Trends in Food Science & Technology 89 (2019) 100–109

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Trends in Food Science & Technology

journal homepage: www.elsevier.com/locate/tifs

Review

Mechanisms of action of novel ingredients used in edible films to preserve T

microbial quality and oxidative stability in sausages - A review
Johana Bolívar-Monsalvea,c,∗, Cristina Ramírez-Toroa,b, Germán Bolívara,
Carlos Ceballos-Gonzáleza,c,∗∗
a
Grupo de Investigación en Microbiología y Biotecnología Aplicada (MIBIA), Departamento de Biología, Universidad del Valle, Calle 13 # 100-00, Cali, Colombia
b
Grupo de Investigación en Ingeniería de Procesos Agroalimentarios y Biotecnológicos (GIPAB), Escuela de Ingeniería de Alimentos, Departamento de Ingeniería,
Universidad del Valle, Cali, Colombia
c
Centro de Biotecnología - FEMSA, Escuela de Ingeniería y Ciencias, Tecnológico de Monterrey, Monterrey, Mexico

A R T I C LE I N FO A B S T R A C T

Keywords: Background: Loss of organoleptic and microbial quality of sausages is a phenomenon mostly given by lipid
Shelf life oxidation, enzymatic autolysis, and presence of pathogenic microorganisms in meat, as well as inappropriate
Bioactive compound handling and storage conditions. The fast shortage of meat products unleashed by these processes may cause
Microbial spoilage foodborne diseases and productivity losses. Therefore, the use of natural bioactive agents has been proposed as a
Lipid oxidation
suitable solution to address this problem. However, the phenomena exerted by specific molecules of these
compounds to prevent the depletion of sausage quality are not clear enough.
Scope and approach: This review encompasses an overview of oxidative and bacterial spoilage in sausages, and a
description of some biochemical mechanisms of novel bioactive compounds that have been included in film
formulations to delay the shortage of these meat products.
Key Findings and Conclusions: Edible films containing bioactive compounds have been reviewed in order to
analyze ingredients that may improve organoleptic and/or microbiological quality of sausages over time,
highlighting relevant features concerning their antibacterial and antioxidant properties. Some of the main
mechanisms of action have been related to pore formation and permeation of bacterial membrane, as well as
depletion of its proton motive force, causing down-regulation of its metabolic functions and even cell death. On
the other hand, hydroxyl groups from plant extracts may help to neutralize free radicals involved in lipid oxi-
dation.

1. Introduction
Sausages are comminuted meats, seasoned with spices, which are
stuffed in casings, and processed by salting and drying. Due to their
complex matrix mostly composed of water, proteins and lipids, they are
prone to deterioration in terms of sensory and nutritional quality during
processing and storage (Hugo & Hugo, 2015). The most common non-
microbial spoilage in sausages consists in oxidation, leading to the use
of chemical substances such as sulphur dioxide or synthetic anti-
oxidants, e.g. butylated hydroxytoluene (BHT), butylated hydro-
xyanisole (BHA), and propyl gallate (PG) to avoid oxidative reactions in
lipids and proteins (Hugo & Hugo, 2015; Islam, Khan, & Islam, 2017;
Kim, Cho, & Han, 2013). Moreover, pathogenic and spoilage micro-
organisms take advantage of rich nutrients to grow in food, but a
technique commonly used to retard the growth of undesirable microbes
is to subject sausages to a curing process, adding nitrite salts to impair
(Honikel, 2008; Marco, Navarro, & Flores, 2006; Mortensen et al.,
2017). However, several studies have shown a correlation between high
intake of these synthetic preservatives and possible carcinogenic and
allergenic effects (Honikel, 2008; Islam et al., 2017; Kim et al., 2013).
Nowadays, a growing demand for meat products without synthetic
chemical preservatives has boosted the utilization of additives obtained
from natural sources, and they have become popular tools to extend the
shelf life of foods due to their antimicrobial and antioxidant properties

∗
Corresponding author. Grupo de investigación en Microbiología y Biotecnología Aplicada (MIBIA), Departamento de Biología, Universidad del Valle, Calle 13 #
100-00, Cali, Colombia.
∗∗
Corresponding author. Grupo de investigación en Microbiología y Biotecnología Aplicada (MIBIA), Departamento de Biología, Universidad del Valle, Calle 13 #
100-00, Cali, Colombia.
E-mail addresses: johana.bolivar27@gmail.com (J. Bolívar-Monsalve), carlos.ceballos28@gmail.com,
carlos.ceballos.g@correounivalle.edu.co (C. Ceballos-González).

