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Collection Highlights

Food Waste Recovery: Processing Technologies, Industrial

Techniques, and Applications 2nd Edition Charis M.
Galanakis

Regulation of Innovative Technologies: Blockchain,

Artificial Intelligence and Quantum Computing Rosario
Girasa

Food Processing Technology: Principles and Practice 5th

Edition P.J. Fellows

Microbiological Analysis of Foods and Food Processing

Environments Osman Erkmen
Electromagnetic Technologies in Food Science Vicente M.
Gómez-López

Food processing technology: principles and practice 4th ed

Edition Peter Fellows

Starch-based materials in food packaging processing,

characterization and applications Barbosa

Innovations in Thermochemical Technologies for Biofuel

Processing 1st Edition Sonil Nanda

Netter Atlas of Human Anatomy-Classic Regional Approach,

8e (Mar 29, 2022)_(0323793738)_(Elsevier) NOT TRUE PDF
Frank H. Netter
 1.01 Overview of Research Needs, Future and Potential Applications of High-
Pressure Processing
Robert Sevenich, Cornelia Rauh, and Dietrich Knorr, Department of Food Biotechnology and Food Process Engineering,
Technische Universität Berlin, Berlin, Germany
© 2021 Elsevier Inc. All rights reserved.

1.01.1 Introduction 1
1.01.2 Vegetative Microorganisms, Spores, Viruses, Parasites, Bacteriophages and Nematodes 2
1.01.2.1 Vegetative Microorganisms 2
1.01.2.2 Spores 3
1.01.2.3 Viruses, Bacteriophages, Parasites and Nematodes 4
1.01.3 Chemical Reactions: Influence on Allergens, Toxins (Food Borne and Agriculturally Based) 5
1.01.3.1 Allergens 5
1.01.3.2 Toxins 6
1.01.3.2.1 Food Processing Contaminants 6
1.01.3.2.2 Aflatoxins, Pesticides and Herbicides 7
1.01.4 Process-Structure-Relationship 7
1.01.4.1 Starch 8
1.01.4.2 Pectin 8
1.01.4.3 Proteins 9
1.01.5 Packaging Material 10
1.01.5.1 Polylactides (PLA) 11
1.01.6 Data Reporting and Experimental Design 11
1.01.7 Conclusion 11
References 14

1.01.1 Introduction

Today, the consumers’ demand for high quality safe foods requires the development and application of emerging processing tech-
nologies for the gentle pasteurization and sterilization of foods. Therefore, the food industry is looking for new ways to produce
safe, healthy and stable foods. One way to meet this aim is applying high pressure, in the order of 600 MPa, to foods, which is
referred to in the literature as cold pasteurization (Matser et al., 2004; Knoerzer et al., 2010; Mújica-Paz et al., 2011). Currently,
high-pressure processing (HPP) in the food industry is solely used for pasteurization purposes. The trend of using high pressure
as a technology for pasteurization of different kinds of foods, e.g., juices, ham, sauces and seafood is a growing sector in the
food industry since the 1990’s (Hogan and Kelly, 2005). Pasteurization of foods with high pressure (HP) is well established in
the food industry. In 2018, 500 industrial-scale high pressure systems were in use worldwide, producing approx. Four million
metric tons annually of pressurized foods. There is still a need for research in this field, e.g., considering the impact on food struc-
ture, food quality, microorganisms, enzyme activity, and nutrients. Furthermore, there is a high acceptance of pressurized foods on
the consumer side due to the clean label and health promoting attributes the technology offers (Olsen et al., 2010), without which
industrial application would be less attractive. Despite the steadily increasing commercial production of high pressure treated food,
some important scientific and technological questions, as well as some potential other applications of HPP are still unresolved. One
of these issues is the impact of different intrinsic and extrinsic factors on the inactivation mechanisms of vegetative bacteria and
bacterial spores under pressure. To unravel the impact of the different pressure and temperature combinations on a possible cell
death or recovery, detailed analyses about the physiological state of the cells and how they are influenced by different food compo-
nents are needed. According to Le Chatelier’s principle, in a system facing a shift of equilibrium, all cellular components are affected
by high pressure, including the cell membrane and its membrane proteins, enzymes and ribosomes as well as the entire cell metab-
olism (Winter and Jeworrek, 2009; Georget et al., 2015). In general, prokaryotic cells show a higher resistance toward pressure than
eukaryotic cells. Yeasts and molds are in general more pressure sensitive, although ascospores of some molds such as Byssochlamys
and Talaromyces can be very pressure resistant (Smelt, 1998; Considine et al., 2008; Georget et al., 2015). Within prokaryotes, gram
positive microorganisms such as Bacillus, Listeria, Staphylococcus and Clostridium have a thicker peptidoglycan layer and are therefore
more pressure resistant than gram-negative microorganisms (Smelt, 1998; Considine et al., 2008; Dumay et al., 2010). The mech-
anisms leading to cell death have been investigated in several bacterial species (Huang et al., 2014). However, the particular events
leading to inactivation are not well understood (Cheftel, 1995; Buckow and Heinz, 2008; Klotz et al., 2010). High pressure between
300 and 800 MPa at ambient temperatures can lead to the unfolding and denaturation of important cell enzymes and proteins in
vegetative microorganisms (Rastogi et al., 2007; Knorr et al., 2011a,b), but the specific pressure effects on microorganisms are more

Innovative Food Processing Technologies, Volume 1 https://doi.org/10.1016/B978-0-08-100596-5.22991-0 1

2 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

complex and several different mechanisms leading to cell death can interact when high pressures are applied. Primarily, pressure at
a sufficiently high level, can induce enzyme inactivation, membrane proteins denaturation and cell membrane rupture caused by
a phase transition of the membrane and change in its fluidity (Ananta et al., 2005; Georget et al., 2015). The pressure level needed to
achieve a 5 log10 reduction of pathogenic microorganism in different food-products ranges from 300 to 800 MPa(Hendrickx et al.,
2001) and often synergy between pressure and temperature is observed (Buckow and Heinz, 2008). By increasing the process pres-
sure, it is possible to decrease the temperature needed to achieve the same inactivation. According to the literature, the pressure
induced effects leading to cell death of vegetative microorganisms can be attributed to four factors:
(i) Protein and enzyme unfolding, including partial or complete denaturation
(ii) Cell membranes undergoing a phase transition and change of fluidity
(iii) Disintegration of ribosomes in their subunits
(iv) Intracellular pH changes related to the inactivation of enzymes and membrane damage (Knorr et al., 2011a,b; Molina-
Höppner et al., 2013; Georget et al., 2015)
Further, the influence on the food matrix still needs clarification in terms of what compounds are formed or destroyed in compar-
ison to the conventional process. Also, a clear assessment of what the potential and limits of HPP are, is still missing to this point.
Some well-known potential and limits are:
(i) Advantages and Potentials
Rapid, quasi-instantaneous uniform distribution throughout the sample; Minimal or reduced thermal exposure; Instant
temperature increase and subsequent cooling upon depressurization; Suitable for high moisture–content foods; in package
processing; Suitable for both liquid and pumpable foods; Independent of product shape and size; Opportunity for novel
product formulation; Distinct products through pressure effects such as protein denaturation, carbohydrate gelatinization, and
fat crystallization; Within some pressure-thermal boundary conditions, pressure accelerates microbial inactivation; Consumer
acceptance as a physical process.
(ii) Limits
Batch or semi-continuous operation; Preheating step for pressure-assisted thermal processing (PATP) required; Thermal
nonuniformity during PATP; Not suitable for products containing dissimilar compressibility materials such as foams;
Throughput limited due to batch operation; Variable efficacy in enzyme inactivation; pressure alone cannot inactivate bacterial
spores; Higher processing costs.
Some authors have looked beyond the obvious application of HPP, other than just mimicking of thermal processing. An overview
of some of the applications can be found in the literature (Smelt, 1998; Oey et al., 2008; Verbeyst et al., 2010; Van Der Plancken
et al., 2012). Most of these authors are referring to the promising use of high-pressure processing as a gentle preservation technique
but are also mentioning the possible applications, if the food safety is given, to alter texture, increase the bioavailability of certain
health promoting compounds, allergenicity reduction as well as vitamin retention.
Initially, the empiric approach of trial an error was used, which drove the evolution of high pressure processing from inactivation
studies of vegetative microorganism (Hite, 1899; Timson and Short, 1965; Sale et al., 1970; Metrick et al., 1989; Cheftel, 1995; Fior-
etto et al., 2005; Georget et al., 2015) to inactivation of enzymes (Sila et al., 2008; Rauh et al., 2009; Knorr et al., 2011b; Grauwet
et al., 2012) to shucking of crustaceans (Kingsley 2014) to spore inactivation (Sale et al., 1970; Reddy et al., 2003; Black et al., 2007;
Olivier et al., 2012; Setlow and Markland, 2012; Reineke et al., 2013a,b; Olivier et al., 2015; Sevenich and Mathys, 2018) to struc-
ture/texture induced by high pressure (Oey et al., 2008; Sila et al., 2008; Sikes and Warner, 2016; Balasubramaniam et al., 2016; Thai
Union Group, 2017; Sim et al., 2019) to currently the latest frontier of the influence on chemical reactions pathways (e.g., degra-
dation of vitamins, oxidation of poly unsaturated fatty acids and mitigation of food process contaminants) (Oey et al., 2008; Ver-
beyst et al., 2010; Van Der Plancken et al., 2012; Sevenich et al., 2013). To see beyond the obvious, one must ask what is different
compared to the thermal processes and why. How can this difference be used as a potential and advantage to create for example new
functional food ingredients, food properties and health foods? Some of these aspects are tackled and covered by some recent reviews
(Balasubramaniam et al., 2016; Huang et al., 2017).
Another issue that needs further research focus could be to think of novel combination processes as for example done in the past
with HPP/PEF, high pressure thermal sterilization, pressure assisted thermal sterilization (PATS), pressure induced freezing (PIF),
pressure assisted freezing (PAF), high pressure homogenization (HPH) and high pressure extraction (Volkert et al., 2008; Park et al.,
2013; Huang et al., 2013; Sevenich et al., 2015a,b; Sevenich and Mathys, 2018).
This article will review the recent literature and will dive into different areas where the authors consider high pressure processing
to have a significant impact in the coming years.

