3

Current Expansions in Microbiology for

Food Preservation
K. Ranjitha
Scientist (Microbiology), Division of Post Harvest Technology & Agricultural
Engineering, ICAR- Indian Institute of Horticultural Research, Hessaraghatta
All rights reserved. May not be reproduced in any form without permission from the publisher, except fair uses permitted under U.S. or applicable copyright law.

Lake (PO), Bengaluru- 560 089, Karnataka, India

Introduction
Advances in food preservation methods have supported the journey of food science
from that of a cookery science to a fast growing industrial technology. Food
preservation knowledge deals with the way to prevent spoilage and maintenance of
quality in foods. Spoilage is the deterioration in quality brought about by inherent
biochemical and chemical changes, as well as microbial metabolism during food
storage. Basic principles of food preservation (Frazier et al., 2008) remains the
same, but newer methods for achieving the goal is added through research fuelled
by a consumer demand for fresh-like foods with convenience and safety. Scientific
information on novel processing methods are beautifully compiled (Cullen et al., 2011;
Da-Wen Sun, 2014; Tokuþoðlu and Swanson 2015; Doona, 2018).
In-depth knowledge of physiological activities of micro-organisms in presence of
preservation agents is essential for preparation of safe foods with prolonged shelf life.
Besides this, it is necessary to understand the effect of food constituents on microbial
resistance against inactivation agents, to identify critical dose requirement. This
profound knowledge would also help to understand kinetics of microbial inactivation
and to develop mathematical models suitable for a realistic design of process
parameters. For better appreciation of the following sections, readers are encouraged
Copyright 2021. [NEW INDIA PUBLISHING AGENCY] NIPA.

to have fundamental knowledge in microbiology and food processing. The sections in

this chapter cover advanced microbiological knowledge pertaining to the preservation
aspects of food.

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 72  Advances in Processing Technology

Food spoilage microorganisms

Microbes use food as a source of nutrients for cell growth and energy. In the process,
the food texture, odour, appearance and quality changes. Multiple types of microbial
species are often found in spoiled food but specific spoilage organisms (SSO) are
spoilage initiators responsible for deteriorative; changes making food unfit for
consumption. Common specific spoilage organisms in foods are summarized in Table
1. With newer development of molecular typing methods, scientific names of certain
microorganisms have changed in recent years, and some older names are no longer
in use.
Table 1: Common microorganisms associated with food spoilage
Bacteria Yeasts Molds
Acetobacter Candida Aspergillus
Alicyclobacillus Dekkera Botrytis
Alcaligenes Rhodotorula Ceratocystis
Bacillus Saccharomyces Colletotrichum
Clostridium Zygosachharomyces Diploidia
Desulfotomaculum Fusarium
Erwinia Monilinia
Flavobacterium Mucor
Lactobacillus Penicillium
Leuconostoc Phomopsis
Moraxella Rhizopus
Pediococcus
Photobacterium
Proteus
Pseudomonas
Serratia
Streptococcus
Weissella

Metabolic properties of food spoilers

Correlation of food spoilage characteristics with the functional properties of spoiler
organisms is a difficult task. This is due to the diversity in the food composition and
metabolic characteristics of microbes. Spoilage microorganisms are responsible
for visual, texture and flavor defects, but these sensory descriptors are not directly
correlated with enzymatic functions and metabolic pathways. Some studies reported
the detection and measurement of volatile molecules, amines, organic acids etc. in food
spoiled by known microorganisms (Table 2). However, correlating the production of
these molecules to the metabolic functions of spoilage microorganisms is not always
possible; the reason being complexity of reactions in spoilage, serial changes in the
first formed degradation products (Eg. meat discolouration), enzymatic reactions

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 Current Expansions in Microbiology for Food Preservation 73

originating from both the spoilage organisms and the food matrix (Eg. lytic enzymes
of muscle cells) etc. Some of the spoilage enzymes are not well characterized. Usually,
the end product molecule in a biological system is formed through multiple pathways
and enzymes and identifying the entire enzymatic chain in the context of insufficient
knowledge on metabolic properties of the microbes is a challenge (Remenant et al.,
2015).

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 Table 2: Examples for spoilage compounds formed in food due to metabolism of specific spoilage organisms.

Sensory defect Spoilage product Spoilage substrate Food product Specific spoilage organism
Slime Dextran Sugars Fermented vegetables Leuconostoc
Fishy off odour Trimethyl amine Trimethyl amine oxide Fish Shewanella putrefaciens,
Photoobacterium
phosphoreum,
Medicinal flavor 2-methoxy phenol Sugars Juice Alicyclobacillus acidoterrestris
Cheesy/buttery off flavor Acetoin Sugars Meat Enterobacteriaceae
Fruity smell Esters Fish Pseudomonas fragii
Sweet curdling Proteinaceous fat particles Phospholipids Milk Bacillus cereus
74  Advances in Processing Technology

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 Current Expansions in Microbiology for Food Preservation 75