https://doi.org/10.1016/j.tifs.2019.05.011
Received 7 December 2018; Received in revised form 27 March 2019; Accepted 20 May 2019
Available online 23 May 2019
0924-2244/ © 2019 Elsevier Ltd. All rights reserved.
J. Bolívar-Monsalve, et al.
(Aziz & Karboune, 2017; Echegaray et al., 2018; Lorenzo et al., 2018a,
2018b; Pateiro et al., 2018; Salgado, Ortiz, Musso, Di Giorgio, & Mauri,
2015; Umaraw & Verma, 2017). Nevertheless, direct incorporation of
these bioactive compounds into the sausage matrix may significantly
affect its organoleptic quality (Fernandes, Trindade, Lorenzo, & de
Melo, 2018; Lorenzo, González-Rodríguez, Sánchez, Amado, & Franco,
2013). As a consequence, packages have been proposed as media to
permeate chemical substances to food matrix through controlled release
(Benbettaïeb, Debeaufort, & Karbowiak, 2018; Santos et al., 2018), in
which edible films and casings are the alternatives most studied in meat
products.
In nature, biofilms are formed by microbial communities as a pro-
tective mechanism against hostile environments, and as a tool to settle
on surfaces such as stainless steel, packaging materials, or food matrices
(Firmino et al., 2018). In the case of edible films, they can be composed
of polysaccharides, proteins, and/or lipids, and they exhibit a viscoe-
lastic behavior that provides them mechanical resistance (Giteru et al.,
2015). The latter offer a variety of advantages in sausages such as
prevention of aroma loss, and regulation of permeability to water, li-
pids, gases, and solutes (Fakhouri, Martelli, Caon, Velasco, & Mei,
2015).
Edible films are used to allow continuous diffusion of natural
bioactive composites onto sausage surface over time, in order to help to
preserve, or even to improve, their quality. Bioactive peptides are often
used to mitigate growth of pathogenic bacteria, and some of them
correspond to bacteriocins and enzymes (Barry-Ryan, 2015; McMillin,
2017; Pisoschi et al., 2017; Ukuku, Mukhopadhyay, Juneja, &
Rajkowski, 2015). In the same way, plant extracts such as phenolic
compounds (flavonoids and phenolic acids); pigments; and essential
oils may be used as tools to avoid undesirable oxidative reactions in
meat products (Catarino et al., 2017; Majid, Thakur, & Nanda, 2018;
Zamudio-Flores et al., 2015).
Up to now, many authors have explained relevant mechanisms that
confer antimicrobial and antioxidative capacity to edible films, though
most of them do not provide clear explanations about how the chemical
compounds constituting these films can help to delay deteriorative
processes in sausages, in order to enhance the performance of anti-
microbial or antioxidant films. In this regard, the present review ana-
lyzes the roles of some current natural components used to preserve
sausages, focusing on biochemical mechanisms involved in slowing
down the growth of pathogenic and spoilage microorganisms, and de-
laying oxidative reactions.
The search of relevant studies and reviews was conducted based on
the concept map previously designed for this manuscript. Firstly,
Scholar Google and Scopus databases were used to find primary studies.
Secondly, Scimago Journal & Country Rank was entered to make sure
that these reports and their citations were published in high-qualified,
peer-reviewed journals (mainly Q1). Studies published before 2008 or
that referred to topics out of the core-subject of this review (sausages)
were discarded, e.g. chicken nuggets, meat slices, etc. This search
methodology left 43 papers to be analyzed.
2. Materials used to create film matrix and their main roles
Polymers used in manufacture of edible films may be obtained from
agricultural resources and microorganisms, as well as through chemical
synthesis, although to use just one type of polymer could not be enough
to preserve food quality. Therefore, the mixture of some of them, and
addition of plasticizers is often required (Aziz & Karboune, 2017).
Table 1 summarizes relevant features of each one.
2.1. Polysaccharides
Polysaccharides such as starch, alginate, cellulose, chitosan, pectin,
gums, and carrageenan can confer good barrier properties to gases, in
particular against oxygen, because of their ordered hydrogen bonds
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Trends in Food Science & Technology 89 (2019) 100–109
network, although most of these saccharides are sensitive to moisture
due to their hydrophilic structure. They are used in films to prevent
dehydration, oxidative rancidity, and surface browning in food
(Hassan, Chatha, Hussain, Zia, & Akhtar, 2018; Sánchez-Ortega et al.,
2014). However, polysaccharide-based films do not have mechanical
properties as good as those containing synthetic substances, though
high amylose starch seems to provide flexible and stretch resistant films
(Aziz & Karboune, 2017; Hamedi, Kargozari, Shotorbani, Mogadam, &
Fahimdanesh, 2017).
Alginate-based films can reduce meat shrink and improve juiciness
and texture, but free calcium and metal cations needed to fix alginate
coatings may induce undesired proteolytic activity (Khan, Adrees,
Tariq, & Sohaib, 2013).
2.2. Proteins
Functional properties of protein-based films depend on the degree of
extension of protein chains, and the sequence of residual amino acids.
Some materials such as collagen, keratin and proteins from quinoa,
milk, egg, whey, zein, and soy are widely used (Cazón, Velazquez,
Ramírez, & Vázquez, 2017). Denatured proteins of the three last sources
allow to obtain more cohesive structures than polysaccharides, and
their adherence to hydrophilic meat surfaces help to create a barrier
against oxygen and carbon dioxide. Nonetheless, protein-based films
may exhibit low resistance to both mechanical stress and water diffu-
sion, and they may become fragile under dried conditions. In addition,
the use of these macromolecules is a topic for discussion because they
may be susceptible to native enzymes, or cause adverse reactions on
consumer metabolism due to allergenic protein fractions (Galus &
Kadzińska, 2015; Sánchez-Ortega et al., 2014).
2.3. Lipids
The manufacture of lipid-based films is called larding (Khan et al.,
2013). Many waxes and oils obtained from petroleum have been used to
create moisture barriers, and improve the cohesiveness and flexibility
of films. In meat products, sensory properties, such as tenderness, have
been improved by using lipid coatings (Galus & Kadzińska, 2015;
Sánchez-Ortega et al., 2014). The main compounds used to produce
these films are usually natural waxes such as beeswax and paraffin wax;
resins; acetoglycerides; fatty acids; and both mineral and vegetable oils
(Galus & Kadzińska, 2015). Nevertheless, these films may confer an
undesirable taste to meat products.
2.4. Plasticizers
Plasticizers are characterized by low migration and low toxicity,
and they are used in combination with biodegradable polymers to en-
hance mechanical properties of edible packaging materials. Their low
molecular weight allow them to accommodate easily within inter-
molecular spaces of polymeric chains. However, low energy, vapor
pressure and diffusion rate are required for the plasticizer to solvate and
remain in the polymeric structure. Also, after a critical concentration of
plasticizer, a phase separation may be observed (Vieira, da Silva, dos
Santos, & Beppu, 2011).
The main role of plasticizers is to improve flexibility, reduce brit-
tleness, and decrease both porosity and tendency to crack. High con-
centration of hydrophilic plasticizers may increase water uptake of the
polymeric structure, meanwhile, hydrophobic plasticizers can reduce
its moisture permeability by closing micro-voids (Hamedi et al., 2017;
Vieira et al., 2011). Sorbitol, mannitol, and polyethylene glycol are
plasticizers frequently used in edible films, but polyols such as glycerol
are the most common due to its stability and compatibility with hy-
drophilic compounds (Catarino et al., 2017; Cazón et al., 2017; Hamedi
et al., 2017).
J. Bolívar-Monsalve, et al. Trends in Food Science & Technology 89 (2019) 100–109

Table 1
Properties of biopolymers used in films for packaging.
Polymer Barrier properties Mechanical properties Films characteristics

Water Gases Oil and Lipids

Polysaccharides • Transparent, tough and flexible films.