1.01.2 Vegetative Microorganisms, Spores, Viruses, Parasites, Bacteriophages and Nematodes

1.01.2.1 Vegetative Microorganisms
The use of HPP to inactivate pathogenic vegetative microorganisms has been largely investigated for the pasteurization of commer-
cial products for decades (Buckow and Heinz, 2008). In 1899, Hite (1899) was first to conduct experiments using high pressure in
combinations with foods to extend shelf life, and reported that milk stayed sweet longer after the treatment with high pressure. Since
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 3

then, significant research efforts have focused on understanding the underlying mechanisms of the inactivation of microorganisms
under high pressure conditions. HPP offers a lower thermal input into the product by comparison with conventional thermal treat-
ment and therefore increases the quality of the food while maintaining food safety (Smelt, 1998; Hogan and Kelly, 2005; Balasu-
bramaniam et al., 2008; Bermudez-Aguirre and Barbosa-Canovas, 2011; Barba et al., 2012). Despite the steadily increasing
commercial production of high pressure pasteurized food with several millions of tons per year (Aganovic et al., 2017), some
important scientific and technological questions are still unsolved.
One of these issues is the impact of different intrinsic and extrinsic factors on the inactivation mechanisms of vegetative bacteria
and bacterial spores under pressure. To unravel the impact of the different pressure and temperature combinations on a possible cell
death or recovery, detailed analyses about the physiological state of the cells and how they are influenced by different food compo-
nents are needed, In recent years, a common world-wide concern has been foodborne outbreaks with pathogens like EHEC
(O157:H5; O104:H4) and others (Adley and Ryan, 2016). The contamination of the food supply with spoilage and pathogenic
microorganisms continues to be a global problem, despite the wide range of preservation methods employed (Kaczmarek et al.,
2019). Despite significant advances in food processing technologies (hurdle concept, new innovative non-thermal technologies),
an annual estimated 76 million cases of foodborne illnesses occur in the US alone, resulting in approximately 5000 deaths (Scallan
et al., 2011). The report by the US Center for disease control and prevention center states that most (58%) illnesses were caused by
norovirus, followed by nontyphoidal Salmonella spp. (11%), Clostridium perfringens (10%), and Campylobacter spp. (9%). Leading
causes of hospitalization were nontyphoidal S. spp. (35%), norovirus (26%), C. spp. (15%), and Toxoplasma gondii (8%). Leading
causes of death were nontyphoidal S. spp. (28%), T. gondii (24%), Listeria monocytogenes (19%), and norovirus (11%). In the year
2000, e.g., approximately 2.4 million pounds of beef were recalled due to possible contamination with Escherichia coli O157:H7
(Kaczmarek et al., 2019). Newest studies from the WHO (2015) on foodborne diseases caused by pathogenic microorganisms,
such as Salmonella, Campylobacter, EHEC and Norovirus, show that worldwide 1 in 10 people fall ill every year from eating contam-
inated food and 420,000 die as a result. Here, the application of high pressure in combination with mild temperatures at pressures
between 300 and 700 MPa could possibly be used to inactivate vegetative pathogenic microorganisms as well as several enzymes,
which cause food deterioration. The resistance toward temperature of the pathogenic Escherichia coli strains is usually higher than
its non-pathogenic counterpart (Garcia-Graells et al., 1998). Hence, a more intense heat treatment needs to be applied. The more
complex and resistant the microorganism the more intense must be the treatment. In comparison to the pathogenic E. coli, higher
temperatures and higher pressures must be applied to inactivate spores (Reineke et al., 2013a,b). This becomes even more complex
if this is conducted in a real food system since here baro-protective effects can occur and the severe heat treatment can lead to
unwanted changes in the food matrix, leading to the formation of unwanted and possibly unhealthy compounds in the foodstuff.
The comparison of pressure resistance among vegetative food-borne pathogens revealed that strains of Escherichia coli O157:H7 were
the most resistant so far encountered. The USDA (2012) has requirements of E. coli O157:H7 as the indicator strain for reprocessing,
an HPP process that achieves a 5-log10 E. coli O157:H7 reduction should be sufficient enough to produce microbially safe products.
Other possible indicators to monitor if HPP is sufficiently applied during processing is by copper tablets in combination with
Heckel value (rate of density change of e.g., chopper under pressure). The increase in density over the entire pressure range is linear.
The tablets can be placed inside the vessel as well as directly into the food. Measurements at 400 MPa revealed that for trials with
ham the product experienced 9 MPa less than the surrounding water. This could lead to short comings in terms of sufficient inac-
tivation. The trial was not repeated at 600 MPa (Minerich and Labuza, 2003).
Another phenomenon that could lead to false results in terms of inactivation kinetic is agglomeration, which is very common for
vegetative microorganisms as well as spores. The effectiveness of inactivation processes is often verified by challenge tests using
foods that have been spiked with highly concentrated microbe suspensions. Due to preparation, storage and handling of those
suspensions, the clumping and the formation of aggregates can hardly be avoided. This phenomenon is well known, first reported
on the effect of spore agglomerations on inactivation. However, the importance for the quantitative assessment of survivors in inac-
tivation experiments has rarely been addressed. Its importance becomes evident by the following: Agglomerates produce one colony
for a high number of cells up to 103 CFU’s. Consequently, agglomerates of unknown cell numbers are always counted as one spore
until all spores in the agglomerate are inactivated. Beyond this, agglomeration and disintegration can change the colony forming
units per milliliter. In this context a model for the discussed phenomenon was developed (Mathys and Heinz, 2006).

1.01.2.2 Spores
The inactivation of bacterial endospores by pressure is generally considered to rely on pressure-induced spore germination, followed
by inactivation of germinated spores (Setlow, 2003; Margosch et al., 2004; Margosch et al., 2004). In the past decades, other
possible non-physiological pathways of spore germination have been detected. Non-nutrient germination can be further catego-
rized into a (recently discovered) second physiological and several non-physiological routes. The physiological routes include
germination initiated by eukaryotic-like serine/threonine kinase, which is located in the inner spore membrane like nutrient recep-
tors. This kinase, which is present in Bacillus and Clostridium species, recognizes peptidoglycan fragments. Non-physiological germi-
nation pathways initiate spore germination by bypassing individual germination steps. This could be stimulated by physiochemical
agents, such as exogenous Ca-DPA (Paidhungat et al., 2002; Moir, 2006), which directly activates the CLE CwlJ, or cationic surfac-
tants such as dodecylamine (Setlow, 2003), which interferes with the inner spore membrane and causes a direct release of Ca-DPA.
At pressures between 100 and 400 MPa it has been shown that the nGeR of Bacillus subtilis and Bacillus cereus are triggered. The spores
germinated quite well between pressures of 100–200 MPa and led to a maximum 4 log10 inactivation but the pressure induced
4 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

physiological germination decreased for higher pressures, indicating that other pathways must be active (Wuytack et al., 1998; Paid-
hungat et al., 2002; Reineke et al., 2013a,b). A treatment of 200–400 MPa at 40
C for 30 min showed germination of the spores but
negligible inactivation (Wuytack et al., 1998).
To achieve a quick and sudden inactivation of spores, which is relevant for food processing, pressures above 500 MPa must be
applied in combination with temperatures above 60
C. Under these conditions, Paidhungat et al. (2002) were able to germinate
B. subtilis spores that lack all major nutrient receptors. This suggests a direct opening of the spores’ Ca–DPA channels, a germination
mechanism identified as active at 200 MPa and moderate temperatures (<50
C) using B. subtilis mutant strains that lack the nGeR.
These findings were also verified by Reineke et al. (2012) for pressures  600 MPa and temperatures  60
C. The following step,
which is rapid release of Ca–DPA under pressure, is accompanied by core hydration. This step of germination is the crucial step with
regard to loss of resistance, and it is therefore of great interest for a variety of sterilization techniques and research (Reineke et al.,
2013a,b). Therefore, the ability of the spore to retain the DPA as long as possible under these conditions becomes the rate limiting
step of the spore inactivation (Margosch et al., 2006; Reineke et al., 2013a,b). This suggests that the structure most susceptible to HP
(600 MPa) and high temperatures (60
C) is the inner spore membrane or its associated membrane proteins (Reineke et al.,
2013a,b). At pressures above or equal to 400 MPa, when an opening of the Ca - Dipicolinic acid (DPA) - channels occur, the
following happens: (i) DPA is released from the spore core; (ii) the spore core gets hydrated; and (iii) the spore becomes thermo-
and pressure sensitive and can be inactivated (Reineke et al., 2012). Further, a threshold pressure of 600 MPa was established; at and
above this pressure level the DPA-release is dominated by temperature. To guarantee a successful inactivation of spores by pressures
of 600 MPa, many researchers recommend a treatment at 90–121
C (Margosch et al., 2004; Juliano and Barbosa-Canovas, 2005;
Margosch et al., 2006; Mathys et al., 2007; Knorr et al., 2011b; Georget et al., 2015; Sevenich et al., 2016) due to the synergistic effect
pressure and temperature have on the spore inactivation (Olivier et al., 2015). In comparison to conventional retorting, this could
reduce the thermal load applied to the product without affecting the safety or the quality of the food. Two sterilization approaches
can be derived from this, both of which need the adiabatic heat of compression to reach the target temperature:

