently
used as antimicrobials in foods. Novel enzymes such as papain, a
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 55
peptidase extracted from papaya (Carica papaya), and bromelain a
proteolytic enzyme derived from pineapple (Ananas comosus) have
been proposed as antimicrobials for foods due to their proteolytic
capacity. No toxic or mutagenic activity was reported for papain in
bacteria strains of Escherichia coli, conversely antioxidant activity
was observed (Da Silva et al., 2010). Bromelain is considering a food
supplement and is consumed commonly in the United States and
Europe; although it has shown low toxic and mutagenic potential (Dos
Anjos et al., 2016). Bromelain inactivated Alicyclobacillus acidoterrestris 0244T (5 × 104 CFU/mL) in reconstituted orange juice at 500 μg/
mL after 48 h at three different storage temperatures (28°C, 35°C, or
45°C), while papain inactivated microbial load only at 45°C and 48 h
of storage when 7.84 μg/mL were utilized (Dos Anjos et al., 2016).
Also, mixtures of both enzymes were evaluated and a synergistic effect was obtained for Alicyclobacillus acidoterrestris inactivation. Due
to the protein nature of papain and bromelain, industrial applications
should be after filtration (sterilization process), since pasteurization
(80°C, 10 min) of enzymes reduced 64 and 8 times their antimicrobial
activity, respectively (Dos Anjos et al., 2016).
2.2.2.4
Others Novel Preservatives
Other proposals of novel antimicrobials include plant extracts,
oleoresins, and specific substances derived from plants (saponins and
vanillin) or produced by microorganisms. The addition of 40 ppm of
citrus extract (a commercial mixture containing bioflavonoids from
five Citrus species) inhibited the growth of Z. bailii (105 CFU/mL) in
a red fruit juice mixture (20% red orange, 20% blueberry, 10% pomegranate, and 11.3% sugars) after 17 days at 4 °C; the same extract was
ineffective to inhibit Saccharomyces cerevisiae under the same conditions (Bevilacqua et al., 2013b). Lyophilized aqueous extracts of Ilex
paraguariensis (yerba mate) (40 mg/mL) inactivated 4 log CFU/mL of
a cocktail of Escherichia coli O157:H7 (ATCC 13894 and “Cider”) after 24 h in apple juice (pH 6.0) at 35°C (Burris et al., 2012). Oleoresins
from Cinnamomun tamala leaves utilizing methanol and ethanol as
solvents showed low antimicrobial activity against native microbiota
(<1 log CFU/mL) of pineapple juice, during 28 days of storage (Kapoor
et al., 2008).
Commercial or purified fraction of saponin from Sapindus
saponaria L. was evaluated as antimicrobials against spores of
Alicyclobacillus acidoterrestris CCT 49028 in concentrated or reconstituted orange juices; both saponins at 300 or 500 mg/L and thermal
treatment (99°C for 1 min) inactivated 4 log CFU/mL of target microorganism after 120 h of incubation at 45°C (Alberice et al., 2012).
Saponins could be an additive to inactivate Alicyclobacillus acidoterrestris in orange juices when they are combined with heat treatments
56 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
like pasteurization; however, due to bitter flavor of saponins, further
sensory evaluation is necessary prior to their application.
Vanillin at 40 mM was able to inactivate Saccharomyces cerevisiae
NCYC 956 (104 CFU/mL) or Candida parapsilosis (104 CFU/mL) in organic apple juice or peach-flavored soft drink after 1 week at 8°C or
25°C, except for Saccharomyces cerevisiae at 8°C in apple juice that required 3 weeks. Amounts or vanillin could be ≤20 mM if the storage is
prolonged up to 8 weeks at 25°C (Fitzgerald et al., 2004). Due to flavor
properties of vanillin, organoleptic attributes should be further investigated prior to its use.
Surfactin (a biosurfactant cyclic lipopeptide produced by Bacillus
subtillis) nano-emulsified in sunflower oil promoted a significant reduction of naturally occurring bacteria and fungi, in apple juice (Joe
et al., 2012). Piramicin (a polyene macrolide fungicide produced by
submerged aerobic fermentation of Streptomyces natalensis) loaded
in nano-hydrogel devices inhibited Saccharomyces cerevisiae CECT
growth in grape juice under slow and controlled released of the antimicrobial (Fuciños et al., 2015). Natamycin (a polyene macrolide produced from the controlled fermentation of dextrose by Streptomyces
natalensis) had been assessed as a natural preservative to extend the
shelf life of Niagara grape juice; antimicrobial activity against yeast inoculums (cocktail of Zygosaccharomyces, Kluveromyces, Dekkera, and
Brettanomyces) was level dependent and inhibition of fungi was effective only at 102 CFU/mL for 109 days at 25°C at 20 ppm of natamycin; in
spite of these results, natamycin is an interesting natural preservative
for grape juice (Siricururatana et al., 2013).