Stress adaptation of food microorganisms

Microbes in food are able to outgrow the preservation factors due to the gradual
fine tuning of their metabolisms to adapt the changes and thus to maintain cellular
homeostasis. The change from a normal physiological situation to a situation of
stress is a gradual one. Processing steps present multiple stresses to the organisms
by changing the water availability, heating, pH changes, antimicrobial additives etc.
Spoilage of processed foods is caused due to a set of organisms capable of overcoming
these stresses. Most often, the stress adaptation is achieved through modifications of
the cell wall and membrane, which represents ‘outer and inner barriers’ for the action
of antimicrobial agents. Cell membrane adaptations in Clostridium botulinum cells
resistant to heat and the bacteriocin nisin have been reported (Mazzotta and Montville,
1999). Acid-adaptation of Listeria monocytogenes resulted in enhanced tolerance
against the biological preservatives nisin and lacticin 3147; and this observation
correlated with changes in fatty acid profile of cell membrane (van Schaik et al., 1999).
Induced changes in bacterial cell wall have also been reported as a response to the
presence of antibacterial compounds. These studies on these aspects were focused
on food borne pathogens, rather than on the spoilers, but it is logical to assume that
similar mechanisms exist across similar bacterial genera.
Yeast cells adapt against membrane active peptides by significantly increasing the chitin
and cell wall protein levels, specifically of cell wall mannoproteins. Role of proton-
pumping ATPase, in resistance development against weak organic acids in yeasts
are well established. Brul and Coote (1999) have shown that in adapted organisms,
transportation of preservatives, sorbic acid, benzoic acid, acetic acid from the cytosol
to the extracellular environment occurs. Microarray and proteomics analysis indicated
mechanisms such as the activation of heat-shock proteins and indications for the
activation of the cell integrity pathway as adaptive responses in weak organic acid
resistant cells (Smelt and Brul., 2014).
The phenomenon of adaptation to one type of stress improving resistance of other
unrelated stresses is called cross adaptation or cross protection. For example, acid
adaptation and starvation induces thermotolerance in Listeria monocytogenes,
Escherichia coli and Salmonella. Thus, initial adaptation of the microbes to one type of
stress helps in resistance development and survival of the microorganisms in food.
A study by Gahan et al. (1996) revealed that acid adaptation improved the survival
of L.monocytogenes in low-pH dairy products, including cottage cheese, yogurt, and
whole-fat cheddar cheese, and low pH foods such as orange juice and salad dressing.
This is because, many stresses can have similar effects on bacterial cells, and bacteria
have evolved to build up an all-purpose general stress response. The best-known
general stress responses are controlled at the gene expression level by controlling
transcription by alternative transcription initiator proteins (alternative sigma factors).
In bacteria, a constitutive sigma factor controls housekeeping genes under non-stress

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 76  Advances in Processing Technology

conditions, but an alternative sigma factor binds under stress conditions, and this
changes specificity of RNA polymerase to achieve the transcription a different set of
genes that protect the cell against adverse conditions. This coordinated regulation
allows the simultaneous expression of numerous genes in response to a single stimulus
in the environment. These regulators are key players in raising a coordinated stress
response involved in tolerance of stresses such as acids, low water activity, temperature,
bacteriocins, antibiotics, ethanol, and starvation. Most of the proteins formed as a
result of stress adaptation responses have role in biofilm formation and sporulation.
(Begley and Hill, 2016).

Microbiology of food preservation methods

Thermal inactivation of microorganisms
Application of heat treatment to destroy pathogenic and spoilage microorganisms is
one of the most successful food preservation method used since centuries, and is widely
used even today. Based on the extent of heat treatment, the processes are classified into
major groups-
1. Low heat processing or pasteurization: The purpose is to destroy the vegetative
growth of all spoilage microbes and the time temperature combination required
for this treatment should be sufficient to destroy the most heat resistant
pathogens viz., Mycobacterium tuberculosis and Coxiella burnetti. The shelf life
of pasteurized foods is further maintained through refrigeration.
2. High heat processing or sterilization: This heat processing is meant for achieving
the complete destruction of microorganisms. Bacillus stearothermophilus spores,
being extremely heat resistant are used as evaluator for efficiency of sterilization.
But the old type ‘canning’ sterilization is replaced now by ultra high temperature
(UHT) processing, which combines continuous flow thermal processing with
aseptic packaging. Some times to denote this combination, the term ‘aseptic
processing’ is used. By UHT processing, quality of the products can be retained
due to the low heating time and fast cooling time. UHT process is followed in
milk industry, beverage industries etc.
Process design suitable for thermal inactivation of microorganisms in foods is
achieved through insights gained so far on the inactivation pattern through heating.
This pattern help to decide the temperature-time combination required for the target
organisms in heat processing.
When a microbial population is heated at a specific temperature, the cells die at a
constant rate (first order reaction). To describe the thermal inactivation kinetics,
two parameters viz., D value and Z value is vital. D value is the time at particular
temperature necessary to destroy 90% of the population of a specific organism. Z
value is the change in temperature needed to change the D value by 90%. The thermal

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 Current Expansions in Microbiology for Food Preservation 77

deactivation kinetics with respect to foods is well worked out (Stumbo, 1973; Singh & 
Heldman, 2014). Although the results of D value experiments in buffer are not always
the same as in real food, the data are comparable taking into account the simulation
of pH and water activity in the studies. For most vegetative cells, the reported z values
are generally around 5 to 6 with a range of 4 - 9. For Listeria monocytogenes, D 55 values
of 3.2 minutes are reported for vacuum-packed minced meat to 47 minutes in ground
pork with z values between 4 and 5. For E. coli O157:H7 and Salmonella, similar values
have been reported ranging about six minutes in minced meat and in ground beef or
ground pork. Yersinia enterocolitica in ground beef and milk or in liquid egg (Toora et
al., 1992., Bolton et al., 2000, Hunag and Juneja., 2001; Favier, 2008.).
Variation occurs from the standard thermal inactivation model in real situations. The
variation occurs as appearance of a shoulder and tailing in the log survivor Vs time
graph. (Fig. 1). Shoulder is the lag period contributed by poor heat transfer of the
heating medium (food) and due to the increase in temperature required to bring out
sufficient injury before the first order inactivation kinetics in the log number of survivors
with time. The tailing effect may be due to either the stress adaptation response of the
cells or due to the heterogeneous flora with difference in their heat resistance levels.
Heat shock also activate physiological responses leading to the synthesis of an array
of proteins known as heat shock proteins (HSPs), which provide resistance to further
heat treatment. In general, heat shocked cells have to be heated twice than that of non-
heat-shocked cells to achieve the same extent of lethality. Increased heat resistance due
to heat shock proteins have been reported in a variety of Enterobacteriaceae members,
spore formers like Clostridium spp., Bacillus stearothermophilus etc., Most of the HSPs
acts as molecular chaperones to helping in proper folding of proteins denatured by heat.