• Pressure assisted thermal sterilization (PATS): pressure is neglected and only seen as the method to reach the end temperature
faster (Dunne et al., 2010)
• High Pressure Thermal Sterilization (HPTS) which takes into account the impact of pressure on the spore inactivation (Mathys,
2008; Knorr et al., 2011)
Recently, the differences in germination under pressure between Clostridium spores and Bacillus were revealed, showing interesting
insights (Paredes-Sabja et al., 2011; Lenz and Vogel, 2015; Doona et al., 2016). In Bacillus spp., DPA release triggers cortex lytic
enzyme (CLE) activation; CLE action is not essential for DPA release, but it can accelerate it. In Clostridium difficile or
C. perfringens, the initiation of cortex hydrolysis by SleC precedes and triggers DPA release. Using C. difficile spores, which lack inner
spore germinant receptors, 550 MPa treatment triggered DPA release. However, spore germination was not completed because cortex
hydrolysis is not activated by DPA release. C. perfringens spores became activated by high pressures during the come-up time to
550 MPa. It could be shown that (i) the action of 550 MPa directly stimulated DPA release and (ii) activation of inner membrane
germinant receptors during the come-up time to 550 MPa and that this activated state of germinant receptors is maintained for a while
and activates cortex lytic enzymes (Doona et al., 2016). This is only valid if temperatures below 80
C are used; if a heat shock T
>80
C is applied, spore germination can be completed. These findings were very interesting since they showed how different spores
are and how important process knowledge and microbiological knowledge are to create safe processes, such as the HPTS.
The question of what kind of indicator strain should be used is to this point still not settled. Some researchers use the non-
pathogenic Bacillus amyloliquefaciens, instead of Clostridium botulinum, as a surrogate to check for sufficient inactivation respectively
sterilization success (Olivier et al., 2015; Margosch et al., 2006; Sevenich et al., 2016). Since other strains usually used to test for
sterilization like Geobacillus stearothermophilus and Clostridium sporogenes are very high pressure high temperature sensitive (Sevenich
et al., 2016). It is also important to note that one cannot conclude based on inactivation kinetics obtained in buffer or model food
systems how the inactivation of spores will be in real food systems (Georget et al., 2015; Sevenich et al., 2015a,b; Sevenich and
Mathys, 2018). The US FDA recommends testing for a 4 log10 inactivation of Clostridium botulinum (Dunne, 2009; Balasubrama-
niam and Lelieveld, 2016).

1.01.2.3 Viruses, Bacteriophages, Parasites and Nematodes

In total, there are 31 known pathogens that cause food borne diseases, 21 are bacteria related (E. coli, Salmonella, Listeria etc.), five
viruses and five parasites (Adley and Ryan, 2016). Especially viruses and parasites are generally underrecognized if it comes to food
borne diseases. The growing global interconnection of the food supply chain and more sophisticated analytical tools are reasons for
the increased diagnosis of food borne diseases (Dorny et al., 2009). Food borne diseases also have an impact on the economy, each
incident was calculated with 1500 $/person and a total annually estimated cost of $75 billion, in the United States in 2015 alone
(Moye et al., 2018). Before going into further detail on how high-pressure processing might have an impact on the inactivation of
these organisms, a short introduction on what defines these groups, where they can come in contact with foods and what disease
they can cause, is given.
(i) Viruses/Bacteriophages: A virus is a small infectious vehicle that cannot grow or reproduce without a living cell. A virus invades
living cells and uses their chemical machinery to keep itself alive and to replicate itself. It may reproduce with fidelity or with
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 5

errors (mutations); this ability to mutate is responsible for the ability of some viruses to change slightly in each infected person,
making treatment difficult. In common usage, the term virus is used to describe viruses that can only affect humans. Viruses
cause many common human infections and are also responsible for several rare diseases. The two main food borne viruses of
concern in the food industry are the Norovirus and Hepatitis A virus. In 2011, the Norovirus was the main cause of food borne
illnesses in the United States (Adley and Ryan, 2016; Pan et al., 2016).
There are mainly two food types that have a high risk for contamination during production or harvest. One would be fruits and
vegetables, where the contamination can be due to the use of non-potable water for cleaning or fecally contaminated fingers
(Kingsley, 2013). The second food type having elevated risk of containing high amounts of viruses are shellfish (oysters, clams,
cockles etc.). Due to their nature as filter feeders and the high amounts of viruses found in seawater (9  108 virons/mL), they
can accumulate viruses as much as 1000-fold (Kingsley 2013, 2014). Bacteriophages are viruses that can only attack and
replicate in bacteria and archaea. Phages have been used since the late 19th century as an alternative to antibiotics in the former
Soviet Union and Central Europe, as well as in France. They are seen as a possible therapy against multi-drug-resistant strains of
many bacteria (phage therapy). Since 2006, the United States Food and Drug Administration (FDA) and United States
Department of Agriculture (USDA) have approved several bacteriophage products. LMP-102 (Intralytix) was approved for
treating ready-to-eat (RTE) poultry and meat products. In that same year, the FDA approved LISTEX (developed and produced
by Micreos) using bacteriophages on cheese to kill Listeria monocytogenes bacteria, in order to give them generally recognized as
safe (GRAS) status. In July 2007, the same bacteriophage was approved for use on all food products. In 2011, USDA confirmed
that LISTEX is a clean label processing aid. Research in the field of food safety is continuing to see if lytic phages are a viable
option to control other food-borne pathogens in various food products (Moye et al., 2018).
(ii) Parasites including nematodes: Parasitism is a relationship (consumer-resource interaction) between species, where one
organism lives on or in another organism on the expense of the host. Parasites include protozoans, tapeworms, nematodes etc.
Some of the major parasites that are of concern for the food industry are e.g., Echinococcus multilocularis associated with berries
and water; Cryptosporidium spp. are beginning to be associated not only with water, but also with salads; Trypanosoma cruzi is
found in juices; Trichinella spp. are nowadays found in game animals; Anisakiasis is becoming a global problem as the world
develops a taste for sushi (Robertson, 2018).
Literature on the pressure inactivation of viruses is scarce and there are only a handful of papers that are dealing with inactivation
studies of viruses in foods (Kova et al., 2010; Kingsley 2013, 2014; Balasubramaniam et al., 2015). Especially research dealing with
the inactivation mechanism are limited (Pan et al., 2016). Research with human norovirus strains and viruses in generalis difficult
due to the lack of suitable laboratory animals and the inability to propagate the virus in vitro. A study conducted by Wiezorek
(2012)with Pixuna virus, which is a surrogate for many human parthenogenic alphavirus, showed that Pixuna could be inactivated
by 109 with a treatment at 150 MPa/37
C.
The inactivation of viruses via high pressure processing is associated with altering the proteins located at the capsid of the virus.
Due to conformation change of these proteins the virus is not able to dock onto the host cell and release the RNA into the cell
(Moroni et al., 2002; Kova et al., 2010; Kingsley, 2013; Lou et al., 2015). Non-enveloped viruses are generally more sensitive toward
high pressure processing in comparison to enveloped viruses, which have a lipid envelope. The inactivation of viruses is depending,
as valid for spores and bacteria, on the temperature, pressure and time applied (Pan et al., 2016). Pressures above 400 MPa are effi-
cient enough to inactivate most viruses (Kova et al., 2010). Although, higher pressures do not necessarily need to lead to a more
efficient inactivation of a more resistant virus.
The studies found in the literature suggest that viruses like human norovirus (Norwalk 8fIIb) inoculated in oysters (104) can be
completely inactivated by 600 MPa and 5 min (Leon et al., 2011). Studies with Hepatitis A virus also inoculated in oysters with 106
showed an inactivation of more than 3 log10 after 400 MPa and 1 min (Kingsley, 2013). Rotavirus in buffer solution was inactivated
by 8 log10 at 300 MPa and 2 min (Khadre and Yousef, 2002). Very pressure resistant are the viruses Aichivirus and Poliovirus, which
were completely resistant at 600 MPa and 5 min (Kingsley et al., 2002).
For viruses, the USFDA is proposing a maximum desired reduction in virus level of 5 log10 (Pan et al., 2016).
The literature on nematodes and other parasites is very scarce and virtually non-existing. There is one publication on the inac-
tivation of a pinewood nematode in wood chips, inoculated with 1.2  105 by high pressure processing. Here it was shown that the
nematodes were completely inactivated by 30 MPa at a holding time of 5 min (Fonseca et al., 2014). This suggests that the bigger the
organism the easier it can be inactivated by high pressure processing. The question that still needs to be answered is if that is also
valid for other parasites like Cryptosporidium, Trypanosoma etc.
The research on the influence of high pressure processing on viruses and parasites needs to be driven forward since it is a prom-
ising alternative to guarantee the safety of usually non-processed or gentle processed fresh products.

