Adding Molecules to Food, Pros and Cons: A

Review on Synthetic and Natural Food Additives

Márcio Carocho, Maria Filomena Barreiro, Patricia Morales, and Isabel C.F.R. Ferreira

Abstract: The pressing issue to feed the increasing world population has created a demand to enhance food production,
which has to be cheaper, but at the same time must meet high quality standards. Taste, appearance, texture, and
microbiological safety are required to be preserved within a foodstuff for the longest period of time. Although considerable
improvements have been achieved in terms of food additives, some are still enveloped in controversy. The lack of uniformity
in worldwide laws regarding additives, along with conflicting results of many studies help foster this controversy. In this
report, the most important preservatives, nutritional additives, coloring, flavoring, texturizing, and miscellaneous agents
are analyzed in terms of safety and toxicity. Natural additives and extracts, which are gaining interest due to changes in
consumer habits are also evaluated in terms of their benefits to health and combined effects. Technologies, like edible
coatings and films, which have helped overcome some drawbacks of additives, but still pose some disadvantages, are briefly
addressed.
Future trends like nanoencapsulation and the development of “smart” additives and packages, specific vaccines for
intolerance to additives, use of fungi to produce additives, and DNA recombinant technologies are summarized.
Keywords: natural food additives, antimicrobial, antioxidant, conservatives, 34 Colorants

Historic Background of Food Additives century, processed food became an important part of human nu-
Since the dawn of man, our species searches for better ways to trition, and legal chemical additives became increasingly prevalent
feed itself, by developing more efficient methods of hunting, ani- in them, fostering tight regulation, which still remains controver-
mal/vegetable domestication, food preservation by physical meth- sial due to the high number of studies concerning food additives
ods, and finally, by adding molecules to food in order to enhance that produce conflicting results and different interpretations by
flavors or to preserve it. governments (Fennema 1987).
Back in the 1800s, food additives were intentionally used for Today, more than 2500 additives are intentionally added to food
food adulteration. This practice was widespread due to the cen- in order to keep certain properties or to extend shelf life, while
tralization of food processing, decline of personal accountability, many others were banned throughout the years, some of them
birth of analytical chemistry, and inadequate governmental regu- at a global level and others only in specific countries (Branen
lation. The consequence of such uncontrolled tampering of food and others 2001). The definition of food additive has changed
led to a serious worldwide problem with concern about food qual- during time, being today defined as “any substance not normally
ity rising gradually. In 1920, the availability of effective methods consumed as a food by itself and not normally used as a typical
for food analysis, together with regulatory pressures, started to re- ingredient of the food, whether or not it has nutritive value, the in-
duce the significance of this problem. In the middle of the 20th tentional addition of which to food for a technological (including
organoleptic) purpose in the manufacture, processing, prepara-
tion, treatment, packing, packaging, transport or holding of such
food results, or may be reasonably expected to result (directly or
MS 20131556 Submitted 10/28/2013, Accepted 2/8/2014. Authors Carocho indirectly), in it or its by-products becoming a component of or
and Ferreira are with Mountain Research Center (CIMO) ESA, Polytechnic Inst.
of Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855 Bragança,
otherwise affecting the characteristics of such foods. The term does
Portugal. Author Morales is with Dept. of Nutrition and Bromatology II, Faculty of not include contaminants or substances added to food for main-
Pharmacy, Complutense Univ. of Madrid, Pza Ramón y Cajal s/n, E-28040 Madrid taining or improving nutritional qualities” (Codex Alimentarius).
s/n, E-28040 Madrid, Spain. Author Barreiro is with Laboratory of Separation and This definition was proposed in 1995 by the joint panel, com-
Reaction Engineering (LSRE), Associate Laboratory LSRE/LCM, Polytechnic Inst. prised by the Food and Agriculture Organization (FAO) of the
of Bragança, Campus Santa Apolónia Apartado 1134, 5301-857 Bragança, Portugal.
Author Carocho is also with Dept. of Nutrition and Bromatology II, Faculty of Phar- United Nations and by the World Health Organization (WHO)
macy, Complutense Univ. of Madrid, Pza Ramón y Cajal, s/n, E-28040 Madrid, and being revised during all the subsequent years, with the last
Spain. Direct inquiries to authors Morales (E-mail: [email protected]) revision in 2012. Today, the Codex Alimentarius gathers all the
and Ferreira (E-mail: [email protected]). information regarding standards, codes of practice, and guidelines


C 2014 Institute of Food Technologists®

doi: 10.1111/1541-4337.12065 Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 377
Adding molecules to food, pros and cons . . .

about food and its processing. Worldwide, the 2 major regulators The 1st evaluation includes food colorants and preservatives (in-
of food additives are the European Food Safety Authority (EFSA) cluding antimicrobials and antioxidants), which has to be con-
and the Food and Drug Administration (FDA) of the United cluded before 2015. The 2nd group to be evaluated, comprising
States. texturizing agents (including emulsifiers, stabilizers, and gelling
agents) by 2018, and the last group, sweeteners, to be revised until
Food Additives in the XXI Century 2020 (Lodi and others 2011). This re-evaluation of food addi-
Today, mankind depends on food additives; in fact, the indus- tives brings an opportunity to pursue research toward the use of
trialized world would not have been possible without them. The natural additives, while clarifying doubts that still might persist
citizens of industrialized societies are not often involved in the cul- in food producing companies, the scientific community, among
tivation, harvesting, and processing of the food they eat. Due to stakeholders, regulating bodies and consumers.
this fact, processed food has to be transported across large distances In the European Union (EU), all food additives, whether ap-
to reach consumers. In order to ensure that the food reaches its proved or not in the EU, are labeled with the letter “E” (repre-
destination in good conditions, special requirements are needed, senting Europe) and a specific number. This nomenclature was
mainly to prevent contamination and spoilage (Cheng and others extended to the Codex Alimentarius Commission to easily iden-
2010; Lerner and Lerner 2011; Becerril and others 2013). These tify food additives worldwide. A huge effort was put in motion to
requirements involve the correct packaging and environmental gather knowledge toward the creation of a single database of legal
conditions, as well as the incorporation of additives to preserve or additives to be used within the EU, and in the Regulation 1129,
enhance different parameters. Although research concerning food of 2011, all the approved additives as well as their acceptable daily
additives (Patel and others 2010; Maqsood and others 2013), their intake (ADI) were listed (Council Regulation (EC) 1333/2008;
impact on health and behavior (Gultekin and others 2013; Martyn Council Regulation (EC) 1129/2011). In the United States, as
and others 2013), as well as the methods to detect them (González from 1961, the FDA determined that all food components were
and others 1999; Ferreira and others 2000; Frazier and others labeled as generally regarded as safe (GRAS). This term is still
2000; Watson 2002; Saad and others 2005; Isaac and others 2006; in use today, but for an additive to be considered in it, vari-
Wang and others 2006; Garcı́a-Jiménez and Capitán-Vallvey 2007; ous toxicology assays must be performed, and the list has seen
Yoshioka and Ichihashi 2008; Cantarelli and others 2009; Chen changes throughout the years. The FDA, although quite resistant
and Ni 2009; Xiu-Qin and others 2009; Merusi and others 2010; to approve new food additives has an online database covering
Yoshikawa and others 2011; Ohtsuki and others 2012; Pundir and “Everything Added to Food in the United States” (EAFUS) were
Rawal 2013) is growing exponentially, much speculation, incon- all the compounds added to food, including additives, are com-
sistencies, controversy, and health risks still remain unclear and are piled, as well as the FDA Redbook with guides and legislation
object of ample debate. Adding to this fact is the growing pressure for the food industry (Branen and others 2001; FDA EAFUS;
to produce new and more effective additives pushing the regula- FDA Redbook)
tions to become tighter to detect adulterations and excessive use The highest dose at which no adverse effects are observed in
of certain additives as fast as possible. the most susceptible animal species is identified as the No Ob-
In 2001, the Centers for Disease Control and Prevention of the served Adverse Effect Level (NOAEL). This parameter is used as
United States estimated that there were about 76 million cases of the basis for setting the human safety standards for food additives.
foodborne illnesses in the United States alone, each year, resulting NOAEL is measured in milligrams per kilogram of body weight
in about 5000 deaths. The associated cost of foodborne illnesses per day. The acceptable daily intake, which was introduced by
with specific strains of bacteria is estimated to be between 6.5 the Joint FAO/WHO Expert Committee on Food Additives in
and 34.9 billion dollars. For the same year, in England and Wales 1961, postulated that it should measure the amount of an addi-
100000 cases were reported, and authorities believe this number tive in food that could be ingested orally on a daily basis over a
was not accurate, since many of the occurrences were not reported lifetime without an appreciable health risk. To calculate the ADI,
(Cleveland and others 2001; Smith-Palmer and others 2001). NOAEL is corrected by 2 certainty factors, one for extrapolating
The FDA has been blamed by the Council for Responsible from animal to human, and another to account for interindividual
Nutrition for creating a very strict and unachievable draft guid- variability in humans (Lu 1988; Branen and others 2001; Watson
ance in order to approve new dietary ingredients, comparing their 2002). Although widely used, certain problems and limitations of
approval to those of food additives, which are tested to a higher the NOAEL have been pointed out by several authors (Leisenring
threshold than the reasonable expectation of safety prescribed by and Ryan 1992; Dorato and Engelhardt 2005).
the Congress (Mister and Hathcock 2012). In 2011, the FDA’s ex- The ADI is the starting point to establish the maximum amount
posure assessment approaches were reviewed, and it was concluded of a certain additive to be included in each foodstuff, which can
that the agency should develop a science-based framework to pri- vary from a few milligrams to “quantum satis,” and expressed as
oritize and reassess prior safety decisions, and conduct more exten- milligrams of additive per kilogram body weight (mg/kg bw).
sive postmarket monitoring while communicating and exchanging Quantum satis is a Latin word employed by the EFSA which de-
scientific information with stakeholders (Alger and others 2013). termines that there is “no maximum numerical level specified
The EFSA also gathered a scientific forum in 2008 to join scien- and substances shall be used in accordance with good manufac-
tists around food safety issues in Europe. The authority revealed turing practice, at a level not higher than necessary to achieve
that consumer confidence was growing since its foundation in the intended purpose and provided the consumer is not misled.”
2002, a year in which it was low due to the 1990s’ food-related (Council Regulation (EC) 1333/2008).
issues. In that forum, food additives were considered desirable due In order to approve new additives or extend the usage of an
to the function they carried out, but could, in some cases, have approved one within the EU, a series of procedures has to be
negative effects on health (Bronzwaer 2008). Since 2010, both the carried out, divided into 4 parts. The 1st regards the “Chem-
European Commission Regulation along with EFSA have started istry and specifications,” where the additive must be rigorously
a program to re-evaluate all the existing approved food additives. identified according to its origin as a single substance, a simple

378 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
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Adding molecules to food, pros and cons . . .