Fig 1. Thermal inactivation pattern of microorganisms

Bacterial endospores are heat resistant. Heat resistance of bacterial spores have positive
correlation with DNA content, dipicolinic acid and calcium level, mineralization and
dehydration, while the vegetative cells heat resistance is associated with high level of

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 78  Advances in Processing Technology

saturated fatty acids in the cell membrane. Apart from these inherent heat resistance,
stage of growth, growth temperature and growth medium affects the heat resistance
of organisms in food. pH of the medium is recognized as one of the most important
factors affecting the heat resistance of bacteria, typified by reduction in heat resistance
at acidic pH. Logarithm of heat- destruction rate shows first order reaction curve at
acid to alkaline range with a minimum rate at the optimum pH for growth.
Fatty materials in foods prevent the faster heat penetration and thus provide
protective effect to the microorganisms against heat treatment. Soluble solids in the
medium also affect the heat resistance of microbes. For example, dissolved sodium
chloride increases heat resistance by stabilizing membrane proteins, increase melting
temperature of membrane phospholipids. By doing so, cell membrane damage and cell
leakage is retarded. The heat resistance is also influenced by oxygen concentration. In
certain cases, eight fold increase in heat resistance was recorded when the organisms
are heated at low redox potential conditions. Stress adaptation of Listeria cells to
starvation, ethanol, hydrogen peroxide and acid significantly increase the resistance to
heat through cross protection mechanisms (Juneja and Sofos, 2001).

Novel thermal methodologies for inactivation of microbes in foods

Food technologists have adopted the concepts of alternative methods for heat generation
such as radiofrequency (RF), microwave (MW), infrared (IR) and ohmic heating (OH)
methods, for faster heating of foods and thus to retain the better sensory qualities along
with microbial inactivation. Novel thermal food-preservation technologies, causes self
heating of the product, helping to reduce the heating time. Lower heating time reduce
the loss of sensory attributes, making these method as potential next generation food
processing methods.

Ohmic heating
Ohmic or electrical resistance heating involves an application of a low-voltage
alternating current to a continuously flowing food product. Heat is generated within
the food product during this process due to electrical conductivity of food. This method
has potential applications in inactivation of microbes in highly viscous products and
liquid-particle mixtures. Earlier studies on microbial inactivation kinetics have shown
that D values due to ohmic heating is usually less than conventional heating, showing
additional mechanisms of microbial inactivation operating synergistically with heat
generation. The mechanisms have been reported as thermal, chemical and mechanical
effects. The thermal effect is due to self heating of foods, while chemical effects are due
to generation of highly oxidizing compounds such as hydrogen peroxide, chlorine etc.
Extent of chemical damage depends on type of electrode, current density, frequency
and the medium used. More recent studies suggest that the electricity flow results in
pores in cell membrane (electrical permeation mechanism or electroporation) causing
leakage of cellular constituents and cell death (Cunha et al., 2017; Cappato et al., 2017).

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 Current Expansions in Microbiology for Food Preservation 79

Microwave heating
Microwave heating is a very popular, extensively studied technology in food processing
worldwide. This process has advantage of high heating rates, which result in reduced
processing time, easy operation, cleaner work environment, lower maintenance
requirements, and reduced use of plant space. Microwave pasteurization or sterilization
can be explained by various mechanisms, such as selective heating, electroporation, cell
membrane rupture and magnetic field coupling to cause reactivity of biomolecules. For
example, due to the microwave selective heating, microbial bodies can reach a higher
temperature than the surrounding fluid, leading to faster microbial destruction (Tang,
2015).

Nonthermal physical methods of microbial inactivation

Increased consumer demand for fresh-like foods has promoted the development
of nonthermal methods for microbial inactivation for food preservation. These
technologies are named according to the main processing parameter leading to cell
inactivation. The alternative physical technologies food processing with high microbial
inactivation properties are ultrasound, high hydrostatic pressure (HHP), pulsed
electric field (PEF), and electromagnetic radiations such as gamma rays, electron beam
& UV radiations.

Ultrasound
Sound waves with frequencies above the threshold for human hearing (>16 kHz)
are called ultra sound. High-power ultrasound (>1 W/cm2  and & 20 - 500 kHz) is
a green substitute to the traditional methods of  food preservation. The cavitation
force produced due to ultrasound device in the liquid food medium causes lethality
to microbes. The antimicrobial efficiency of high-intensity ultrasound in foods is
determined by contact time with the microorganism, microorganism type, food
quantity, composition, and treatment temperature. Intracellular cavitation ruptures
cell membranes, and produces free radicals and H2O, which also assist in microbial
inactivation. Greater surface area of the cell, more is the lethality Thus in general
rod shaped bacteria are more vulnerable to death by sonication than cocci. Bacteria
that form spores of Clostridium and Bacillus species have shown greater resistance to
ultrasonic treatment than vegetative cells, these bacteria are more resistant to thermal
treatment as well as to ultrasound. Microbial lethality improves when sonication is
combined with heat (thermosonication) or pressure (manosonication). Unlike many
other types of cross adaptations, the stress adaptation of microorganism to heat does
not affect their sensitivity to ultrasound. This characteristic makes manosonication and
manothermosonication as a potential tool in processing. The other advantage is that
damage to cells caused by heat can be reversible, in contrast to this, damage caused by
mano sonication is irreversible (Piyasena et al., 2003) .

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 80  Advances in Processing Technology

