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Sequencing, Bioinformatics Tools, and Databases in Food Microbiology

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CHAPTER 13

Sequencing, Bioinformatics Tools, and

Databases in Food Microbiology
SUSHMA PRADEEP, ANISHA S. JAIN, CHANDAN DHARMASHEKARA,
ASHWINI PRASAD, KOLLUR SHIVA PRASAD,
CHANDRASHEKAR SRINIVASA, and CHANDAN SHIVAMALLU

ABSTRACT

Food is an essential component of our everyday lives. Before reaching the

market, many food products are processed in some way, ranging from fermen-
tation to packaging. Microorganisms play an important role in all of these
processes, whether they are converting the food or spoiling or contaminating
it. Bioinformatics is becoming more important in predicting and evaluating
the desirable and unwanted effects of microorganisms on food. It combined
with laboratory confirmation of selected findings is especially effective.
This chapter discusses about how bioinformatics plays an important role in
predicting and assessing the desired and undesired effects of microorganisms
on food, genomics, and proteomics study to meet the requirements of food
production, food processing, improving the quality and nutritive value of
food sources and many others.

13.1 INTRODUCTION

Food consists of ample microbial populations composed of fungi, bacteria,

and viruses. It might even contain detrimental microorganisms, which can
reduce the quality of foods (spoilage microorganisms) or adversely affect
the food safety (Mayo et al., 2019). Thus, food microbiology involves the
study of microbes that are present or involved in the food, which may have
Food Microbial and Molecular Biology: From Fundamentals to Applications. Saher Islam, Devarajan Thangadurai,
Jeyabalan Sangeetha, Zaira Zaman Chowdhury (Eds.)
© 2023 Apple Academic Press, Inc. Co-published with CRC Press (Taylor & Francis)
414 Food Microbial and Molecular Biology

both desirable and undesirable effects on the safety and quality of the food
production (Mari et al., 2006).
Culturing method is the most common or the outmoded style of deter-
mining the composition of food-related microbiota, which relay on the culti-
vation and isolation of the microorganisms before typing and sequencing,
but 98% of the environmental bacteria cannot be cultured under the routine
laboratory techniques. Nevertheless, culturing technique is not always used
for the overall microbial characterization of ecosystems, including foods
(Liu et al., 2008). The isolation and cultivation of particular microorganisms
require unknown growth factor and condition.
Foods may be stored or kept under harsh conditions, or it may have a low
pH level, etc., due to which the microbes leave in a physiologically viable
state than in cultivable state (Abee et al., 2004). Over the decades to over-
come these problems, many culture-independent, molecular approaches have
been identified; most of which are extensively used in food systems. These
techniques help us in identifying, quantifying the food linked microbes, and
offer sensitive and quick methods to determine the diversity and composition
of microbial populations (Lemay et al., 2007). Culture independent methods
are usually based on the microbial genetic material sequence (DNA and/or
RNA) analysis (Figure 13.1).

FIGURE 13.1 The sequential aspects involved in the food microbiology.

Sequencing, Bioinformatics Tools, and Databases 415

13.2 PCR GENE AMPLIFICATION TECHNIQUE

Most of which rely on the amplification of the DNA or RNA using the
polymerase chain reaction (PCR) technique (Liu et al., 2008). These nucleic
acids amplifying techniques majorly target the rRNA or rDNA genes. By
matching or comparing the sequences obtained to that held in databases,
establishes the phylogenetic relationships between the microbes (Mari et al.,
2006) (Figure 13.2).

FIGURE 13.2 Flow chart representing one complete cycle of PCR.

The PCR-based techniques such as DGGE (denaturing gradient gel

electrophoresis), TTGE (temporal temperature gradient gel electrophoresis),
qPCR (real-time quantitative PCR), TRFLP (terminal restriction fragment
length polymorphism), as well as single-stranded conformation polymorphism
(SSCP) are available to date for analyzing the quality of food (Kumar et
al., 2017). Out of which, qPCR (quantitative analysis), DGGE (qualitative/
416 Food Microbial and Molecular Biology

semi-quantitative analysis) and have been extensively used in microbial

characterize food backgrounds and in the field of food microbiology.