Figure 1–Groups and subgroups of food additives. Adapted from Branen and others (2001), Watson (2002), and Sarikaya and others (2012).

or complex mixture, a polymer, a derivative of botanical sources, Types of Additives

or a nanomaterial. The manufacturing process, the methods of Within the EU, food additives are divided into 26 func-
analysis in foods, and stability must also be clearly explained. tional classes, depending on their function in food: sweeteners,
The 2nd part, “Existing authorizations and evaluation,” refers colorants, “preservatives,” antioxidants, carriers, acids, acidity
to the extension of authorizations of already approved additives regulators, anticaking agents, antifoaming agents, bulking agents,
and intends to gather previous data regarding it. The 3rd part emulsifiers, emulsifying salts, firming agents, flavor enhancers,
“Proposed uses and exposure assessment” must estimate the di- foaming agents, gelling agents, glazing agents, humectants, mod-
etary exposure based on the proposed uses and use levels consid- ified starches, packaging gases, propellants, raising agents, seques-
ering age groups and the population of all the EU member states. trants, stabilizers, thickeners, and flour treatment agents (Council
Finally the “Toxicological Studies” encompass studies regarding Regulation (EC) 1333/2008). The American approach of food
the additive’s effect on in vitro and in vivo scenarios. The latter in- additives narrows down the number of classes and allows additives
cludes metabolism/toxicokinetics, acute subchronic and chronic to be mentioned in 2 or more classes. According to the FDA, there
toxicity, as well as genotoxicity, carcinogenicity, reproduction, ab- are more than 3000 food additives allowed in the United States,
sorption, developmental toxicity, immunotoxicity, and hypersen- which are distributed into 6 groups: Preservatives, nutritional
sitivity/allergy in various animal models. Human trials are only additives, coloring agents, flavoring agents, texturizing agents,
allowed after adequate data from animal and other related studies and miscellaneous agents (Figure 1). The preservatives group is
are deemed safe, and always used to define adjustment factors be- divided into 3 subgroups, although some additives may serve
tween animals and humans. Other specific studies may be required more than 1 function in foods: antimicrobials, antioxidants, and
for certain cases (EFSA 2012b). The premarket testing of additives antibrowning agents. Within the flavoring agents group, there are
may not be enough to conclude their safety toward humans, fos- 3 subgroups: the sweeteners, the natural or synthetic flavors, and
tering posterior studies, and if necessary, eventual banning, when the flavor enhancers. The texturizing agents comprise emulsifiers
their toxicity is proven. Adding to this, is the controversy sur- and stabilizers. Finally, the miscellaneous agents are composed of
rounding some additives that are approved in some countries and many classes: chelating agents, enzymes, antifoaming agents, sur-
restricted or banned in others and the public concern regarding face finishing agents, catalysts, solvents, lubricants, and propellants
the use of animals for toxicological assays. These factors, among (Branen and others 2001). Despite the various classes of additives
others, induce distrust in some people toward additives, pressur- and the different classifications used, they can be divided in 4
izing companies to look for natural substitutes for chemical ad- fundamental groups with regard to their origin and manufacture:
ditives, even though these natural compounds must logically be natural additives (obtained directly from animals or plants); similar
subjected to the same treatment as chemical ones. Lack of knowl- to natural additives (produced synthetically imitating natural ones);
edge in the population toward food additives also affects their modified from natural (natural additives that are then modified
acceptance. chemically); and finally artificial additives (synthetic compounds).


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Adding molecules to food, pros and cons . . .

The authors recognize that it would be impossible to totally others 2007). The same hyperactivity behavior was reported in a
review the more than 3000 food additives, thus the review will be study involving college students who consumed sodium benzoate-
focused on the most important ones, either based on their high rich soft drinks, validating the need for further studies regarding
consumption or due to the important functional properties they this compound (Beezhold and others 2014). In Portugal and Italy,
can provide to foods in which they are incorporated. Only the surveys were carried out in order to discover if soft drinks con-
most import additives per class are described here. tained benzoic acid or benzene residues. The results pointed out
that both benzoic and sorbic acids were present above legal val-
Preservatives ues in some cases, although not exceeding the acceptable daily
This group is composed of antimicrobials, antioxidants, and intakes. In the case of benzene, it was found in some Italian soft
antibrowning agents. The E numbers of the preservatives range drinks. Other surveys from Turkey analyzed both these antimicro-
from E200 to E399. bials in various foods and revealed that, in certain cases, benzoic
Antimicrobials. The antimicrobials are added to food for 2 pur- and sorbic acids were detected in some samples above the max-
poses, (1) to control natural spoilage of food (food control) and/or imum limit of the Turkish Food Codex (Lino and Pena 2010;
(2) to avoid/control contamination by microorganisms, including Bonaccorsi and others 2012; Cakir and Cagri-Mehmetoglu 2013;
pathogenic ones (of food safety concern) (Tajkarimi and others Ulca and others 2013). Adams and others (2005) reported that the
2010). The main chemical antimicrobials used in food with quan- safety of benzyl derivatives in food was supported by the higher
tum satis status are acetic acid (E260), potassium acetate (E261), intake of those compounds in traditional foods rather than in the
calcium acetate (E263), lactic acid (E270), carbon dioxide (E209), intentionally added flavorings. Benzyl alcohol and its derivatives
and malic acid (E296). The antimicrobial additives with restricted (benzoic acid and sodium benzoate, among others) belong to the
uses are benzoic acid and benzoates (E210-E219; ADI 5 mg/kg aryl alkyl alcohols, which apart from being food additives are also
bw), sorbic acid, and sorbates (E200-E209; ADI 25 mg/kg bw), commonly used in fragrances, cosmetics, shampoos, soaps, and
propionic acid and propionates (E280-E289; quantum satis), ni- other toiletries as well as in household cleaners and detergents.
trites (potassium nitrite E249; ADI 0.07 mg/kg bw, sodium ni- Scognamiglio and others (2012) reviewed all the toxicological and
trite E250; ADI 0.1 mg/kg bw), nitrates (sodium nitrate E251 dermatological research concerning in vitro, animal models, and
and potassium nitrate E252; both with ADI 3.7mg/kg bw), and human dermatological assays regarding absorption of benzyl al-
parabens (E214-E219; ADI 10 mg/kg bw), which are depicted in cohol, and referred to Belsito and others (2012) for conclusions.
Table 1. Benzoic acid (E210), produced by oxidation of toluene, These authors reviewed the toxicological and dermatological as-
is a widespread antimicrobial agent, employed against yeast, bac- pects of aryl alkyl alcohols used in fragrances, and determined that
teria, and fungi. It acts through membrane disruption and inhi- with all the data gathered, these compounds do not pose safety
bition of metabolic reactions, stress, and accumulation of toxic concerns in the declared levels of exposure. Still, very little research
anions inside the microbial cell (Brul and Coote 1999). It may focuses on occupational exposure to these compounds, and future
be coupled to calcium, potassium, or sodium for different an- studies should take into consideration the combined effects of oc-
timicrobial targets and effects. The main applications of sodium cupational and nonoccupational (cosmetics, perfumes, shampoos)
benzoate (E211) are soft drinks, fruit juices, sauces, pickles, ed- quantities absorbed by the skin (Schnuch and others 2011).
ible coatings, seafood products, toothpastes, lotions, creams, and Although these reports seem unsettling, benzoates are necessary
some pharmaceutical products (WHO 2000). This antimicrobial and the only way they will be removed as additives is when a
compound has been tested in vitro, and was regarded as nontoxic, substitute with the same effect and no toxicity is found. Without
but some authors found toxicity in the Drosphila SMART (so- these compounds, food spoilage and poisoning would have a much
matic mutation recombination test) test, root tips of garlic (Allium higher incidence.
sativum), as well as a clastogenic, mutagenic, and cytotoxic ef- While benzoates are used with acidic foods, sorbates can be
fect in human peripheral blood lymphocytes (Yilmaz and others employed in foods with higher pH values. Sorbic acid (E200), an
2008, 2009; Zengin and others 2011). In murine models, sodium organic natural compound, is the base molecule of 3 important an-
benzoate decreased the release of leptin, helping to contribute to timicrobials: potassium sorbate (E202), sodium sorbate (E201), and
obesity, while also leading to malformation of zebrafish (Danio re- calcium sorbate (E203). Among these molecules, sodium sorbate,
rio) larvae (Tsay and others 2007; Ciardi and others 2012; Mangge although being allowed in the United States, is banned in the EU.
and others 2013). Sodium benzoate has also been reported to in- Some in vitro studies have related the conjugated double bonds
tercalate with bovine thymus DNA at concentrations as low as present in sorbic acid’s structure as being prone to nucleophilic
4.5 × 10−5 mol/L (Zhang and Ma 2013a). Nair (2001) reviewed attack, turning it into a mutagenic compound. The interaction
the risk of exposure of benzyl alcohol, benzoic acid, and sodium between sorbic acid and various amines was tested by Ferrand
benzoate and concluded that, although being safe, in some studies and others (2000) for mutagenic and genotoxic activities on HeLa
regarding mice, malformations and toxicity effects were detected cells and plasmid DNA, resulting in negative values, while another
and dermal complications were known to occur in humans. Fur- study found sodium sorbate toxic toward human lymphocytes at
thermore, due to the various applications of these compounds, 400 and 800 μg/mL (Mamur and others 2012). Sorbic acid forms
the risk of inhalation could not be determined, and remained as mutagenic compounds when in contact with nitrites, which are
an important and urgent matter to be further studied. In a study another kind of widely used antimicrobials (Binstok and others
involving children (a group of 3-y-olds and another of 8/9-y-olds) 1998), while potassium sorbate is also genotoxic to human pe-
exposed to sodium benzoate, a global hyperactivity aggregate was ripheral blood lymphocytes at 1000 μg/mL (Mamur and others
reported when compared to the control groups. Although point- 2010), although these claims are controversial (Mpountoukas and
ing out interesting conclusions, this was not well accepted because others 2008). Jung and others (1992) also described sodium sorbate
sodium benzoate was mixed with food colorants, being difficult as being easily oxidizable, and therefore leading to the formation
to determine which of the compounds was the responsible one of 4,5-oxohexenoate, which is mutagenic, while sorbic acid and
for the hyperactivity or if a synergistic effect exists (McCann and its potassium sorbate are not.

380 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
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Adding molecules to food, pros and cons . . .