Irradiation
Gamma irradiation for inactivation of insect pests, pathogens and sprouting etc. is
already a technology, even though the public acceptances is slightly less. Medium
doses (0.75- 2.5 kGy) impart a pasteurising effect in food (radurization). High doses
(30- 40kGy) can be applied to commercial sterilization for purposes similar to canning
(radappertization).
Mechanism of action of gamma rays occurs through both direct and indirect effects of
irradiation on cellular components. Irradiation results in radiolysis of water resulting
in hydrogen and hydroxyl free radicals generation. In the direct action, rays cause
damage to the DNA of of organisms (Lacrox, 2005).
Anaerobic conditions reduce the lethality due to irradiation. This is due to the lowered
rate of oxidizing reactions which produce free radicals and toxic oxygen derivatives.
Prokaryotes are more resistant to irradiation damage than eukaryotic microorganisms.
Viruses exhibit higher resistance to irradiation treatments than bacterial spores, which
are more resistant than vegetative cells. Vegetative cells of bacteria are more resistant
than yeasts and molds. Among bacteria, gram negative bacteria are more sensitive to
irradiation than gram positives. Researchers have demonstrated that irradiation doses
of at least 1.0 kGy, which could virtually eliminate gram negative bacteria in foods, have
a much little effect on gram positive bacteria. Similar to heat resistance pattern, spore
formers and stationary phase cultures offer more resistance to irradiation. A radiation
dose of 1.5kGy to 3.0kGy is required to inactivate spoilage fungi such as Aspergillus,
Penicillium etc. in commodities like pulses.
Proteins, antioxidants and low water activity of food matrices protect the microbes
from the lethality due to irradiation, while fat content does not affect the sensitivity of
microbes in foods. The vast majority of scientific literature on effect of temperature
on ionizing radiations indicates that microbial sensitivity to irradiation is higher at
ambient temperatures than at subfreezing temperatures. For example, 8.5 fold higher
irradiation doses were required for a 90% reduction in Pseudomonas spp. at subfreezing
temperatures than an ambient temperature. The D value for E coli O157:H7 was 0.28kGy
at 5 °C and 0.44 kGy at -5°C. This is due to low water activity subfreezing temperatures,
and thus low level of radioloysis, resulting in less production and movement of free
radicals. Free radicals such as hydroxyl radicals and hydrogen radicals accounts for
85% of lethality factors linked to irradiated organism when E colichallenge tests
were conducted. The atmospheric composition in contact with microbes influences
their inactivation by irradiations. For example, Hastings et al. (1986) observed that
Lactobacillus sake, Lactobacillus alimentarius, Lactobacillus curvatus were more
sensitive to gamma irradiation in ground meat packaged under 100% carbon dioxide
than under nitrogen.

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 Current Expansions in Microbiology for Food Preservation 81

Electron beam Irradiation(EBI) is another kind of irradiation used for inhibit microbial
growth in food. The energy transfer within the body is due to EBI destruction of
chemical and molecular bonds of the cells. Ultra violet radiations of the wavelength
ranging between 200- 280 nm (UV C) are also germicidal, and finds application in
very popularly water purification systems. UV light exposure causes the formation of
covalent links between adjacent bases, resulting in thymine dimer ormation. These
thymine dimers inhibit DNA functions and cell growth stopped. Ultra violet exposure
reduces the heat resistance in microbes. Therefore UV light in combination with
thermal treatment might be an alternative to thermal sterilization (Gayan et al., 2013)

High hydrostatic pressure (HHP) processing

HHP processing, the application of high pressures (100–800 MPa) to foods, is a well-
developed non-thermal treatment, to prolong shelf life of solid or liquid foods. The
pressure is transmitted uniformly within the product through a liquid medium, usually
water (Isostatic Principle). As the pressure build up in the medium, adiabatic heating
occurs and water temperature increases around 3 ˚C every 100 MPa. Therefore, HHP
should be applied in high-moisture foods; while porous and dry foods are not suitable
for this process. Along the years, successful results on microorganism inactivation by
HHP in plant- and animal based food products have been reported.
HHP acts a microbial agent by inducing the structural changes in the cell membrane and
enzymes. For example, 50 MPa of pressure inhibit protein synthesis in microorganisms
and reduce the number of ribosomes. A pressure of 100 MPa can induce partial protein
denaturation, and 200 MPa causes damage to the cell membrane and internal cell
structure. Increasing the pressure to 300 MPa or more induces irreversible denaturation
(Srinivas et al., 2018). Fungi are the most sensitive microorganisms to HHP, while some
fungal spores are highly resistant. Vegetative cells are killed at 400-600 MPa, spores can
survive upto 1000 MPa. Temperature of sporulation determines pressure resistance, as
they are most resistant when sporulated at lower temperatures. Initiation of pressure
induced germination is also affected by sporulation at lower temperatures (Barba et
al., 2015).
Eukaryotic microorganisms are more pressure sensitive than prokaryotes, and
consequently fungi are easy to be killed than bacteria using HPP. However, heat
resistant fungus Byssochlamys produce pressure resistant spores. Since fungi are easily
killed, HPP is commercially used in food products such as fruit juices, where bacterial
spoilage is lesser concern.
Besides intrinsic parameters of foods, pressure, temperature, and treatment time are the
most important parameters to be considered for processing optimization using HPP.
HHP has efficiency similar to conventional pasteurization in microbial inactivation,
with the advantage of not modifying nutritional and sensory properties of the product.

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 82  Advances in Processing Technology

The intrinsic parameters refer to the type of substrate and composition of the food.
All major nutrients like carbohydrates, proteins, lipids confer a protective effect to the
microbes. The higher protective effect due to the presence of vitamins is attributed
to the faster recovery of organisms from sub lethal injury with the help of these
nutrients (Black et al., 2007). Baroprotective effect of ions like calcium, magnesium
and phosphates etc. is also known. Black et al. (2007) reported the increased survival
of Listeria innocua in simulated milk ultra filtrate with added calcium, magnesium,
citrate and phosphate. The authors concluded that magnesium stabilises ribosomes,
calcium stabilizes cell membranes. Sucrose, a very common ingredient many of the
fruit based and cereal based products, can impart protection to bacterial cells from the
damaging effects of HPP stabilizing membrane protein functionality (Manas & Pag ˜
an, 2005). Foods with low water activity (aw) exerts a protective effect to microbial cells
against HPP. On the other hand, microbes injured through HPP are more sensitive to
death by lowering the water activity as a strategy for further preservation. It is not only
the osmolarity and water availability that has an impact on resistance to HPP but also
the nature of the solute. Application of high pressure brings down the pH of the food,
which can also inhibit the proton pumping force and energy generation. Oscillatory
high pressure treatments (several cycles of high pressure) are more effective than
equivalent continuous pressure of comparable holding time. For example, Hayakawa et
al (1994) compared continuous and oscillatory (cycles of 5 min each) pressurizations in
Bacillus stearothermophilus spore suspensions. Treatments at 800MPa for 60 minutes
at 60 °C and 70 °C decreased spore counts by 3.5 and 4.5 log CFU/mL., Oscillatory
pressurization at 70 °C could achieve 6 log reductions (Hayakawa et al., 1994).
Numerous HHP treated products are available in the market. Important challenge in
HHP processing is the development of basic research and in the equipment design of
high temperature and high pressure (HTHP) processing to achieve food sterilization
levels (Considine et al., 2004).