Chitosan Poor Good Good Poor • Barrier properties are improved using higher content of amylopectin.
Starch
Cellulose
• High amylose starch contributes to mechanical resistance.
Alginate
Pullulan
Proteins • Transparent, flexible and highly brittle films.
Casein
Zein
Poor Excellent Good Poor • Crosslinking agents may improve its water vapor barrier.
Gelatin
Wheat gluten
Whey
Lipids • Brittle and thicker films due to their hydrophobicity.
Waxes and paraffin Excellent Good Poor Poor • Improve the outer surface appearance of meals.
Acetoglyceride
Plasticizersa
• Present organoleptic problems.
Plasticizers are added to polymers to:
Glycerol • Decrease brittleness.
Polyethylene glycol
Sorbitol
– – – – • Improve flexibility and elasticity.
a
They are used to improve barrier and mechanical properties in films made up of polysaccharides, proteins or lipids.

3. Some factors involved in sausage quality decline
3.1. Microbial spoilage
Meat is a highly perishable food not only because of its chemical
composition, but also due to inadequate handling in post slaughter and
further steps such as packaging, transport and storage. In addition, a pH
value ranging from 5.5 to 7.0 is favorable for the growth of undesirable
microorganisms in meat products (Addis, 2015). Pathogenic bacteria
can be classified according to the composition of its cell wall: Gram-
positive or Gram-negative. The latter are more resistant because their
cell wall is composed of 90%–95% peptidoglycan along with teichoic
acid and proteins linked to it. However, hydrophobic molecules can
easily penetrate cell wall of Gram-positive bacteria and cause damage
in their organelles (Nazzaro, Fratianni, De Martino, Coppola, & De Feo,
2013).
Inclusion of bacteriocins, enzymes, and other polypeptides into the
packaging matrix has been proposed to delay the growth of pathogenic
and spoilage microorganisms in meat products (Aziz & Karboune, 2017;
Irkin & Esmer, 2015). Table 2 shows recently studies about bioactive
compounds used in edible films to help to control microbial quality of
sausages. It is important to mention that these substances may undergo
proteolytic degradation when they are included in food formulation, or
their preservative effect may be reduced due to interactions with some
food components (Santos et al., 2018).
A general schematic representation of the mechanism of action of an
antimicrobial polypeptide over a Gram-positive and a Gram-negative
bacteria is shown in Fig. 1. The favorable role of the mentioned com-
pounds has been attributed to some of the following events: ability of α-
helical antimicrobial peptides to modify the bilayer of phospholipids
and induce the pore formation, which is given by the presence of hy-
drophobic and hydrophilic portions that bind polar heads of phospho-
lipids from the microbial membrane (Santos et al., 2018), promoting
disintegration of the cytoplasmic membrane, and thereby, increasing its
permeability; inactivating extracellular enzymes by dissociation of
disulfide bonds; and oxidizing unsaturated fatty acids when they pe-
netrate the lipid structure of the cell wall. Finally, these phenomena
lead to cell death (Aziz & Karboune, 2017).
3.2. Lipid oxidation
Oxidative rancidity is considered one of the most detrimental
mechanisms in meat products quality because this phenomenon causes
nutritional-value loss, off-flavours, and formation of chemical com-
pounds that can be harmful to human health. Oxidation occurs when
there is a disruption of meat muscle membranes caused by grinding,
cutting, or during heat treatment and storage, whose exposition to
metal ions (the free iron in muscles, for instance) give rise to reactions
with pro-oxidants and unsaturated fatty acids (Addis, 2015; Aziz &
Karboune, 2017; Hugo & Hugo, 2015; Islam et al., 2017).
Lipid oxidation may be initiated by enzymatic reactions involving
enzymes naturally present in the product and/or from microbial origin,
as well as, autoxidation due to presence of polyunsaturated fatty acids
(Alparslan et al., 2016). Moreover, this phenomenon depends on factors
such as light, concentration of vitamin E (antioxidant), and oxygen flux
(Addis, 2015; Aziz & Karboune, 2017; Hugo & Hugo, 2015; Islam et al.,
2017).
Enzymatic hydrolysis of lipids is governed by lipases, esterases and
phospholipases, being phospholipase A1 and A2 the main enzymes in-
volved in lipolysis of meat. There is also non-enzymatic hydrolysis
caused by heme proteins such as hemoglobin, myoglobin or cytochrome
(Addis, 2015). The oxidation of unsaturated fatty acids (Fig. 2) is
brought about by formation of lipid radicals which increases along with
the number of bis-allylic carbons. The formed compounds (R*) react
with oxygen, producing peroxy radicals (ROO-) which pull H+ out
from another lipid molecule to form lipid hydroperoxide. Secondary
derivatives of the last one can be decomposed to produce volatile and
non-volatile compounds, such as alcohols, hydrocarbons, carbonyls,
furans, aldehydes and ketones, which contribute to rancid odor, and
loss of both color and nutritive value (Islam et al., 2017; Kim, Nahm, &
Choi, 2010; Šojić et al., 2014). In addition, formation of aldehydes
during oxidation or from smoking process can cause diseases related to
the digestive tract, and stimulate carcinogenic phenotypes (Šojić et al.,
2014).
On the other hand, protein oxidation occurs as a consequence of
covalent modifications made by reactive oxygen species or secondary
by-products from oxidative stress (Aziz & Karboune, 2017; Hromiš
et al., 2017; Noor, Bhat, Kumar, & Mudiyanselage, 2018). In the whole
muscle, proteins can be oxidized during storage due to the depletion of
endogenous antioxidants (Islam et al., 2017). For example, under
chilling, oxymyoglobin (which give the red color to the meat) reacts
with oxygen molecules from hydroperoxides and free radicals, and
thereby the Fe2+ from oxymyoglobin is oxidized into the Fe3+ present
in metmyoglobin. This reaction triggers oxidative rancidity that causes