Table 1–Antimicrobial food additives with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E210 Benzoic acid Code of Federal 5 mg/kg bw WHO (2000)
Regulations 21 Nair (2001)
Sec.184.1021 Yilmaz and others (2008)
— Yilmaz and others (2009)
EU Regulation No. Zengin and others (2011)
1129/2011 Cakir and Cagri-Mehmetoglu (2013)
E211 Sodium benzoate Code of Federal 5 mg/kg bw Nair (2001)
Regulations 21 McCann and others (2007)
Sec.184.1733 Tsay and others (2007)
— Beezhold and others (2014)
EU Regulation No. Ciardi and others (2012)
1129/2011 Mangge and others (2013)
Zhang and Ma (2013a)
E214 Ethyl-p-hydroxybenzoate Code of Federal 10 mg/kg bw
(paraben) Regulations 21
Sec.175.105
—
EU Regulation No.
1129/2011
E215 Sodium ethyl Code of Federal 10 mg/kg bw Oishi (2002)
p-hydroxybenzoate Regulations 21 Darbre and others (2004)
(parabens) Sec.175.105 Harvey and Everett (2004)
— Tavares and others (2009)
EU Regulation No. Meeker and others (2011)
1129/2011 Aubert and others (2012)
Castelain and Castelain (2012)
E218 Methyl Code of Federal 10 mg/kg bw
p-hydroxybenzoate Regulations 21
(parabens) Sec.150.141
—
EU Regulation No.
1129/2011
E219 Sodium methyl Banned in the United 10 mg/kg
p-hydroxybenzoate States
(paraben) —
EU Regulation No.
1129/2011
E200 Sorbic acid Code of Federal 25 mg/kg bw Binstok and others (1998)
Regulations 21 Ferrand and others (2000)
Sec.182.3089
—
EU Regulation No.
1129/2011
E201 Sodium sorbate Code of Federal 25 mg/kg bw Jung and others (1992)
Regulations 21 Mamur and others (2012)
Sec.182.3089- Not
approved in the EU
E202 Potassium sorbate Code of Federal 25 mg/kg bw Mpountoukas and others (2008)
Regulations 21 Mamur and others (2010)
Sec.182.3640
—
EU Regulation No.
1129/2011
E220 – E228 Sulfites Code of Federal 0.7 mg/kg Rencüzoğullari and others (2001)
Regulations 21 Suh and others (2007)
Sec.182.3616, 3637, Cressey and Jones (2009)
3739, 3766, 3798 Vally and others (2009)
— Vandevijvere and others (2010)
EU Regulation No. Ulca and others (2011)
1129/2011 Garcı́a-Gavı́n and others (2012)
E249 Potassium nitrite Code of Federal 0.07 mg/kg bw Cammack and others (1999)
Regulations 21 Sebranek and Bacus (2007)
Sec.172.160 Hord and others (2009)
—
EU Regulation No.
1129/2011
E250 Sodium nitrite Code of Federal 0.1 mg/kg bw Cammack and others (1999)
Regulations 21 Chow and Hong (2002)
Sec.172.175 Sebranek and Bacus (2007)
— Hord and others (2009)
EU Regulation No. Chan (2011)
1129/2011 Hord (2011)
E281 Sodium propionate Code of Federal Not specified Dengate and Ruben (2002)
Regulations 21 Türkoğlu (2008)
Sec.184.1784 Maier and others (2010)
—
EU Regulation No.
1129/2011


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Propionic or propanoic acid (E280) is a naturally occurring car- Castelain and Castelain (2012) concluded that methyl and ethyl
boxylic acid that is used in food, especially bakery products, to parabens are safe to be used and did not find evidence of related
avoid mold and other fungal contamination. There are not many health problems, while pointing out that propyl and butyl parabens
studies regarding the toxicity of propionic acid or its salts, (sodium should still undergo more studies in order to correctly assess their
propionate, E281), calcium propionate (E282), and potassium pro- toxicity and potential harm to human health.
pionate (E283), although it has been considered to suppress, in a Sulfites are a group of molecules (the most common are sulfur
dose-dependent manner, Th1-type immune response in human dioxide, sodium and potassium bisulfite, and, sodium and potas-
peripheral blood mononuclear cells in vitro. Sodium propionate sium bisulfate) used in foods as antimicrobials and antibrowning
has been stated as inducing abnormalities on the root tips of onion, agents. Their antimicrobial effect is carried out by the uptake of SH
while calcium propionate has been related to irritability, restless- groups from sulfites into the microorganism’s cell where they react
ness, inattention, and sleep disturbance in some children (Dengate with proteins, DNA, enzymes, while the antioxidant effect occurs
and Ruben 2002; Türkoğlu 2008; Maier and others 2010). Ni- by inhibiting both Maillard reactions and the enzyme polyphe-
trates (E240-E259) and nitrites (E249, E250) in which sodium nol oxidase. Sulfites can act freely or be combined with organic
nitrate (E251), potassium nitrate (E252), potassium nitrite, and acids, being used in wine making and in many other foodstuff that
sodium nitrite are the most important compounds. They are used is prone to microbiological decay. The negative effects of sulfites
in the meat industry, namely for curing. While nitrate was widely are related with the destruction of vitamin B1 (thiamine) and to
used in the past, nowadays it is restricted to specific slow meat cur- cause skin and respiratory sensitivities, such as dermatitis, urticaria,
ing. On the other hand, nitrites are used for various applications in angioedema, abdominal pain, diarrhea, bronchoconstriction, and
several types of meat, namely for color formation, flavor enhance- fatal anaphylaxis (Rencüzoğullari and others 2001; Vally and oth-
ment, and antimicrobial activity. Nowadays, nitrites are considered ers 2009; Garcı́a-Gavı́n and others 2012). These symptoms can
the only food additive that can inhibit the development of the bo- become more prevalent due to the large quantity of foodstuffs
tulinum toxin, thus justifying their use in a benefit/risk scale in treated with sulfites, like canned goods, seafood, and dried fruits.
the food industry. The EFSA allows its use at the minimum pos- In the EU and United States, all products that contain sulfites
sible dosage. Apart from being used as food preservatives, nitrites must show this information on the label. Many surveys have been
are also present in considerable quantities in nontreated vegetables carried out in various countries, with Belgium and New Zealand
and fruits. These compounds are also known to take part in the reporting danger to individuals who consume high quantities of
formation of nitrosamines (carcinogenic molecules resulting from sulfited wine (Cressey and Jones 2009; Vandevijvere and others
the reaction of nitrites with secondary amines) posing a threat 2010). In Turkey, some foodstuffs intended for export displayed
to consumers (Sebranek and Bacus 2007; Sindelar and Milkowski quantities of sulfites above the legal limit, while Korea reported
2012). Nitrites are considered by some authors as being carcino- that the consumption of sulfites for individuals aged between 30
genic, while others refute this possibility and consider plant ni- and 64 is above that of the other age groups (Suh and others 2007;
trites important for some physiological roles, such as supporting Ulca and others 2011). More research should be carried out to
cardiovascular health and gastrointestinal immune function (Hord determine the real effect of sulfites to human health, especially
and others 2009). Although evidence supports both theories, it when used as a food additive, since food is one of the main, but
is widely accepted that the excess intake of nitrite is dangerous not the only, source of exposure to these compounds.
and has deleterious effects on human health, by oxidation of oxy- Antioxidants. The antioxidants are another subgroup of the
hemoglobin to ferrihemoglobin, leading to methemoglobinemia preservatives, essential to extend the shelf life of many foodstuffs.
(Cammack and others 1999). In order to counter these adverse Antioxidants prevent the oxidation of molecules by donating a
effects, much research is being carried out to find alternatives to hydrogen atom or an electron, becoming themselves reduced, in
nitrites (Chow and Hong 2002; Chan 2011; Hord 2011). Paraben the radical form, but contrary to other radicals, antioxidants when
is a generic name for a group of food additives, which are alkyl in radical form are stable and do not allow further reactions to
esters of p-hydroxybenzoic acid. These compounds are widely take place, therefore preserving the status quo of the system (Caro-
used in food as antimicrobials, especially due to their absence of cho and Ferreira 2013a). Food antioxidants are used for extending
odor or/and taste (Tavares and others 2009). Its effectiveness in- shelf life and impeding decay while not adding taste or odors
creases as a function of the alkyl group length. The most used com- to food or modify appearance (Nanditha and Prabhasankar 2009).
pounds of this group are methyl paraben (E218; ADI 10 mg/kg Lipid peroxidation and rancidification are the most common types
bw), ethyl paraben (E214; ADI 10 mg/kg bw), and propyl paraben of oxidation occurring in foodstuffs while they are stored. The
(E216; ADI 10 mg/kg bw). In the past, parabens were not consid- most commonly used antioxidants with quantum satis status are
ered mutagenic, but were known to cause chromosomal aberra- ascorbic acid (E300), sodium ascorbate (E301), calcium ascor-
tions and contact allergy (Darbre and others 2004; Tavares and oth- bate (E302), fatty acid esters of ascorbic acid (E304), tocopherols
ers 2009). Today, not without debate, they have been linked with (E306), α-tocopherol (E307), γ -tocopherol (E308), δ-tocopherol
reproductive decrease in men by interacting with the mitochon- (E309), lecithins (E322), sodium lactate (E325), potassium lac-
drial function of testicular cells. Epididymis sperm reserves and tacte (E326), calcium lactate (E327), citric acid (E330), sodium
sperm concentration has also been reported to decrease in a dose- citrate (E331), potassium citrate (E 332), calcium citrate (E333),
dependent manner when males are exposed to parabens. Other tartaric acid (E334), sodium tartrate (E335), potassium tartrate
authors have reported that these compounds have no relation with (E336), sodium potassium tartrate (E337), sodium malate (E350),
the male reproductive system, and are readily metabolized into potassium malate (E351), calcium malate (E 352), calcium tartrate
p-hydroxybenzoic acid, inside the body, which is not a toxic com- (E354), and triammonium citrate (E380). As shown in Table 2,
pound. Parabens have also been described as estrogenic disruptors the most common chemical antioxidants added to food to in-
and to be related with breast cancer in women, although, to this hibit lipid peroxidation and rancidification are butylated hydrox-
date, many conflicting theories exist (Oishi 2002; Harvey and Ev- yanisole (BHA, E320; ADI 0.5 mg/kg bw), butylated hydroxy-
erett 2004; Meeker and others 2011; Aubert and others 2012). toluene (BHT, E321; ADI 0.05 mg/kg bw), propyl gallate, (PG,

382 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Adding molecules to food, pros and cons . . .