Pulsed Electric Fields (PEF)

PEF process involves the application of voltage pulses of the range 15–80 kV/
cm for a short time to pumpable foods passing between two electrodes. Therefore,
only homogeneous liquids can be pasteurized by PEF treatment. Unlike other non-
thermal methods, PEF has several critical parameters to be considered for processing
optimization. This includes electric field strength (E), treatment time, pulse-shape,
pulse-width, pulse-frequency, pulse-polarity, and temperature. Electric field strength
is considered the most influencing factor on producing cell damages and thus
microbial death. By increasing E and treatment time, higher microbial inactivation is
expected. Likewise, PEF effectiveness is highly impacted by the type of microorganism
and medium characteristics. PEF is not suitable for food sterilization, but it can be a
potential option for acid products pasteurization, like fruit juices.

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 Current Expansions in Microbiology for Food Preservation 83

Inactivation rates of microorganisms using PEF increase with decreasing conductivity

of the treatment medium. Several investigators have reported protective effect of food
constituents such as xanthan gum, proteins and fats. The presence of nonconductive
large solid particles and air bubbles also reduce the efficacy of PEF mediated microbial
inactivation. The main microorganism inactivation mechanism is the electroporation,
which is the formation of pores on the cellular membrane. Nonetheless, structural
arrangement of microbial enzymes, electromechanical compression, and osmotic
imbalance also could explain microbial death due to PEF. (Vega-Mercado et al., 1997).
Sublethal effects by PEF with subsequent high resistance of microbial cells to PEF have
been reported. For example, exposure of the yeast S. cerevisiae to PEF induced oxidation
stress responses which activated glutathione synthesis enzymes. Such a response results
in protection against the injury and the recovery of the cells. Subsequently, it was
shown that the glutathione-dependent biochemical defense system against oxidation
stress in sublethally injured cells is closely related to their level of resistance toward PEF
stress and PEF can be used for improving this antioxidant in foods (Wang etal., 2014).

Non-thermal Plasma (NTP)

Plasma, fourth state of matter where increases in the material’s energy levels converts
matter to an ionised state. Cold plasma (CP) is comprised of several excited atomic,
molecular, ionic, and radical species, co-existing with numerous reactive species,
including electrons, positive and negative ions, free radicals, gas atoms, molecules
in the ground or excited state and quanta of electromagnetic radiation (UV photons
and visible light). In recent years, NTP has emerged as a  potential  alternative zero-
residue, nonthermal processing technology for decontamination of foods, which does
not leave any toxic residuals on processed foods (Misra et al., 2011). Plasma can be
generated by different methods, like gas discharge, photo ionization, heat radiation,
radio frequencies etc. Among these methods, gas discharge is the most common way
to create NTP.
Since plasma is a complex mixture, several mechanisms applicable to each individual
component is attributed to contribute in antimicrobial action. Some researchers
hypothesize that UV-C radiation present in plasma plays an important inactivating role
result; while various other researchers identifies the mechanical or oxidative damage
caused to cellular envelopes as the main cause of death. NTP cause an uncontrolled
process of lipid peroxidation resulting in cell membrane rupture. Reactive species, such
as singlet oxygen, hydrogen peroxide, nitric oxide and excited atoms and molecules,
can rapidly and easily diffuse into the cells, even when the membrane is intact, and
oxidize many macromolecules. The oxidation of DNA bases leads to substitution of a
purine for a pyrimidine or vice versa, leading to cell death. The reactive species are able
to break peptide bonds, oxidize amino acid side chains, produce protein cross links to
produce protein aggregate. All these effects result in modifications in the conformation

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 84  Advances in Processing Technology

and three-dimensional structure of proteins and enzymes. In addition, the enzymes are
inhibited also by the oxidation of their cofactors (Lopez et al., 2019).
Environmental factors, such as pH, food matrix, and relative humidity, have significant
influences on the sterilization efficacy of NTP. Kayes et al. (2007) found that the Bacillus
cereus reduction at pH 5 was about 4.9 logs, while it was only 2.1 logs at pH 7 after
30  s plasma treatment. Muranyi et  al. (2008)  demonstrated that increasing relative
humidity had improved the rate of NTP inactivation of Aspergillus niger because of the
decomposition of additional water molecules into more hydroxyl radicals.

Preservation of foods through additives

One of the fastest growing areas of research on food science is pertaining to the
antimicrobial food additives. Preservatives are preferred by the processors due to
their wide availability, broad spectrum antimicrobial activity and low cost. Besides
the conventional preservatives, novel molecules with antimicrobial activities are also
explored and introduced for food preservation. The microbiological knowledge of
preservatives is summarized below.

Organic acids and their derivatives

Organic acids and their derivatives are the conventional, vastly used preservatives.
Acetic, propionic, lactic, sorbic, benzoic, citric, malic, fumaric, tartaric and adipic
acids, esters of hydroxyl benzoic acids (parabens) and glucono-delta-lactone. Benzoate,
propionate and sorbates are used in a wide variety of foods, where as lactate and acetates
are used often in meat and meat products.
Among the organic acids, benzoic acids and parabens have good antimicrobial effect
against bacteria, yeasts and molds, while propionic acid is more effective against yeasts
and molds. Acetic acid and citric acids are more effective against bacteria than yeasts
and molds. The mechanisms of antimicrobial activity of organic acids and their salts
are mainly by the inhibition of ATP generation by affecting the proton motive force
(pmf) of membrane.