1.01.3 Chemical Reactions: Influence on Allergens, Toxins (Food Borne and Agriculturally Based)
1.01.3.1 Allergens
Allergy is a false, hypersensitive and intense immune response toward typically harmless substances in the environment. Food can
be a trigger for the immune system to overreact, especially dairy, egg, soy, peach, cherry, selfish, wheat, nuts and apple can cause an
allergic reaction (Somkuti and Smeller, 2013). Food allergy is more prevalent in young children (5%) but 3%–4% of adults also
exhibit a form of food allergy.
6 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

The beginning of a type I allergic reaction is the sensitization to an allergen. The initial contact of an allergen with the mucosa of
a susceptive organism is followed by a complex series of events, leading to the production of allergen specific immunoglobulin E
(IgE). The effector phase of an allergic reaction is initiated upon second exposure to the allergen. Allergen binding to the specific IgE
antibody on the mast cells will cause an inflammatory response due to the release of histamine and other mediators. Not all aller-
gens are able to sensitize, i.e., induce the production of specific IgE-type antibodies (Somkuti and Smeller, 2013; Ontiveros et al.,
2015; Ekezie et al., 2018). Allergens which are capable to both sensitize and trigger allergic reactions are called complete allergens.
Cross reactive allergens bind to IgE antibodies that are present in the body due to an earlier sensitization by another allergen. There
are more than 6000 allergens which can have a negative impact and there are currently no accepted therapeutic approaches other
than avoiding the foods that contain the possible allergen (Ontiveros et al., 2015; Meinlschmidt et al., 2016). Here, the targeted
treatment by food processing technologies, such as high pressure, enzymatic hydrolyzes, heat treatment or combined processes,
could be a promising strategy to create hypoallergenic foods. To gain knowledge if a food protein is allergenic or not it needs to
be characterized by the allergic (epitopes) and non-allergic components; this can be done with the help of immune-informatics
(Ontiveros et al., 2015). The main sequence/pattern in allergenic proteins’ secondary structure are (1) antiparallel b-strands; (2)
antiparallel b-sheets; (3) aþb structures and (4) a-helical proteins (Somkuti and Smeller, 2013). Since high pressure processing
can have an impact on the functionality of proteins by altering the secondary, tertiary and quaternary structure of the protein
due to changes in electrostatic and hydrophobic interactions it surely influences the reactive properties of allergenic proteins (Jimé-
nez-Saiz et al., 2015). The application, also in combination with other processes, is an interesting tool to create foods with reduced
food allergenicity. Somkutti and Smeller (2013) mentioned in their review that three questions need to be discussed to find out if
high pressure is suitable: (1) How high is the pressure needed to unfold the protein? (2) Is the unfolding reversible? and (3) How is
the binding of IgE to the allergen?
In general, it can be said that for some of the major food allergens like Mal D1 (apple), Bos d 5 (Milk), Gal d 2 (Egg), Ara h 2
(Peanut), Gad m 1 (fish), pressures and temperatures of 150–600 MPa and 30–80
C are needed (Somkuti and Smeller, 2013). The
mechanism of a potential allergenic reduction while applying high pressure processing is either the masking of epitopes due to
conformational changes induced by high pressure, or the higher exposure of epitopes but therefore leading to higher enzymatic
hydrolyzes (Peñas et al., 2008; Meinlschmidt et al., 2016). The second mechanism will only work if the food/allergens are treated
with enzymes like pepsin, chymotrypsin or papain after the treatment, otherwise it would make the food even more allergenic. It
could be shown that the allergenic potential of the walnut allergens Jug r 1–5 can be reduced by pressures ranging from 550–
650 MPa(Ekezie et al., 2018). Further a decrease of 40%–60% of alpha-casein in milk and Pru p 3 in peach could be shown in a pres-
sure range of 200–600 MPa (Lavilla et al., 2016; Ekezie et al., 2018).
High pressure alone could not modify the allergenic properties of carrots, peanut, milk, celery, apple (Jiménez-Saiz et al., 2015).
As it is suitable for other technologies, the most effective way to reduce the allergenicity by high pressure processing is the combi-
nation with other processes that act synergistically. One possibility is to combine pressure and heat, which works well, but more
effective seems to be the combined treatment of high pressure and enzymic hydrolyzes during or after the treatment (Somkuti
and Smeller, 2013; Jiménez-Saiz et al., 2015; Meinlschmidt et al., 2016). Meinlschmidt (2016) studied the effect on soy protein
isolate with an enzymatic hydrolysis during and after high pressure processing at pressures ranging from 100 to 600 MPa at
50
C and a dwell time of 15 min. It was shown here that a combined process at pressures from 300 to 600 MPa could lead to
a nearly complete loss of immunoreactivity of the mentioned allergen. Further, the oil binding and foaming activity were enhanced.
Similar results and studies are summarized by Somkutti and Smeller (2013), e.g., beta-lactoglobulin and ovalbumin in combina-
tion with pepsin led to an increased hydrolysis at 600 MPa and a few minutes of treatment time. For peanut allergen Ara h 6 no
changes could be shown at 700 MPa at 20 or 80
C.
The pressure stability of allergen proteins varies greatly, dependent on their secondary structure, the surroundings and treatment
conditions applied. Further, combined studies are needed to assess the influence and the effectiveness of pressure-enzymatic hydro-
lyzes processes. Eventually, only prick test studies in combination with structural analysis of the allergen will validate the effective-
ness of the reduction.

1.01.3.2 Toxins
There are two types of toxins that are of interest in food. One being those incorporated due to the use of chemicals during growth of
crops (pesticides and herbicides) as well as the generation of microbiological toxins, such as aflatoxins, C. botulinum toxin, etc. The
second one being toxins that arise during the processing of the foods due to heat treatment, the so-called food processing contam-
inants (furan, acrylamide, HMF, MCPD-esters etc.)

1.01.3.2.1 Food Processing Contaminants

The occurrence of FPCs in foods is not new, as they have always existed since the first day humans started to prepare and conserve
their foods by fire or heat. Nowadays, the awareness of what these compounds can do within the human body is more advanced and
the analytical tools are available to analyze the amounts of these compounds within foods in the mg kg1 range (Sevenich, 2016;
Sevenich et al., 2016).
The high temperatures at >110
C needed during thermal sterilization for the inactivation of spores lead to the formation of
unhealthy compounds within the food. Since the beginning of the new millennium, more attention has been given to the mech-
anisms and mitigation strategies of these compounds. The way of our foods from farm to fork or from raw material to better
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 7