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Table 2
Antimicrobial action in sausages using edible films.
Film ingredients Type of sausage Microorganism Antimicrobial activity Reference

Asparagus racemus and calcium alginate Chevon sausages Total viable count, psycrophilic bacteria, yeast, Edible films using A. racemus had lower values for Noor et al. (2018)
mould, coliforms microbial count than films without extract and control
sample
Chitosan and essential oils (thyme, rosemary) Sucuk (Turkish Enterobacteriaceae, micrococcaceae, LAB, yeast Essential oils reduced microbial growth. Higher Soncu et al. (2018)
fermented sausages) and mould. concentrations were most effective.
Calcium alginate and Tinospora cordifolia Goat meat sausages Total viable count, psychrophilic, yeast, mould Films made from 1.0% T. cordifolia showed a Kalem et al. (2018)
significatively reduction in microbial count
Chitosan and gallic acid Fresh pork sausages Total viable count Antimicrobial activity was probed. Highest Fang, Lin, Warner, & Ha (2018)
concentration of gallic acid (0.4%) was better inhibiting
microorganisms
Chitosan and silver substituted zeolite (AgZeo) Sucuk Aerobic plate count and LAB Chitosan significantly reduced microorganisms in the Çelebi Sezer & Bozkurt (2018)
product along 15 days
Eucalyptus globulus essential oil (EGO) Sliced sausages E. coli, S. entertidis, B. cereus, S. aureus 1.5% EGO in liquid medium produced 3.98, 4.22, 4.55, Azadbakht et al. (2018)
and 4.71 log reduction, respectively
Turmeric starch residue and gelatin, Gelatind and curcumin Commercial chicken L. innocua Edible coatings showed potential to inhibit L. innocua Tosati, de Oliveira, Oliveira, Nitin,

103
sausages and prevent cross-contamination of sausages & Monteiro (2018)
Collagen and Sakacin G Artesanal dry sausages L. monocytogenes Collagen was an effective carrier of sakacin G. It last Rivas et al. (2018)
showed bactericidal action against L. monocytogenes
Turmeric starch residue and gelatin Frankfurt sausages LAB and psychrotrophic bacteria The components of film reduced microbial growth in a Tosati et al. (2017)
period of 30 days
Poly-lactic acid (PLA), Zataria multiflora Bioss. essential oil Cooked beef sausages S. aureus, total viable count, psychrotrophic The mixture PLA and ZME was able to limit microbial Rezaeigolestani et al. (2017)
(ZME), propolis ethanolic extract (PEE), cellulose bacteria, LAB, mould, yeast, enterobacteriaceae. growth in sausage without affect barrier and mechanical
nanofiber properties
Chitosan and poly-vinyl alcochol Cooked Sichuan Total viable count and LAB A reduction in microbiological count of sausage was Feng, Liu, Makino, García Martín,
sausages observed for 29 days & Cummins (2017)
Chitosan and clove oil Cooked pork sausages Total viable count, psychrotrophic bacteria The treatment with better microbial stunting was 2% Lekjing (2016)
chitosan and 1.5% clove oil
Oxidized cellulose and chitosan Commercial sausages Total viable count Films made from grafting chitosan onto oxidized Wu et al. (2016)
cellulose had an excellent antimicrobial action
Garlic and oregano essential oils Dry cured-sausages Salmonella spp., L. monocytogenes, S. aureus 0.05% garlic and oregano essential oils inhibited García-Díez et al. (2016)
Salmonella and L. monocytogenes growth, and reduced
S. aureus count
Trans-cinnamaldehyde, carvacrol, thymol, eugenol and Frankfurt sausages L. monocytogenes Incorporation of phytochemicals in a chitosan matrix Upadhyay et al. (2015)
chitosan inhibited the growth of the pathogenic bacteria
Trends in Food Science & Technology 89 (2019) 100–109
J. Bolívar-Monsalve, et al.
Fig. 1. Effect of an antimicrobial peptide over the bacterial membrane.
meat browning, undesirable flavors and changes in product texture
(Kim et al., 2013).
Therefore, antioxidants have been used because they allow to pre-
vent lipid oxidation by means of scavenging radicals, breaking chain
reactions, decomposing peroxides, decreasing localized oxygen con-
centrations, and binding chain initiating catalysts (Hugo & Hugo,
2015). Table 3 shows recent studies about edible films containing an-
tioxidant compounds that may increase the shelf life of sausages.
3.3. Enzymatic autolysis
Autolysis is a phenomenon that occurs in postmortem breakdown of
macromolecules present in the tissues. A decrease in pH value of meat
makes the membrane more permeable, releasing a wide variety of en-
zymes. For example, lypolytic enzymes are responsible for fat oxida-
tion, amylolytic enzymes cause conversion of glycogen into lactic acid,
and proteolytic enzymes such as calpains, cathepsins and amino pep-
tidases perform the degradation of z-line proteins from myofibril. These
processes cause softening and greenish coloration of meat, and subse-
quently, they facilitate spoilage reactions mediated by microorganisms
and biogenic amines (Addis, 2015).
Fig. 2. Steps involved in lipid oxidation.
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Trends in Food Science & Technology 89 (2019) 100–109
4. Some benefits and mechanisms of action of novel bioactive
ingredients
4.1. Microbial-derived compounds
4.1.1. Bacteriocins
These composites have been defined as non-self-propagating pro-
teins synthesized by the ribosome, and seem to be effective against
some types of bacteria (Hugo & Hugo, 2015). There are four classes of
bacteriocins depending on their molecular weight, physicochemical
properties, and their amino acid sequence. Nevertheless, only class I
and II bacteriocins are used in food formulation. Class I bacteriocins are
membrane-active peptides whose molecular weights are less than
5 kDa, and contain lanthionine; whereas, those from Class II do not
have lanthionine and are heat-stable (O'Bryan et al., 2018).
Bacteriocins and the cell wall precursor Lipid II can form a complex
through electrostatic interactions (cationic peptide and the negatively
charged cell envelope) that inhibit peptidoglycan biosynthesis. These
aggregated complexes incorporate further peptides, and form pores at
the cell membrane, being this event the primary killing mechanism due
to rapid efflux of ions, amino acids and ATP, which causes depletion of
the transmembrane proton motive force (PMF), leading to cell death
(Liu et al., 2017; O'Bryan et al., 2018; Sánchez-Ortega et al., 2014).
J. Bolívar-Monsalve, et al. Trends in Food Science & Technology 89 (2019) 100–109