Plant derived compounds

Apart from the above, traditionally used organic acids or their derivatives, long
chain fatty acids, phenolic acids such as oleic, chlorogenic, hydroxyl cinnamic,
caffeic, p-coumaric, ferulic and lauric acids are currently being investigated for their
antimicrobial activities in foods. Medium and long chain unsaturated fatty acids are
also antimicrobial compounds and they are more efficient against Gram positive, than
against Gram negative bacteria (Desbois et al., 2010, Pisoschi et al., 2018).
During last century, the antimicrobial principles from numerous plants have been
elucidated. These include essential oils of cinnamon, clove, thyme, sage, rosemary,
oregano, allspice, nutmeg, coriander, lemon grass, cumin, black pepper etc. The essential

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 Current Expansions in Microbiology for Food Preservation 85

oil components mainly include terpenes, phenols, aldehydes, esters and ketones. Plant
based antimicrobials show a broad spectrum activity against gram positive and gram
negative bacteria and fungi, although majority of the studies show better activity
against gram positive than gram negative bacteria. Even though very good in vitro
antimicrobial activity is shown by essential oils, the efficacy is weak in food sytsems,
probably due to interference. Thus, appreciably high amount of the essential oils have
to be added for inactivation of microbes in food. In majority of the cases, this will lead
to unacceptable sensory effects.
Plant derived phenolic compounds acts by altering microbial cell membranes. This is
accomplished through the interaction of phenolics with membrane proteins inducing
their structure and function alteration, creating membrane instability. So Membrane
functions like electron transport,  nutrient  uptake,  synthesis of proteins  and  nucleic
acids, and  enzyme activity  are affected. (Bajpai et al., 2008, Hayek et al., 2013).
Alkyl  substitution  into the aromatic nucleus  contributes the antibacterial activity  of
phenolics. The  antimicrobial activity  of phenolics depends on concentration at low
concentration,  phenols inhibit microbial  enzyme activity, while high concentrations
induce denaturation of proteins.
Presence of functional groups such as hydroxyl groups promote electron delocalization,
and can act as proton exchangers, diminishing the pH gradient across the cytoplasmic
membrane of bacterial cells. This causes the diminished proton motive force,
energy depletion, and subsequent  cell death. Isothiocyanates present in crucifers,
garlic etc. another group of plant derived secondary metabolites with potential
use as antimicrobials in foods. These inactivate extracellular enzymes through
oxidative dissociation of -S-S- bonds.

Chitosan
Chitosan, the biopolymer obtained by deacetylation of chitin, is a molecule approved by
several food regulatory agencies across the globe, is another prospective antimicrobial
for food preservation. The net charge in the chitosan molecule (extent of acetylation)
and pH are the main factors affecting the antimicrobial efficacy of chitosan. The charge
density on the cell surface is the microbial characteristic affecting the sensitivity to
chitosan (Kong et al., 2010, Zou et al., 2016). Several studies have shown that the
biological activity of chitosan depends significantly on its molecular weight (MW)
and degree of acetylation (DA). Both parameters affect the antimicrobial activity of
chitosan independently, though it has been suggested that the influence of the MW
on the antimicrobial activity is greater than the influence of the DA. An increase in
the DA of the amino group in the chitosan molecule cause less activity. Minimum
inhibitory concentration (MIC) of chitosan has been found to range from 0.05% to
0.1%, depending on the bacterial species and the molecular weight of chitosan. At
present, chitosan has gained more importance as an edible coating with antimicrobial
properties, rather than as a standalone preservative.

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 86  Advances in Processing Technology

Bacteriocins
Bacteriocins are ribosomally-synthesized cationic and hydrophobic peptides with
approximately 20-60 amino acids length, possesses anti-microbial peptides (AMP) and
secreted by the producer-organism. Among these, nisin, an AMP produced by LAB
such as Lactococcus lactis, has been granted “generally regarded as safe” (GRAS) status
for certain applications by Food and Drug Administration (FDA), and is now permitted
in more than 50 countries for food use. A multitude of other bacteriocins are also
reported from lactic acid bacteria. Some other bacteriocins include piscicolin, subtilin,
lichenicidin, divergicin, cinnamycin, actagardine, epidermin, lacticin, carnobacteriocin,
mutacin, mesenterocin, enterocin, sakacin, leucocin, curvacin, lysostaphin,enterocin,
duramycin, brevinin, mundticin, ruminococcin, curvaticin, and columbicin (Kumariya
et al., 2019). Most of the food applications involving bacteriocins can be divided into
three categories Partially purified bacteriocins (e.g., Nisaplin®, containing 2.5% nisin),
dairy and other food-grade fermented products containing bacteriocins in the form of
a crude fermentate (e.g., MicroGARD® series of bacteriocin-containing products and
bacteriocin-producing protective cultures. One of the biggest challenge in bacteriocins
use is their suceptability to proteases/peptidases and narrow spectrum of activity.
Researchers are continuously working to genetically engineer the producer strains to
overcome these limitations to obtain ideal bacteriocins (Chikindas et al., (2018).
The anionic molecules on bacterial cell surface are receptors for cationic bacteriocins.
The anionic molecules include phospholipids, lipopolysaccharides, lipotechoic acid and
cardiolipin. The bacteriocins align themselves on the membrane and positively charged
groups interact electrostatically with the negatively charged bacterial cell surface, while
the hydrophobic  surfaces  align toward the membrane to traverse the  lipid bilayer.
After piercing into the lipid bilayer, the peptides self-associate or polymerize to form
complexes. These changes cause pore formation in bacterial membrane and finally cell
death. Of late, there are scientific reports available on the resistance development by
microbes against bacteriocins. Since  charge  and  fluidity  of membranes are the two
bacterial properties exploited by bacteriocins during the attack, manipulation in these
properties renders the bacteriocins ineffective, resulting in bacteriocins resistance.
Bacteriocin combinations is a good way of tackling the bacteriocins resistance. Since
bacteriocins are ribosomally-synthesized peptides, they can also be bioengineered at
specific amino acid residues to make them more effective against the food-spoilage
bacteria (Johnson et al., 2019).