digestible safe foods is made possible by processes such as heat treatment. Those treatments often result in an over-processing and,
due to chemical changes in the product, giving rise to the food processing contaminants (FPCs); synonyms are, process-induced
toxicants and neo-formed contaminants. FPCs only involve those compounds formed during the processing (heating, roasting,
frying, baking, grilling etc.) of the food. FPCs are substances present in food because of food processing/preparation that are consid-
ered to exert adverse physiological (toxicological) effects in humans, i.e., substances which pose a potential or real risk to human
health. Ingredients commonly occurring in food formulations are excellent substrates for chemical reactions occurring under the
conditions encountered in food processing. The reaction products formed depend on the processes and conditions used, such as
fermentation, irradiation, or heat processing (Stadler and Lineback, 2009). The compounds formed play a vital role in food prop-
erties such as flavor, aroma and color. Precursors of FPCs are sugars and amino acids; other reaction pathways can involve poly-
unsaturated fatty acids (PUFAs; linoleic acid), ascorbic acid, sugars (glucose, fructose), glycerol, chloride or carotenoids (Crews
and Castle, 2007; Vranová and Ciesarová, 2009; Lachenmeier and Kuballa, 2010; Bravo et al., 2012; Crews, 2012). Therefore,
changing the recipe of foods is not an option since some of the most potent precursors of FPCs are essential nutrients, like poly-
unsaturated fatty acids, reducing sugars, carotenoids, proteins etc. Compounds formed during the processing of food are, for
example, acrylamide, furan, 3-MCPD esters etc. These show carcinogenic, mutagenic (genotoxic), or neurotoxic properties at
high doses in animal studies (BfR, 2004, 2012; Studer et al., 2004; Märk et al., 2006; Vranová and Ciesarová, 2009; Jestoi et al.,
2009; Larsen, 2009; Stadler and Lineback, 2009; Lachenmeier and Kuballa, 2010; Palmers et al., 2014; Sevenich et al., 2015a,b; Ket-
tlitz et al., 2019). Therefore, a risk for humans, especially for infants, the elderly and immune suppressed person is not neglectable.
The risk of the exposure to FPCs is not a new one, since humans always have been exposed to these kinds of compounds from the
moment “they caught fire”. Nevertheless, there is a public concern and those risks must be minimized (Curtis et al., 2013). For gen-
otoxic and carcinogenic substances, such as furan and acrylamide, the ALARA principle (as low as reasonably achievable) is applied
to foods as a possible risk assessment (Crews and Castle, 2007). Nevertheless, the data shown here clearly indicates that the FPCs of
major concern in foods are acrylamide, furan and 3-MCPD. The European Commission and the European Food Safety Authority
(EFSA) have been monitoring different FPCs, especially acrylamide, furan (and its derivates), 3-MCPD and -esters and glycidyl
esters, in all kinds of foods over the last years and have already issued benchmark levels for acrylamide in 2017 (EU, 2017). The
creation and introduction of new guidelines in the future is likely and will be a difficult task for the food industry (EFSA, 2013;
Kettlitz et al., 2019). Therefore, other technologies and research is needed to find mitigation strategies which lead to the same
quality without affecting the safety of the product. Here the high-pressure thermal sterilization or high-pressure pasteurization
(instead of conventional heat treatment), ohmic heating and vacuum baking, just to name a few innovative technologies, could
be powerful tools to achieve this aim. Research in all these areas is progressing at a rapid pace and these selected examples show
that process toxicants have in the past few years gained significant attention on a global scale in terms of potential human health
risk. In March 2018, EFSA issued a call for the continuous collection of chemical contaminants occurrence data in food and feed
(EFSA, 2018). These data are used in EFSA’s scientific opinions and reports on contaminants in food and feed. This last call shows
how important it is to legislative organizations to be on top of this sensitive matter. There are only a few studies available that show
the mitigating influence of high-pressure thermal sterilization on the reduction in real food systems. Sevenich et al. (2013) and Sev-
enich et al. (2015a,b) showed that a reduction of furan in different food systems (vegetable baby food and sardine in olive oil) is
possible with HPTS (600 MPa, 90–121
C) by up to 95% in comparison to thermal sterilization. Palmers et al. (2014) also showed
a reduction of furan in various vegetable blends under high pressure thermal sterilization conditions (600 MPa, 117
C) in compar-
ison to thermal treatment. To this point, the question if the pressure, following the Le Chatelier principle, has an influence on the
formation pathway of these compounds is still unanswered. In the opinion of the authors the reduction of FPCs can be solely attrib-
uted to the lower thermal load applied to the product. For high pressure pasteurization the formation of food processing is not an
issue since the threshold temperatures of above 110
C or higher are not reached.

1.01.3.2.2 Aflatoxins, Pesticides and Herbicides

To this date there is no literature available how high-pressure processing affects or destroys either aflatoxins or pesticides and herbi-
cides. It will be interesting to investigate these compounds and how they may be altered by high pressure processing. Aflatoxin for
example is very temperature stable and starts decomposing at temperatures between 237 and 306
C (Kumar et al., 2017) but a heat
treatment between 90 and 120
C can already reduce the amount of aflatoxin as much as 25%–65%. There are some studies in the
public domain on processing by extrusion cooking of peanut and rice meal at temperatures of 140–200
C and pressures in the
extruder of 30–60 bar. The highest aflatoxin reduction was found to be 51%–95% with a moisture content of 35% in peanut
meal, and the extrusion variables did not significantly affect its nutritional composition (Castells et al., 2006; Kumar et al.,
2017). It is worthwhile to investigate the effect of HPTS and HPP on aflatoxins. There was no publication found on influence of
high-pressure processing on pesticides or herbicides.

1.01.4 Process-Structure-Relationship

High pressure processing can alter the properties of macromolecules like starch, pectin and proteins and combinations of the afore-
mentioned compounds. Therefore, it can be of great interest to create new textures, which could lead to the development of new and
exciting structures. This is not only to design new foods but also to evaluate how the process influences products in terms of texture
and structure that usually would have been thermally treated. There is significant interest in understanding the effects of high pres-
sure on food and food ingredients, to anticipate further applications of the technology (ETP, 2007; Sun and Holley, 2010). The
following section will summarize ongoing research activities and needs in this field.
8 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

1.01.4.1 Starch
Starch is one of the major carbohydrates used in the food industry and in human diet. As a source of energy and a major source of
carbon, starch is an essential part of human nutrition. In the food industry, starch serves as a thickening and gelling agent, e.g., for
sauces and puddings. Starch and its derivatives are also important in the chemical, textile and paper industries. It is obtained from
the reserve organs of plants. Wheat, potatoes, maize and tapioca are the most important raw materials in global starch production.
As already mentioned, ultra-high pressure has a significant impact on the physical and chemical properties of macromolecules. In
terms of carbohydrates, ultra-high pressure leads to gel formation, a lowering of the gelation temperature, a reduced enzymatic
stability of starch and little or no Maillard reaction at room temperature (Hendrickx et al., 2001). Partial swelling and gelatinization
of starch can occur under high pressure conditions even at room temperature (Stute, 1999; Douzals et al., 2001; Katopo et al., 2002).
The required treatment parameters, such as pressure, temperature, holding time and water concentration depend to a large extent on
the type of starch (Autio, 1998; Bauer and Knorr, 2005). The gelatinization under ultra-high pressure at room temperature is accord-
ing to Rubens et al. (1999) described similar to thermal gelatinization by a 2-step mechanism. First, the amorphous areas are
hydrated, which loosens the crystalline structures and swells the grain. In the second step, the crystalline areas are increasingly
broken up and hydrated. Only a small amount of amylose leakage from the grains can be observed. Stolt et al. (1997) also describe
stabilization of the crystalline areas through interactions with the remaining amylose. Hydrogen bonds and van der Waals forces are
likely stabilized by ultra-high pressure, which in turn favors the double helix (Buckow et al., 2007). The granular character of the
grains is largely retained during the ultra-high pressure treatment (Hayashi, 1992) and there is no complete disintegration of the
structure as in the case of thermal gelatinization. Thus, the pressure-induced, pasty to solid starch gels can only be referred to as
particle gels, which do not form a real gel network. In addition, the thermally formed starch gels and paste have a much higher
strength or viscosity (Buckow et al., 2007). Interestingly, the starch grains lose the double refraction despite no degradation of
the crystalline structures. It becomes clear that the process described requires an excess of water molecules and is therefore heavily
dependent on the water content. Additions of water-binding substances such as sugar or salts can therefore have a significant impact
on the high-pressure-induced swelling. Rumpold and Knorr (2005) showed that the previously described influence of the number
of equatorial groups also exists in the case of swelling and gelatinization induced by high pressure. Starches with a high amylose-
amylopectin ratio, such as wheat or potatoes, have very high pressure resistance (Hendrickx et al., 2001). Waxy starches therefore
show little resistance to pressure (Simonin et al., 2009). Stute (1999) used loss of birefringence to show that pressure resistance is
also visible in the X-ray diffraction pattern. Accordingly, mostly B-type starches are more pressure-resistant than those of the A and C
types, but due to overlaps, a clear distinction cannot be made in this regard (Rubens et al., 1999; Katopo et al., 2002). X-ray structure
analyses also showed that an X-ray diffraction pattern change during ultra-high-pressure treatment can occur. For example, native A-
type starches show the X-ray diffraction pattern of B-type starches after treatment, with the latter undergoing no change (Hibi et al.,
1993; Katopo et al., 2002). According to the authors mentioned, this change is due to the structure of the amylopectin. As already
mentioned, B-type starches have channels between the double helices in which a large number of water molecules can be
embedded. Under ultra-high-pressure conditions, these interact with and stabilize the double helices. Due to the molecular flexi-
bility of the A-type double helices, these can form channels under ultra-high-pressure conditions. In combination with the thermal
process, gelatinization under ultra-high pressure can be achieved below the atmospheric gelatinization temperature since ultra-high
pressure lowers it. As the temperature rises, this pressure effect becomes weaker (Buckow et al., 2007).
The features high pressure treated starch of different plant-based origins offer are not fully assessed and used. One possibility
could be to use the texture to design and build low fat foods since the gels have a mouth feel like fat. Further starch in combinations
with e.g. proteins could be used to create a hybrid networks of starch and proteins to develop meat free plant-based protein rich and
fiber rich foods. Due to the nature of the starch after the treatment, the starch would be resistant and therefore the food would have
a low glycemic index. Papathanasiou et al. (2015) showed that 5% starch solutions of wheat, tapioca, potato, corn, waxy corn and
resistant starch (RS3) released less glucose after 120 min of enzymatic digestions if they were pressurized at 600 MPa for 15 min at
room temperature in comparison to heat treated starch solutions.