Table 2–Antioxidant food additives with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E310 Propyl galate (PG) Code of Federal 1.4 mg/kg bw Jacobi and others (1998)
Regulations 21 Branen and others (2001)
Sec.184.1660 Tayama and Nakagawa (2001)
— Chen and others (2011)
EU Regulation No. Zurita and others (2007)
1129/2011 Amadasi and others (2009)
Han and Park (2009)
Tian and others (2012)
E319 tert-butylhydroquinone Code of Federal 0.7 mg/kg bw Whysner and Williams (1996a)
(TBHQ) Regulations 21 EFSA (2004)
Sec.172185
—
EU Regulation No.
1129/2011
E320 Butylated hydroxyanisole Code of Federal 0.5 mg/kg bw Whysner and Williams (1996a)
(BHA) Regulations 21 Jeong and others (2005)
Sec.172.110 EFSA (2011)
— Ali and Suziki (2012)
EU Regulation No. Carocho and Ferreira (2013a)
1129/2011 Vandghanooni and others (2013)
E321 Butylated hydroxytoluene Code of Federal 0.05 mg/kg bw McFarlane and others (1997)
(BHT) Regulations 21 Williams and others (1999)
Sec.172.115 Botterweck and others (2000)
— Engin and others (2011)
EU Regulation No. EFSA (2012a)
1129/2011 Carocho and Ferreira (2013a)
E324 Ethoxyquin (EQ) Code of Federal 0.005 mg/kg bw Błaszczyk and others (2003)
Regulations 21 Rodrı́guez-Trabado and others
Sec.172.140 (2007)
— Błaszczyk and others (2013)
EU Regulation No.
1129/2011

E310; ADI 1.4 mg/kg bw), ethoxyquin (EQ, E324; ADI 0.005 thors. Some claim PG as chemotherapeutic, nephroprotective, and
mg/kg bw), and tert-butylhydroquinone (TBHQ, E319; ADI 0.7 cytotoxic to HeLa cells, among other beneficial activities, while
mg/kg bw). BHA has been used since the 1970s as an effective others point out its effect as a xenoestrogen, a contact dermatitis
antioxidant in food with numerous studies describing its toxic- precursor, mutagen inducer, and that its antioxidant potential can,
ity against lab models. Whysner and Williams (1996a) described under certain conditions turn prooxidant (Tayama and Nakagawa
BHA’s toxicity as being species-specific for murine models and safe 2001; Zurita and others 2007; Amadasi and others 2009; Han and
for human consumption due to the lack of a forestomach in the Park 2009; Chen and others 2011; Tian and others 2012). Ethyl
human species that is sensitive to this antioxidant. More murine gallate, octyl gallate, and dodecyl gallate are very similar to PG
assays were carried out until 2011, when the EFSA reviewed the and execute the same effect in food, although being less used and,
literature and published a revised acceptable daily intake (ADI) therefore, less researched. EQ (E324; ADI 0.005 mg/kg bw) is a
that was not likely to be exceeded by the population. Still, much quinolone-based antioxidant that is not permitted to be added to
controversy arises from the consumption of BHA and studies have human food, only used in domestic and farm animal feed. This
to continue to bring more facts into the discussion (Jeong and oth- compound has been reported to induce dermatitis in animals and
ers 2005; EFSA 2011; Ali and Suzuki 2012; Carocho and Ferreira humans, as well as being a promoter of certain types of cancer.
2013a; Vandghanooni and others 2013). BHT, due to its similarity Although there is no immediate danger from EQ to humans, since
with BHA has suffered the same fate, with many studies pointing it is not allowed in food, there is still a latent risk, which derives
out its carcinogenity and deleterious effect on murine and human from the excess present in the ingested animal tissue, thus further
health. The EFSA also reviewed the daily intake of BHT, placing it studies should be carried out to regulate its indirect potential haz-
at 0.05 mg/kg bw, which is low if compared with the one of BHA ard (Błaszczyk and others 2003; Rodrı́guez-Trabado and others
(0.5 mg/kg bw). Still, as with BHA, some researchers continue to 2007; Błaszczyk and others 2013; EFSA 2013a).
find deleterious effects with BHT, while others have demonstrated Antibrowning agents. Antibrowning agents are used as food
its anticarcinogenic effects (McFarlane and others 1997; Williams additives to prevent food browning, which can occur at any mo-
and others 1999; Botterweck and others 2000; Engin and others ment during handling, processing, and storage. There are 2 types
2011; EFSA 2012a; Carocho and Ferreira 2013a). PG is used to of browning, enzymatic and nonenzymatic browning. In the for-
prevent rancidity in meat products, and due to its water solubility mer, the enzyme polyphenol oxidase catalyzes the conversion of
it can form complexes with iron salts and darken some food- polyphenols to quinones with further breakdown of these com-
stuffs, and therefore is usually added with citric acid to inhibit this pounds, ultimately darkening the color of the food (Branen and
phenomenon (Jacobi and others 1998; Branen and others 2001). others 2001). Nonenzymatic browning occurs in sugar carameliza-
PG can also act in synergism with BHA and BHT, but not with tion and in the Maillard reaction between carbonyl and free amino
TBHQ. PG is prepared by esterification of gallic acid with propyl groups, producing melanoidin pigments in various foodstuffs (Bra-
alcohol, and, since its discovery in 1948, and ever since, it has nen and others 2001). Among the most used antibrowning agents
had controversial and antagonistic effects, as reported by many au- are sulfites, covered in the previous section. The alternatives to


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 383
Adding molecules to food, pros and cons . . .

Table 3–Azo compounds of dyes with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E102 Tartrazine- FD&C Yellow Code of Federal 7.5 mg/kg bw Tanaka (2006)
No. 5 Regulations 21 Moutinho and others (2007)
Sec.74.1705 Tanaka and others (2008)
— Poul and others (2009)
EU Regulation No. Amin and others (2010)
1129/2011 Mpountoukas and others (2010)
Gao and others (2011)
Axon and others (2012)
E110 Sunset yellow- FD&C Code of Federal 2.5 mg/kg bw Axon and others (2012)
Yellow No. 6 Regulations 21 Sayed and others (2012)
Sec.74.1706 Ceyhan and others (2013)
— Yadav and others (2013)
EU Regulation No.
1129/2011
E122 Carmoisine No permission sought 4 mg/kg bw Amin and others (2010)
in the United States Arvin and others (2013)
— Datta and others (2013)
EU Regulation No.
1129/2011
E123 Amaranth Banned in the United 0.8 mg/kg bw Sasaki and others (2002)
States Mpountoukas and others (2010)
— Poul and others (2009) Sarikaya and
EU Regulation No. others (2012)
1129/2011
E129 Allura red- FD&C Red No. Code of Federal 7 mg/kg bw Tsuda and others (2001)
40 Regulations 21 Shimada and others (2010)
Sec.74.340 Ceyhan and others (2013)
— Abramsson-Zetterberg and Ilbäck
EU Regulation No. (2013)
1129/2011

sulfites are natural compounds, like ascorbic acid-based forma- EU, while fast green (FD&C Green No. 3) is forbidden to be used
tions, such as erythorbic acid (E315; 5 mg/kg bw); cysteine (E920; within the EU and legally added to food in the United States.
quantum satis), which is an aminoacid and reacts with quinolone There are 5 groups of coloring agents: the azo compounds, the
intermediates inhibiting further formation of compounds; some chinophthalon derivatives of quinoline yellow, the triarylmethane
phenolic acids, and finally 4-hexylresorcinol (E586; ADI not spec- group, xanthenes, and the indigo colorants (Sarikaya and others
ified), an organic compound which is only regulated in shrimp 2012).
(Branen and others 2001; Oms-Oliu and others 2010). Azo compounds. Regarding the azo group, it has many dif-
ferent colors, and all of them display the functional group R-N
Nutritional additives = N-R , in which R and R can be either aryl or alkyl (Stolz
Although considered by some authors, nutritional additives 2001; Chudgar and Oakes 2003). Among them, some of the most
should not be considered as such, since they confer nutritional used are tartrazine, known in the United States as FD&C Yellow
value to the food where they are incorporated, and, should rather No.5 (E102; ADI 7.5 mg/kg bw), sunset yellow, known in the
be considered as enrichments to foodstuffs. The consumption of United States as FD&C Yellow No. 6 (E110; ADI 2.5 mg/kg
these enriching compounds has increased in recent years due to bw), allura red, known in the United States as FD&C Red No. 40
health concerns of the population and its relation with nutrition. (E129: ADI 7 mg/kg bw), amaranth (E123; ADI 0.15 mg/kg bw),
The functional and nutraceutical activities of some nutrients have and carmosine (E122; ADI 4 mg/kg bw) (Fennema 1996). These
been gaining interest from both the scientific community and the compounds are described in Table 3. The azo compounds, with
food industry, resulting in its incorporation in certain foodstuffs, the N = N functional group and aromatic rings linked to them
adding value to them. These nutritional enrichments are natural are reductively cleaved into aromatic amines, with some of these
ones, and can be vitamins, amino acids, fibers, fatty acids, and aromatic amines being toxic, mutagenic, and carcinogenic (Chung
polyphenols, among others. The sources can vary depending on 2000; Zhang and Ma 2013b). Tartrazine is probably one of the
plant, mushroom, animal, or even synthetic origin (Branen and most controversial colorants, with some studies classifying it as a
others 2001). DNA binder, toxic to human lymphocytes (4 mM), a contributor
to primary biliary cirrhosis, lipid peroxidation promoter by pro-
Coloring agents duction of malondialdehyde, and reducer of superoxide dismutase
Coloring agents or food dyes are used to alter or confer colors to and glutathione peroxidase in mice (500 mg/kg) (Amin and oth-
food, in order to increase its attractiveness toward consumers. The ers 2010; Mpountoukas and others 2010; Gao and others 2011;
only dye with quantum satis status is calcium carbonate (E170), Axon and others 2012). Other studies regard tartrazine as safe to
which confers a white color to food. Dyes have been used for be consumed in the acceptable daily intake, posing no harmful
a long time in the food industry, but not without controversy effect in murine models and in humans (Tanaka 2006; Moutinho
and disagreement regarding their health effects (Branen and others and others 2007; Tanaka and others 2008; Poul and others 2009).
2001; Msagati 2013). Some dyes, like amaranth (E123; ADI 0.15 Another widely used azo dye is sunset yellow, which is produced
mg/kg bw), carmosine (E122; ADI 4 mg/kg bw), and others are from aromatic compounds derived from petroleum hydrocarbons.
banned in some countries, but not in others; for instance, both This compound, as tartrazine, has been related to genotoxicity in
these compounds are banned in the United States and not in the murine models with memory and learning deficits on the offspring

384 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Adding molecules to food, pros and cons . . .

Table 4–Triarylmethane compounds of dyes with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E131 Patent blue Banned in the United 15 mg/kg bw Sarikaya and others (2012)
States Lucová and others (2013)
—
EU Regulation No.
1129/2011
E133 Brilliant blue - FD&C Blue Code of Federal 10 mg/kg bw Tanaka and others (2012)
No. 1 Regulations 21 Lucová and others (2013)
Sec.74.101
—
EU Regulation No.
1129/2011
E142 Brilliant green No permission sought 5 mg/kg bw EFSA (2010b)
in the United States
—
EU Regulation No.
1129/2011
E143 Fast green - FD&C Green Code of Federal 25 mg/kg bw Ha and others (2013)
No. 3 Regulations 21
Sec.74.203- Banned
in the EU
E151 Brilliant black No permission sought 5 mg/kg bw Macioszek and Kononowicz (2004)
in the United States May and others (2010)
—
EU Regulation No.
1129/2011

