CHAPTER ONE

1.0. Introduction
Food microbiology evolved as tools and techniques revealed problems and solutions. When
Antonie van Leeuwenhoek first observed ‘animalcules’ in 1676 with his microscope it was a tool
that revealed there was microscopic life around us. When Louis Pasteur demonstrated that
fermentation was caused by yeasts, not spontaneous germination, and later that heat could
inactivate microbes, he was building on the work of earlier scientists, such as Girolamo
Fracastoro, Agostino Bassi, Friedrich Henle, and others. But, Pasteur understood the mechanism
that heat killed bacteria and publicized it. Similarly, Robert Koch built on the work of others,
developed a cadre of microbiologists, and provided tools for future food microbiologists. Before
Pasteur and Koch, people had practical knowledge about food spoilage and fermentations from
experience. Brewers and bread makers knew that successful ferments could be transferred to new
batches. The effect of heat on spoilage was known years before Nicholas Apert won his 12 000
Franc prize, but he developed a reliable method for food preservation. Centuries prior, humans
had discovered that salting or drying would prevent food spoilage; however, food
microbiologists began understanding the mechanisms of how these methods worked and
developed more successful and reliable methods. An array of microorganisms has long been
known to be of major importance in the food industries, from public health perspectives as well
as for the biotechnological applications. Despite the ongoing development in the serological and
molecular methods in food protection consideration over the traditional cultural methodology,
the assurance of food safety and quality is still an essential issue as well as has appeared as a
major confront at present time urging more effective microbial controls along the food
production chain. Indeed, foods are usually prone to microbial attack (mostly by Salmonella
spp., Campylobacter jejuni, Escherichia strains including the Shiga toxin-producing E. coli-
mostly the O157 serogroups, Listeria monocytogenes, Vibrio spp., Clostridium botulinum,
Bacillus cereus, Penicillium expansum, Alicyclobacillus. Due to foodborne hazards, public
health concerns have been projected due to several foodborne pathogenic microorganisms mostly
the toxigenic Escherichia coli, Salmonella spp., Shigella spp., and Campylobacter spp., non-
typhoidal Salmonella enterica (NTS), Listeria spp., Yersinia spp., and Staphylococcus aureus
Guerra MM, de Almeida AM, Willingham AL. Conventional microbial control technologies
(replying on chemical exposure, application of radiation, and thermal approaches) have been
noticed to pose some major drawbacks like the degradation of quality and texture of the food
items; high energy cost; wastage of foods; occupational and health hazards Eleftheriadou M,
Shen C.
Therefore, the chemical-free approaches rise with its popularity among the consumers and hence
pondering to develop novel, more efficient, sustainable, and low-cost anti-microbial methods
Newell DG, Koopmans M. The availability of safe food items is probably the most important
global issue as well as it is challenging for the corresponding food authorities and the food
industries Pyrgiotakis G. The frequency and the dreadful impact of the foodborne disease are
globally fatal with significant morbidity. Progression in food safety research around the globe is
projecting the total knowledge of the foodborne microorganisms as well as the associated disease
onset. Besides the wet-lab experiments, the construction of food safety based databases has
drawn a huge improvement in the field of food microbiology. The present review attempted to
concentrate on the ongoing application and necessity of involving the advanced approaches
currently employed globally.
 CHAPTER TWO
2.0. Literature review
We have reviewed key literature, in particular documents related to the proposal that include the
evidence used in the 2008 impact assessment and evidence published since this date, to provide a
more up to date assessment. Where relevant, peer-reviewed scientific journals have also been
analysed. It is important to highlight here that very little scientific literature has been published
on the subject of Novel Foods since 2008.
Other sources included position papers from the food industry to illustrate the views of industry
stakeholders on the proposed changes to the Novel Food Regulations. Some of these position
papers were published after 2008, but make reference to the previous Commission proposal of 14
January 2008. These position papers were useful in informing our research when the points
under discussion related to certain provisions which are still present in the 2013 proposal.
Finally, in carrying out the interview programme (see below), we also used the content of some
of these position papers as a basis for engaging in discussions with the associations who had
published them, as well as for substantiating the information gathered during the interviews.
2.1. Interview programme
In order to accurately assess the impact of the proposals on relevant stakeholders, including
applicants, consumers and public authorities, a number of representative consultations were
carried out.
Table 1 lists the types of organisations consulted. The study team consulted a mix of stakeholder
types to get the broadest possible view on the impacts of the proposed regulation. These included
both public and private stakeholders to get the views of regulators, authorities, consumers and
applicants who will be affected by the Regulation. Within the time frame available for this
assessment it was not practical to interview all Member States’ competent authorities. As such,
the Member States initially targeted, were those who have the most experience in dealing with
applications under the current system. For the industry member bodies interviewed, this included
large organisations and SMEs (one-member body had 22% of its industry members being SMEs,
two in which SME membership was 70-80%, and one in which SME membership was over
90%). The European umbrella organisation for consumer associations (BEUC) was interviewed
and one of its member associations also provided additional feedback.
Table 2.1: Organisations interviewed
Type of interviewee No. of
interviews
Member state competent authorities currently responsible for Novel Food 6
authorisations
EU body responsible for risk assessment under the new proposals (EFSA) 1
EU Membership bodies representing applicants in different food sectors 3
European and national consumer associations 2
Representative from a third country 1
Relevant EU Commission DGs 2
Total 15