Table 3
Antioxidant edible films used in sausages.
Film ingredients Type of sausage Antioxidant Activity Reference

TBARS (mg malonaldehyde/kg), DPPH (%), Free Fatty Acids (% FFA)

Asparagus racemus and calcium alginate
Calcium alginate and Tinospora cordifolia
Chitosan and gallic acid
Chitosan
Microalgae crude extract and bacterial
cellulose
Cinnamon, clove and anise extracts
Kaffir lime leaf essential oil
Green tea extract and collagen fiber
Starch and clove essential oil
Rose polyphenols
Chitosan and clove oil
Trans-cinnamaldehyde, carvacrol,
thymol, eugenol and chitosan
Chevon sausage A. racemus had strong antioxidant properties. 2.0% of the extract Noor et al. (2018)
showed the lowest values for TBARS.
% oleic acid was reduced using 2.0% A. racemus
Goat meat sausage 1.0% T. cordifolia showed a significantly reduction in TBARS Kalem et al. (2018)
compared with 0.5%, 0% and control sausages. Also, these films had a
better effect in delaying the %FFA in the samples
Fresh pork sausage 0.4% gallic acid was able to stabilize free radicals. It exhibited lower Fang et al. (2018)
lipid, myoglobin and pro-protein oxidation
Turkish dry- Lower TBARS values were achieved with chitosan coatings due to its Arslan and Soyer (2018)
fermented sausages antioxidant activity and low oxygen permeability
Chinese sausage Active film showed the lowest TBARS value (2.6 mg/kg). Ochaikul, Suklampoo, Choochote, &
4 mg/mL extract exhibited high DPPH radical scavenging activity in a Suwanposri, (2018)
24 h period
Harbin dry sausages Spice extracts had similar TBARS value among them, and lower values Sun, Zhao, Chen, Zhang, & Kong
than control. Anise extract showed the lowest value (2018)
Beef sausage Samples had acceptable TBARS values and there were not differences Utami, Khasanah, and Solikhah
among them (2018)
Beef sausage Lower TBARS values was obtained using 0.5% collagen and 1% green Shokraneh, Ariaii, Rasouli
tea extract Ghahrodi, Hasannia, &
Sabbaghpour, (2017)
Commercial sausages 1% clove essential oil was able to maintain TBARS value lower than Ugalde et al. (2017)
only starch film and the control sample
Dry fermented Higher concentrations (3 mg/g) of rose polyphenols can reduce Zhang et al. (2017)
sausages effectively oxidation of sausages
Cooked pork sausage TBARS values increased along the time. However, they were lower Lekjing (2016)
using 2% chitosan and 1.5% clove oil. In addition, similar behavior in
peroxide values was obtained
Frankfurters Coating with phytochemicals and chitosan reduced lipid oxidation 10- Upadhyay et al. (2015)
fold than control

The use of chelating agents such as ethylenediamine tetra-acetic
acid (EDTA) makes more permeable the cell walls of Gram-negative
microorganisms, such as Escherichia coli O157:H7 and Salmonella ty-
phimurium, and induces the release of most of the lipopolysaccharides
from the outer membrane. This phenomenon causes an increased sen-
sitivity in bacteria when they are exposed to bacteriocins (Benbettaïeb
et al., 2018; Liu et al., 2017). In addition, films containing nisin, lyso-
zyme and EDTA have showed a better inhibition against both Gram-
positive and Gram-negative bacteria (Benbettaïeb et al., 2018).
Lungu and Johnson (2005) argued that nisin films reduced 6.6 log
CFU of Listeria monocytogenes per gram on frankfurters stored at 4 °C,
and bacteriocins extended the shelf life of the sausage over 28 days. In
addition, Benbettaïeb et al. (2018) found that hydrophobic films made
up of whey and soy exhibited a good delivering capacity of nisin on the
surface of food products. Hence, they proposed that these films were
suitable antimicrobial packages. On the other hand, Rivas, Cayré,
Campos, and Castro (2018) reported that casings made of collagen and
sakacin G exhibited bactericidal properties against L. monocytogenes in
meat emulsions for 35 days at 5 °C.
4.2. Organic acids
Organic acids are natural antimicrobial produced during lactic acid
fermentation. Most of them have pKa ranging from 3.0 to 5.0 which
limits their antimicrobial functionality when food has a pH value lower
than 5.5. They may inhibit the growth of microbial cells and fungi, and
sorbic acid can prevent the germination of spores (Khaneghah,
Hashemi, & Limbo, 2018).
Undissociated organic acids have the capacity to penetrate the mi-
crobial cell membrane, and dissociate into free proton and acid anion,
causing acidification of the cytoplasm. As bacteria have a neutral cy-
toplasmic pH, they must extrude protons to the outer layer but high
amount of ATP is used during this process, and therefore, the PMF is
halted (Sánchez-Ortega et al., 2014; Calo, Crandall, O'Bryan, & Ricke,
2015). In addition, cytoplasmic accumulation of anions causes damage
of both nucleic acid and protein functionality (Setlow & Johnson,
2013).
Edible films containing 2% polylactic acid from lemongrass oil have
been developed, showing antilisterial activity in pork sausages during
12 days at 4 °C, due to the disruption of microbial cell wall (Yang &
Song, 2016). Another type of films containing salts derived from levu-
linic- and lactic acid has been made, in which 2.7% of sodium levuli-
nate and sodium lactate had the best inhibitory capacity against aerobic
microorganisms during 14 days in both pork and turkey sausages.
Meanwhile, alterations in thiobarbituric acid reacting susbtances
(TBARS) and color sausages were not observed (Vasavada, Carpenter,
Cornforth, & Ghorpade, 2003).
4.3. Plant-derived compounds
4.3.1. Phenolic compounds
Phenolic compounds (PC) are the main composites involved in plant
defense against insects and fungi. Moreover, they have been widely
used in food industry for variety of applications. Their antimicrobial
and antioxidant properties are associated with their role as reducing
agent, hydrogen donor, oxygen quencher and metal chelator
(Benbettaïeb et al., 2018). Moreover, these authors have stated that the
antioxidant activity of PC depends on their molecular weight, number
of aromatic rings and hydroxyl radical substitution of functional
groups.
Low concentrations of PC can affect enzymes and thereby, down-
regulate energy-dependent mechanisms. For example, anthocyanins
can increase the Gibbs free energy necessary to adhere to epithelial cells
and hence, bacterial colonization is delayed. Meanwhile, high con-
centrations of PC can denature proteins (Nazzaro et al., 2013). On the
other hand, Noor et al. (2018) and Sharma, Mendiratta, Agarwal,
Kumar, and Soni (2017) have argued that PC can delay oxidation
processes either when they get oxidized in chemical reactions, or when