Food preservation using microorganisms: Fermentation and protective

cultures
Fermented foods are forming an important part of diet globally. Preservation of
foods using fermentation by various microorganisms resulted in the vast expansion
of dairy science, enology, brewing and lactic fermentation science. Primarily, lactic
acid bacteria, yeasts and certain molds are used for food fermentations. Qualities of

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 Current Expansions in Microbiology for Food Preservation 87

starter culture for different fermentations are well defined and scientists are trying to
either isolate or to genetically modify the existing strains to materialize the ‘ideal strain
concept’ for different fermentation. Extensive literature is available on microbiology
of wine, beer and lactic fermented products. Delfini and Formica (2001), Ameen et
al (2017), Bokulich and Bamforth (2017). All fermentations result in consumption
of sugars present in raw food, induces flavour changes and either acid or alcohol
production, thus resulting in formation of distinct category of food products. Apart
from the role of preservation, fermentation is a means to improve the functionality
of foods. For example, soybean fermentation produces different bioactive peptides
with health benefits such as antihypertensive and antimicrobial activities. The type of
bioactive peptide formed is largely dependent on the fermentation agent (Sanjukta and
Rai, 2016).
Apart from the fermented product, the use of protective cultures for preservation of
fresh and fresh-cut products are also gaining importance. These microbes bring in
the control of pathogens through competition for nutrients and niches, antibiosis,
lytic enzymes, volatile inhibitory metabolites, pH decrease, organic acid production,
parasitism and induction of defense responses from the harvested plant product and
others (Montesinos et al., 2015). These antagonistic organisms used for preservation of
fresh and fresh-like products. Lactic acid bacteria of genera Lactococcus, Lactobacillus,
Lactosphaera, Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Vagococcus, and
Weissella are reported to have biopreservation properties.
In certain cases, yeasts are also used for biopreservation. Mode of action of antagonistic
yeasts is different than lactic acid bacteria. Studies suggest that the action is related to
production of antifungal hydrolases, siderophores (iron binding molecules), induction
of defense related proteins, production of volatile organic compounds and induction
of ROS. Among them, volatile organic compound production has been adjudged as the
major mechanism of antagonism. A volatile-producing fungus of Ceratocystis fimbriata
was used to control two postharvest diseases caused by M. fructicola and P. digitatum
on peaches and citrus respectively. The exposure to VOCs significantly inhibited the
test fungi in vitro and in vivo trials. The most abundant VOCs that accounted for 97%
of the total volatile compound yield were butyl acetate, ethyl acetate and ethanol (Ribes
et al., 2017)

Microbiological implications of food packaging methods

Advances in food packaging system have revolutionized methods of preservation.
Quality maintenance by packaging is achieved by maintaining the gas, moisture,
water activity and sunlight exposure at optimal level. Among this in-pack atmospheric
modification is the most important factor deciding the microbial proliferation in
foods. Low oxygen level and high CO2 in the packs either formed as a result of tissue
respiration as in case of fresh fruits and vegetables (passive), or by flushing with suitable
gas mixtures (active) prevents the spoilage by aerobic microbes especially fungi. But,

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 88  Advances in Processing Technology

very high CO2 levels can support the proliferation of organisms such as Clostridium.
Thus, apart from the sensory quality mainatance, the microbiological safety concern
is an important parameter to be taken care in modified atmosphere packaging. Ample
evidences are generated on this regard. (Zahra et al., 2016, Oliveira et al., 2015).
Another packaging strategy to control undesirable microorganisms is the incorporation
of antimicrobial substances onto food packaging materials. Natural antimicrobial
agents, silver, volatile compounds and bacteriocins are usually incroprporated for this
purpose. The silver zeolite is laminated as a thin layer on the food contact surface
of the laminate. Commercial examples of silver substituted zeolites include Zeomic®,
Apacider®, AgIon, Bactekiller and Novaron etc. Volatile antimicrobials generating
systems to release chlorine dioxide, sulphur dioxide, carbon dioxide and ethanol are
also proved efficient in controlling microflora in packed foods. The packaging material
used for the volatile antimicrobial system should possess high barrier properties to
prevent the loss through permeation. Ethicap® and Antimold® sachets release ethanol
absorbing moisture and releasing ethanol vapour. Ethanol vapour generators are
commercially used for packaging high moisture bakery products, cheese and fish.
Edible materials such as carbohydrates (Eg. methyl cellulose, starch), proteins (Eg.
Gelatin, casein), lipids (Eg Bees wax, carnauba wax) incorporated with antimicrobial
substances are directly applied on the food material to make edible films. It makes
use of organic acids, salts of organic acids, bacteriocins, fungicides, enzymes, and
compounds like silver zeolites etc. as antimicrobial for incorporating to films (Biji et
al., 2015).

Enhancing the efficacy of individual preservation factors: Hurdle concept

Earlier discussions have shown that adaptation of pathogens to environmental and
processing stresses can result in survival of the cells in the foods even after the processing
is completed. Therefore, food industry considers multiple barriers (hurdles) to improve
the microbial stability of foods. Application of a single factor alone can bring in stress
adaptations, ultimately making the product unstable. Better shelf stability results from
the synergism of combination of factors. The hurdle technology produces minimal
sensory changes, which makes the products more acceptable than those obtained by
conventional methods (Aguilera and Chirife, 1994). Hurdle technology represents the
application of well known strategies in reduced “doses” to interact synergistically to
prolong overall quality of foods. Some of the well known hurdles are pH control, water
activity reduction, mild heat, natural antimicrobials, aseptic packaging, rapid cooling,
maintenance of refrigerated storage, modified atmosphere packaging, active packaging
etc. Therefore all antimicrobial agents discussed earlier in this chapter can be used
as a tool for developing suitable hurdle technologies for specific food products. Vast
number of combinations of hurdles such as lowering water activity, pH, mild heating
(blanching), chemical preservatives at low dose etc., have been successful in food
preservation conventionally. In fact, most of the processed products available today

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 Current Expansions in Microbiology for Food Preservation 89

in the market use the principle of hurdle technology deliberately or inadvertently

(Singh and Shalini, 2016). At present, hurdle combinations of physical non thermal
processes and natural preservatives and packaging are receiving significant attention
as hurdles with an objective to reduce the use of chemical preservatives and heat. For
example, nisin has a synergistic effect with PEF treatment, and an additive effect with
HPP treatment (Yuste et al., 2002; Black et al., 2005). This phenomenon is explained
as facilitation of the entry of a nisin molecule through the membrane during HPP
treatments, as the combination helps cause membrane pores rapidly in bacteria.