The interview checklist we used was structured around the key issues outlined in the European
Parliament’s terms of reference for the assignment. The checklist was sent to respondents prior
to interview so they could prepare. A copy of the interview checklist is provided in Appendix B.
The interviews were conducted by telephone in a semistructured manner in order to elicit the
greatest amount of detail from respondents. Each interview typically lasted between 60-90
minutes.
2.2. Reporting
The findings from the assessment have been written up in way that reflects the principles in the
European Commission's Impact Assessment Guidelines of 2009. The analysis consisted of
presenting all evidence gathered from literature and interviews with organisations under each of
the five key research questions outlined above. The viewpoints of different organisations
interviewed (consumers, industry and regulatory) were summarized for each research question
and a conclusion reached on the impact of the proposal for a new Regulation on novel foods.
Recommendations were based on the conclusions reached in each section and aimed to outline
further actions which might be needed on the proposal to address the concerns identified.
2.3. Novel Foods & the 2008 Impact Assessment
Novel Foods – as defined in Article 1 of Regulation (EC) 258/97 – are foods which were not
consumed in the EU to a significant degree before 15 May 1997, i.e. before Regulation (EC)
258/97 entered into force. This is in particular relation to food produced using new techniques
and technologies, such as nanomaterials. Currently, foods falling under the scope of Regulation
(EC) 258/97 are subject to pre-market approval, based on a safety assessment. In other words, to
market a Novel Food or ingredient, companies must apply to an EU Member State authority for
authorisation, presenting the scientific information and safety assessment report.
Under the existing assessment procedure, the competent body of the Member State which
receives an application, must make an initial assessment and determine whether or not an
additional assessment is required. If neither the Commission nor the Member States raise an
objection, and if no additional assessment is required, the Member State informs the applicant
that they may place the product on the market. In other cases an authorisation decision is
required. This decision is adopted in accordance with the measures proposed by the Commission
within the Committee on Food Safety and Animal Health. The decision defines the scope of the
authorisation and specifies, as appropriate, the conditions of use, the designation of the food or
food ingredient, its specification and the specific labelling requirements. Finally, any decision or
provision concerning a Novel Food or food ingredient which is likely to have an effect on public
health must be referred to the Scientific Committee for Food. The safety assessment and product
authorisation procedure for Novel Foods is currently described as very lengthy, by both the food
industry and relevant Member State authorities. The decentralised procedure (i.e. on a Member
State basis) duplicates the work and often generates unnecessary delays in the authorisation
process.
A first proposal streamlining the approval process of the 1997 Novel Food Regulation was issued
by the Commission on 14 January 2008. Inter-institutional negotiations on the content of this
proposal started in 2009. The legislative discussions on that proposal mainly focused on the
provisions applicable to nanomaterials and traditional foods from third countries, but also the
cloning of animals for food production. Further discussions concentrated on the criteria to be
examined for the risk assessment and risk management, and the procedure for the authorisation
of Novel Foods in accordance with the Lisbon Treaty.
However, the discussions reached a stalemate on a number of issues. More specifically, no
agreement could be reached between the Council and the European Parliament on any of the
issues linked to cloning. The Conciliation Committee did not reach a final agreement at its last
meeting on 28 March 2011, and the proposal was not therefore adopted. Following this failure,
the Commission undertook to present a separate proposal on cloning based on an impact
assessment.
Thus, the proposal of December 2013 on Novel Foods (the ‘2013 proposal’) is limited to the
safety of Novel Foods and is said, by the Commission, to be based on the overall agreement,
which had been achieved in Conciliation in that area. It aims to streamline the authorisation
procedure and to improve its efficiency and transparency, whilst maintaining a high level of
public health protection. Under the proposed Regulation, Novel Food would be subject to a
simpler, clearer and more efficient authorisation procedure, fully centralised at EU level, which
should enable safe and innovative food to be placed on the EU market faster, without
compromising a high level of public health.
The proposal also clarifies the definition of a Novel Food, including new technologies which
have an impact on food, by replacing existing categories of Novel Food laid down in Article 1 of
Regulation (EC) No 258/97, with reference to the general definition of food provided for in
Article 2 of Regulation (EC) No 178/2002. Special provisions are also made for food which has
not been marketed in the EU, but which has a history of safe use in non-EU countries. The aim
here is to create a more balanced system and a positive environment for trade.
Protecting and stimulating innovation, so as to improve the competitiveness of the European
food industry, is also a feature of the proposal. Under the new approval system put forward in the
proposal, in case of innovation supported by new scientific developments, the food company
which submitted the application may be given the authorisation to market the food for five years
before it can be produced and marketed by others.
2.4. Key points in the 2008 Impact Assessment
The Impact Assessment published by the Commission in 2008 presented four key measures that
were identified during the consultations as having a major impact, and in this regard,
recommended four key policy actions intended to amend and replace provisions already covered
under Regulation (EC) No. 258/97. These are presented below:
Policy Action 1: Adjusted safety assessment and management for traditional food from third
countries
Current problems: at present, uniform criteria apply for the safety assessment of all kinds of
food, including traditional food from third countries and newly developed innovative food.
However, the strictness of the requirements is not always proportional to the potential risks,
which mean that the costs of application, could in turn be considered disproportionate. This is
perceived, for example, by third countries as unjustified barriers to trade in their traditional food
with a history of use.
Conclusions: for traditional food from third countries, a procedure setting out essential criteria
and guidelines, that would allow food, with a history of safe food use, to be subject to an
adjusted safety assessment and management procedure, should be introduced.
Policy Action 2: Safety assessment and authorisation procedure
Current problems: at present the initial risk assessment is carried out by a Member State’s
competent assessment body within three months of receiving the application. The initial
assessment report is circulated to the other Member States. If no objections are presented within
the 60 days' commenting period, the Member State’s competent authority informs the applicant
that it may place the Novel Food product in question on the market. An application is only
assessed and authorised at EU level if member state objections have been raised. In practice, this
is generally what has happened. So the system has proved to be time-consuming and has
imposed a high administrative burden, as applications are assessed twice.
Conclusions: the decentralised procedure should be replaced by a centralised procedure at EU
level. The safety assessment should be carried out by EFSA, and the authorisation decision taken
by comitology procedure, combined with time limits to be respected.
Policy action 3: Authorisation decision
Current problems: at present the authorisation decision is linked to the applicant, thus only
initially allowing this applicant to market the Novel Food in the EU, and making it necessary to
have an additional administrative notification procedure (simplified procedure). This allows food
to be marketed in the EU which is substantially equivalent to food already authorised in the EU.
This system is held in high regard by industry, but causes duplication of work for food that has
already been authorised and is regarded as safe.
Conclusions: the applicant-linked authorisation needs to be replaced and the present simplified
procedure abolished by granting generic authorisations as a general rule. In order to support
innovation and to ensure food safety, consideration could be given, in justified cases, to an
applicant-linked authorisation for a newly developed food for a certain period of time. Data
protection could be a further consideration.
Policy action 4: Submission of application for several food uses
Current Problems: at present separate applications need to be made within the respective legal
frameworks for a substance with different food uses (e.g. additives, flavourings, extraction
solvents or Novel Foods). The regulation, assessment and authorisation of one and the same
substance under different sectoral legislation leads to repetitive work and an additional
administrative burden. Industry is also seeking the simplest possible regulatory framework.
Conclusions: the present system should be simplified and applicants should be able to apply for
an approval by a single application covering Novel Food and food uses regulated under various
regulatory frameworks.
In addition, the Commission Impact Assessment of 2008 explored possibilities for bringing the
revised Novel Foods Regulation into line with other EU food safety policies.
Amongst the possibilities that were explored were the following:
• Making use of the food definition in the General Food Law and abandoning the
categories of describing foods;
• Setting out definitions or criteria for significant human consumption as food, traditional
food from third countries and history of safe use;
• Defining the role of EFSA and introducing deadlines for its opinions;
• Updating and formulating provisions on confidentiality and data protection;
• Creating a register of Novel Foods.
The findings of the 2008 Impact Assessment served as the basis for the Commission’s proposal
of 18 December 2013 for a Novel Food Regulation.
2.5. Developments since 2008
As discussed previously, very little research relating to the EU Novel Foods Regulation has been
published since 2008. A number of position papers were published after 2008, but these relate to
the previous proposal of January 2008. Most of these position papers were thus published prior
to the failure of the Conciliation procedure in March 2011.
EFSA and the Member State authorities interviewed reported that no change has been observed
since 2008 as regards the volume of Novel Food applications submitted. Similarly, the
Commission has not observed any particular changes since 2008 in terms of Member States'
voting patterns in the context of the decentralised system and whether, as a consequence, the
decentralised system has become even more burdensome since 2008.
As mentioned earlier, the failure of the conciliation procedure in March 2011 was mainly due to
disagreements over food produced from cloning. As such, cloning has been excluded from the
scope of the 2013 proposal. The second major disagreement was over the notion of ‘delegated
acts’ for the procedure of authorisation of Novel Foods. Delegated acts allow the European
Parliament and Council to delegate, to the European Commission, the power to adopt non-
legislative acts of general application and to supplement or amend non-essential regulatory
requirements. In this case, the Council would not agree to delegated acts in relation to the
authorisation procedure, as it may mean that Member States national experts are unable to vote
on the authorisation of particular Novel Foods.
Delegated acts can be vetoed by either the European Parliament or the Council. In this regard, it
is worth pointing out that in February 2014 the European Parliament rejected the Commission’s
proposal for a delegated act amending the definition of nanomaterials contained in Regulation
1169/2011 on the Provision of Food Information to Consumers, as its views were that this could
lead to existing nano-materials not being labelled.
In summary, very little research has been published on the topic of novel foods and no changes
to the volume of applications or Member States’ voting patterns have been reported since 2008.
Since the failure of the Conciliation procedure in March 2011, the use of Delegated Acts by the
Commission, as a legal instrument, has remained a rather sensitive issue for both the European
Parliament and the Council. This particular issue is likely to be further debated in inter-
institutional negotiations on the 2013 Novel Foods proposal.
2.6. Disrupting the Quorum Sensing (QS) pathway: A metabolic
approach
Food spoilage takes place principally due to cell-to-cell communication among the foodborne
pathogens, a phenomenon known as quorum sensing (QS) Skandamis PN, Nychas GJ. Quorum
sensing in the context of food microbiology. Several studies pondered the bacterial QS system to
be strongly linked to the biofilm formation followed by foodborne disease onset Skandamis PN,
Stopforth JD, Ashton LV, et al. Escherichia coli O157:H7 survival, biofilm formation and acid
tolerance under simulated slaughter plant moist and dry conditions. A number of QS signaling
compounds have been reported to be present in different food items in the association of the
dominance of Pseudomonas spp., enteric bacteria, and Lactic Acid Bacteria (LAB). Thus, the
information on the QS system would aid in developing approaches for disrupting such cell-to-
cell communication systems; and hence preventing the spoilage network possible through
controlling the expression of the genes encoding the virulence factors required to disseminate the
foodborne diseases within hosts as shown in FIG. 1. For cell-to-cell communication with the
concomitant virulence factor production, bacterial cells have been reported to employ several
classes of signaling molecules namely the N-acyl homoserine lactones (AHLs, also called
autoinducer-1 or AI-1, produced by Gram-negative bacteria), a furanosyl borate diester (a
universal signal for interspecies and intraspecies communications), also known as autoinducer-2
(produced by both Gram-positive and Gram-negative bacteria), the autoinducer-3 (AI-3, serving
as the QS signal for enterohemorrhagic Escherichia coli), the autoinducing peptides (AIPs),
produced by Gram-positive bacteria, and the 2-heptyl-3-hydroxy-4-quinolone (PQS) in
Pseudomonas aeruginosa. Development of the Quorum-sensing inhibitors (QSIs) in order to
target and block the synthesis of the cell signaling molecules would also be helpful to prevent the
biofilm-forming food spoilage bacteria as evident from Bacillus spp., Enterococcus spp.,
Staphylococcus spp., Streptococcus spp., Streptomyces spp., and Rhizobium spp. QSIs can be
regarded of significant values in terms of food safety through their capacity to regulate virulence
factors of pathogens with the biofilm formation traits (FIG. 1). This approach is well known to
identify the virulent microorganisms especially within the meat (isolation of Enterobacteriaceae
strains, Aeromonas hydrophila, Yersinia enterocolitica, Pseudomonas fragi, E. coli O157:H7,
Lactic acid bacteria) and vegetable products (isolation of Enterobacteriaceae strains,
Pseudomonads, and Vibrionaceae strains, Yersinia enterocolitica, E. coli O157:H7).

Figure. 1. Quorum sensing (QS) aspects in food safety management.

With the aid of different QS signals like N-acyl homoserine lactones (AHLs, also called the
autoinducer-1 or AI-1), furanosyl borate diester, the autoinducer-3 (AI-3), the Autoinducing
Peptides (AIPs), and the 2-heptyl-3-hydroxy-4-quinolone (PQS), food spoilage may take place
due to the strong cell-to-cell communication among the foodborne pathogens triggered by those
signals. A number of QS signaling compounds are actually present in different food items, and
hence the information on the QS system would help in developing approaches for disrupting the
cell-to-cell communication systems and thereby preventing the food spoilage. Such an ingenious
mechanism can be induced by the Quorum-Sensing Inhibitors (QSIs) which target and block the
synthesis of the QS signaling molecules.

2.7. Molecular Tools and Microbial Food Biotechnology: The Prime

Approach
The use of microorganisms in food industries has long been practiced because of their
fermentative metabolism, and due to their health-promoting effects; i.e., the probiotic products
Rau MH, Zeidan AA. As commonly known, the commercial starter and secondary cultures (of
bacteria, yeasts and molds, produced in the industrial-scale bioreactors) are applied in the
modern fermentation based food processing. The cultures, typically consisting of a single type of
microorganisms or a complex consortium of microorganisms are formulated (by possible
genetic- or metabolic engineering) to achieve the desired features and quality of the finished
product(s) (FIG. 2). The microbial diversity in food items has already been analyzed using High-
Throughput Sequencing (HTS) approaches and by several Next-Generation Sequencing (NGS)
technologies Ercolini D.
However, an interesting molecular approach using the genome-scale reconstruction of metabolic
networks combined with constraint-based modeling (CBM) became widely acceptable which
was basically projected through the genomics data of the organism of interest; and further
converting the network reconstruction into a mathematical model known as the genome-scale
model (GEM) as shown in FIG. 2. Such an approach can be used to assess the metabolic
potential of industrial strains used in food processing, and to understand the existing interactions
in microbial consortia and the simulating community phenotype which are actually required for
designing culture blends for food fermentations. Very recently, an approach namely the RedCom
approach, regarding the building of appropriate microbial consortium for food fermentation
technology, has been introduced where reduced community models were constructed from net
conversions of the linear single-species models.

Figure 2. Application of molecular tools and the implementation of microbial food

biotechnology for food safety

industrial food item production accompanied by the microbial culture improvement and the
bioprocess (fermentation) optimization in order to achieve safe food products. The integration of
genome-scale model (GEM, where the comprehensive information is available) and CBM
together with the omics data analysis (through the relevant databases) further ponders to the
global qualitative and quantitative microbial metabolic traits within a given environment (i.e., in
a chemostat or in an industrial fermenter) to achieve high-quality products.
Knowledge and Applications of Genomics in Food Safety: The Database Approach
As shown in FIG. 2, the whole genome sequencing (WGS) to provide detailed characterization
of foodborne pathogens (as evident from Salmonella spp., Escherichia coli, Listeria spp.,
Campylobacter spp. and Vibrio spp.), and the applications of shotgun metagenomics for their
taxonomic profiling and the possible drug-resistance traits have emerged as novel approaches in
the field of food safety. The genetic make-up of diverse pathogens causing foodborne illness will
doubt go a long way to resolve the public health issue related to food-related problems. Such a
genomic approach is expected to function as the foodborne disease outbreak detection,
identifying the reason for food contamination, accumulating all the virulence traits, etc. Thus the
new technologies and tools in genomics and metagenomics, and obviously about bioinformatics
in food microbiology would be a great impact to ensure food safety as well as maintaining public
health sustainability.