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they donate electrons to finish an oxidation cycle or remove free radical
initiators.
Kim et al. (2013) have proposed that the hydroxyl (-OH) group is
responsible for suppressing the formation of free radicals. This func-
tional group can donate hydrogen atoms with electrons to aromatic
rings, and subsequently, the neutralization of free radicals is achieved.
Moreover, –OH group may bind to the cell wall and trigger conforma-
tional changes in certain molecules present at the bacterial membrane
and, as a consequence, enzymes are inactivated, and bacterial pro-
liferation is delayed.
Zhang et al. (2017) found that rose polyphenols can prevent bio-
genic amine formation, increase of pH value, and lipid oxidation in
fermented sausages, along with a selective modification of bacterial
diversity. In particular, these authors reported an increased growth rate
of lactic acid bacteria when these polyphenols were added to the sau-
sage matrix. Although these PC were not added onto an edible film,
promising outcomes can be envisaged if they are. Kalem, Bhat, Kumar,
Noor, and Desai (2018) and Noor et al. (2018) used polyphenols-con-
taining extracts from Terminalia arjuna and Asparagus racemosus in
Chevon sausages, respectively, and they attributed the inhibition of
lipid oxidation reactions to the neutralization of radicals by bioactive
compounds through the supply of hydroxyl groups.
4.3.2. Essential oils
Essential oils (EOs) are secondary metabolites produced by aromatic
plants which may have antioxidant effects, medicinal properties and
fragrance. Their antimicrobial activity depends on their chemical
composition and the amount of single compounds, and their efficacy
rely on the growth stage of spoilage microorganisms (Chivandi,
Dangarembizi, Nyakudya, & Erlwanger, 2016). Some examples are the
alkyl group in the case of limonene and p-cymene; some functional
groups and the presence of delocalized electrons in most terpenoids;
and free hydroxyl groups and substitutions on the aromatic ring in
phenylpropenes (Nazzaro et al., 2013).
The most studied essential oils have been extracted from rosemary
and thyme (Soncu et al., 2018), eucalyptus (Azadbakht, Maghsoudlou,
Khomiri, & Kashiri, 2018) and Syzygium aromaticum (Ugalde et al.,
2017). Although the capacity of these substances to efficiently reduce
the microbial population present in the meat matrix has been demon-
strated, high concentration of essential oils may modify sensory prop-
erties of sausages due to their intense aroma (Aziz & Karboune, 2017;
Domínguez et al., 2018; McMillin, 2017). Therefore, it is necessary to
design controlled-release mechanisms of these composites from the film
onto food, considering the microbial growth kinetics (Irkin & Esmer,
2015). In addition, Benbettaïeb et al. (2018) have stated that the rate of
release of bioactive compounds should be similar, or slightly faster,
than the growth rate of pathogenic or spoilage microorganisms.
Cell membrane provides a barrier to regulate the metabolism and
maintain transport of solutes and energy-transducing processes
(Chivandi et al., 2016). However, regular cell operation is affected
when EOs diffuse through the lipid bilayer of the cell membrane, or
bind to periplasmic proteins by means of hydrogen bonds and hydro-
phobic interactions, causing alterations in membrane permeability
(Benbettaïeb et al., 2018; Khaneghah et al., 2018). Thereby, this de-
stabilization of the phospholipid bilayer produces a release of K+, and
an increase in ATP inside the cell, which may disrupt the PMF of the
cytoplasmic membrane, and cause uncontrolled active transport, de-
naturation of enzymes and cellular proteins, as well as loss of meta-
bolites, and coagulation of cytoplasm. Nonetheless, Gram-negative
bacteria exhibit better resistance due to their outer hydrophilic cell wall
composed by lipopolysaccharides molecules which provide them buf-
fering capacity against protonation (Azadbakht et al., 2018;
Benbettaïeb et al., 2018; Kim et al., 2013).
Some authors have argued that thymol and carvacrol are the most
potent EOs because they may trigger a change in membrane fatty acid
composition when they get into contact with microbial cells, and hence
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Trends in Food Science & Technology 89 (2019) 100–109
increase the permeability of the cytoplasmic membrane (Khaneghah
et al., 2018). This phenomenon may facilitate the loss of K+ ions, li-
popolysaccharides and ATP, affecting cellular functions. In addition,
carvacrol may reduce PMF, and thereby decrease flagellar movement
(Nazzaro et al., 2013).
All plant extract does not always cause damage to bacteria through
affecting the membrane permeability, for example, p-cymene can im-
pair the membrane potential of microbial cells by reducing the en-
thalpy, melting temperature of the membrane and affecting the PMF
(Hayashi, Bizerra, & Junior, 2014). On the other hand, eugenol is used
against microbial enzymes due to the double bond in α, β positions of
the side chain and a functional group (methyl) placed in the γ position.
Isoeugenol has antimicrobial activity not only against bacteria, but it
can also impair molds and yeast (Nazzaro et al., 2013). Finally, cin-
namaldehyde may be used to inhibit enzymes involved in cytokine
interactions and some cell functions, disturb functionality of ATPase,
and perturb the membrane (Chivandi et al., 2016).
Araújo, Gumiela, Bordin, Luciano, and de Macedo (2018) have de-
monstrated that antimicrobial activity of essential oils is a function of
the amount of oxygen available in a packed meat. In this way, low