as well as immunomodulatory and xenoestrogenic effects (Axon brilliant blue, known in the United States as FD&C Blue No.
and others 2012; Sayed and others 2012; Ceyhan and others 2013; 1 (E133; ADI 12.5 mg/kg bw), fast green (E143; ADI 12.5
Yadav and others 2013). Petroleum-derived allura red has been mg/kg bw), patent blue (E131; ADI 1 mg/kg bw), and bril-
subject to many toxicologic studies. Some authors consider it as liant black (E151; 1 mg/kg bw) among others (Table 4). Of
an impeding factor of memory and learning in infant rats, induc- these, brilliant blue and patent blue are the most common ad-
ing also damage to rodent colons (10 mg/kg) (Tsuda and others ditives, although patent blue is banned in the United States, and
2001; Shimada and others 2010; Ceyhan and others 2013). The no permission was sought for brilliant black, while fast green
EU has reviewed its opinion on allura red twice, concluding in the is banned in the EU. These colorants are not readily absorbed
2nd statement that there is a possibility that it can be genotoxic, by our bodies, in fact, 95% of them are present in feces, and
nevertheless some existing studies point otherwise (Abramsson- there are no reports of deaths due to human improper absorption
Zetterberg and Ilbäck 2013). Amaranth, another dye derived from (Gaur and others 2003). Reports on changes in mitochondrial
petroleum, has been extensively investigated in the past and was respiration have been described (Reyes and others 1993), as well
banned in the United States for being allegedly carcinogenic. It is as somatic mutation in Drosophila melanogaster wing spot test (25
approved in the EU and some other countries. Recent studies, us- mg/mL of patent blue, 12.5 mg/mL amaranth) (Sarikaya and oth-
ing comet assay, have described it as inducing damage in the colon ers 2012; Tanaka and others 2012). Some authors consider that
of rats (10 to 100 mg/kg). Moreover, using a somatic mutation the consumption of lollipops with brilliant and patent blue col-
and recombination test, amaranth proved to induce genotoxicity orants by children is dangerous due to their absorption through
to human lymphocytes (8 mM) (Sasaki and others 2002; Mpoun- the lingual mucosa (Lucová and others 2013), and with nega-
toukas and others 2010; Sarikaya and others 2012), while others tive effects on children who are the greatest consumers of these
have found no evidence of these effects in murine models (Poul products.
and others 2009). Carmoisine, another widely used dye which Xanthenes group. The xanthenes group is comprised of ery-
is forbidden in the United States has also been reported as be- throsine, known in the United States as FD&C Red No. 3,
ing responsible for biochemical markers alteration in murine vital (E127; ADI 0.1 mg/kg bw), fluoresceine, eosines, and rhodamines.
organs, modification of the secondary structure of serum pro- Erythrosine is allowed in both the United States and EU, while
teins (human serum albumin and bovine serum albumin), as well fluorescine is banned in the EU. This dye is a polyiodinated com-
as promoting conformation changes in DNA of bovine models pound that has been related to alteration in childhood behavior
(Amin and others 2010; Arvin and others 2013; Datta and others and thyroid function due to high iodine content (Chequer and
2013). others 2012; Lakshmi and Buchwald 2013). Other studies pointed
Chinophthalon derivatives. Quinoline yellow (E104; ADI 10 out that this compound could induce toxicity to mice testicles (689
mg/kg bw) is a quinophthalone synthetic dye chemically pre- mg/kg) and alter behavior patterns in these models. Recently, it
pared by mixing sodium disulfonates, monosulfonates, and trisul- has also been found to alter DNA structures, to display toxicity on
fonates. This compound has been reported to cause urticaria, human lymphocytes, HepG2 (50 μg/mL), and to promiscuously
asthma, rashes, and hyperactivity. It also alters the conformation inhibit protein–protein binding (Aziz and others 1997; Tanaka
of bovine serum albumin. A case of quinoline yellow-derived 2001; Mpountoukas and others 2010).
skin eruption was documented in 2013 (Branen and others 2001; Indigo colorants. The indigo colorants derive from the indigo
Macioszek and Kononowicz 2004; Shahabadi and others 2012; dye, known as FD&C Blue No. 2 in the United States, which was
Leleu and others 2013). initially extracted from the shrub Indigofera tinctoria. Today they
Triarylmethane group. The triarylmethane group relies on are chemically produced, and adverse reactions rarely occur in the
triphenylmethane backbones to produce different compounds like typical quantities added to food.


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 385
Adding molecules to food, pros and cons . . .

Food colorants have been subject of many extensive studies. One food without adding calories, making them edible by children,
of the most controversial was “The Southampton Study,” in which diabetic patients, and people who want to reduce calorie intake.
a cocktail of food colorings was given to a group of children in their Despite this noble objective, various studies have related saccha-
meal, with posterior comparison of the behaviour patterns to a rin with an increase in weight gain of mice consuming saccharin
placebo group. The researchers found evidence of attention deficit when compared to groups fed with sucrose (Swithers and others
hyperactivity disorder for the group consuming the colorants. This 2010; Feijó and others 2013). Cohen-Addad and others (1986)
study was highly contested by both the advisory body of the U.K. pointed out the capacity of saccharin to cross the human placenta,
Food Safety Authority and the EFSA, but the public advocacy and hypothesized that the presence of this compound, both in utero
groups and the media promoted a voluntary ban on the implicated and ex utero, could increase the incidence of neoplasms. Although
colors. A couple of years later, a follow-up study concerning food the carcinogenicity effects of this compound have been ruled out,
additives in Irish children pointed out that, comparatively to the it is still advisable to continue studying different types of activi-
Southampton study, children would never be exposed to such high ties of saccharin in the human body taking advantage of the new
doses of additives, helping their readmission. In 2008, the EU technologies that can help to uncover potential new dangerous
published regulation 1333 pointing out that some additives should interactions (Bandyopadhyay and others 2008).
clearly state possible negative effects on concentration activity of Cyclamate is another low-calorie sweetener; it was discovered
children (Council Regulation (EC) 1333/2008). in 1937 and was joined with saccharin in a 10:1 blend to remove
The conflicting results for virtually all the food colorants are its unpleasant metallic aftertaste. In the United States, cyclamates
quite preoccupying and should motivate the competent institu- are not allowed in food. After its discovery, cyclamate was related
tions to proceed with regulation, in order to dissipate consumer to bladder cancer in mice, which was later ruled out after many
doubts. Although many studies have been published regarding studies with different animals (Takayama and others 2000). Today,
colorants, very few considered interactions among colorants and some studies have described retardation of fetal development and
their dangerous effects on health. Two studies have pointed out hypertrophy in the exocrine pancreas of rat fetuses when exposed
the deleterious synergistic effects of colorants in neuronal cell lines to cyclamate (Martins and others 2010). Cyclamate is differently
in vitro (Lau and others 2006; Park and others 2009). Although metabolized in humans. Some individuals are able to convert cy-
subject of much controversy, colorants play an important role in clamate to cyclohexylamine, which is known to have some car-
the food industry, and the pressure for natural ones has been in- cinogenic effects; nevertheless others simply do not metabolize it
creasing. There are some alternatives with good results that are (Renwick and others 2004). This difference in metabolization and
slowly approved in both the United States and EU. Carotenoids, the fact that cyclohexylamine has been proven to induce testicular
anthocyanins, annatto, and paprika are examples of natural col- atrophy in rats are the main concern among scientists.
orants that can substitute their synthetic counterparts. The main Aspartame, discovered in 1965, is another widespread low-
drawbacks of these natural compounds are instability due to pH calorie sweetener. It is composed of phenylalanine and aspartic
and temperature, loss of color by oxidation, the need for higher acid linked to methanol. Extensive studies have been carried out
quantities in comparison to chemical ones, and the higher cost of regarding aspartame, and while some vouch for its safety others
manufacture (Hendry and Houghton 1996; Calvo and Salvador find troubling conclusions (Ashok and others 2013; EFSA 2013b;
2000; Delgado-Vargas and others 2000; Downham and Collins Rycerz and Jaworsaka-Adamu 2013). In a brief communication
2000; Giusti and Wrolstad 2003; Scotter 2009; Msagati 2013). Al- after the 1st conference on aspartame, Renwick and Nordmann
though colorants are better regulated and safer, further research is (2007) pointed out that the risk assessment gave an unbalanced im-
still needed to mitigate the faults in colorants, either by improving pression to regulators and consumers, and that future quantitative
the safety of approved ones or by discovering new. risk-benefit analyses should be able to provide more comprehen-
sive advice. After this, other studies have shown that long-term
Flavoring agents consumption of aspartame may lead to hepatocellular injury and
The flavoring agents are additives used to alter the taste of food alterations in liver antioxidant status while also altering behavior
either by enhancing it, sweetening, or completely changing the in rats (Abhilash and others 2011; Ashok and others 2013), al-
final taste of the produced foodstuff. The flavoring agents group is though not without intense debate. Aspartame present in stored
divided into 3 subgroups, the sweeteners, the natural and synthetic food can, at high temperatures and pH above 6, break down into its
flavors, and the flavor enhancers. metabolite, deketopiperazine, which is a major carcinogen to the
Sweeteners. The sweeteners are a group of compounds that central nervous system and is being actively investigated (Rycerz
confer sweetness to food. These can be nutritional sweeteners like and Jaworsaka-Adamu 2013).
sucrose, fructose (high-fructose corn syrup), and glucose or ar- Acesulfame K, or acesulfame potassium was discovered in 1967.
tificial nonnutritive sweeteners. There are no sweeteners with a It is not metabolized by the human body, therefore does not con-
quantum satis level. The most widespread nonnutritive sweeteners tribute to potassium uptake. Mukherjee and Chakrabart (1997)
are saccharin (E954; ADI 2.5 mg/kg bw), cyclamates (E952; ADI described this compound 1st as being genotoxic in rats, but later,
350 mg/kg bw), aspartame (E951; ADI 50 mg/kg bw), acesulfame after repeating some assays, refuted the previous conclusions and
K (E950; ADI 15 mg/kg bw), and sucralose (E955; ADI 15 mg/kg considered acesulfame K as safe, although some authors pointed
bw), as depicted in Table 5 (Branen and others 2001). Saccharin out that a long-term study should be carried out (Karstadt 2006;
is the oldest low-calorie sweetener, discovered in 1878, being 300 Soffritti 2006). Sucralose is an artificial sweetener very similar to
times sweeter than sugar. In the past, saccharin was thought to sucrose, in fact, only 3 OH groups are substituted by Cl atoms.
be related to human bladder cancer, but after intense research this Discovered in 1976, after several years of research, it was approved
idea was abandoned, with only a specific species (mouse) tumori- for consumption with no adverse effects reported in the vari-
genicity prevailing, with the results being corroborated in 2002 ous animal model studies performed, even at long-term exposure,
(Whysner and Williams 1996b; Dybing 2002). Saccharin’s main seen as though the human body does not recognize sucralose as a
objective, like all noncaloric sweeteners, is to confer sweetness to sugar and therefore does not metabolize it (Baird and others 2000;

386 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Adding molecules to food, pros and cons . . .