CHAPTERB THREE
3.0. Food safety management: preventive strategies and control of
pathogenic microorganisms in food
In current times, food security has emerged as one of the major global concerns, driven by the
growing awareness of the health risks associated with consuming foods contaminated by
pathogenic microorganisms Vågsholm I 2020. The presence of these microorganisms in food
poses a significant threat to public health, as it can result in foodborne illnesses with serious
consequences for individuals and communities Bhunia AK 2018. Given this scenario, the
implementation of preventive and control strategies becomes vital to ensure the quality and
safety of food products, safeguarding the health and well-being of the population.
This review article proposes a comprehensive approach to food safety management, highlighting
its significance as a preventive and control strategy against pathogenic microorganisms in food.
A profound understanding of these aspects becomes essential, as improper handling, inadequate
processing, and incorrect storage of food can facilitate the proliferation of these harmful agents.
In this context, it is crucial to adopt rigorous measures throughout the entire food chain, from
production to consumption, in order to mitigate risks and ensure food safety.
This article examines the key pathogenic microorganisms that can contaminate food, focusing on
bacteria, fungi, parasites and virus that are often associated with outbreaks of foodborne diseases
Braz J Dev. 2021. Additionally, it emphasizes the importance of utilizing advanced techniques
for microbiological identification, such as Real-Time PCR, Next-Generation Sequencing (NGS),
and MALDI-TOF mass spectrometry, to ensure early and accurate detection of these pathogens.
The environmental and intrinsic factors affecting microbial growth in food are also explored in
this study. Elements such as temperature, pH, water activity, and additives are analyzed in detail,
as they play a crucial role in inhibiting the proliferation of these pathogenic microorganisms
Peleg M. Furthermore, the article underscores the importance of applying quality management
systems, such as the Hazard Analysis and Critical Control Points (HACCP) system, as an
effective approach to identify and control risks throughout the food chain.
The collaboration among various stakeholders, including regulatory bodies and international
entities, is presented as a fundamental component in promoting global food safety. The synergy
between different sectors and shared responsibility emerge as indispensable pillars to ensure that
the food offered to consumers is safe and healthy.
Finally, consumer education is highlighted as a crucial element for the success of food safety
strategies. Consumer awareness of safe food handling and storage practices plays a vital role in
preventing foodborne illnesses and promoting healthy eating habits.
In the current context, where food safety is a complex global issue, this scientific article provides
a comprehensive and detailed analysis of food safety management as a preventive and control
strategy against pathogenic microorganisms in food. By understanding and applying the
measures and knowledge presented in this study, society will be better equipped to address the
inherent challenges of food safety, ensuring a continuous supply of safe and high-quality food
for human consumption.

3.1. Pathogenic Microorganisms

Pathogenic microorganisms, commonly referred to as pathogens, are microscopic organisms that
have the potential to cause disease in humans, animals, and plants. These microorganisms
encompass a diverse range of biological entities, including bacteria, fungi, parasites, and viruses
(Table 1). When introduced into the body or a host organism, they can disrupt normal
physiological functions, leading to a spectrum of illnesses, ranging from mild infections to
severe, life-threatening conditions.
 Bacteria
The bacteria responsible for foodborne intoxications and infections, for example, are
microorganisms that can result in serious health problems when present in foods consumed by
humans.
Table 3.1. Pathogenic microorganisms in food: examples and associated illnesses
Type of Sources of
Specific species Associated illnesses
contamination contamination
 Diarrhea, fever, stomach cramps, nausea, vomiting, chills and
headache. Rarely, infection spreads to bloodstream and other
Salmonella sp.
organs, posing life-threatening risks without prompt antibiotic
treatment.
Improper
Resemble those of Salmonella, including diarrhea, fever,
handling,
Escherichia coli stomach cramps, nausea, and vomiting. However, in some
inadequate
O157:H7 cases, E. coli infection can lead to more severe complications
Bacteria cooking,
such as kidney failure, especially in vulnerable populations
contaminated
water and Fever, muscle aches, nausea, and diarrhea are common
surfaces symptoms of Listeria infection. In severe cases, Listeria
Listeria infection can lead to neurological complications, making it a
monocytogenes serious health concern, especially for those at higher risk, such
as pregnant women, the elderly, and individuals with
weakened immune systems.
Production of aflatoxins mycotoxins, associated with a
Aspergillus
spectrum of illnesses, including acute toxicity manifested
flavus and A.
through symptoms such as vomiting and liver damage, as well
Improper parasiticus
as long-term carcinogenic effects
storage, high
Fungi humidity, Produces mycotoxins that are commonly found in corn and
inadequate cereals, such as wheat and barley, as well as in cereal-based
processing products. These mycotoxins pose potential health risks to both
Fusarium sp.
humans and animals, leading to neurological problems and
gastrointestinal issues, and causing reproductive system issues
in animals
It can lead to flu-like symptoms, including fever, swollen
Toxoplasma lymph nodes, headache, muscle aches, and skin rash. Poses a
gondii particular risk to pregnant women and individuals with
weakened immune systems
Gastrointestinal distress, including symptoms like diarrhea
Cryptosporidium
and abdominal cramps, with more severe consequences for
Inadequate parvum
individuals with compromised immune systems
cooking,
Parasites It can include muscle pain, fever, and inflammation, with
contaminated
water and food Trichinella spp. severe cases potentially leading to complications such as
myocarditis and encephalitis
After ingestion, the larvae develop into adult tapeworms in the
human intestines, which can lead to symptoms such as
Taenia solium
abdominal discomfort, weight loss, and nausea. Infections
and T. saginata
with Taenia solium can indeed result in neurocysticercosis, a
severe condition affecting the central nervous system
Viruses Poor It can lead to rapid and severe symptoms, including nausea,
Norovirus
sanitation, vomiting, diarrhea, and abdominal pain
contaminated Infection manifests as acute hepatitis, with symptoms that can
food and include jaundice, fatigue, abdominal discomfort, unusual
water, personto- tiredness and weakness, sudden nausea, vomiting, diarrhea,
person Hepatitis A virus and in some cases, dark-colored urine. Vulnerable individuals,
transmission such as older adults and those with preexisting liver
conditions, can experience more severe illnesses, such as liver
failure and other liver diseases
Infection can lead to acute hepatitis, presenting symptoms
similar to other forms of hepatitis, including jaundice and
Hepatitis E virus
malaise. Chronic infection indeed poses significant risks,
particularly for pregnant women
Rotavirus Infections can range from mild to severe, with symptoms
including severe watery diarrhea, vomiting, fever, and
abdominal pain. In vulnerable populations, such as infants,
young children, the elderly, and those with weakened immune
systems, infections can potentially lead to hospitalization due
to the risk of severe dehydration
 Infections include symptoms such as vomiting, diarrhea,
stomach cramps, chills, nausea, headache, and myalgia. They
Sapovirus
can cause more severe illness in vulnerable individuals, such
as those who are immunocompromised