oxygen diffusion avoids oxidative reactions in oils, and anaerobic mi-
croorganisms becomes more sensitive to the toxicity of these sub-
stances.
Azadbakht et al. (2018) have attributed the reduction of L. mono-
cytogenes in sausage cuts to the activity of many compounds such as 1,8-
cineole and oil monoterpene hydrocarbons. They observed that Gram-
negative bacteria exhibited greater survival than those Gram-positive,
due to the presence of hydrophilic lipopolysaccharides in the external
membrane that act as a barrier against these hydrophobic compounds.
On the other hand, Portuguese sausages packaged in active coatings
made of whey protein and Origanum virens essential oil were storage for
4 months, and they were preserved until 20 days (paínhos) and 15 days
(alheiras) more in comparison with uncoated samples, allowing to have
a permissible microbiological count (Catarino et al., 2017). In the same
way, the shelf life of sausages packaged in coatings with 2% chitosan
and 1.5% clove oil was 10 days longer compared to a control (without
coating), and 2% chitosan coating. This behavior was attributed to the
presence of eugenol from clove oil, which inhibits production of cellular
enzymes and hence, induce cell damage. Moreover, chitosan modifies
the permeability of the cytoplasmic membrane (Lekjing, 2016).
Regarding oxidation mechanisms in food, it has been demonstrated
that bioactive coatings containing Origanum virens positively con-
tributed to prevent chain oxidative reactions in alheiras sausages, which
contains a high fat content (23 g/100 g). Nevertheless, this packaging
did not have a significant effect on lipids of paínhos sausages (Catarino
et al., 2017).
4.4. Animal-derived compounds
4.4.1. Chitosan
Chitosan is a deacetylated form of chitin, derived from the shell of
shrimps and the cell wall of fungi, and is made up of N-acetyl-D-glu-
cosamine residues joined by β-(1–4) glycosidic. This substance exhibits
high solubility under acidic conditions and presents antimicrobial ac-
tivity when the pH value is lower than its pKa (6.2–7.0) (Hu & Gänzle,
2018; Khaneghah et al., 2018). This polycationic polymer has been
tested as a carrier for active substances like organic acids, essential oils,
and derivative organic compounds (Hu & Gänzle, 2018; Šuput et al.,
2012).
The antimicrobial activity of chitosan depends on several factors
such as its molecular weight (MW), degree of deacetylation (DDA),
conformational structure, interactions between positive charges (C-2
position of the glucosamine monomer) of the polymer, and anionic
components of the bacteria cell surface such as sialic acid in phos-
pholipids which limit the flow of microbial substances resulting in the
membrane leakage (Arslan & Soyer, 2018; Hugo & Hugo, 2015). High
J. Bolívar-Monsalve, et al.
MW of chitosan forms a shield and prevent the movement of nutrients
through the cell wall in Gram-positive bacteria, whereas its ability to
attach to Gram-negative bacteria is enhanced when its MW is low,
causing flocculation and disruption of physiological processes
(Khaneghan et al., 2018).
High DDA values, which are obtained from alkaline treatment
around 80 °C, enhance antimicrobial activity of chitosan due to stronger
electrostatic interaction between the positive charge of this compound
and the negative charge of cell surface (Hu & Gänzle, 2018). This event
with the subsequent loss in the barrier function due to penetration of
oligomeric chitosan into the cell wall, make proteins and electrolytes to
come out of cell, and impair transcriptional and translational me-
chanisms of the microorganisms, as well as inhibition of some enzymes
(Alemán et al., 2016; Hugo & Hugo, 2015; Zivanovic, Davis, & Golden,
2015).
Films made up of a chitosan-gelatin matrix inhibited both Gram-
positive and Gram-negative bacteria in Alaska pollock sausages for 42
days, and it was attributed to hydrophobic amino acid residues found in
shrimp cooking juice (Alemán et al., 2016). Arslan and Soyer (2018)
reported interesting results on suppression of fungal growth during ri-
pening when chitosan was applied to fermented sausages, achieving
around 2 log cycle decrease of mould and yeast count. Fungal pro-
liferation on casings of sausages was also mitigated when chitosan at
1% level was applied, exhibiting the same effectiveness as potassium
sorbate at 20%.
On the other hand, antioxidant action of chitosan is associated with
chelation of free iron and other essential ions driven by thermal treat-
ment of meat, which may destabilize the microbial cell wall and in-
terfere with their enzymes (Zivanovic et al., 2015). Chitosan can also
modify the oxygen barrier through interactions with ionic functional
groups that restrict the PMF, though the antioxidant effect of this
polysaccharide is strongly related to its molecular weight, and thereby,
to its concentration (Arslan & Soyer, 2018; Šuput et al., 2012).
The advantages of coatings made of chitosan with oregano for
sausages was shown in pork sausages, in which lipid oxidation in the
coated meat product (9.64 mg TBARS/kg) was lower than in the un-
coated sample (29.23 mg TBARS/kg) at 15 °C and 75% relative hu-
midity (RH) for 150 days. Besides, no statistical changes in color were
found between the tested products (Šuput et al., 2012). Moreover,
Arslan and Soyer (2018) found that TBARS values at the end of ripening
period of sausages were significantly lower for those treated with
chitosan solutions in comparison with the control group.
Lekjing (2016) evaluated peroxide values and TBARS in cooked
pork sausages wrapped with films containing chitosan and clove oil