2.2.2.5 Mode of Action
Antimicrobial mechanisms depend on the novel preservative,
type of microorganism, microbial load, and fruit beverage condition
(pH, temperature, and suspended solids). For EOs, their mechanisms
of action in bacteria include the loss of ions, permeabilization of cell
membrane, changes in membrane potential, the coagulation of cytoplasm, and damage to lipids and proteins (Tianli et al., 2014). When
EOs are combined with others technologies lower concentrations of
EO are required to inactivate target microorganisms, therefore, mild
thermal treatment, HIPEF, high-pressure homogenization (HPH), or
FTT destabilize cell membrane or elicit pore membrane formation
(membrane damage) allowing EOs to penetrate the cell and leading
to its death.
Nisin is recognized as a pore formation bacteriocin due to its ability to interact with cell membrane peptides of bacteria. After enough
pores are formed, losses of ions and cytoplasm material lead the
bacterial cell to death. Bacteriocins, such as bificin C6165 damage
cell walls causing loss of cytoplasmic content, cell disorganization,
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 57
and ­formation of vesicles in Alicyclobacillus acidoterrestris after 24 h
of treatment (Pei et al., 2014). Paracin C (50 μg/mL) showed similar
damage (cell wall destruction and loss of cytoplasmic content) in
Alicyclobacillus acidoterrestris cells after 24 h of exposition in apple
juice at 30°C (Pei et al., 2017).
For papain and bromelain enzymes, Dos Anjos et al. (2016) observed that antimicrobial activity against Alicyclobacillus acidoterrestris did not relate to their proteolytic action; instead these authors
suggested amidase and esterase activity of the enzymes as main
modes of action.
More studies are necessary to clear the action mechanism of other
novel preservatives such as saponins, vanillin, and ICs or EOs; furthermore, studies regarding mechanisms of action of novel antimicrobials
are necessary in fungi cells since nowadays very scarce information is
available.
2.2.2.6
Quality Factors
An ideal antimicrobial substance should not change the physicochemical and sensory properties while ensuring inhibition or inactivation of target microorganisms and extending the shelf life of the fruit
beverage. However, most novel antimicrobials could have a negative
impact on sensory or physicochemical properties of fruit beverages;
meanwhile, fruit beverage safety is first, thus optimal combinations or
concentrations of natural preservatives have to be examined.
Depending on the novel preservative, specific quality changes can
be recognized. For EOs, main concerns about its application are the
modification or rejection of fruit beverages due to changes in flavor
and taste, since EOs have intense and typical sensory profiles. In order to diminish these sensory effects, an adequate combination of EOs
and fruit beverages should be performed, thus EOs can enhance the
flavor while performing their antimicrobial action. In fact, one of the
main uses of EOs is as flavorings, thus we need to take advantage of
this to incorporate them in specific fruit beverages. Other strategy is to
combine EOs with mild thermal treatments, HIPEF, HHP, and/or FTT
as previously mentioned to reduce sensory changes of fruit beverages.
Selected studies with interesting results about the addition of EOs or
ICs of EOs are described below.
Orange juice added with ≤100 ppm of (+)-limonene or 200 ppm
of orange EO and treated with heat (60°C for 2.1–3.9 min) had similar sensory acceptance than orange juice without antimicrobial
tested substances; however, when (+)-limonene was incorporated
at 200 ppm lower sensory acceptance was observed (Espina et al.,
2014a). Supplemented apple juice (clear or cloudy) with 0.25 μL/mL
of lemon EO was well accepted by panelists that stated juices as refreshing and harmonic, but odor of juices with EO was perceived as
58 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
unpleasant (Tserennadmid et al., 2011). In tomato juice, four EOs
(lemon, pennyroyal mint, thyme, or rosemary) and two ICs (carvacrol
or p-cymene) at different concentrations (20, 100, or 200 μL/L) were
tested on their taste acceptance. Lowest concentrations of pennyroyal
mint or lemon EO did not change the taste acceptance of juice; furthermore high concentrations of pennyroyal mint increased the taste
acceptance of juice; tomato juice with other studied EOs or ICs was
rejected by panelists (Espina et al., 2014b). The mint EO (1.13 mg/mL)
incorporated to apple-orange mixed beverage did not alter its odor or
color (sensory acceptance) after 8 days of refrigerated storage (Tyagi
et al., 2013). At bactericidal concentrations of Mentha arvensis and
Mentha piperita EOs (0.625 or 1.25 μL/mL) sensory attributes such as
appearance, odor, and viscosity of cashew, guava, mango, and pineapple juices were not affected while their taste, aftertaste, and overall
acceptability were unpleasant for the panelists, probably due to burning, stinging, and cold sensation of menthol (De Sousa Guedes et al.,
2016). Low scores were assigned to taste and aftertaste of pineapple
juice preserved with Cymbopogon citratus EO at 1.25 μL/mL (Leite
et al., 2016).
Sensory effect of bacteriocin (paracin C) in apple juice was concentration dependent and no changes were observed at levels ≤100 μL/
mL, but at 200 μL/mL the flavor of apple juice was not pleasant (Pei
et al., 2017).