1.01.4.2 Pectin
Pectin is mainly found in plant-based products. Here the highest amounts are found in apple (1.5% w/w) and carrots (1.4% w/w).
In processing, pectin, which is mainly found in the middle lamella, can be altered by either chemical or biochemical conversions.
This can be in favor of the texture (demethoxylation) or detrimental (depolymerization/beta-elimination) (Van Der Plancken et al.,
2012). Demethoxylation of pectin can either be triggered by non-enzymatic or enzymatic demethoxylation (Pectin methyl esterase,
PME). In the case of high-pressure processing, both mechanisms could be present simultaneously since the process does not affect
PME to such an extent that it would be fully inactivated. The rate of this reaction is accelerated with increasing degrees of methyl-
ation, temperature, and pH (4–6) (Diaz et al., 2007). Especially the pH for fruit and vegetable-based products is at an optimum. For
non-enzymatic degradation of pectin there are two mechanisms that could apply:beta-elimination or demethoxylation by acid
hydrolysis (Van Der Plancken et al., 2012; Chen et al., 2015). The ß-elimination reaction is primarily base-catalyzed, but can
also become dominant at pH >4, and leads to the cleavage at a glycosidic linkage next to an esterified galacturonic acid; as a result,
pectin with a high degree of methoxylation (DM) is more subject to ß-elimination than pectin with a low DM (Chen et al., 2015). At
low degree of methoxylation (DM), demethoxylation can also occur by acid hydrolysis, enhanced by temperature but not by pres-
sure. Two possible explanations were suggested for the improved retention of hardness under pressure by Van der Plancken et al.
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 9

and Chen et al. (2015): (i) inhibition of the beta-eliminative depolymerization, either directly by pressure or indirectly by the
observed extensive demethoxylation and (ii) formation of fortifying networks of the low methoxylated pectin with endogenous
Ca2þ ions. In the case of HP pasteurization, only limited texture loss was observed as processing temperatures were not high enough
to support beta-eliminative pectin degradation. High hydrostatic pressure treatment did not cause degradation of the main chain
(covalent bonds) of pectic substance. In addition, when combining high temperature with high pressure, ß-elimination that
predominates in high temperature was retarded or even stopped, whereas demethoxylation was stimulated (Chen et al., 2015).
These results are very promising in the texture preservation of high-pressure sterilized fruits and vegetables, as ß-elimination is
accepted to be one of the main causes of thermal softening and low methoxylated pectin can enhance tissue strength by forming
cross-links with calcium ions present.

1.01.4.3 Proteins
The literature of protein denaturation and changes to the nature of the protein structure in real food systems (mainly meat) is exten-
sive (Cheftel and Culiolib, 1997; Galazkaa et al., 2000; Hendrickx et al., 2001; Hogan and Kelly, 2005; Sun and Holley, 2010;
Escobedo-Avellaneda et al., 2011; Barba et al., 2015; Santos et al., 2017). Limited knowledge is present on how specific proteins
could be used and changed to increase the techno functional abilities or create new food systems in combination e.g. with starch.
This part will try to highlight the possibilities to use proteins as structuring agents to form gels in protein only solutions or to form
three-dimensional networks in combination with other macromolecules.
For protein solutions, one of the main protein group of interest concerning their structuring abilities were whey proteins, and
here in particular beta-lactoglobulin (b-LG). A book chapter by Patel and Huppertz (2014) summarizes the current knowledge well.
At high protein concentration of >10%, aggregation and amount of disulfide bonds are favored and lead to the formation of high
pressure induced protein gels. The intensity of pressure, holding time, protein concentration, pH of the medium (the closer to the
isoelectric point the better) and temperature have a severe influence on the characteristic of the formed gel. The threshold concen-
tration of whey protein to form a self-supporting gel is 8%–12% w/v, if treated at pressures of 600–800 MPa and 20
C. Gels can
also be formed at lower pressures 200–400 MPa, but here the concentration needs to be up to 20%. The induced gels differ to heat
induced gels by lower firmness and a more porous structure. The mechanism behind the high pressure induced gel formation is that
pressure favors aggregate formation of whey proteins which are stabilized/crosslinked by intermolecular disulfide bonds and non-
covalent interactions, which lead to an increased viscosity and opacity of the solution. It was proposed that beta-LG plays a vital role
in the formation of whey protein gels, since the formation of beta-LG disulfide bonds proceeds the formation of other bonds within
the protein solution. Therefore, the major whey protein defines and determines the functional behavior under high pressure.
An indirect way how pressure processing can have an impact on the texture/structure of a food is the treatment of milk with
pressure prior to yoghurt fermentation (Guamis et al., 2005). High pressure treatment of milk has the following effects. (i) As
with heat treatment at temperatures greater 70
C, high pressure treatment of milk leads to denaturation of whey proteins; beta-
lactoglobulin is primarily denatured by pressure (López-Fandiño, 2006). (ii) The casein micelles are broken down into smaller
pieces at higher pressures (>250 MPa). This effect is more pronounced than with a thermal treatment (Huppertz et al., 2004; Patel
and Huppertz, 2014). (iii) An increase in the soluble content of calcium was observed in part, which was attributed to the removal
of calcium phosphate from the micelles (Schrader et al., 1997). This has several consequences in the production of yoghurt from
high-pressure milk. Due to the high level of destruction of the casein micelles, there is a significantly more uniform distribution and
closer crosslinking through disulfate bonds between denatured beta-lactoglobulin and kappa-casein than in the untreated and also
the thermally treated milk (Harte et al., 2003; Aguilera and Park, 2016). If high pressure treated milk is fermented, small sub
micelles are obtained during gel formation, which are arranged in larger groups. The increased proportion of soluble calcium, which
is essential for the formation of the structure, is also helpful here. These can bind water extremely well, which leads to a reduced
syneresis in the resulting gel.
Another focus in the light of meat alternatives is currently also given to local and sustainable protein sources, such pea protein.
One study from Sim et al. (2019) has been looking into the structuring of pea protein concentrates. They applied pressure between
250 and 500 MPa, temperatures of 20–33
C for 15 min and used pea protein concentrations of 8%–24% (w/v). Gel formation
started at 16% at pressures of 250 MPa for the HPP-treated samples, and at 12% ion for the heat-treated samples. Gel strength
increased with both pressure level and protein concentration. Heat-treated samples exhibited greater gel strength than pressure-
treated samples at the same protein concentration. A greater extent of protein denaturation, aggregation, and network formation
occurred with increasing pressure level, due to protein tertiary and quaternary conformation changes. Starch granules present in
PPC retained their structure and were not gelatinized even at 550 MPa. The authors also mentioned the ability of creating novel
pea-based products, such as puddings or tofu analogs. The low temperature of the treatment will help preserve the sensory and
nutritional properties of such products.
Further structuring and texturing approaches by high pressure, which are discussed in the literature is the combination of high
pressure processing and transglutaminase (TG) during the production of gels. In surimi and other fish meat products, high pressure
readily induces gelation at low temperatures. This process can be enhanced by TG, which can induce crosslinking of proteins and the
process is called setting or suwari. TG catalyzes a covalent bond between the e-amino group of lysyl residues and the c-carboxamide
group of glutaminyl residues of adjacent proteins (Ashie and Lanier, 1999; Uresti et al., 2006; Trespalacios and Pla, 2007). It was
further observed that even after a treatment of 600 MPa and 60 min that the TG kept 60% of their activity indicating the pressure
resistance of this enzyme (Trespalacios and Pla, 2007).
10 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

Urestie et al. (2006) looked at the usage of TG and high pressure to produce restructured fish products out of Arrowtooth
flounder. They found that fish paste inoculated with TG and treated at 600 MPa and 5 min had better mechanical properties in
comparison to heat treated gels. They concluded that pressures of 600 MPa induce an aggregation characterized by side-to-side inter-
actions of proteins with a low degree of denaturation and not by aggregation of proteins with large changes in molecular confor-
mation, allowing TG to improve mechanical properties of heat-induced gels.
Trespalicos and Pla (2007)investigated the simultaneous effects of microbial TG (MTG) and high pressure (500, 700 and
900 MPa) on chicken meat and egg. The proposed mechanism of protein-TG interaction during pressure is the following: irrevers-
ible changes in the beta structure of the protein above 400 MPa by HP treatment. The pressure causes the protein hydrophobic core
to become more exposed and thus more readily available for the MTG action. The increase in cutting force, hardness and chewiness
of gels suggests that a heterologous complex of meat and egg proteins was formed when high pressure processing and MTG were
combined. Although the enzyme and higher pressure at 700 and 900 MPa considerably improved the yield, color and texture, there
were no differences between the two pressure levels. The synergistic effect of combining enzymatic crosslinking with high pressure
treatment at 700 MPa is of interest for food manufacturers, in order to generate high quality products, which exhibit many desirable
characteristics that satisfy the consumers’ demands, such as low-fat, low-sodium content, phosphate-free and improved sensorial
attributes, while assuring the nutritive value and microbiological safety. Ashie and Lanier (1999) treated turkey breast in combina-
tion with TG and the samples were pressurized at 250 or 300 MPa at 4
C, 40
C, or 50
C for 15 min. They concluded that high
pressure processing is rendering myosin to make it more accessible to TG catalyzed cross linking. The effects of prior pressurization
on gel strengthening by TG were most pronounced in uncooked gels. Thus, the greatest commercial application maybe in enhancing
TG-mediated crosslinking of uncooked proteins.