These pathogenic microorganisms can proliferate in food due to improper handling, preparation,
storage, and cooking practices. Ingesting food contaminated with these bacteria can lead to
gastrointestinal illnesses, ranging in severity from mild symptoms to potentially fatal conditions.
One of the most well-known bacteria causing food poisoning is Salmonella. It is often associated
with animal-derived foods such as raw or undercooked meat, eggs, and unpasteurized dairy
products. Salmonella contamination occurs during the production, processing, or inadequate
preparation of these foods. When ingested, Salmonella can cause symptoms such as diarrhea,
nausea, vomiting, abdominal cramps, and fever. In severe cases, Salmonella infection can spread
to the bloodstream and lead to more serious complications.
Another concerning bacterium is Escherichia coli. Some strains of E. coli, such as E. coli
O157:H7, are notorious for causing severe illnesses. This bacterium is often linked to raw or
undercooked meat as well as unpasteurized dairy products. E. coli infection can result in
symptoms similar to those of Salmonella, but in some cases, it can lead to more serious
complications like kidney failure.
The Campylobacter bacterium is another common cause of foodborne infections. It is often
found in raw or undercooked poultry, such as chicken. Ingesting food contaminated with
Campylobacter can lead to gastrointestinal symptoms, including diarrhea, abdominal cramps,
and fever.
Listeria monocytogenes is a bacterium that can be found in processed foods such as soft cheeses,
deli meats, and ready-to-eat vegetables. It is particularly dangerous for vulnerable groups such as
pregnant women, the elderly, and people with weakened immune systems. Listeria infection can
lead to symptoms like fever, muscle aches, nausea, and diarrhea. In severe cases, it can cause
neurological complications.
Clostridium perfringens is widely found in soil, water, and the intestinal tracts of animals and
humans. It is often associated with ready-to-eat foods such as cooked meats, stews, and sauces.
Food poisoning caused by C. perfringens occurs when prepared foods are kept at room
temperature for an extended period, allowing the bacteria to multiply and release toxins into the
food. Symptoms include abdominal pain, diarrhea, and nausea.
Additionally, Staphylococcus aureus is a bacterium that produces toxins that cause food
poisoning. Contamination occurs due to improper handling of food, such as leaving it at room
temperature for too long. Symptoms include nausea, vomiting, and abdominal cramps. Vibrio
cholerae, the bacterium responsible for cholera, has also been considered. This infection is
primarily transmitted through the consumption of water and food contaminated with infected
human feces, posing a common concern in areas with limited access to clean water and basic
sanitation.
 Fungi
In addition to the mentioned bacteria, fungi can also cause foodborne illnesses. Fungi are
eukaryotic organisms belonging to the kingdom Fungi. They are distinct from plants, animals,
and bacteria, forming a diverse group of living beings. Fungi are characterized by their cells with
a defined nucleus, a cell wall primarily composed of chitin, and a lack of chlorophyll, the green
pigment responsible for photosynthesis in plants.
Fungi play an essential role in nature, acting as decomposers, breaking down dead organic matter
and recycling nutrients in the environment. They can also establish symbiotic associations with
other life forms, such as mycorrhizae, which benefit both fungi and plants by enhancing nutrient
absorption. Although the majority of them are beneficial, some fungi can produce toxic
substances known as mycotoxins during their growth and development in food. These
mycotoxins can be harmful to human and animal health, causing acute or chronic poisoning and
even being carcinogenic. Several mycotoxins are of particular interest in food. Among them,
aflatoxins, ochratoxin A, fumonisins, deoxynivalenol (DON) or vomitoxin, and zearalenone are
notable.
Aflatoxins are primarily produced by fungi of the genus Aspergillus, notably A. flavus and A.
parasiticus. These mycotoxins are often found in agricultural commodities such as peanuts, corn,
grains, and seeds, especially under inadequate storage conditions, high humidity, and elevated
temperatures. Aflatoxins are highly toxic and carcinogenic, posing a serious risk to human and
animal health. Ochratoxin A is another concerning mycotoxin, produced by some species of
fungi, including Aspergillus and Penicillium. It can be found in various foods like grains, coffee,
grapes, and meat products. Ochratoxin A has the ability to accumulate in the body, primarily in
the kidneys, and is known to cause renal damage, besides being classified as potentially
carcinogenic.
Fumonisins, primarily produced by fungi of the genus Fusarium, are commonly found in corn
and its derivatives. These mycotoxins have been associated with diseases in livestock such as
horses and can cause neurological problems and even death in severe cases. Although their effect
on humans is not as well established, there are concerns regarding potential health risks.
Deoxynivalenol (DON), also known as vomitoxin, is another mycotoxin produced by Fusarium
fungi, such as F. graminearum and F. culmorum. This mycotoxin is often found in cereals,
especially wheat, barley, and corn. Exposure to DON can cause gastrointestinal problems like
nausea, vomiting, and diarrhea, in both humans and animals.
Zearalenone is another mycotoxin produced by Fusarium fungi. It can be found in cereals and
cerealbased products. Zearalenone is known for its ability to mimic the estrogen hormone and
can cause reproductive system issues in animals, negatively affecting production and
reproductive health. The consequences of mycotoxin contamination in food are severe. They can
cause organ damage, such as to the liver and kidneys, and negatively impact the nervous and
immune systems. Some mycotoxins also exhibit estrogenic effects, affecting the reproductive
system of animals and potentially causing hormonal imbalances in humans.
 Parasites
Parasites present yet another class of microorganisms that demand attention. Parasites are
organisms that rely on other living hosts to sustain themselves, often at the expense of the host's
well-being. In the context of foodborne illnesses, the ingestion of contaminated food can
introduce parasites into the human body, leading to a range of health complications. Parasites
encompass a diverse range of organisms, including protozoa and helminths, more commonly
known as worms. These organisms find their way into the food chain through various avenues,
such as mishandling, improper cooking, or even contaminated water sources. Once ingested,
parasites can establish themselves within the host's gastrointestinal tract or other organs,
potentially triggering gastrointestinal, hepatic, and even neurological disorders. Several parasites
stand out due to their impact on food safety and public health. Toxoplasma gondii, a protozoan
parasite, can be transmitted through the consumption of undercooked or raw meat from infected
animals, particularly pork, lamb, and game meats. It can also spread through contact with
contaminated soil, water, and vegetables. Toxoplasma infection can lead to flu-like symptoms,
posing a particular risk to pregnant women and those with weakened immune systems.
Cryptosporidium parvum and Giardia duodenalis, both protozoan parasites, often find their way
into the human body via contaminated water sources. However, they can also hitch a ride
through contaminated food, such as fresh produce irrigated or washed with tainted water.
Ingesting food or water housing these parasites can lead to gastrointestinal distress, including
diarrhea and abdominal cramps, with more severe consequences for those with compromised
immune systems.
Trichinella spp, parasitic worms, can lurk in undercooked or raw meat from infected animals,
particularly pork and game meats. Infection occurs through the consumption of larvae, which
then mature in the host's muscles. The result can be muscle pain, fever, and inflammation, with
severe cases potentially causing myocarditis and encephalitis.
Tapeworms, such as Taenia solium and T. saginata, are also consired significant. These parasites
are contracted through the consumption of undercooked or raw beef or pork, respectively. After
ingestion, the larvae develop into adult tapeworms in the human intestines, leading to symptoms
like abdominal discomfort, weight loss, and nausea. T. solium infection can even result in
neurocysticercosis, a severe condition affecting the central nervous system.
 Viruses
Distinct from other microorganisms, viruses consist of genetic material enclosed within a protein
coat. Lacking the cellular structure of living organisms, viruses rely on host cells for replication
and survival. Viral contamination of food can occur through various routes, including poor
sanitation practices, improper handling, and contact with infected food handlers. Several
prominent foodborne viruses merit close attention due to their potential impact on public health:
Norovirus stands as a leading culprit behind foodborne illnesses. Often associated with
contaminated food, water, and person-to-person transmission, norovirus ingestion can trigger
rapid and severe symptoms, including nausea, vomiting, diarrhea, and abdominal pain. Notably,
outbreaks of norovirus can escalate quickly in crowded environments such as cruise ships and
institutional settings.
The hepatitis A virus (HAV) primarily gains entry through the consumption of contaminated
water and food, particularly shellfish and produce. HAV infection manifests as acute hepatitis,
marked by symptoms such as jaundice, fatigue, and abdominal discomfort [80]. Although most
individuals recover fully, certain populations, such as older adults and those with preexisting
liver conditions, can experience more severe illness.
Rotavirus is a leading cause of severe diarrhea and dehydration in infants and young children.
While most infections are associated with person-to-person transmission, contaminated food can
also contribute to the spread of this virus. Rotavirus symptoms can range from mild to severe,
potentially leading to hospitalization in vulnerable populations.
Hepatitis E virus (HEV) is transmitted primarily through the consumption of contaminated water
and undercooked meat, particularly pork and game meats. HEV infection can lead to acute
hepatitis, causing symptoms similar to other forms of hepatitis, such as jaundice and malaise. In
certain regions, chronic HEV infection can pose significant risks, especially for pregnant women.
Sapovirus, like norovirus, is associated with gastroenteritis and often spreads through
contaminated food and water. Symptoms of sapovirus infection include vomiting, diarrhea, and
stomach cramps. While sapovirus infections are generally self-limiting, they can cause more
severe illness in vulnerable individuals.
3.2. FOOD SAFETY MANAGEMENT
Food contamination arises from a multitude of sources, resulting in the introduction of harmful
substances or pathogens that undermine the safety and quality of food products [87-89]. A
comprehensive understanding of these sources is essential for the implementation of effective
food safety measures. Pathogenic microorganisms, encompassing bacteria, fungi, parasites, and
viruses, can infiltrate food during various stages of production, processing, handling, and storage
(Table 2). For this reason, efficient food safety management is a shared responsibility among
producers, industry, distributors, retailers, and regulatory authorities.
Table 3.2. Sources of foodborne pathogenic contamination and descriptions.
Contamination Source Description
Contamination can originate from raw materials used in food production, such as
Raw Ingredients
contaminated soil or water for crops and livestock.
Transfer of pathogens from one food to another, usually through shared utensils, cutting
Cross-Contamination
boards, or hands during food preparation.
Poor hygiene practices by food handlers, such as inadequate handwashing, can introduce
Improper Handling
pathogens into the food.
Insufficient cooking temperatures or inadequate cooking times may leave harmful
Inadequate Cooking
microorganisms alive in the food.
Equipment used in food processing, packaging, or storage can harbor pathogens if not
Contaminated Equipment
cleaned and sanitized properly.
Contaminated water used for washing produce, diluting beverages, or making ice can
Water and Ice
introduce pathogens to the food.
Inadequate temperature control during storage can lead to the growth of harmful
Poor Storage Conditions
microorganisms.
Pests Insects, rodents, and other pests can carry and transmit pathogens to food products.
Environmental Factors Airborne contamination, dust, and pollution can introduce pathogens into food during
 processing or handling.
Contaminated food preparation surfaces, storage areas, and facilities can contribute to
Unsanitary Facilities
microbial growth.
Inadequate Packaging Improperly sealed or damaged packaging can allow contaminants to enter the food.
During transportation, food can be exposed to unsanitary conditions, leading to
Improper Transportation
contamination.
Thawing food at room temperature can allow bacteria to multiply, leading to
Improper Thawing
contamination.
Inadequate Supplier
Contaminated ingredients from suppliers can introduce pathogens to the final product.
Verification

One of the key aspects to be considered is hygiene in production. In the field, it's essential to
adopt
good agricultural practices that minimize the presence of contaminants in food [90]. This
includes the responsible use of pesticides, proper pest and disease control, ensuring the personal
hygiene of workers involved in production, and protecting planting areas against potential
external contaminations. In food processing industries, it's crucial to follow good manufacturing
practices (GMP). These guidelines encompass various aspects, such as strict equipment and
facility hygiene, precise control of temperature and processing time, proper training of
employees for safe food handling, and product traceability, which enables the identification of
the origin and destination of each batch, facilitating corrective actions in case of issues. Another
crucial point is quality control carried out throughout the entire food chain. It's important to also
verify the presence of pesticide residues and other chemicals that could be harmful to health.
Additionally, microbiological testing and laboratory analyses are essential to monitor the
presence of pathogens and other contaminants in food.
3.3. Microbiological Identification Techniques
3.3.1. Collection and Sampling
The first step in this process, in this context, is the careful collection of representative samples of
the food in question. These samples can be obtained from different batches, production areas, or
storage locations to ensure that the analysis is comprehensive and reflects the potential diversity
of microorganisms present. Accuracy at this stage is crucial as the quality of the result depends
on the sample's representativeness. Once the samples are collected, they need to be prepared for
analysis. This may involve homogenizing the food and breaking up any clumps to achieve a
uniform mixture. Additionally, in some cases, the samples may need to be diluted in sterile
solutions to allow for a manageable number of microorganisms on culture plates, ensuring
reliable results.
3.3.2. Conventional Identification Methods
The subsequent step, isolation and enrichment, is crucial to promote the growth of the target
microorganisms. For this purpose, samples are incubated on specific culture media that provide
optimal conditions for the growth of particular groups of microorganisms. Upon the growth of
microorganisms on the culture plates, the preliminary identification stage begins. At this stage, it
is observed morphological characteristics, Gram staining, and biochemical tests to differentiate
the microorganisms present. These initial tests help identify general groups of microorganisms.
Furthermore, microscopy is a fundamental tool in this process. It enables direct visualization of
the microorganisms present in the samples, which can provide important clues about their
identity. By observing characteristics such as shape, size, and cellular arrangement, technicians
can gather valuable information to aid in microorganism identification. Quantification is also
important to determine the quantity of microorganisms present in the samples. This can be done
through colony counting on Petri dishes or more modern techniques like flow cytometry, which
allows rapid and accurate assessment of microorganism quantities in a sample. For more precise
identification, however, more specific tests are conducted. This can include more detailed
biochemical tests, serological tests to detect the presence of specific antigens, metabolism tests to
analyze the production of characteristic chemicals, and even antibiotic susceptibility tests to
determine the microorganism's response to different treatments. An example is the Enzyme-
Linked Immunosorbent Assay (ELISA), which is used to detect specific pathogens like
Salmonella and Listeria.
To ensure the accuracy and reliability of results, the confirmation step is crucial. This may
involve advanced molecular techniques, such as genetic sequencing, which provides detailed
information about the microorganism's DNA and allows highly specific identification.
3.3.3. Advanced Identification Methods
The research and identification of relevant microorganisms in food have significantly evolved in
recent years, thanks to the development of alternative methods that provide greater efficiency,
speed, and accuracy compared to traditional techniques. These advancements are crucial to
ensuring food safety and the quality of consumed products, as well as monitoring and controlling
outbreaks of foodborne diseases.
Polymerase Chain Reaction (PCR), for example, is a powerful molecular technique that
selectively amplifies specific DNA sequences present in a sample, making it one of the most
widely used methods in microbiology and molecular biology for the identification of
microorganisms in food. The principle of PCR is based on the use of heat-stable enzymes, such
as Taq DNA polymerase, to repeatedly amplify a specific DNA sequence in a chain reaction.
The reaction involves successive cycles of heating (DNA denaturation), cooling (primer
hybridization), and extension (new DNA chain synthesis).
Another widely employed modern approach is Real-Time PCR (qPCR). This revolutionary
technique enables the amplification of target DNA from specific microorganisms present in food,
such as pathogenic bacteria. Providing results within hours, qPCR is essential for the rapid
detection of pathogens like Salmonella, Escherichia coli, and Listeria, allowing immediate
actions to contain potential risks to public health.
Furthermore, Next-Generation Sequencing (NGS) has played a transformative role in food
microorganism research. With its ability to simultaneously sequence multiple genomes, NGS
offers a comprehensive view of the microbial composition of a food sample. This approach not
only identifies pathogenic microorganisms but also contributes to the study of normal
microbiota, enabling a deeper understanding of microbial interactions in food products.
Another promising technique is Matrix-Assisted Laser Desorption/Ionization Time-of-Flight
Mass Spectrometry (MALDI-TOF MS). This approach analyzes protein or peptide patterns
present in microorganisms and compares these patterns to a database for identification. MALDI-
TOF mass spectrometry offers an efficient and accurate way to identify microorganisms in food,
becoming a valuable tool for food analysis laboratories.
Biosensors have emerged as innovative devices for the detection of microorganisms in food.
These sensors use biological components to interact with target microorganisms, generating
detectable signals, such as color changes. This approach is especially useful for portable and
field applications, enabling rapid and convenient identification of microorganisms.
Metagenomics, in turn, has revolutionized our understanding of microbial diversity in food. This
approach directly analyzes the genetic material extracted from a sample, allowing the
identification of microorganisms without the need for prior isolation or cultivation.
Metagenomics is particularly valuable for studying complex microbial ecosystems in food,
highlighting the interconnection between different microorganisms.
Nanotechnology has also been explored to develop sensitive and specific detection methods for
microorganisms in food. In this technology, functionalized nanoparticles interact with target
microorganisms and generate detectable signals, providing an innovative and highly sensitive
approach for the identification of pathogens and contaminants.
Furthermore, the use of automation and artificial intelligence has enabled the development of
automated microorganism detection systems in food. These systems, so called Automated
Detection Systems, are capable of analyzing large volumes of data and providing accurate, real-
time results, making the identification process more efficient.