Table 5–Sweeteners with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E950 Acesulfame K Code of Federal 15 mg/kg bw Mukherjee and Chakrabart (1997)
Regulations 21 EFSA (2000)
Sec.172.800 Karstadt (2006)
— Soffritti (2006)
EU Regulation No.
1129/2011
E952 Cyclamates Banned in the United 11 mg/kg bw Takayama and others (2000)
States Martins and others (2010)
— Renwick and others (2004)
EU Regulation No.
1129/2011
E954 Saccharin Code of Federal 5 mg/kg bw Whysner and Williams (1996b)
Regulations 21 Dybing (2002)
Sec.180.37 Bandyopadhyay and others (2008)
— Swithers and others (2010)
EU Regulation No. Feijó and others (2013)
1129/2011
E955 Sucralose Code of Federal 5 mg/kg bw Abou-Donia and others (2008)
Regulations 21 Baird and others (2000)
Sec.172.831 Roberts and others (2000)
— Sims and others (2000)
EU Regulation No. Brusick and others (2009)
1129/2011 Brusick and others (2010)
Viberg and Fredriksson (2011)
E962 Aspartame Code of Federal 40 mg/kg bw Renwick and Nordmann (2007)
Regulations 21 Abhilash and others (2011)
Sec.172.804 Ashok and others (2013)
— Rycerz and Jaworsaka-Adamu (2013)
EU Regulation No.
1129/2011
- Fructose (high- fructose Code of Federal No limitation CFR (2003) Bray (2004)
corn syrup) Regulations 21 EFSA (2008)
Sec.184.1866- Not
considered a food
additive in the EU
- Sucrose Code of Federal No limitations CFR (2003) EFSA (2008)
Regulations 21 Anton and others (2010)
Sec.184.1854- Not
considered a food
additive in the EU
E957 Thaumatin EAFUS Doc No. 2849 Not specified Gibbs and others (1996)
— Branen and others (2001)
EU Regulation No. Watson (2002)
1129/2011
E960 Stevia GRAS No. 000468 4 mg/kg bw Koyama and others (2003)
— Kroger and others (2006)
EU Regulation No. Carakostas and others (2008)
1129/2011 EFSA (2010a)
Lemus-Mondaca and others (2012)

Roberts and others 2000; Sims and others 2000). In 2008, a study within the union “Food additives are substances that are not nor-
carried out with rats fed with sucralose pointed out various adverse mally consumed as food itself but are added to food intentionally
effects, including reduction in beneficial fecal microflora, increased for a technological purpose described in this Regulation, such
fecal pH, and enhanced expression levels of P-glycoprotein and as the preservation of food” (EFSA 2008). In the United States,
cytochrome P450 (Abou-Donia and others 2008), although this these sugars are approved, and added as additives to food “Food
study was highly contested by an expert panel (Brusick and others additives includes all substances not exempted by section 201(s) of
2009). Recent research has corroborated the previous findings of the act, the intended use of which results or may reasonably be
nontoxicity or carcinogenicity (Brusick and others 2010; Viberg expected to result, directly or indirectly, either in their becom-
and Fredriksson 2011). The subgroup of sweeteners offers many ing a component of food or otherwise affecting the characteristics
conflicting results and different points of view, but have led to of food” (CFR 2003; Bray and others 2004; Anton and others
several research articles assuring the safety of some artificial sweet- 2010). Thaumatin is a polypeptide extracted from the Thauma-
eners and pointing out others still demanding further research tococcus danielli plant, and 1st isolated in 1972. It is about 3000
(Weihrauch and Diehl 2004; Kroger and others 2006; Brown and times sweeter than sucrose and consists of 2 protein components,
others 2010; Swithers and others 2010; Tandel 2011; Shankar and thaumatin I and thaumatin II. It is defined as GRAS in the United
others 2013). In the meantime, natural sweeteners have become States, has an E number of 957 and a “not specified” ADI. Thau-
increasingly prevalent in modern diets, for their reduced caloric matin is used as a sweetener, but is also applied as a flavor enhancer,
contribution. Among them, sucrose, fructose (high-fructose corn and due to being a protein, it is rapidly digested. This compound
syrup), thaumatin, and stevia are some of the most important. In has been studied and no reports of allergic reactions, mutagenic
the EU, sucrose and fructose are not considered as food addi- or teratogenic effects or toxic toward rats, dogs and humans were
tives, rather as ingredients, due to the definition of food additives reported (Gibbs and others 1996; Branen and others 2001; Watson


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 387
Adding molecules to food, pros and cons . . .

2002). Stevia is a natural sweetener, extracted from the plant Stevia searched, although some claims exist against their use in children’s
rebaudiana, a shrub endemic to North and South America. Stevia is food.
300 times sweeter than sucrose, and is composed of 8 sweet diter-
pene glucosides (stevioside, steviolbioside, rebaudiosides [A, B, C, Texturizing agents
D, E], and dulcoside A). In the United States, stevia is a GRAS Texturizing agents are chemicals added to food in order to
sweetener and is used in beverages, deserts, and sauces, among modify the overall texture or mouthfeel of foodstuffs. The 2 main
others. In the EU, stevia is known as steviol glucosides, and uses groups within the texturizing agents are emulsifiers and stabilizers.
the identifier E960. Stevia has tested negative for carcinogenic Emulsifiers. The primary role of emulsifiers is to maintain
and genotoxic tests, as well and not having negative effects for re- emulsions (mixtures of 2 immiscible liquids) in good dispersion.
productive and developmental studies (Koyama and others 2003; By presenting a balance between hydrophobic and hydrophilic
Kroger and others 2006; Carakostas and others 2008; EFSA 2010a; groups, they surround the oil and other immiscible substances
Lemus-Mondaca and others 2012). present in the foodstuff avoiding their “clumping.” Emulsifiers
Natural and synthetic flavors. Natural and synthetic flavors are with quantum satis status are: lecithins (E322), calcium tartrate
mixtures of several chemicals used to substitute the flavor of foods. (E354), alginic acid (E400), sodium, potassium, ammonium, and
In most cases, these mixtures mimic the flavor of natural ones. calcium alginates (E401–404), agar (E406), carrageenan (E407),
Some additives of this group also carry out other functions in food, processed euchema seaweed (E407a), locust bean gum (E410),
like antimicrobial activity, gelling properties, and others. There are guar gum (E412), tragacanth (E413), gum arabic (E414), xantham
more than 1700 natural and synthetic compounds available to fla- gum (E415), tara gum (E417), gellan gum (E418), glycerol (E422),
vor foods. Within this large number, acidulants play an important konjac (E425), pectins (E440), celluloses (E461-466 and 469),
role by reducing the overall pH of the food. Most of these acidu- sodium, potassium, calcium, magnesium, mono- and diglycerides
lants are organic acids, some of them do not present uses restriction of fatty acids (E470a, 470b), acetic, lactic, citric, tartaric acid esters
(quantum satis) like acetic (E260), lactic (E270), malic (E296), cit- of mono- and diglycerides of fatty acids (E472a-472d), mono- and
ric (E330), propionic (E280), succinic (E363; ADI not specified), diacetyl tartaric acid esters, and mixed mixed acetic and tartaric
while others present maximum amounts permitted with ADI es- acid esters of mono- and diglycerides of fatty acids (E472e, E472f)
tablished, as fumaric (E297; ADI 6 mg/kg bw), tartaric (E334; (Branen and others 2001; Council Regulation (EC) 1129/2011;
ADI 30 mg/kg bw), and adipic (E355; ADI 5 mg/kg bw) acids Rohman and others 2013). Lecithins are naturally occurring phos-
(Branen and others 2001). pholipids that have excellent emulsifying capacities, cholesterol-
Flavor enhancers. Flavor enhancers are used to magnify, supple- lowering properties, and no reported toxicity (Iwata and others
ment, or enhance food flavor, but do not contribute with their own 1993; Wilson and others 1998). Sorbitan, also known as polysor-
flavor. The most used flavor enhancers are glutamic acid (E620; bate, consists of compounds containing polyoxyethylene ethers of
ADI not specified), monosodium glutamate or MSG (E621; ADI mixed partial oleic acid esters of sorbitol anhydrides and related
not specified), disodium inosinate (E631; ADI not specified), and compounds. Sorbitan is efficient as an emulsifying agent in food,
disodium guanylate (E627; ADI not specified). Monosodium glu- although there are some reports of neuron and cytotoxicity in
tamate is a salt of glutamic acid, a naturally occurring aminoacid. some types of cells (10 μg/mL) and increased susceptibility to
It is used in food to enhance its natural flavor and to produce the oxidative stress in murine models among other aspects (Tatsuishi
umami flavor (pleasant savory taste), either alone or in synergy and others 2005; Ema and others 2008; Eskandani and others
with disodium inosinate or disodium guanylate. Glutamic acid is 2013). Carboxymethyl cellulose is a cellulose derivative with car-
a known excitotoxin (molecules that can impair or destroy nerve boxymethyl groups bound to some of the glucopyranose monomer
cells by excessive stimulation), therefore it was involved in a long- hydroxyls, frequently used in various foodstuffs. No records of
term controversy. After this effect was proven in murine models, toxicity have been found for this emulsifier. Polyglycerol esters
scientists extrapolated to primates and considered that these were are also used for their emulsifying properties, with, polyglycerol
not affected by it. Others considered that humans were affected by polyricinoleate one of the most widespread. Various assays were
them, especially at a young age, and that, even though glutamic conducted with this compound in murine and human models,
acid alone could not induce damage, the overall daily consump- and no toxicity or carcinogenicity was detected (Smith and oth-
tion of all possible excitotoxins should be taken into consideration ers 1998; Wilson and Smith 1998). Propylene glycol is another
(Olney 1990, 1994). In 2000, a review of previous safety evalua- emulsifier produced from the reaction of propylene oxide with an
tions by the FAO, WHO, and the Scientific Committee for Food alcohol of choice in the presence of a catalyst. Propylene glycol
toward monosodium glutamate was published. It concluded that, is used as a food emulsifier, but also employed in the cosmetic
despite a part of the population being sensible to it, monosodium and pharmaceutical industries. Despite some controversial claims
glutamate could be consumed without any concern (Walker and regarding toxicity in the 1970s and 1980s, this compound can be
Lupien 2000). Recently, other effects on murine models and on consumed without concern (Thomas and others 2004; Spencer
humans have been attributed to monosodium glutamate, namely, 2005; Werley and others 2011).
induction of lipid peroxidation, impairment of synaptic plasticity Stabilizers. Stabilizers and emulsifiers are often the same com-
of mice neurons, deleterious effects of murine oocytes, and in- pound displaying both effects, although one of them is carried
crease in the overweight development of Chinese adults (Sanabria out more effectively. Quantum satis stabilizers are alginic acid
and others 2002; Eweka and Om’Iniabohs 2011; He and others (E400), sodium, potassium, ammonium, and calcium alginates
2011; Singh and Ahluwalia 2012), while other scientists found no (E401-404), carrageenan (E407), locust bean gum (E410), guar
correlation between monosodium glutamate and Chinese popu- gum (E412), tragacanth (E413), gum arabic (E414), xantham gum
lation obesity and that supplementation of food of postweaning (E415), gellan gum (E418), pectin (E440), invertase (E1103),
pigs is safe and improves growth performance (Shi and others polydextrose (E1200), oxidized starch (E1404), and monostarch
2010; Rezaei and others 2013). Disodium inosinate and disodium phosphate (E1410). Some of the most used stabilizers are listed
guanylate are used in fewer foodstuffs and therefore are less re- on Table 6. These compounds are added to confer and maintain

388 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Adding molecules to food, pros and cons . . .

Table 6–Stabilizers with use restrictions and their respective ADI quantities (mg/kg bw).