Indicator microorganisms for testing for efficacy of processing and

preservation
Studies on identification and behavior of indicator organisms are very important for
processing parameter optimization of food. The classical example is successful process
design in canning due to the scientific understanding of Clostridium botulinum as
an indicator organism to check the efficiency of canning process, and design of
12D (time required to reduce 12 log population of C. botulinum) process. Similarly,
safe pasteurized milk is ‘virtually free’ of Mycobacterium sp. In heat inactivation
itself, the indicator organism has to be different based on the food properties. For
example, Silva and Gibbs (2004) suggested  Alicyclobacillus acidocaldarius  as the
reference microorganism to design heat processes for food products. USDA (2012)
has requirements E. coli O157:H7 as the indicator strain for reprocessing, an HPP
process that achieves a 5-log E. coli O157:H7 reduction should be sufficient for product
produced to ensure microbial safety (Sonaliben et al., 2017). Deinococcus radiodurans,
due to its high resistance to irradiation, has been used to design radiation processing. It is
known that some of the human pathogens in food enters to a metabolic inactive state
called viable but non culturable state (VBNC). VBNC form is also a stress adaptation
of the organisms. With suitable environmental stimuli, these organisms inititate their
growth. Micrococcus luteus, a milk spoilage bacteria has become a model organism for
studying this growth initiation process, often called resuscitation (Galina et al., 1998).
Challenge and validation studies using food-borne pathogens in the food processing
facility pose contamination risk, and are prohibited by law. In such situation, surrogate
biological indicators have been proposed as alternatives to pathogens in these studies.
Clostridium sporogenes spore is the surrogate to predict the heat inactivation of C.
botulinum spores while Listeria innocua is a non-pathogenic strain that grows in
environments similar to those suitable for L. monocytogenes and this surrogate is
suitable for studying efficacy of milk pasteurization. Surrogate microorganisms chosen
should possess a slightly higher resistance than the targeted pathogens, in order to
parctically estimate the pathogens surviving in the treated food. The rapid detection
of low levels of pathogens or their corresponding nonpathogenic surrogates may be
facilitated with markers such as bioluminescence, resistance to antibiotics etc. In the
wake of increased use of alternative thermal and nonthermal processing methods for
microbial inactivation, suitable indicators and their surrogates are to be clearly defined.

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 90  Advances in Processing Technology

Measurement of cell survival and physiological state of microorganisms

Another remarkable microbiological expansion which contributed towards the food
preservation science is the advances in detection of viability and physiological state of
food organisms post food processing. Several methods are available to directly assess the
extent and nature of cell injury within a microbial population. The conventional ‘gold
standard’ method is to culture the organisms in selective mediase include culturing
on selective agar media. But nowadays modern microscopic, immunological and
molecular biology tools are used for rapid detection of food microbes, which in turn will
give real-time status of microbiological load and physiological state in foods and thus
to assess the efficacy of preservation methods. For example, epifluorescence staining
using propidium iodide can be used to detect cells that lost selective permeability after
HPP. Surface hydrophobicity studies and ATPase activity help for understanding the
mechanism of action through membrane damage (Huang et al., 2014). The combination
of fluorescent dyes viz., ethidium bromide, bis-oxonol and propidium iodide can be
used to quantify, the populations that are reproductively viable, metabolically active
and/or with intact polarized membrane (Von-caron, 1998). Acridine orange staining
differentiates viable and dead cells based on their relative proportion of DNA and RNA.
Commercial kits such as Live/Dead BacLightTM® provide information on viable cells
in a population. The molecular biology applications are not useful so far in spoilage
microbiology and preservation per se; but have found high application in food safety
microbiology and fermentation microbiology for studying the population changes of
a targeted microbe and gene expression changes due to stresses in the microbial cell.

Modelling and predictive microbiology as a tool for designing microbial

inactivation in food
Predictive Microbiology (PM) is the prediction of microbiological consequences in
a system achieved through the mathematical modelling. This has high application
in food technology for evaluation of microbiological shelf life, and prediction of
microbiological hazards connected with foods in HACCP and Microbiological
risk assessment programmes. The most important death models used in predictive
modelling are negative Gompertz, log-linear, shoulder/tail, Weibull, Weibull+tail, re-
parameterized Weibull, biphasic approach, etc. . The details of mathematical models
in foods were reviewed by Bevilacqua et al. (2015). One constraint with these models
is the use of same types of  mathematical models for different microbes, although
the parameters of the models are specific to species or even strains. For example, the
common model organisms used are Morganella sp (for histamine production), Listeria
monocytogenes (as psychrotrophic organism) and a few lactic acid bacteria, E. coli
etc. Several modelling packages are already available towards this purpose. Common
ones are ComBase (www.combase.cc), Food Spoilage and Safety Predictor (http://fssp.
food.dtu.dk). ARS Pathogen Modelling Program (https://pmp.errc.ars.usda.gov) and
Symprevious (https://symprevius.eu), 

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 Current Expansions in Microbiology for Food Preservation 91

Future prospects
This chapter summarized the role of advancements in understanding the antimicrobial
mechanisms by different preservation strategies, their practical uses and the methods to
study the microbiological dynamics in food preservation. Advancement of every aspect
of microbiology has implications due to the fact that the knowledge can be applied
in monitoring and curbing the microbial proliferation in foods. Every new methods
of food processing has to be checked for their implications on microflora. Microbial
stress adaptation is a tricky issue for the scientific world, considering the diversity of
foods and food borne microbes or their strains. Having elucidated the physiology of
microbial stress adaptation, the ultimate role of food microbiologists is to incorporate
this knowledge into concepts that can be used by the food processors to warrant supply
of safe foods coupled with better shelf life. Lot more scientific information need to
be generated for developing real time quality monitoring system and process design
for better preservation and implementation of the same through a quality control
system. Additionally, fermentation is an area of food preservation now gaining more
importance due to their acceptance as functional foods. More knowledge on microbe
mediated biotransformations is required to establish the potential of fermented foods
as functional foods.

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