3.3.4. Prevention and Control of Microbial Proliferation

Microbial proliferation is influenced by optimal growth conditions, such as temperature,
humidity, pH, and nutrient availability in food. Contamination occurs during various stages of
the production process, including cultivation, harvesting, processing, improper storage, and
handling.
Cross-contamination is another common source of microorganism spread, where bacteria or
other pathogens are transferred from one food to another.
Prevention and control of microbial spoilage are essential to ensure food safety. Measures of
personal hygiene and sanitation are crucial to prevent food contamination by unwanted
microorganisms. This includes practices such as frequent handwashing, wearing clean and
appropriate attire, as well as maintaining facilities and equipment properly sanitized.
The personal hygiene of food handlers is one of the primary pillars in contamination prevention.
Regular and proper handwashing, the use of clean clothing, and being mindful of coughing and
sneezing near food are critical practices to avoid the transmission of unwanted microorganisms.
Correct sanitation of facilities, equipment, and utensils is also paramount. Regular cleaning and
proper sanitization ensure the elimination of potential sources of cross-contamination,
minimizing the proliferation of microorganisms in food.
Proper storage is another critical factor in preventing microbial spoilage. Keeping raw and
processed foods at appropriate temperatures, such as refrigeration and freezing, slows down the
growth of microorganisms, preventing them from reaching dangerous levels. Additionally,
controlling humidity and using suitable packaging helps prevent the entry of microorganisms.
Thermal treatment, such as cooking, pasteurization, and sterilization, is one of the key measures
to eliminate pathogenic microorganisms and reduce microbial load in processed foods, making
them safer for consumption. pH and water activity control are also important to prevent the
growth of unwanted microorganisms. Adjusting these parameters creates a less favorable
environment for the development of bacteria and fungi, extending the shelf life of foods. Adding
additives, such as preservatives and natural or artificial antioxidants, is a common strategy to
inhibit microbial growth and ensure the preservation of processed foods for a longer period.
Regularly monitoring and analyzing the presence of pathogenic microorganisms and indicators
of
spoilage is essential to identify potential issues before they become a threat to food safety.
Traceability, in this context, is also an important tool in preventing food safety-related issues.
The ability to trace the origin of foods facilitates the rapid identification and resolution of
potential contamination cases. Furthermore, education and training of staff on good practices for
food handling and storage are crucial for the correct adoption of these preventive measures.
It is important to emphasize that, during food transportation, it is essential to ensure proper
conditions to preserve their safety. Temperature and hygiene during this stage must be rigorously
controlled to prevent the growth of microorganisms and product deterioration. Adequate
packaging and protection against contamination are essential to maintain the integrity of food
throughout its journey to the consumer.
In addition to ensuring food safety at all stages of the supply chain, it is necessary to inform
consumers about the necessary precautions to maintain food safety in their homes. This includes
guidance on proper food hygiene before consumption, appropriate storage and handling to avoid
cross-contamination, and recognizing signs that the product may be compromised.

3.3.5. EFFICIENT FOOD SAFETY MANAGEMENT

To enable efficient food safety management, solid cooperation among the various stakeholders is
essential. The implementation of quality management systems, such as the Hazard Analysis and
Critical Control Points (HACCP) system, is an effective approach to identify critical control
points along the chain and systematically take preventive and corrective actions [8]. The HACCP
is a systematic approach to food safety management that aims to prevent, eliminate, or reduce
potential hazards that could pose risks to food safety (Figure 3).
 Figure 3. Principles of Hazard Analysis and Critical Control Points (HACCP).
Conduct a Hazard Analysis: Identify potential biological, chemical, and physical hazards that
may occur in the production process; Identify Critical Control Points (CCPs): Determine the
points in the process where hazards can be controlled or eliminated; Establish Critical Limits: Set
specific criteria for each CCP to ensure that hazards are effectively controlled; Monitor CCPs:
Regularly monitor and measure the CCPs to ensure they are within the established critical limits;
Establish Corrective Actions: Define steps to be taken if a deviation from critical limits occurs,
including identifying the cause and taking appropriate actions; Establish Verification Procedures:
Regularly review and verify the effectiveness of the HACCP system and its control measures;
Establish Record-Keeping and Documentation: Maintain thorough records of the HACCP plan,
monitoring activities, corrective actions, and verification procedures.
Governmental agencies play a central role in regulating and overseeing food safety. Establishing
norms and quality standards, as well as conducting regular inspections of facilities and products,
are essential to ensure compliance with requirements and safeguard public health.
For instance, the World Health Organization (WHO) assumes a pivotal role in setting global
guidelines that direct safe practices in hygiene, quality, and microbiological food safety on an
international level. Its recommendations are crucial for shaping food policies and regulations in
many countries around the world.
Furthermore, the Codex Alimentarius, a joint program of the WHO and the Food and Agriculture
Organization of the United Nations (FAO), plays a crucial role in setting international standards
and guidelines for food safety. The Codex works to harmonize regulations and ensure uniformity
of food standards across different countries, thus promoting safe and equitable international trade
of food. At the national level, regulatory agencies play a vital role in implementing and enforcing
food regulations. In the United States, the Food and Drug Administration (FDA) is a reference
point, establishing stringent regulations that cover everything from production to the marketing
of food. The FDA is known for its detailed guidelines, rigorous testing, and risk assessments
aimed at safeguarding public health and maintaining food quality standards.
In this complex network of global and national organizations, it's important to carefully balance
the established international rules with the need to adapt them to different situations in order to
ensure food safety. Collaborating effectively among countries is crucial to align how we handle
food safety, while also considering the impact of unique cultural and local factors on how we put
these rules into action. As technology keeps advancing and smart strategies driven by data
become more common, these new ideas offer a big potential to greatly improve how we manage
food safety across borders. Innovations like Internet of Things (IoT), and artificial intelligence
(AI) are increasingly being used to strengthen our ability to track where food comes from,
monitor it in real-time, and quickly catch any safety issues. Looking ahead at what might happen,
like changes in the environment due to climate change or shifts in what consumers want,
highlights even more why it's crucial to be proactive and flexible in how we make sure our food
is safe to eat.
3.4. Microbial Fermentation and Its Role in Quality Improvement of
Fermented Foods
Fermentation is a process that helps break down large organic molecules via the action of
microorganisms into simpler ones. For example, yeast enzymes convert sugars and starches into
alcohol, while proteins are converted to peptides/amino acids. The microbial or enzymatic
actions on food ingredients tend to ferment food, leading to desirable biochemical changes
responsible for the significant modification to the food. Fermentation is a natural way of
improving vitamins, essential amino acids, anti-nutrients, proteins, food appearance, flavors and
enhanced aroma. Fermentation also helps in the reduction of the energy needed for cooking as
well as making a safer product. Food Sci. Nutr. 2018, Food Sci. Hum. Well. 2019. Therefore,
microorganisms’ activity plays a significant role in the fermentation of foods by showing
changes in the foods’ chemical and physical properties. Fermented foods have several
advantages [Nutrients 2019, . Rev. Food Sci. Nutr. 2019]:
 a) Fermented foods have a longer shelf life than the original foods.
b) The enhancement of organoleptic properties; for example, cheese has more enhanced
organoleptic properties in terms of taste than its raw substrate viz. milk.
c) The removal of harmful/unwanted ingredients from raw materials—for example, during
garri preparation, there is a reduction in the poisonous cyanide content of cassava, and the
flatulence factors in soybeans are removed by fermentation.
d) The enhancement of nutritional properties due to the presence of fermenting
microorganisms. For example, yeast in bread and yeast and lactic acid bacteria in garri
add to its nutritive quality.
e) The fermentation process reduces the cooking time of food. For example, West African
food, i.e., Ogi (prepared from fermented maize), and soybean products.
The fermented products consist of higher in vitro antioxidant capacity. For example, fermented
milk and yogurt consist of higher antioxidant properties compared to milk, as there is a release of
biopeptides that follow the proteolysis of milk proteins, particularly α-casein, α-lactalbumin, and
β-lactoglobulin.
The composition of the substrates used and the fermenting microorganisms are the major factors
that influence fermented food. Moreover, food treatment and the length of fermentation during
processing also affect food fermentation [Fundamentals in Biotechnology; Eolss Publishers:
Oxford, UK, 2009]. For all the fermented foods and beverages that have been identified, lactic
acid bacteria (L.A.B.) is the dominant microbiota, which has been considered the most critical
part contributing to beneficial effects in fermented foods/beverages [A mini-review. Molecules
2017]. The fermenting microorganisms mainly involve L.A.B. like Enterococcus, Streptococcus,
Leuconostoc, Lactobacillus, and Pediococcus and yeasts and molds viz. Debaryomyces,
Kluyveromyces, Saccharomyces, Geotrichium, Mucor, Penicillium, and Rhizopus species. The
fermentative sugar pathway for Lactobacillus and yeasts is mentioned figure 4. A list of some of
the most commonly prepared fermented foods/beverages with their fermenting microorganisms
is also discussed in table 3.4. Despite adding beneficial effects during fermentation,
microorganisms in food also help prevent many harmful chemicals and microorganisms during
the fermentation process. These microorganisms are also responsible for the production of new
enzymes that assist with digestion.
 a. Lactobacillus b. Saccharomyces

Figure 4. Sugar metabolism by Lactobacillus and Saccharomyces as representatives of