E number Name Legislation ADI References

E400–E404 Alginates Code of Federal — Bouyer and others (2012)
Regulations 21
Sec.184.1011
—
EU Regulation No.
1129/2011
E407 Carrageenan Code of Federal — Tobacman (2001)
Regulations 21 Weiner and others (2007)
Sec.172.620 Yang and others (2012)
—
EU Regulation No.
1129/2011
E440 Pectin Code of Federal — Akhtar and others (2002)
Regulations 21 Leroux and others (2003)
Sec.184.1588
—
EU Regulation No.
1129/2011

the desired food texture, as well as to prevent evaporation and along with the demand for less additives in food has helped this
deterioration of volatile flavor oils. Pectin is a naturally occurring area of research to develop. The main difference between coatings
heteropolysaccharide contained in plant cell walls. This compound and films is that the latter are used as covers, wraps, or separation
has demonstrated excellent stabilizing properties in foodstuffs and layers, while the coatings are considered part of the final prod-
no reported toxicity has been found (Akhtar and others 2002; uct, and are designed to protect or enhance it. The main benefits
Leroux and others 2003). Alginates (potassium, sodium, and of edible coatings and films are the extension of the shelf life of
calcium) derive from alginic acid, and have gained much interest the foodstuff, the addition of beneficial compounds like vitamins
in recent decades for their natural origin in brown seaweeds or and antioxidants in the films, the environmental friendliness of
algae and no reported cases of toxicity. Alginates are hydrophilic the materials used, and the potential enhancement of the food
colloidal carbohydrates that are used in the food industry due taste. In terms of disadvantages, the main issues with this tech-
to their unique colloidal properties, which include thickening, nology are related to the uneven thickness of the films, which
stabilizing, suspending, gel producing, and emulsion stabilizing. In can lead to irregular dispersion of the active constituents. This
recent years, alginates have been used for microencapsulation and can also occur if the food is not spatially uniform, which can
production of biofilms (Bouyer and others 2012). Carrageenans lead to unprotected spots, leaving the foodstuff prone to con-
are a family of polysaccharides, which are also extracted form sea- tamination or decay. In terms of their components, edible films
weeds (kelp) and widely used in the food industry as a thickener, are divided into 3 categories: hydrocolloids (containing proteins,
stabilizer, and texturizer. Up to 2001, very few reports on their polysaccharides, or alginates), lipids (containing fatty acids, acyl-
toxicity existed until Tobacman (2001) described carrageenan glycerols or waxes), and finally composites, which contains com-
as posing a carcinogenic risk to humans after detecting tumors pounds from both categories (Guilbert and others 1996; Debeau-
in colons of animal models. Immediately after this study, many fort and others 1998; Lin and Zhao 2007; Bourtoom 2008; Skurtys
others disproved the carcinogenicity of carrageenan, concluding and others 2010; Han 2013). There are some commercially avail-
that the carrageenan added to food is not carcinogenic, suggesting able coatings, like chitosan, calcium pectinate, calcium ascorbate,
a confusion between carrageenan and poligeenan (degraded wheat gluten, calcium acetate, sucrose esters, and corn protein
carrageenan) which is not used in the food industry (Weiner and which are already added to foodstuffs (Dutta and others 2009;
others 2007). Recently, other studies have described carrageenan Embuscado and Huber 2009; Elsabee and Abdou 2013). One of
as altering sulfatase activity in cells, therefore changing vital cell the most researched compounds for edible films is chitosan, due
processes (Yang and others 2012). to its combined antibacterial and antifungal effects, although it
does have some limitations in terms of mechanical properties and
Miscellaneous agents vapor permeability. Pectins have also displayed interesting results
Miscellaneous agents are additives that are added to certain food- (Espitia and others 2014). Other individual compounds extracted
stuffs for a specific outcome and that are not included in the other from plants, like carvacrol and methyl cinnamate have been tested
described additive groups. Examples of miscellaneous additives are in edible films, along with natural extracts of fruits and vegetables
chelating agents, enzymes, antifoaming agents, surface finishing (lemon, orange, oregano, thyme, among others) with satisfactory
agents, catalysts, solvents, lubricants, and propellants (Branen and results (Ponce and others 2008; Iturriaga and others 2012; Peretto
others 2001). and others 2014).
In order to overcome the limitations of this technology, future
Moving the Additives to the Exterior: Edible Coatings research should focus on tailor-made coatings and films, which
and Films are specific for the foodstuff rather than find the ideal compound
Since the early 1990s, edible coatings and films containing an-
or compounds for different foodstuffs (Valencia-Chamorro and
timicrobials, antioxidants, and other bioactive compounds have
others 2011).
been researched and used to extend shelf life of food and avoid
spoilage. This technology is usually applied in minimally processed Natural Additives: The Future or the Source of More
food, vegetables, fruit and meat, but also to other matrices like Controversy?
medication pills, sweets, and even French fries. The advantages of The era of natural food additives has started, some consumers
this technology and the recent developments in food processing deliberately choose minimally processed foods over processed


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 389
Adding molecules to food, pros and cons . . .

ones, and when they have to choose processed food they will gated with apparent success (Hagiwara and others 2010; Malheiros
generally select one with fewer additives and/or containing natu- and others 2012; Boualem and others 2013; Kallinteri and oth-
ral additives. Although the natural additives do not always represent ers 2013). Many other types of naturally occurring molecules are
a benefit compared to chemical ones, in most cases they are be- effective against food microbial contaminants, namely peptides,
lieved to be healthier, can carry out various functions in the food, which are also known as bacteriocins. Bacteriocins should not
and confer added value (bioactivity, nutraceutical). Natural addi- be confused with antibiotics, their use is strictly for food, while
tives are compounds, groups of compounds, or essential oils from antibiotics are for clinical/medical use. These molecules have no
plants that are already used empirically by the population for taste reported toxicity or secondary effects, unlike antibiotics (Cleve-
purposes. Fungi, seaweeds, and algae are also interesting sources of land and others 2001).
natural additives. These natural compounds have been around for
some time, but in recent years they have gained more interest from Natural antioxidants
the food industry for direct application or in synergy with other Antioxidants present in plants, algae, and mushrooms are excel-
natural or chemical additives. Among the many effects, the most lent natural additives to be added to foodstuffs for their ion or hy-
studied natural additive activities are their antimicrobial and an- drogen donating, metal chelating, and chain breaking capabilities.
tioxidant powers (Rasooli 2007; Tiwari and others 2009; Brewer Among others, the most antioxidant natural molecules are vita-
2011; Pillai and Ramaswamy 2012). mins, polyphenols, and carotenoids. These groups of molecules,
although being antioxidants can also exhibit additional properties
Natural antimicrobials (Ferreira and others 2009; Shahidi and Zhong 2010; Brewer 2011;
Natural antimicrobials that can be added to food are mainly ter- Carocho and Ferreira 2013a).
penes, peptides, polysaccharides, and phenolic compounds, among The main vitamins with antioxidant potential already in use
others with less expression. Examples of terpenes and their relatives as food additives are vitamin C (ascorbic acid) and vitamin E
include carvacrol, thymol, and menthol. Carvacrol is a monoter- (tocopherols). Vitamin C is an essential vitamin for humans that
penoid phenol, present in large quantities in oregano with great can only be acquired through diet (Davey and others 2000). This
antimicrobial (Escherichia coli, Bacillus cereus, and Staphylococcus) and molecule is an effective scavenger of the superoxide radical anion,
antifungal activities, even at low concentrations (Ultee and Smid hydrogen peroxide, hydroxyl radical, singlet oxygen, and reactive
2001; Ultee and others 2002; Nostro and others 2006; Xu and nitrogen oxide, avoiding oxidative stress in the human body. In the
others 2008). Carvacrol can act in synergy with cinnamaldehyde food industry, ascorbic acid is one of the most used antioxidants,
and nisin potentiating its effects. This compound has also been being used in the meat, beverage, fish, and bread industries, among
described as antimutagenic in the Ames Salmonella/microsomal others. By absorbing oxygen in the food, and oxidizing itself to
test (0.5 μL/plate) (Ipek and others 2005). Finally, positive results dehydroascorbic acid, the available oxygen is reduced, therefore
were obtained from the microencapsulation of carvacrol, allowing preserving the food. Apart from this antioxidant mechanism,
a slower spreading of its antimicrobial activity, therefore potentiat- ascorbic acid also acts as an antibrowning agent by reconverting
ing its use (Periago and Moezelaar 2001; Liolios and others 2009; quinones back to the phenolic form and avoiding flavor deterio-
Guarda and others 2011; Ye and others 2013). Thymol is an isomer ration in beverages (Davey and others 2000; Carocho and Ferreira
of carvacrol and it displays the same antibacterial and fungicidal 2013a). Vitamin E is composed of 4 isoforms (α-tocopherol, β-
activity (Ahmad and others 2011; Anderson and others 2009). tocopherol, γ -tocopherol, and δ-tocopherol) and 4 tocotrienols
Eugenol, a polyphenol used in various food and pharmacological (α-tocotrienol, β-tocotrienol, γ -tocotrienol, and δ-tocotrienol)
applications is effective against the carcinogenic aflatoxin B1 (300 (Hussain and others 2013). This vitamin exerts its activity espe-
ppm) produced by Aspergillus flavus, as well as an inhibitor of other cially against lipid peroxidation and rancidification by donating its
species of Aspergillus in vitro (150 ppm) (Komala and others 2012; phenolic hydrogen to the peroxyl radicals forming tocopheroxyl
Pillai and Ramaswamy 2012). Natural polysaccharides that are al- radicals that, despite also being radicals, are unreactive and unable
ready used as food additives have positive effects on health with no to continue the oxidative chain reaction. Both these vitamins can
reported toxicity. Chitosan and its derivatives, chitooligosaccha- work in synergism with the regeneration of vitamin E through vi-
rides, extracted from the shells of crustaceans, are used in the food tamin C from the tocopheroxyl radical to an intermediate, reinstat-
and pharmaceutical industries for their beneficial effects, namely ing once again its antioxidant potential. This is why they are usually
hypocholesterolemic, antimicrobial, immunity enhancing, antitu- employed together to extend shelf life of foodstuffs (Carocho and
mor, anticancer, anti-inflammatory, calcium-absorbing improve- Ferreira 2013a; Hussain and others 2013). Polyphenols, secondary
ment, and antioxidant power among others (Xia and others 2011). metabolites, of plants are also excellent antioxidants. Among
It is used as a food additive, but it also takes part in the manu- their various effects (antimicrobial, antimutagenic, anticancer,
facture of antimicrobial biofilms and can be used combined with antitumor, anti-inflammatory), they also scavenge radicals, chelate
other molecules, like xylan and glucose (Kanatt and others 2008; metals, quench oxygen atoms, and can act as ion or hydrogen
Dutta and others 2009; Kong and others 2010; Li and others donors. The 8000 described polyphenols are divided into 8 groups:
2011). Nisin, a peptide comprised of 34 amino acids has long hydroxybenzoic acids, hydroxycinnamic acids, coumarins, lignans,
been used in the food industry due to its excellent antibacterial chalcones, flavonoids, lignins, and xanthones. Some polyphenols
properties, namely against Clostridium perfringens, Clostridium sporo- exhibit good antioxidant activity as pure compounds incorporated
genes, and others (Jamura and others 2005; He and Chen 2006; in foodstuffs, while others depend on synergism to carry out
Udompijitkul and others 2012). It has been used with good results the protective effects. This is a drawback and at the same time
in meat, Galotyri cheese, and other foodstuffs. This natural com- an opportunity for the industry. For one, there is an imposing
pound has also reported toxicological effects on mice and has been demand to get to the compound that exerts the effect, but at the
described as arresting the progression of squamous cell carcinoma same time synergisms can be research opportunities, which could
in human cell lines (Joo and others 2012). The synergic effects be beneficial for the food industry (Carocho and Ferreira 2013a;
of nisin, as well as its encapsulation viability, have been investi- Carocho and Ferreira 2013b). Polyphenols have been used as