compared to those in which the last one was not present in the coating,
both stored at 4 °C. All samples showed an increase in the mentioned
parameters, but the product covered with 2% chitosan and 1.5% clove
oil had the lowest values. This phenomenon was related to the sy-
nergistic effect of the mixture, in which chitosan act as chelator of ions
that promote oxidative reactions in lipids. Furthermore, the L* value of
control samples increased, whereas the mixture composed of chitosan
and clove oil exerted antioxidant activity over the sausage, which is
related to the TBARS value. Finally, a* and b* values were not affected
by any treatment.
4.4.2. Enzymes
Enzymes are very sensitive to environments and substrates, for ex-
ample, they can only be subjected to low-temperature processes to
avoid their denaturation (Irkin & Esmer, 2015; Sánchez-Ortega et al.,
2014). However, they exhibit inhibitory activity against Gram-positive
bacteria and oxidative processes.
These chemical compounds are immobilized to be used in food
packaging in order to inhibit microorganisms, being lysozymes the most
used enzymes for this purpose (Benbettaïeb et al., 2018; Sánchez-
Ortega et al., 2014). Their mechanism of action relies on the splitting of
β-1-4-glycosidic bonds between C1 of N-acetylmuramic acid and C4 of
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Trends in Food Science & Technology 89 (2019) 100–109
N- acetylglucosamine of the peptidoglycan of bacterial cell walls, and
consequently, a lysis of bacteria is produced. Some enzymes can inhibit
Gram-negative bacteria by modifying their lipopolysaccharide layer
using chelators that bind to Ca++ or Mg++ (Zivanovic et al., 2015),
making these microorganisms more susceptible to some of the bioactive
compounds mentioned before.
On the other hand, enzymatic antioxidants have primary defenses
that hinder the formation of free radicals. Some examples are glu-
tathione peroxidase, catalase and superoxide dismutase. In the same
way, there are secondary enzymes like glutathione reductase and glu-
cose-6-phosphate dehydrogenase which are reducing agents to coun-
teract free radical formation (Islam et al., 2017).
L. monocytogenes was inhibited in bologna sausages when myr-
osinase from yellow mustard extract was added to a film, and the de-
crease in the number of pathogenic viable cells was attributed to the
hydrolysis of glucosinolate (Lara-Lledó, Olaimat, & Holley, 2012). On
the other hand, Marcos, Aymerich, Garriga, and Arnau (2013) have
stated that non-thermal processes such as high-pressure processing
(HPP) may not be required to inhibit microorganisms when bioactive
films are used. However, some authors have argued that HPP is vital to
improve the effect of antimicrobial films, since HPP causes lethal or
sub-lethal injuries in the cell membrane of microbes.
4.5. Pigments
Edible films produced using oxidized banana starch, glycerol and
5.17 mg/g betalains allowed to keep constant TBARS values in
Frankfurt turkey sausages at 4 °C and 5% relative humidity from 5 to 20
days, whereas the control group exhibited an exponential increase in
this parameter. Lipid oxidation and the production of malondialdehyde
was correlated with the decrease in L* value, although samples covered
with these films did not exhibit a significant change. On the other hand,
coatings containing betalains kept constant a* and b* values, whereas
an increase of these values in the control samples was reported.
Moreover, the unusual brown color of sausages is often due to light-
dependent reactions, because nitric oxide is separated from the hemo
group of hemoglobin and then, the hemo group is oxidized by oxygen
present in the environment (Zamudio-Flores et al., 2015).
5. Conclusions
Understanding the mechanisms of action of chemical compounds
used to mitigate deteriorative process in sausages plays a crucial role in
the design of strategies to achieve this aim, since the effect of different
treatments can be combined in a synergetic way, and at the same time,
the addition of more than one substance for the same aim can be
avoided, unless a significant improvement is exhibited over time.
Therefore, when the key features of food preservatives are well-un-
derstood, a wide variety of strategies to mitigate microbial deteriora-
tion and lipid oxidation may come into mind. However, some tradi-
tional methods such as high-temperature treatments should not be
replaced by the addition of these substances.
Furthermore, physicochemical properties of the sausage-film matrix
should be analyzed in order to avoid using bioactive compounds that
lose their bioactive properties when are exposed to specific conditions,
or that may lead to the formation of any chemical complex that impair
the nutritional or sensory quality of the product.
Funding
This work was supported by COLCIENCIAS (Convocatoria 745,
2016).
Acknowledgements
Authors acknowledge the funding received from COLCIENCIAS
J. Bolívar-Monsalve, et al. Trends in Food Science & Technology 89 (2019) 100–109

(Convocatoria 745 - 2016), the partial funding provided by Industrias doi.org/10.1155/2018/7405736.

de Alimentos Zenú S.A.S., and the support from both Grupo MIBIA and Galus, S., & Kadzińska, J. (2015). Food applications of emulsion-based edible films and
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(GIPAB) at Universidad del Valle. We highlight the experimental and García-Díez, J., Alheiro, J., Pinto, A. L., Soares, L., Falco, V., Fraqueza, M. J., et al. (2016).
theoretical contribution of members of MIBIA group. Behaviour of food-borne pathogens on dry cured sausage manufactured with herbs
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