L.A.B. and yeasts.
Table 3.3. Some of the most commonly prepared fermented foods/beverages with their
fermenting microorganisms.
 Fermented
Substrates Used Microorganisms Involved in Fermentation
Foods/Beverages
Dairy products Milk and milk casein Lactobacillus bulgaricus, Lactococcus lactis, L. acidophilus, L. cremoris,
Curd, Yogurt, Cheese, L. casei, L. paracasei, L. thermophilus, L. kefiri, L. caucasicus,
Yakult, Kefir Penicillium camemberti, P. roqueforti, Acetobacter lovaniensis,
Kluyveromyces lactis, Saccharomyces cerevisiae
Leuconostoc mesenteroides, Aspergillus sp., Rhizopus oligosporus,
Vegetable products R. oryzae, L. sakei, L. plantarum, Thermotoga sp., L. hokkaidonensis,
Soybean, cabbage, ginger,
L. rhamnosus, Rhodotorula rubra, Leuconostoc carnosum,
Kimchi, Tempeh, Natto, cucumber, broccoli, radish Bifidobacterium dentium, Enterococcus faecalis, Weissella confusa,
Miso, Sauerkraut Candida sake
Cereals L. pantheris, L. plantarum, Penicillium sp., S. cerevisiae, L.
Bahtura, Ambali, Chilra, Wheat, maize, sorghum, mesenteroides, E. faecalis, Trichosporon pullulans,
Dosa, Kunu-Zaki, millet, rice Pediococcus acidilactici, P. cerevisiae, Delbrueckii hansenii, Deb.
Marchu tamari
Beverages Aspergillus oryzae, Zygosaccharomyces bailii, S. cerevisiae,
Wine, Beer, Kombucha, Grapes, rice, cereals Acetobacter pasteurianus, Gluconacetobacter, Acetobacter xylinus,
Sake Komagataeibacter xylinus
Meat Products L. sakei, L. curvatus, L. plantarum, Leuconostoc carnosum,
Sucuk, Salami, Arjia, Meat Leuconostoc gelidium, B. licheniformis, E. faecalis, E. hirae, E. durans,
Jama, Nham Bacillus subtilis, L. divergens, L. carnis, E. cecorum, B. lentus

3.4.1. Enhancement of Nutritional Quality in Fermented Foods by

Microorganisms.
It has been known that fermented foods are more nutritious than their unfermented counterparts.
The increased nutritional value in fermented foods is due to the fermenting microorganisms
present in them, and the three different ways of fermentation by microorganisms are as follows:
Microorganisms are both catabolic and anabolic, break down complex compounds, and
synthesize complex vitamins and other growth factors. Indigestible substances liberate the
nutrients that are locked into plant structures as well as cells. This event occurs especially with
individual seeds and grains. In the milling process, cellulosic and hemicellulosic structures
surrounding the endosperm (viz., rich in proteins and digestible carbohydrates) have been
physically ruptured to release nutrients. Crude milling is used in less developed regions to extract
nutritional contents, but it is inadequate to release full nutritional value from the plant products.
Even after the cooking process, a few of the bounded nutrients remain inaccessible to the human
digestive system. At the same time, this issue can be resolved by certain bacteria, molds, and
yeasts that decompose or breaks the cell walls and indigestible coatings of these products both
physically and chemically.
A different mechanism to increase plant material’s nutritional properties is through enzymatic
degradation of polymers that are not digested by humans into simple sugars and their derivatives
like cellulose, hemicelluloses, and a similar form of polymers. Using microbial enzymes, the
cellulose-containing substrates in fermented foods can be improved for human consumption.
Many cereal foods are low in their nutritional content and are consumed as an essential staple
diet for poor people. However, L.A.B. and yeast fermentation were observed to enhance
nutritional content and food digestibility. The fermentation process also increases the microbial
enzyme activity as it provides an acidic environment at temperature 22–25 ◦C. The critical
function of enzymatic hydrolysis in fermented foods includes a reduction in levels of anti-
nutrients viz. tannins and phytic acid (degradation with the help of phytases), resulting in
enhanced bioavailability of simple sugars or polysaccharides (amylases), proteins (proteases),
free fatty acids (lipases), and iron.

3.4.2. Effects of Lactic Acid Fermentation on the Nutritional

Aspects of Food
The main factors contributing to food’s nutritional value include its digestibility and the number
of vital nutrients present. Both nutrients, as well as digestibility, may be improved by the process
of fermentation. During the fermentation process, the fermented microorganisms’ enzymes may
initially digest the macronutrients. The several ways by which the nutritional quality of food can
be affected by fermentation include increasing the amount and bioavailability of nutrients and
enhancing nutrient density. The latter may be achieved by synthesizing promoters for absorption,
the degradation of anti-nutritional factors, influencing the uptake of nutrients by the mucosa, and
pre-digestion of individual food components. The solubility of proteins and the availability of
some micronutrients and limiting amino acids are enhanced by the process of lactic acid
fermentation. By this process, tannins (50%), phytates, and oligosaccharides (90%) are also
reduced. There can be a direct or indirect nutritional impact of fermented foods on nutritional
diseases. The fermentation process of food has a direct curative effect. Likewise, food
fermentation contributes directly to consumers’ health by increasing the number of available
vitamins such as niacin, thiamine, folic acid, or riboflavin. It also enhances iron utilization
through the breakdown of complex substances into inorganic iron with vitamin C.
Food fermentation increases mineral and trace elements’ bioavailability by reducing non-
digestible material in plants such as glucuronic and polygalacturonic acids, cellulose, and
hemicelluloses. It also reduces serum cholesterol by inhibiting cholesterol synthesis in the liver
and dietary and endogenous cholesterol absorption in the intestine. It is robust, stable, and safe
for the product, thereby preempting diseases/infections such as diarrhea and salmonellosis.
3.5. Enrichment and Changes of Biological Components in Fermented
Foods
 Vitamins Bio-Enrichment
As a public health measure, nutrients, mainly vitamins, are fortified in some selected,
manufactured foods; for example, vitamin D is added to milk and riboflavin during bread
production, whereas ascorbic acid (vitamin C) can be fortified in fruit juices (Figure 4).
However, this fortification or enrichment process can only be used in the Western world
because of its high-cost value. Hence, most countries should use this type of food
fermentation for the biological enrichment of foods. There is a deficiency of thiamine
(Vitamin B1) caused by using highly polished white rice. This type of rice can cause beriberi,
a disease that leads to strokes and paralysis. Infants fed by the thiamine-deficient (lead to
beriberi) mothers can also suffer sudden death at three months because of heart failure.
Thiamine is synthesized by the microorganisms involved in the tape Ketan fermentation.
These microorganisms are also responsible for the restoration of the thiamine level found in
unpolished rice. Therefore, this can be of great help to rice-eating individuals.

Figure 4. Nutritional enhancement in fermented foods.

An Indonesian origin-rich dish known as “Tempe” is prepared by soaking, dehulling, and
partial cooking soya beans with the help of Rhizopus oligosporus or similar molds. This
mold forms a firm cake by knitting cotyledons into slices and being cooked. There is the
partial hydrolysis of proteins during the fermentation process; the lipids are hydrolyzed to
their constituent stachyose (a tetrasaccharide indigestible by humans), fatty acids, riboflavin
doubles, niacin increases by seven-fold, and vitamin B-12, usually absent in vegetarian
foods, is synthesized by a fermenting bacterium growing with the essential mold. The
manufacturing process of tempe reduces the cooking time and improves the digestibility and
texture of many bowls of cereal/legume mixtures. The bacterium, i.e., Klebsiella pneumoniae
(nonpathogenic strain), is responsible for producing vitamin B-12 when inoculated into
Indian idli fermentation.
Fermentation of the cactus plant (Agave) juices leads to Mexican pulque production and the
oldest alcohol-containing beverage on America’s continent. Pulque is very commonly
consumed among low-income children of Mexico because of its richness in niacin, thiamine,
pantothenic acid, riboflavin, and p-aminobenzoic acid biotin and pyridoxine. An alcoholic
beverage, e.g., Kaffir beer, has a thin gruel consistency and pleasant sour taste. Kaffir is a
beverage traditionally prepared by the people of Bantu of South Africa with 1 to 8% alcohol
content. Kaffir beverage was prepared from malted and unmalted kaffircorn (Sorghum
caffrorum). The substitution for kaffircorn can be millet or maize. This alcoholic beverage
increases riboflavin, and niacin/nicotinic acid nearly doubles, keeping the thiamin level
constant during fermentation in people consuming maize.
Palm sap is a sweet, plump, milky white suspension of bacteria and yeasts that is a colorless,
transparent liquid containing approximately 10 to 12% fermentable sugar. It is consumed in
the tropics. This type of wine consists of approximately 83 mg of ascorbic acid/L. In
fermented palm
wine, thiamine is increased from 25–150 µg/L, pyridoxine: 4–18 µg/L, and riboflavin: 35–50
µg/L. Surprisingly, there is a considerable amount of vitamin B-12 (190 to 280 µg/mL) in
palm wine. Palm toddies are the cheapest vitamin B source and play a crucial role in nutrition
economically drained in the tropics.
 Antioxidant Activity
Antioxidant activities in the fermented foods comprise the reducing power assay, 20-azino-bis
(3-ethylbenzo-thiazoline-6-sulfonic acid; A.B.T.S.) and 1,1-diphenyl-2-picryl hydrazyl
(D.P.P.H.) radical scavenging activity and total phenol content (TPC) estimation. Many Asian
soyabean fermented foods have antioxidant properties, for example, tungrymbai and bekang
(Indian fermented soybean foods), kinema (Indian and Nepal fermented soybean food), tempe
(Indonesian mold-fermented soybean food), jang and chungkokjang (Korean fermented soybean
foods), thuanao (Thailand fermented soybean food), natto (Japan fermented soybean food), and
douchi (China fermented soybean food). The antioxidant activity has also been found in yogurt
and kimchi. Abubakar et al. determined the antioxidant activity of lactic acid bacteria during
fermentation of skim milk via 1,1-diphenyl-2-picrylhydrazyl (D.P.P.H.) assay and observed free
radical scavenging activity ranging from 14.7 to 50.8% (v/v) after fermentation up to 24 to 72 h,
respectively. It has been noticed that the different species of L.A.B. used and fermentation time
affected the antioxidant activity significantly (p ≤ 0.05). The improvement in the quality of
fermented sausage in terms of antioxidant value by L. curvatus (SR6) and L. paracasei (SR10-1)
was assessed by The sausage’s antioxidant activity was significantly enhanced by the bacteria,
which leads to the overall improvement in the sensory attributes and sausage safety. The
D.P.P.H. scavenging activity and reducing power was better in the case of L. curvatus SR6 viz.
59.67% ± 6.68% and 47.31% ± 4.62%,
whereas O.H. scavenging activity and anti-lipid peroxidation capacity were better with L.
paracasei SR10-1, i.e., 285.67% ± 2.00% and 63.89% ± 0.93%, respectively.
 Peptides Production
The bioactive peptides (B.A.P.s) are produced by proteolytic microorganisms (mostly Bacillus)
during food fermentation. Peptides have some antihypertensive properties and also play a vital
functional role as antithrombic and immunomodulatory agents. Due to the lack of large-scale
production facilities and soaring costs of the enzymes for protein hydrolysis, the production of
B.A.P.s is still below the benchmark. However, microbial fermentation is a more economical and
cost-effective method for the production of B.A.P.s. The health properties associated with dairy
products are existent in B.A.P.s. To date, four different types of cell envelope protienase
(C.E.P.s) have been discovered, which are PrtB from L. delbrueckii subsp. bulgaricus, PrtP from
L. casei and L. paracasei, PrtR from L. rhamnosus, and L. plantarum, and PrtH from L.
helveticus. Generally, most of the Lactobacillus species have only one C.E.P., whereas the
presence of four different paralogs of PrtH (i.e., PrtH1, PrtH2, PrtH3, and PrtH4) has been found
out in L. helveticus, and their distribution is strain-dependent. Due to several C.E.P. paralogs and
different specificities, L. helveticus is the most proteolytic species among the Lactobacillus
genus, which is solely responsible for the generation of various varieties of B.A.P.s.
The activity related to inhibition by angiotensin-converting enzyme (A.C.E.) has also been
observed in some milk products previously fermented such as koumiss, kefir, yogurt, cheese,
fermented milk of camel, and fermented products of fish. Bioactive peptides are produced by
fermentation of soybean products, which helps prevent and treat various metabolic diseases.
 Enzymes Production through Microorganisms
Enzymes such as amylase, proteinase, mannase, catalase, cellulose, etc. are generally produced
from fermenting microorganisms, especially Bacillus, in Asian soya bean product fermentation
of foods that hydrolyze complex substances into simple biomolecules (Table 3.4). Carbohydrates
producing enzymes viz. like amyloglucosidase, α-amylase, maltase, pectinase, invertase,
cellulase, alkaline proteases, lipase, and β-galactosidase are produced from mycelia fungi such as
Amylomyces, Actinomucor, Aspergillus, Mucor, Monascus, Rhizopus and Neurospora in
fermented foods/beverages. The enzyme produced by A. oryzae in koji, i.e., Taka-amylase A
(T.A.A.), has numerous uses in industries. In the Himalayan region, stable, dry, and cake-like
amylolytic starter cultures are used to produce alcohol. These starter cultures have mixed yeast
strains such as Saccharomycopsis capsularis, S. fibuligera, and Pichia burtonii, increasing
amylase.
Table 3.4. Some essential commercial enzymes used in fermented foods/beverages.
Enzymatic
Substrates Enzymes Microbial Source
Action/Process
Protease A. niger, A. orzyae and B. Cheese production
Catalase subtilis Removing H2O2
Dairy
Lactase S. boydii and Bacillus sp. Lactose-free milk
B. subtilis
Amylase B. licheniformis and B. subtilis
Protease A. niger
Pentosanase Trichoderma sp.
Glucose oxidase P. notatum
Phytase A. niger
Pullulanase B. acidopullulyticus
Xylanase A. oryzae and B. subtilis Malting, mashing,
Lipases Aspergillus niger liquefaction, and
B-glucanase B. subtilis, A. niger and P. production of flavor
Cereals funiculosum esters
 A-acetolactate-decarboxylase B. subtilis