390 Comprehensive Reviews in Food Science and Food Safety r Vol. 13, 2014 
C 2014 Institute of Food Technologists®
Adding molecules to food, pros and cons . . .

antioxidants in the fish and meat industries by dipping carcasses Problems, Opportunities, and Future Perspectives
into polyphenolic extracts, allowing oxidation and bacterial con- It is estimated that the world’s population will reach 8 billion by
tamination to be delayed (Fan and others 2008; Kumudavally and 2025. This increase represents a challenge for the whole planet.
others 2008; Maqsood and others 2013). Other approaches have Larger amounts of food must be produced to feed the increasing
been tested with success, by incorporating natural extracts rich in population, especially in underdeveloped countries. Technology
polyphenols or pure compounds into food, and therefore avoiding improves all of the food processing stages, in particular the tech-
rancidity, spoilage, and bacterial colony formation for a longer nologies contributing to food preservation, such as mild-heat-
period of time when compared to the controls (Yao and others processing, modified atmosphere packaging, vacuum packaging,
2004; Serra and others 2008; Day and others 2009; Bansal and and refrigeration. New technologies like pulsed-light, high pres-
others 2013). sure, pulsed-electric, and magnetic fields, high-pressure process-
Carotenoids. Another important group of compounds are the ing, ionizing radiation, and ultraviolet radiation are actively being
carotenoids, defined as the pigments of nature. They occur in investigated in order to overcome their specific limitations and
many colors, from red to yellow, and derive from the secondary costs. Although these technologies are effective in reducing the
metabolism in plants, bacteria, fungi, and algae. Some of the most number of additives in food, these molecules cannot be com-
important carotenoids are α-carotene, β-carotene, and lycopene. pletely removed in the high-demanding worldwide food market.
The carotenoids have been used in the past, are still used presently Therefore, it is imperative to find viable solutions for the future
as food colorants, and are approved for those purposes (Hendry food concern (Lado and Yousef 2002; Tajkarimi and others 2010).
and Houghton 1996; Nguyen and Schwartz 1998; Watson 2002; In the 21st century, where information has no boundaries and
Nelis and Leenheer 2008). Recently, their antioxidant activity has can reach a high number of people in seconds, and with the
been regarded as an added value when they are incorporated in literacy of citizens rising, the demand for clarification regarding
food, and adding to the fact that they do not pose a toxicological food additives has grown considerably. In an inverse proportion,
threat, it makes them excellent natural food additives. β-Carotene, the trust in food safety agencies has declined, and citizens around
the precursor of vitamin A, has successfully been added to different the world are worried about the food they consume. Various
foods, especially functional and nutraceutic beverages, and its usage surveys indicate that some consumers are alarmed about food
for this purpose is estimated to increase in the future. Lycopene, additives and do not feel well informed regarding their role in
one of the major constituents of tomatoes, has been successfully food. The population does not know the difference between some
added to minced meat, increasing its storage stability, adding a groups of additives and is prone to be misled by marketing efforts
natural taste, improving the color, and indicating health benefits that request “clean labels,” which are labels that do not display
(Østerlie and Lerfall 2005). Taking into account that there are more names of compounds that resemble chemical or synthetic ones.
than 600 different carotenoids known, there is a high probability Another troubling fact is the consumption of highly transformed
that many of them can display beneficial health effects (Branen food that is growing considerably due to the exodus of populations
and others 2001; Lerner and Lerner 2011). from the countryside to large cities and a way of life away from self-
Essential oils. Essential oils are hydrophobic liquids obtained sufficiency. Home preparation of food is now substituted by the
by hydrodistillation or Likens–Nickerson extraction of plant parts “ready to consume” meal that just needs to be heated. The high
containing terpenes and phenolic compounds. The health benefits consumption of this type of food imposes 2 important reflections.
of these oils are well documented since the time their activities For one, the need for better, safer, and multipurpose additives
were 1st discovered. Essential oils are antioxidants, fungicidal, and (rather than adding various different ones to the same foodstuff)
bactericidal against Listeria, Staphylococcus, and other genera, im- and, on the other hand, the need to educate the population to
prove the shelf life of perishable foods, and delay spoilage. For on the composition of natural and highly processed foods, as well
some applications, their effects are potentiated by synergisms es- as balanced and unbalanced diets and the necessity of additives.
tablished with bacteriocins or even food constituents. The only So far, only the schooled fraction of the population of developed
downside of essential oils is their potential toxicity to humans, countries has become aware of these issues. Some advocates even
even at low concentrations, and despite the extensive research on plead for a shift to a reduced consumption of highly processed
them, further and more meticulous assays should be carried out foods (Shim and others 2011; Varela and Fiszman 2013).
to determine the real effects of these oils in the human body and The concern about misinformation regarding food additives is
to determine an ADI (Smith-Palmer and others 2001; Holley and also fueled by the blurred separation between natural and synthetic
Patel 2005; Sacchetti and others 2005; Rasooli 2007; Kanatt and additives. Today, additives are usually added to repair damage to
others 2008; Kumar and others 2008; Gutierrez and others 2009; food during its harvesting, storage, and processing, by correcting
Nguefack and others 2009; Tajkarimi and others 2010; Lv and the final foods’ colors, texture, moisture, and flavors. The actual
others 2011; Tyagi and Malic 2011; Turgis and others 2012). truth is that, in the global food market, of 30 billion dollars, 40%
Although the natural additives are beneficial as antimicrobials of all additives are used to keep the taste of the food products, 30%
and antioxidants, some of them have drawbacks; in some cases the employed for texture, 5% for food appearance, 20% for aiding the
amount needed for inhibition of certain contaminants is higher processing phase, and 5% is added to fight bacterial spoilage and
than the amount needed when using synthetic chemical com- rapid deterioration.
pounds. On the other hand, certain natural additives can add fla- The core studies regarding food additive safety in the United
vor to food, which can be a disadvantage for some of the effective States have been unaltered for 40 y, it is now time to review these
chemical antimicrobials. Furthermore, some polyphenols can in- protocols and prepare new ones, incorporating new technolo-
teract with proteins, carbohydrates, minerals, and vitamins, which gies, new knowledge, and leaving space for wider, transversal, and
might not prove to be beneficial or not exert the desired functions undoubted debates within the scientific community and by stake-
in foods. This needs further research (Lule and Xia 2005; Rasooli holders. The EU in its review of food additives until 2020 is taking
2007). these premises into account to gather a transversal understanding


C 2014 Institute of Food Technologists® Vol. 13, 2014 r Comprehensive Reviews in Food Science and Food Safety 391
Adding molecules to food, pros and cons . . .

of both the scientific community and legislators. Although diffi- food can be delivered around the world without losses in an ever-
cult, a unique European law regarding food additives could be the growing competitive market. Their role is becoming more and
best solution to bring confidence to the legislating bodies as well as more important with the increase in consumption of highly pro-
safety to the food that is consumed by the more than 500 million cessed foods due to changing lifestyles of modern world citizens.
people living within the EU. This mission is quite challenging due Despite the visible improvements in legislation and the produc-
to the difficulty to obtain concrete, sound, and irrefutable results tion of safer additives, many issues still remain unsolved, leading
regarding human exposure to additives when taking into account to increasing controversy and constant demand for better ones.
the different diets, lifestyles, ages, and genetic predispositions of The most obvious cases include the prohibition of some addi-
citizens (Bateman and others 2004; Connolly and others 2010; tives in EU, which are used in the United States, and others that
Mepham 2011; Lofstedt 2013). are banned in the United States and added to food in the EU. Al-
The future of food additives is of grave importance for mankind; though technologies will continue to develop and reduce the need
it is related to the well being of the entire human population. Food for additives, while they are still necessary, taking into considera-
additives will definitely change throughout the next decades, ei- tion the habits of consumers, it is expectable that natural additives
ther tending to natural extracts that need to be thoroughly studied will gain even more notoriety when compared to chemical ones.
for interactions or to the synthetic chemical ones that will con- This could be due to their various beneficial effects on health,
tinue to scare the consumers. Future technologies regarding food along with antimicrobial, antioxidant and other effects. The great
additives have been studied and aim to reduce impact on health, number of compounds in nature that still remain unknown, the
manufacture costs, and controversy, having also the minimum ef- power of natural extracts, and the synergisms with other com-
fect on the final appearance of food. Nanotechnology, which is pounds represent unlimited sources of new compounds with new
already widely used for different purposes is also being applied to possibilities. Today, natural additives do not answer all the doubts
food and to packaging, encapsulating the additives, allowing for and issues, being unwise to use them as an immediate alternative
controlled release, increase in stability, and reduction of impact in to chemical additives due to the impact on economies and health.
the final product. In a near future, it is expected that these nanos- Careful studies regarding natural additives must be carried out to
tructures, present within the food or in the package, detect decay not make them a source of even more controversy. What should
or contamination, and only then start the release of specific antiox- occur is a soft transition into the natural additives with a simultane-
idants or antimicrobials (Appendini and Hotchkiss 2002; Duncan ous reduction of additives altogether, relying on new technologies
2011; Cushen and others 2012; Coles and Frewer 2013; Sung and to carry out the same effects. There is no timeframe for this to
others 2013). happen, but it will surely take place, and there are 4 forces that
Filamentous fungi are already used to produce several natural will decide the fate of additives: legislators, scientists, commercial
compounds with color. These could be used as food colorants, enterprises, and ultimately, consumers.
substituting their chemical counterparts. This technology will be
used in the near future for the other classes of food additives, Acknowledgments
taking advantage of the extraordinary capacity of fungi and bac- The authors are grateful to Proder project no. 46577 PlantLact
teria to produce specific compounds (Dufossé and others 2014). for financial support of this work, to Fundação para a Ciência
Genetic biomarkers are being studied in order to early detect po- e a Tecnologia (FCT, Portugal) and COMPETE/QREN/EU for
tential allergic reactions to food additives, and vaccines are being CIMO strategic project PEst-OE/AGR/UI0690/2011 and LSRE
developed to be administered to additive susceptible individuals, strategic project PEst-C/EQB/LA0020/2011. The authors also
allowing potential allergens to be added to food or added in a state that they do not have any conflicts of interest.
higher concentration (Watson 2002). Among all the technologies
used for food additives, recombinant DNA will be the one with
the greatest effect. It is already used in bioprocessing to develop
additives and could even reduce the need for common additives.
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