1.01.5 Packaging Material

Packaging is essential to keep the quality and safety of the product over its shelf-life and can avoid food waste to some extent
(Guillard et al., 2018). Especially for high pressure processing, where suitable packaging materials that can cope with the “extreme”
process conditions to keep the better quality are essential. For the HPP and the HPTS-treatments, flexible (important for pressure
transmission), resilient and high barrier packaging need to be used. That packaging must withstand not only 600 MPa, but also cope
with high temperature, not changing its barrier properties toward the food. Further, the head space of this packaging must be as low
as possible to limit oxidation reactions (Balasubramaniam et al., 2016). The reduction of head space can be accomplished through
vacuum sealing. Stand up, meal-ready-to-eat (MRE) pouches, already used by the food industry for various products (ketchup,
soups, sauces, fish foods and vegetable and fruit purees etc.), have the optimal geometric form to be placed in the high-pressure
treatment baskets to get a maximum filling efficiency. The packaging materials that should be used have been discussed in many
research projects and in literature (Welti-Chanes et al., 2005; Galotto et al., 2009; Juliano et al., 2010; Koutchma et al., 2010; Bala-
subramaniam et al., 2016). Fleckenstein et al. (2010; 2014) divided the damages of polymeric packaging materials that could occur
during HPP into direct effects (caused by high pressure treatment) and indirect effects (generated by compression of other
substances in the package). Direct effects have a temporal and reversible decrease in permeability not negatively affecting the func-
tional properties of the packaging; morphology of the polymers may change and the delamination of the multilayer system (mainly
between inorganic (e.g., Aluminum) and polymeric layers (e.g., Polypropylene). The two last mentioned effects have a negative
impact on the packaging, therefore either the adhesion needs to be improved or systems without inorganic layers need to be devel-
oped. Indirect effects represent all influences of compressed substances inside the package. The higher concentration of gases in the
polymeric matrix at high pressure can lead to a plasticization of polymers, followed by structural changes or an extraction of
constituents from the polymer enabled by the supercritical state. These failures arise in the form of cracks, blistering or other micro-
structures and show mostly in weak zones of the materials. Therefore, packaging material such as EVOH (Nylon based ethylene-
vinyl-alcohol-co-polymer) based materials show a high potential due to their integrity and low oxygen permeability (Fleckenstein
et al., 2014; Sterr et al., 2014). A combination of a three layered system of polyethylene, aluminum foil and 4 mL polyolefin showed
good results for a high pressure high temperature processing between 105 and 121
C at 700 MPa(Juliano and Barbosa-Canovas,
2005; Juliano et al., 2010). Nevertheless, more research is needed to evaluate the influence of HP and HPTS treatment on different
packaging materials and to assess if there is a migration from packaging compounds into the food. Development of suitable pack-
aging will be crucial for a wider application of HP and HPTS processing.
Concerning the sustainability and circular economy of packaging materials, one of the agendas of the European union by 2030 is
to only have 100% recyclable packaging solutions. Therefore, the search for alternatives, which have the aforementioned attributes
is currently ongoing (EU, 2018). The most promising way will be to design a packaging that consists of two layers from the same
material, which make up approximately 90% of the packaging. By only having one material, but still two layers, recycling can be
enhanced, and the quality and safety assured at the same time. Also, a focus of many studies is to look into the possibilities to use
green platform chemicals like polylactides (PLA),hydroxy-hydroxymethylfurfural (HMF) or starch, instead of petroleum-based
plastic polymers, to develop new and green packaging solutions. Guillard et al. (2018) summarizes the difficulties bio-based pack-
aging materials currently are facing: An unavoidable raw material variability and smaller process windows in comparison to
petroleum-based packaging, which hinders their scale up and industrial implementation. Also, there are no tools available that
make a conversion from tailor-made packaging to food requirements possible. Their readiness for high pressure processing needs
to be tested and validated. Following, two of the most promising materials will be introduced.
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 11

1.01.5.1 Polylactides (PLA)

Polymerization of lactic acid produces PLA by two methods, direct polycondensation and ring-opening polymerization. Lactic acid
(LA) can be produced by chemical synthesis and by microbial fermentation, which offers advantages due to the production of opti-
cally highly pure LA, the utilization of renewable carbohydrate biomass, low temperature, and low energy consumption during the
process (de Oliveira et al., 2017). PLA has good biodegradability, biocompatibility, a high mechanical strength, and excellent
shaping and molding properties. Therefore, it becomes a good candidate for packaging materials due to its close similarity with
poly (ethylene terephthalate) (PET) (Ahmed et al., 2009). PLA containers are already in use for bottled water, bottled juices,
and yogurts in European and North American markets. Ahmed et al. (2009)investigated the thermal properties of PLA solutions
under pressure. At pressures of 350, 450 and 650 MPa for 15 min they concluded, based on the results of the performed analysis
(FTIR and DSC), that glass transition temperature decreased, melting temperature remained unaffected while partial shift in crys-
tallinity of L-isomer was detected after high pressure (650 MPa). FTIR spectra did not show any shift of crystalline band after pres-
sure treatment. Their final comment was that PLA seems suitable but more detailed research needed to be conducted.
Further studies with actual PLA-based packaging material was conducted by Galotto et al. (2009). They tested a PET based pack-
aging in comparison to a PLA based packaging, treated at 500 MPa for 15 min at a temperature of 50
C and compared the mechan-
ical, thermal and chemical properties of the different materials. HPP treatment affects significantly the mechanical, thermal, and gas
barrier properties of PET and PLA films. For the PET film, all physical properties were negatively affected. For the PLA film, major
changes occurred when the film was in contact with water (due to the plasticization effect of the latter on PLA) and minimal changes
occurred when oil was inside the packaging. This film can be used in HPP applications while in contact with fatty foods, when
medium gas barrier properties are sufficient.
Another promising compound for biodegradable packaging is starch. Although, challenges involve water solubility and poten-
tial changes in mechanical properties due to molecular rearrangement upon water exposure (retrogradation). Among some reported
HPP applications, Kim et al. (2018) reported that a more compact structure of buckwheat starch films processed at high pressure
(600 MPa, 20 min) improved water vapor permeability when compared to heated films, although properties were not evaluated
during storage.
There are also starch/protein -based bioplastics available but these have, to the best knowledge of the authors, never been tested
for the application with high pressure processing (Gonzalez-Gutierrez et al., 2010). Further research needs to be conducted to use
the many opportunities high pressure in combination with bio-degradable or 100% recyclable packaging could give.

1.01.6 Data Reporting and Experimental Design

Data reporting is the process of collecting and submitting data which gives rise to accurate analyses of the facts. In some articles
involving high pressure processing, the main information is on, e.g., high-pressure system, applied pressures, come-up time,
temperature, temperature-pressure-time profiles, initial temperature (pre-HP application), end temperature (post-HP application),
sporulation temperature of spores, packaging, pressure transmitting medium, pH of growth medium for microorganisms, pH of
tested food, other influential food characteristics, kinetic approach not end point approach, choice of model etc. All the points
mentioned make it difficult to compare and relate the mass of data available on high pressure processing in the literature. Balasu-
bramaniam et al. (2004) came up with a concept called “minimal description to be included” in a publication dealing with high
pressure processing. They also concluded that research articles on new technology assessment should include enough description of
process equipment (or citation), experimental design, methods (or citation if standard methods were used) of data analysis and
reporting.
Raso et al. (2016) published a guideline for reporting studies conducted for pulsed electric fields (PEF) applications. Their table
is more detailed and could also be used for high pressure reporting if the bullet points are changed and adapted to high pressure
processing. The version for HPP and HPTS adapted from Raso et al. (2016) and Balasubramaniam et al. (2004) can be found in
Table 1.
Nevertheless, most published papers on the topics do not provide enough information for other researchers to assess results
properly. A general rule/guidance in reporting experimental data and most of all exposure conditions would be to report details
to the extent that other researchers will be able to repeat, judge and evaluate experiments and the data obtained. To the opinion
of the authors there are still multiple research challenges for high pressure processing that need to be assessed and unraveled in
systematic studies. The variables, options and action are summarized in Table 2.

1.01.7 Conclusion

High pressure processing has been proven very valuable as a decontamination technology, but it has much more to offer than just
being an alternative to thermal processing. Reactions are favored differently under high pressure in comparison to thermal
processes, therefore other activation systems exist under pressure. These new acting principles could be used to develop new
food systems, e.g., raw materials with different functionalities, instead of just being an inactivation tool. Historically, most food
raw materials have been selected empirically based on their edibility and safety as raw, fermented or heated. Since high pressure
12 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing

Table 1 Recommended information to be reported in studies on microbial inactivation and chemical analyses for HPP and HPTS.