Amyloglucosidase A. niger and A. flavus

Cellulase T. longibrachiatum

Pectinase A. niger

Table 2. Cont.
Enzymatic
Substrates Enzymes Microbial Source
Action/Process
Glucose oxidase P. notatum Clarification of juices
Tannase A. niger Removing O2
Beverages
Hydrolysis of
esters
Papain S. aureus
Meat Protease T. longibrachiatum, A. niger, A. Tenderization of meat
oryzae and B. subtilis
The enzyme nattokinase produced by B. subtilis present in natto has been observed for its
fibrinolytic activity. Other bacterial strains isolated from fermented foods like B.
amyloliquefaciens, Vagococcus carniphilus, V. lutrae, P. acidilactici, Enterococcus faecalis, E.
faecium, and E. gallinarum also shows fibrinolytic activity. The SK1-3-7 strain of Virgibacillus
halodenitrificans isolated from fermented fish sauce also showed fibrinolytic activity.
3.5.1. Biochemical Changes during Cereal Fermentation
All over the world, cereals are the most crucial carbohydrates, dietary proteins, minerals, fibers,
and vitamins. However, the major problem that exists with cereals is their acceptance among
consumers in terms of its nutritional quality and sensorial properties of their products compared
to milk and milk products. This is due to the low content of proteins, lack of some essential
amino acids (for example, lysine), low availability of starch, presence of anti-nutrients (such as
polyphenols tannins, and phytic acid), and the coarse nature of the cereals. Several methods have
been employed to alleviate the nutritional contents of cereals. For example, improvement
through genetic modifications and supplementation of amino acids with concentrates of proteins
or alternate rich sources of proteins such as defatted/legumes oilseed meals of cereal grains. In
addition to this, various processing technologies such as sprouting, cooking, fermentation, and
milling enhance the nutritive quality of cereals, among which fermentation is the best one.
Natural fermentation of cereals generally decreases the level of carbohydrates with non-
digestible oligo and polysaccharides, improving vitamin B group availability and the synthesis of
amino acids. Natural fermentation promotes enzymatic degradation of phytates by providing
optimum pH conditions present in a complex form with polyvalent cations such as zinc, iron,
magnesium, and calcium. This decrease in phytate can increase the availability of calcium,
soluble iron, and zinc by large folds. Then, after fermentation, the effect on amino acid and
protein levels is controversial; for example, the concentration of available methionine,
tryptophan, and lysine increases in cornmeal. Likewise, fermentation of cereals such as millet,
maize, sorghum, and other grains significantly enhances the quality of protein in addition to the
levels of lysine. However, while investigating sorghum kisra bread’s nutritional content, no
increase has been observed in lysine values, but there is an increase in methionine and tyrosine.
The tryptophan content increased while the manufacturing of uji, whereas it was also measured
that there was a significant drop in lysine content. It shows that the fermentation effect on foods’
nutritional content is changeable, while the evidence for enhancement is significant.
Fermentation in food results in improvement or enhances the flavor, texture, taste, aroma, and,
most importantly, the product’s shelf life. Different volatile compounds are produced during
cereals’ fermentation, which generates a mixture of complex flavors in the products. In addition
to flavors, aroma-producing compounds like butyric acid and diacetyl acetic acid also upgrade
the appeal of fermented cereal food products.
The most important ingredient used in traditional fermented foods and beverages prepared in
most world regions is cereals (such as wheat, rice, wheat, sorghum, or corn). Some of these are
used as significant foods in the human diet, whereas others are utilized as spices, colorants,
breakfast, and beverages. In maximum cereal-based fermented products, fermentation is done
with natural or mixed cultures such as bacteria, fungi, and yeasts. During fermentation, these
microorganisms act parallel or in a sequential manner, changing dominant microbiota. Species of
Bacillus, Lactobacillus, Micrococcus, Streptococcus, Pediococcus, and Leuconostoc are the
common fermenting bacteria. Moreover, Cladosporium, Aspergillus, Trichothecium,
Paecilomyces, Fusarium, and Penicillium are the common fungal genera found in fermented
products. The predominant yeast species is Saccharomyces, used in the fermentation of alcohols.
These factors are responsible for developing fermenting microorganisms in fermented
foods/beverages pH, water activity, concentration of salt, temperature, and food matrix
composition. The most common microbes mediate food fermentation through L.A.B. This type
of lactic acid fermentation provides enhanced nutritional value, safety, a longer shelf life of the
fermented food products, and wide acceptability. During natural fermentation in cereals, when
cereals are cleaned and soaked in water for a few days, a succession of natural microbiota takes
place, which is dominated by a large number of L.A.B.s. In this fermentation type, amylases
produce sugars fermented by lactic acid bacteria as a source of energy. Apart from fermentation,
other steps like reducing size, salting, or heating also contribute to the final products. Aguirre
and Collins described L.A.B. as a broad group of catalase-negative, Gram-positive, non-motile,
and non-sporing cocci and rods. They utilize fermentative carbohydrates to form lactic acid.
Based on hexose sugars’ utilization, the pathways are divided into two groups, i.e.,
homofermentative and heterofermentative. In homofermentative pathways, the sole end or
primary product of glucose fermentation by some lactic acid bacteria such as Streptococcus,
Pediococcus, Lactococcus, and some lactobacilli is lactic acid. In contrast, in heterofermentative
pathways, microorganisms such as Leuconostoc, Weisella, and some lactobacilli, the end
products are ethanol, lactate, and CO2. Lactic acid fermentation technology has also been
confirmed for its preservative role in some cereals. L.A.B.-mediated antibiosis was characterized
by the formation of hydrogen peroxide, antibiotics, and organic acids.
The production of organic acids by L.A.B. creates stressful conditions for spoilage-causing
microorganisms present in cereals by reducing the pH to below 4.0. The acid action on the
bacterial cytoplasmic membrane results in an antimicrobial effect, as it hinders the maintenance
of the membrane potential, affecting active transport. Besides organic acid production, L.A.B.
also produces hydrogen peroxide with flavin nucleotides, which rapidly react with oxygen and
oxidize reduced nicotinamide adenine dinucleotide (NADH). It can also accumulate catalase
enzyme (as true catalase is not present in the L.A.B. to degrade hydrogen peroxide) and inhibit
several microorganisms. In high tannin-containing cereals, lactic acid fermentation can also
reduce tannin levels in some cereal crops to enhance iron absorption. Fermentation through
L.A.B. also contributes to viricidal activity and antitumor properties.
Most of the legume products produced or consumed in Asia and Africa were fortified with
cereals for improving the overall quality of proteins in the fermented product because legumes
can provide a rich amount of lysine but are deficient in sulfur-containing amino acids. However,
cereals are rich in methionine and cysteine but are deficient in lysine.
3.6. Nutritional Value of Fermented Dairy Products
The values of fermented dairy products viz. sour milk, yogurt, dahi, kumiss, acidophilus milk,
and other similar milk products are much more in demand due to their enhanced nutritional
content compared to simple milk. Proteins, vitamins, carbohydrates, and some fat quality and
quantity change in fermented milk, whereas the composition of minerals remains the same
(Figure 5). The quality of sour milk is determined by the fermenting microorganisms and the
substances formed during milk souring’s biochemical reactions. The substances include alcohol,
lactic acid, antibiotics, carbon dioxide, and vitamins.

Figure 5. Changes in fermented dairy products during fermentation.