Physicochemical properties can also be modified by the EOs or
its ICs; decrements of acidity, ascorbic acid, and total sugar content
were reported in orange juice added with 0.2 μL/mL of black pepper
EO (Kapoor et al., 2014) or 0.1% of cardamom EO in sweet orange
juice (Kapoor et al., 2011), after 28 days of refrigerated storage. EOs of
Mentha arvensis and Mentha piperita at bactericidal concentrations
(0.625 or 1.25 μL/mL) did not modify physicochemical properties such
as total soluble solids, pH, and titratable acidity of cashew, guava,
mango, and pineapple juices (De Sousa Guedes et al., 2016). Similar
results were observed in pineapple juice added with Cymbopogon citratus EO at 1.25 μL/mL (Leite et al., 2016). When a bacteriocin, such as
paracin C (200 μL/mL) was added, reductions in °Brix and color were
reported, while titratable acidity and total sugar were maintained, despite, lower concentrations were required to inactivate target bacteria
(Pei et al., 2017).
2.2.3 Legislation
Most EOs are declared as GRAS by the US FDA and are registered
by the European Commission for use as flavoring substances in foods.
EOs’ permitted uses are exhibited in Title 21 of the Code of Federal
Regulations (CFR), Chapter I, subpart B and corresponding parts
Chapter 2 CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES 59
(Part 172, Part 182, and Part 184). For plant extracts such as oleoresins
and others, the same parts of CFR could be consulted for their applications and uses.
The only approved bacteriocin to be utilized as preservative in
foods by the US FDA is nisin since 1988 (GRAS, CFR 21-184.1538)
while since 1969 by the Joint Food Agriculture Organization/World
Health Organization Expert Committee on Food Additives (E234). Up
to date no one other bacteriocin has been accepted as preservative by
these regulatory institutions. For other novel preservatives legal status
are not defined due to recent or scarce studies about their antimicrobial properties in fruit beverages.
2.3 Actual Trends in the Use of
Preservatives
Lately, the interest of consumers on natural preservatives, minimal
processing steps, unpasteurized, high concentration of antioxidants,
and low contents of salt and fats has increased. Among natural preservatives are EOs, spices, phenolic components, organic acids, and
bacteriocins, among others (Rupasinghe and Yu, 2012). These concerns also have increased in the industry, while mishandling of preservatives has caused a rise in process- and preservative-resistant
­microorganisms (Kregiel, 2015).
Peptides or bacteriocins, such as nisin, pediocin, lacticin, enterocin, and variacin could be natural candidates for fruit drinks preservation, being able to retain most of the sensory and nutrimental
properties (Kregiel, 2015). Another trend in natural preservation
would be the use of EOs or their ICs, which have shown antibacterial,
antiviral, and antifungal properties. However, high concentrations
of EOs and their ICs are generally required to achieve these properties, resulting in undesirable flavor and aroma changes. This problem
could be solved combining them with other preservatives to lower the
undesirable changes (Burt, 2004; Khallaf-Allah et al., 2015). KhallafAllah et al. (2015) studied the combination of cinnamaldehyde and
nisin against Alicyclobacillus acidoterrestris in orange nectar, which
presented a synergic effect extending the shelf life of the unpasteurized nectar.
On the other hand, lactoperoxidase enzyme has been proved to be
antibacterial in tomato juices; however, more information about its
application is required. Chitosan has also shown antibacterial properties in fruit juices, specifically chitosan glutamate, which had an effective result as preservative against spoilage yeast in apple juice, but
more investigation is also needed (Rupasinghe and Yu, 2012).
60 Chapter 2
CHARACTERISTICS AND USES OF NOVEL AND CONVENTIONAL PRESERVATIVES
Finally, the use of a single preservation method, could lead to effective microorganism reduction but, as mentioned previously, could compromise product quality. Therefore, the actual recommendation is using
more than one preservation method to maintain quality and ensure
product safety (García-García et al., 2015; Rupasinghe and Yu, 2012).
2.4 Concluding Remarks
Fruit beverages have become popular drinks due to their nutrimental, antioxidant, and sensory qualities. But these qualities could
be easily loss or reduced because of raw material quality, thermal processing and/or microorganism growth. Therefore, the interest of maintaining the mentioned qualities by investigating new ways to preserve
fruit beverages. Besides quality maintenance of fruit drinks, there is
also the consumers’ demand for more natural products. Conventional
preservatives have been used in fruit drinks and beverages for many
years; however, consumer’s demands for “clean” labels are driving the
development of novel and “natural” preservative systems.
A wide variety of novel preservatives have been proposed to preserve fruit drinks and beverages. Most studied novel preservatives are
EOs or their ICs by direct addition or (nano)-emulsified, followed by
nisin and other bacteriocins. Other novel preservatives such as enzymes and oleoresins still have few reports about its antimicrobial
properties and applications. EOs and its ICs have been mainly tested
against pathogen bacteria and fungi, while bacteriocins have been
mostly assessed against deteriorative thermoacidophilic bacteria
such as Alicyclobacillus spp. Further studies are necessary mainly to
clarify the modes and mechanisms of action of most novel proposed
preservatives.
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