Category Information and description

Microorganism culture and recovery condition Genus, species and strain of the microorganisms/spores
Culture conditions
Initial inoculum
• Description of the procedure for microbial cultivation
• Growth medium composition, growth/sporulation temperature,
incubation time and growth phase (exponential or stationary)
Recovery conditions
• Time and storage conditions between treatment and microbiological
analysis
• Description of the procedure for enumerating microorganisms
• Composition of the recovery medium, incubation time and incubation
temperature
HPP/HPTS equipment HPP/HPTS system
• For commercial: equipment name of the supplier company and model
• For prototypes/lab scale systems: adequate description of the
components
High pressure vessel
• Volume; horizontal or vertical; heating systems (vessel immersed in
bath or jacket with heating)
• Heating medium;
• Pressure transmitting medium
• Thermocouples (where located)
Processing parameters • Pressure, pressure build-up rate
• Starting temperature; end temperature after pressure build up;
temperature shortly before decompression; temperature after
decompression
• p,T,t-diagram, kinetic approach at fixed pressure and temperature at
least 5–6 independent data points (time),
• Description of sample container
Treatment medium Composition (Recipe of the food; Origin and name of the fruit or vegetable /
Degree of ripeness)
• pH
• aw
• Sugar content
• Salt content
• Inoculum / Number of CFU’s per g or mL
Enumeration of survivors • Agar used
• Plating method
• Time between treatment and microbiological analysis (storage
conditions)
• Enumeration method
• Model used to fit inactivation kinetics (including R2; equation and
constants)
Analytical methods Description of methodology (References)
• Analytical System used (e.g. HPLC or GC/MS etc.) with information on
set up, important parts and chemicals
• Storage conditions
• Preparation steps
Other things to consider • To have comparable results to other technologies Ce, Fe or P-values if
possible, should be calculated
• Product of comparison should always the industry processing standard
for the investigated product (if possible)

Based on Raso et al. (2016) and Balasubramaniam et al. (2004).

provides a different principle of action than heat inactivation of antinutritional or toxic food constituents, it should be possible to
identify new raw materials to be made safe and edible via high pressure processing. Ananta and Knorr (2003) used mild high pres-
sure conditions (100 MPa, 37
C, 10 min) to trigger a stress response in Lactobacillus rhamnosus, which led to the production of stress
metabolites (proteins). This effect made the probiotics more resistant toward spray drying and therefore a more optimized produc-
tion of probiotic starter cultures is possible. High pressure processing could also be used to convert certain compounds or activate
certain plant cell systems due to stress induction in foods or food waste streams, so that a higher yield of valuable compounds could
be generated. This was just recently shown by Ramos-Parra et al. (2019), who treated papaya fruit with high pressure (50–400 MPa)
 Overview of Research Needs, Future and Potential Applications of High-Pressure Processing 13

Table 2 Variables for future systematic studies of HPP.

Variables Options Action 1 Action 2 Action 3 Action 4 Action 5 Action 6

Pressure Pressure variation Static Steps (pulses) High p Low p High T Low T
Pressure build up Slow Fast Stepwise – – –
Pressure release Slow Fast Step wise – – –
Temperature High Low Steps Initial low or high – –
Cycle time Short Long Holding Starting at set – –
pressure
Packaging Renewable Chitin Lignin Cellulose PLA Starch –
Antimicrobial Chitosan Essential oils Lactobacillus pH – –
Smart Indicators Integrators
In package media Vacuum Gas/air Functional Plasma Brine Infusion
liquids
Product Changes High Low Gases/plasma Degassing pH [ pH Y
temperature temperature
Product Modifications Food package Foods Food Response of – Indicator
interaction constituents constituents microorganism

and proved that HPP triggers the de novo carotenoid biosynthesis, which is regulated at the transcriptional level, possibly by
inducing oxidative stress signaling in fruit tissue. Another approach includes the use of high pressure as an extraction method
and texture improvement via high pressure treatment. There are different combinations of processes that are worth considering,
such as the combination of high pressure and medium to high temperature (60–80
C). This intensified high-pressure processing
would still be much shorter than the thermal process but in comparison to the ambient high pressure process it would not have the
problem concerning the enzymatic activity. Future equipment should be continuous or at least semi-continuous. This seems
possible since some of the ultra-high-pressure homogenizers can go as high as 450 MPa, but this would only work for pumpable
foods like clear juices, milk etc. High pressure processing has come a long way and there is still a long way to go especially in under-
standing the full range of benefits and applications this technology could offer to food technology. High pressure processing at low
temperatures (below ambient temperature) also received little attention and should be explored further, as well as high pressure
assisted/combined processes such as extrusion, ohmic heating or ultrasound.
High pressure offers numerous benefits (Fig. 1) but there are still key issues in terms of food safety that need closer and further
evaluation: (i) indicator microorganism currently E. coli O157:H7, Salmonella and Listeria are used to distinguish the effectiveness of

Reduced FPCs
formation
Activation of Increased Food
biosynthesis quality

High pressure Cosmetics and

Food safety processing applications medical products

Improved Allergen
bioavailability reduction

Structure

Figure 1 Possible future application of high-pressure processing.

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Unit 1: Study tips and learning strategies

Learning Objective 1: Experimental procedures and results
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Learning Objective 2: Critical analysis and evaluation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 2: Diagram/Chart/Graph]
Learning Objective 3: Interdisciplinary approaches
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Learning Objective 4: Comparative analysis and synthesis
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Learning Objective 5: Current trends and future directions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 5: Best practices and recommendations
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Problem-solving strategies and techniques
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Note: Learning outcomes and objectives
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 8: Research findings and conclusions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Comparative analysis and synthesis
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
[Figure 10: Diagram/Chart/Graph]
Topic 2: Assessment criteria and rubrics
Definition: Current trends and future directions
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Critical analysis and evaluation
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Experimental procedures and results
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Ethical considerations and implications
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 14: Diagram/Chart/Graph]
Note: Research findings and conclusions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Note: Interdisciplinary approaches
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 16: Diagram/Chart/Graph]
Remember: Assessment criteria and rubrics
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 17: Research findings and conclusions
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 18: Diagram/Chart/Graph]
Remember: Learning outcomes and objectives
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 19: Problem-solving strategies and techniques
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Lesson 3: Assessment criteria and rubrics
Definition: Study tips and learning strategies
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 21: Literature review and discussion
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 22: Diagram/Chart/Graph]
Definition: Literature review and discussion
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Practical applications and examples
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 24: Diagram/Chart/Graph]
Note: Learning outcomes and objectives
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 25: Diagram/Chart/Graph]
Definition: Ethical considerations and implications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 26: Ethical considerations and implications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 27: Diagram/Chart/Graph]
Definition: Key terms and definitions
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Note: Theoretical framework and methodology
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Definition: Key terms and definitions
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Results 4: Study tips and learning strategies
Key Concept: Assessment criteria and rubrics
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 31: Diagram/Chart/Graph]
Example 31: Experimental procedures and results
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 32: Literature review and discussion
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Case studies and real-world applications
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Important: Historical development and evolution
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Study tips and learning strategies
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 36: Fundamental concepts and principles
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Definition: Experimental procedures and results
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Fundamental concepts and principles
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 39: Theoretical framework and methodology
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Exercise 5: Best practices and recommendations
Example 40: Ethical considerations and implications
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Interdisciplinary approaches
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 42: Diagram/Chart/Graph]
Note: Comparative analysis and synthesis
• Literature review and discussion
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Best practices and recommendations
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Statistical analysis and interpretation
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Fundamental concepts and principles
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Current trends and future directions
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Historical development and evolution
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Assessment criteria and rubrics
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 49: Diagram/Chart/Graph]
Remember: Interdisciplinary approaches
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
[Figure 50: Diagram/Chart/Graph]
Discussion 6: Literature review and discussion
Practice Problem 50: Study tips and learning strategies
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Important: Best practices and recommendations
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Remember: Study tips and learning strategies
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Current trends and future directions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Research findings and conclusions
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 55: Diagram/Chart/Graph]
Definition: Historical development and evolution
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 56: Statistical analysis and interpretation
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 57: Key terms and definitions
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Remember: Assessment criteria and rubrics
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Fundamental concepts and principles
• Study tips and learning strategies
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Results 7: Ethical considerations and implications
Definition: Theoretical framework and methodology
• Key terms and definitions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Learning outcomes and objectives
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Remember: Ethical considerations and implications
• Learning outcomes and objectives
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Note: Literature review and discussion
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 65: Problem-solving strategies and techniques
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Critical analysis and evaluation
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 67: Ethical considerations and implications
• Historical development and evolution
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Research findings and conclusions
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Comparative analysis and synthesis
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Quiz 8: Current trends and future directions
Definition: Experimental procedures and results
• Best practices and recommendations
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 71: Research findings and conclusions
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Key Concept: Best practices and recommendations
• Research findings and conclusions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Critical analysis and evaluation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Practice Problem 75: Theoretical framework and methodology
• Problem-solving strategies and techniques
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Important: Comparative analysis and synthesis
• Interdisciplinary approaches
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Example 77: Critical analysis and evaluation
• Ethical considerations and implications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Key Concept: Interdisciplinary approaches
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
Formula: [Mathematical expression or equation]
Practice Problem 79: Critical analysis and evaluation
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
[Figure 80: Diagram/Chart/Graph]
Topic 9: Learning outcomes and objectives
Important: Comparative analysis and synthesis
• Statistical analysis and interpretation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Case studies and real-world applications
• Critical analysis and evaluation
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Definition: Critical analysis and evaluation
• Current trends and future directions
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 83: Comparative analysis and synthesis
• Fundamental concepts and principles
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 84: Fundamental concepts and principles
• Experimental procedures and results
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Practice Problem 85: Interdisciplinary approaches
• Comparative analysis and synthesis
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
Remember: Problem-solving strategies and techniques
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Key Concept: Research findings and conclusions
• Practical applications and examples
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Note: Fundamental concepts and principles
• Case studies and real-world applications
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Example 89: Statistical analysis and interpretation
• Theoretical framework and methodology
- Sub-point: Additional details and explanations
- Example: Practical application scenario
[Figure 90: Diagram/Chart/Graph]
Appendix 10: Theoretical framework and methodology
Definition: Ethical considerations and implications
• Assessment criteria and rubrics
- Sub-point: Additional details and explanations
- Example: Practical application scenario
- Note: Important consideration
Formula: [Mathematical expression or equation]
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