The biochemical processes mentioned below are beneficial for increasing the nutritional value of
fermented milk:
a) Milk proteolysis
Proteolysis in milk causes the breakdown of proteins into peptides, amino acids, and peptones,
which offer an increased amount of amino acids like methionine, leucine, phenylalanine
isoleucine, tyrosine, tryptophan, valine, and threonine, which are essential A.A.s providing
particular advantages, especially to physically weak persons. Proteolysis takes place with the
help of Exo and endo peptides of L.A.B. The amount of protein increases from 85.4 to 90% in
fermented milk such as dahi, yogurt, kefir, etc., and has higher digestibility for proteins due to
the precipitation of milk by lactic acid into fine curd, leading to increased nutritional value. The
amounts of free amino acids, especially proline, phenylalanine, lysine, isoleucine, cysteine, and
arginine, increase during fermentation and storage of fermented milk products. Due to
proteolysis in milk and biochemical changes in milk protein, milk products become very dietetic.
 b) Lactose hydrolysis
Lactose hydrolysis in milk is carried out by some bacteria present in the milk. The hydrolysis of
lactose produces 0.6–0.8% glucose, 16–20% galactose, and 45–50% lactose compared to an
average 5% lactose in milk. L.A.B. cause the hydrolysis of lactose through the production of β-
galactosidase. Hydrolysis of lactose is vital for lactic acid production, which lowers the bowel’s
pH, inhibiting the putrefaction microorganisms from growing. Also, lactic acid is necessary for
the absorption of calcium and organoleptic properties.
c) Lipolysis
The process of homogenization decreases the size of the fat globules, making them digestible.
Lipolysis results in physiological effects due to the increase in free fatty acid content by lactic
acid bacteria.
d) Vitamins changes
The vitamin content in fermented milk depends on the bacterial culture present in it. Most
vitamin B groups, especially riboflavin, thiamin, and nicotinamide, are increased two-fold,
whereas vitamins B1, B2, and ascorbic acid decrease approximately by half via utilization by the
growing bacteria in milk.
e) Antibacterial activity
The bactericidal action in fermented milk depends on the growing bacteria’s antibiotic activity,
such as lactobacilli in yogurt and other compounds producing antibacterial properties like
hydrogen peroxide, lactic acid, bacteriocins, and antibiotics.
f) Mineral changes
There is increased and higher bioavailability of minerals in fermented milk, especially in
calcium, potassium, zinc, magnesium, potassium iodide, and phosphorus caused by lactic acid
bacteria during and after fermentation due to the process of fermentation and acidity.
3.7. Biochemical Changes in Meat-Based Fermented Food Products
Lactobacillus, Streptococcus, Pediococcus, Leuconostoc, Lactococcus, and Enterococcus are
some of the facultative/obligate anaerobes belonging to gram-positive and acidogenic (lactic
acid) bacteria that are used to metabolize saccharides with different degrees of efficiency into
alcohols, lactic acid, lipids, amino acids, and aliphatic compounds (Figure 6).
These organisms perform three functions simultaneously in fermented sausages, i.e., producing
nitric oxide by reducing nitrate and nitrite, responsible for the cured color when combined with
myglobin, and reducing pH by producing DL-lactic acid from glucose through anaerobic
glycolysis.
Sausage incubation at optimum temperature with facultative anaerobic conditions causes rapid
growth of the L.A.B. converting simple sugars into lactic acid and reducing the pH. A
postmortem range of 4.5–7 µmol/g is not sufficient to lower down the pH; thus, simple sugars
are added as a substrate for L.A.B., bringing pH to 4.6–5. Reference used 0.62 g glucose/kg of
meat to reduce the pH by 0.1.

Figure 5. Quality changes in fermented meat products.

The primary mechanism for lactic acid metabolism includes fermentation of carbohydrates
coupled with required degrees of phosphorylation. The pathway of homofermentative lactic acid
fermentation is through the Embden-Meyerh of Parnas Pathway with lactic acid as an end
product that leads to lactic acid as the sole end product, producing a sharp, tangy taste.
Heterofermentative lactic acid bacteria also produce lactic acid through the phosphoketolase/6-
phosphogluconate pathway, but it also liberates a small amount of acetic acid (approximately
10%), which is one of the causes of an off taste from hexoses. The essential factors in lactic acid
fermentation are time, temperature, and relative humidity; for example, higher water activity
(aw) and temperature aids in faster growth of L.A.B. and reducing the pH level. Smoke can
contribute thousands of aroma and antimicrobial compounds such as organic acids (e.g., acetic,
formic, butyric, isobutyric, and propionic acids), carbonyls, and phenols (antioxidants),
contributing to the coagulation of surface proteins and inhibition of microorganisms. The pH
change is determined by the conversion of ammonia and lactate into lactic acid. This formation is
done by adding carbohydrates produced from glycerol fermentation by bacteria and ammonia
generated from amino acid fermentation. Acetic acid is also formed in this process of
fermentation. Also, oxygen utilized through the metabolism process and other conditions
influence the type of microorganisms and their metabolism, which affects the number of
fermented carbohydrates and the production of lactose.

CHAPTER FOUR
CONCLUSION
Advancement in food safety research and studies around the world is really escalating the in-
depth knowledge of the foodborne pathogens together with the concomitant disease
complications. The fine-tuned application of genetic engineering in the food microbial
biotechnology, sensitive procedures like the whole genome sequencing or the next-generation
sequencing, constructing the phylogeny of the foodborne pathogens, creating the databases
related to foodborne microorganisms and the associated hazards through the application of
“Omics” concept (i.e., the genomics and bioinformatic analyses), precise detection of the
foodborne pathogens using nanotechnology; and predicting the possible foodborne disease have
emerged in many developed countries to maintain the food safety along with the sustainable
consumer safety as well. In perspective of Bangladesh, such high technological throughput along
with the food industries and the food research laboratories would be quite challenging because of
some limitations like the required expertise to handle these tools as well as the financial deficit
for capacity building to conduct such types of advanced studies. Yet the concurrent knowledge
on the ongoing approaches to maintain food safety would increment our researchers and
professionals to design their experimental strategies regarding food safety in the future.
The significance of food safety management, as demonstrated in this article, is paramount in the
prevention and control of pathogenic microorganisms within the food supply chain, thereby
ensuring public health. Research highlights several key aspects contributing to a more resilient
food chain:
• Food safety management is crucial for preventing and controlling pathogenic
microorganisms in food.
 • Collaboration across the food chain, from production to consumption, is essential for
effective management strategies.
• Factors like temperature, pH, and water activity, alongside advanced identification
techniques, play key roles in microbial control.
• Consumer education is essential in promoting safe food handling practices, thereby
reducing foodborne illnesses and reinforcing confidence in the food system.

REFERENCES
Angelotti, R., Foter, M.J., Lewis, K.H., 1961. Time-temperature effects on Salmonellae and
Staphylococci in foods. III. Thermal death time studies. Applied Microbiology 9, 308–315.
Ammor MS, Michaelidis C, Nychas GJ. Insights into the role of quorum sensing in food
spoilage.
J Food Prot. 2008;71:1510-25.
Baird-Parker, A.C., Boothroyd, M., Jones, E., 1970. The effect of water activity on the heat
resistance of heat sensitive and heat resistant strains of Salmonellae. Journal Applied
Bacteriology 33, 515–522.
Ball, C.O., 1923. Thermal process time for canned food. Part I, No. 37. Bulletin of the National
Research Council 7.
Ball, C.O., 1927. Theory and practice in processing. The Canner 64 (5), 27. (Cited by: Stumbo.
C.R., Longley, R.E., 1966. New parameters for process calculation. Food
Technology 20 (3), 341–345.)
Bartram, M.T., Black, L.A., 1936. Detection and significance of the coliform group in milk.
Journal of Food Science 1, 551–563.
Bergdoll, M.S., 1956. The chemistry of staphylococcal enterotoxin. Annuals New
York Academy of Science 65, 139–143.
Blankenship, L.C., June 1978. Survival of a Salmonella typhimurium experimental
contaminant during cooking of beef roasts. Applied and Environmental Microbiology 35 (6),
1160–1165.
Blankenship, L.C., Davis, C.E., Magner, G.J., 1980. Cooking methods for elimination of
Salmonella typhimurium experimental surface contaminant from rare dry-roasted beef roasts.
Journal of Food Science 45, 270–273.
Bradbury, M., Greenfield, P., Midgley, D., Li, D., Tran-Dinh, N., Vriesekoop, F., Brown, J.L.,
2012. Draft genome sequence of Clostridium sporogenes PA 3679, the common nontoxigenic
surrogate for proteolytic Clostridium botulinum. Journal Bacteriology 194, 1631–1632.
Busta, F.F., Jezeski, J.J., 1963. Effect of sodium chloride concentration in an agar medium on
growth of heat-shocked Staphylococcus aureus. Applied Microbiology 11, 404–407.
Busta, F.F., Suslow, T.V., Parish, M.E., Beuchat, L.R., Farber, J.N., Garrett, E.H., Harris, L.J.,
2003. The use of indicators and surrogate microorganisms for the evaluation of pathogens in
fresh and fresh-cut produce. Comprehensive Reviews in Food Science and Food Safety 2
(Suppl. 1), 79–185.
Calhoun, C.L., Frazier, W.C., Wisconsin, U., 1966. Effect of available water on thermal
resistance
or three nonsporeforming species of bacteria. Applied Microbiology 14, 416–420.
Calicioglu, M., Faith, N.G., Buege, D.R., Luchansky, J.B., 1997. Viability of Escherichia coli
0157:H7 in fermented semidry low-temperature-cooked beef summer sausage. Journal of
Food Protection 60 (10), 1158–1162.
Calicioglu, M., Faith, N.G., Buege, D.R., Luchansky, J.B., 2002. Viability of Escherichia coli
O157:H7 during manufacturing and storage of a fermented, semidry Soudjouk-style sausage.
Journal of Food Protection 65, 1541–1544.
Eleftheriadou M, Pyrgiotakis G, Demokritou P. Nanotechnology to the rescue: using nano-
enabled
approaches in microbiological food safety and quality. Curr Opin Biotechnol. 2017;44:87-93.
Guerra MM, de Almeida AM, Willingham AL. An overview of food safety and bacterial
foodborne zoonoses in food production animals in the Caribbean region. Trop Anim Health
Prod. 2015;48:1095-108.
Noor R. Insight to foodborne diseases. Proposed models for infections and intoxications.
Biomed Biotechnol Res J. 2019;3:135-9.
Newell DG, Koopmans M, Verhoef L, et al. Food-borne diseases-the challenges of 20 years ago
still persist while new ones continue to emerge. Int J Food Microbiol. 2010;139:S3-15.
Podbielski A, Kreikemeyer B. Cell density-dependent regulation: basic principles and effects on
the virulence of Gram-positive cocci. Int J Infect Dis. 2004;8:81-95.
Shen C, Luo Y, Nou X, et al. Dynamic effects of free chlorine concentration, organic load, and
exposure time on the inactivation of Salmonella, Escherichia coli O157:H7, and non-O157 Shiga
toxin-producing E. coli. J Food Prot. 2013;76:386-93.
Skandamis PN, Nychas GJ. Quorum sensing in the context of food microbiology. Appl Environ
Microbiol. 2012;78:5473-82.
Skandamis PN, Stopforth JD, Ashton LV, et al. Escherichia coli O157:H7 survival, biofilm
formation and acid tolerance under simulated slaughter plant moist and dry conditions. Food
Microbiol. 2009;26:112-9.
Soni KA, Jesudhasan P, Cepeda M, et al. Identification of ground beef derived fatty acid
inhibitors
of autoinducer-2 based cell signaling. J Food Prot. 2008;71:134-8.
Smith D, Wang JH, Swatton JE, et al. Variations on a theme: diverse N-acyl homoserine lactone-
mediated quorum sensing mechanisms in gram-negative bacteria. Sci Prog. 2006;89:167-211.
Wade DS, Calfee MW, Rocha ER, et al. Regulation of Pseudomonas quinolone signal
synthesis in Pseudomonas aeruginosa. J Bacteriol. 2005;187:4372-80.
Rau MH, Zeidan AA. Constraint-based modeling in microbial food biotechnology. Biochem
Soc Trans. 2018;46:249-60.
Ercolini D. High-throughput sequencing and metagenomics: moving forward in the culture
independent analysis of food microbial ecology. Appl Environ Microbiol. 2013;79:3148-
55.
Thiele I, Palsson BØ. A protocol for generating a high-quality genome-scale metabolic
reconstruction. Nat Protoc. 2010;5:93-121.
Koch S, Kohrs F, Lahmann P, et al. A strategy for reduced metabolic modeling of complex
microbial communities and its application for analyzing experimental datasets from
anaerobic digestion. PLoS Comput Biol.
2019;15:e1006759.