13.3 NEXT GENERATION SEQUENCING (NGS) TECHNIQUE

The characterization, identification, and typing of the microbial species

or mixed microbial communities can be performed using whole-genome
sequencing (WGS) with the high level of precision (Gomes et al., 2015).
The relevant knowledge about the entire microbial genome can be extracted
using microbial WGS followed by determining the single nucleotide poly-
morphisms (SNPs) provided with the highest resolution. NGS is the other
technique that comes under the culture-independent method, the amount
of nucleic acid sequence analysis using NGS method is extremely higher,
which allows to have a deep description about the microbial constituents
in the ecosystems. Alongside, NGS has aided the development of new
genome-aided methods for linking genotype and phenotype (Kumar et
al., 2017). NGS involves many high throughput “omics” techniques such
as glycomics, proteomics, metatranscriptomics, genomics, metagenomics,
and transcriptomics, etc., that are the new and high-throughput techniques
used for sequencing. This technique has two different ways of analyzing the
nucleic acid sequences, shotgun sequencing technique which considers the
total nucleic acids of the microbe and targeted sequencing technique that
does gene-specific sequencing. All of these techniques involved in NGS
generate a large, genome-scale datasets that needs to be further analyzed
(Abee et al., 2004) (Figure 13.3).
In a very short time frame, the NGS technique is proficient in processing
millions of sequences without lowering its accuracy level; instead, this
technique relies on automation and high throughput technologies. To do this,
it might require a very few instruments runs to undergo a sequencing experi-
ment (Desiere et al., 2001).

13.4 NGS INVOLVED IN FOOD MICROBIOLOGY

NGS in food microbiology is not only used to predict the prevalence of

microbes in food production but has been widely used even to interpret the
molecular level organization of these microbes that respond to different
food-related conditions, which further gives the knowledge as to control or
prevent the undesirable microorganisms (Gomes et al., 2015). Testing this
Sequencing, Bioinformatics Tools, and Databases 417

technique in various platforms of food microbiology has been devoted a lot

of work, usually used for the descriptive studies.

FIGURE 13.3 Next-generation sequencing workflow.

The data’s obtained from the omic techniques would be useful to correlate
between microbial diversity and food evolution that leads to the contribution
of quality of the food products. New microbial genes and their species have
been linked with some food processes. Based on this knowledge, during food
production, the identification of the critical microbial variables would be
easier, and hence control food safety and quality (FSQ). In this regard, NGS
technique might help in gathering the knowledge about the relationships
among the microbial safety and diversity properties (Abee et al., 2004). The
safety and quality issues of the food can be directly related to the presence of
unexpected microbe which is referred as contaminants (Figure 13.4).
Scientific hypothetical testing using NGS have been initiated, such as
exploring the challenges, study of the deep interactions occurring between
the microbial species and their strains, essential in the production of aroma
and taste components, developing the metabolic models. When we consider
foods, it is important to differentiate between inactive, active, and viable cells.
NGS techniques focus on the active microbial groups by producing the cDNA
obtained through the reverse transcription from RNA (Brul et al., 2006).

13.5 FUTURE PROSPECTS OF NGS IN FOOD MICROBIOLOGY

Next-generation sequencing (NGS) techniques will eventually reform food

microbiology. Detecting the pathogen, profiling the microbial populations,
418 Food Microbial and Molecular Biology

data mining, phenotype, and genotype linking, determining the pathogens

over food production and shelf life of the food products are the realm of
this NGS techniques (high-throughput techniques) (Carrasco et al., 2012).
Even the NGS technique must overcome some drawbacks and limitations
that need to be addressed to improve the present understanding of data in
food physiology and microbial diversity in the future.

FIGURE 13.4 Applications of next-generation sequencing (NGS) in food microbiology.

13.6 WHAT IS BIOINFORMATICS?

Bioinformatics is a branch of science with the integration of both informa-

tion technology and computer science into the field of Biology (Desiere et
al., 2001). This field utilizes the resources of computer science to mainly
deal with the biological data related to the nucleic acid sequences (DNA,
RNA, and protein). This field of science actually develops software, tools,
and algorithms that can record and analyze the data related to biology (Mayo
et al., 2019). The biological data will always be in the raw format, and thus
this sort of raw data requires a certain type of storage house in which the data
can be organized, stored, and manipulated. These databases and software
Sequencing, Bioinformatics Tools, and Databases 419

provide the users an opportunity to easily extract the data from the databases
and can be further used for any experiment (Figure 13.5).

FIGURE 13.5 The involvement of various fields in bioinformatics.

13.6.1 APPLICATION OF BIOINFORMATICS

To solve the complex biological problems, the bioinformatics integrates several

other fields into it, such as statistics, information science and technology,
mathematics, and computer science (Bukin et al., 2019). Bioinformatics tools
and software can be applied to various research areas like health and drug
discovery, prediction of protein structure, comparative genomics, sequence
analysis, genome annotation, and much more (Figure 13.6).

13.6.2 BIOINFORMATICS INVOLVED IN FOOD SCIENCE

Food sciences is described to be the combination of biological, physical, and

engineering studies to know the nature of the foods involved during the food
processing and for food quality improvement (Carrasco et al., 2012). Compara-
tive genomic sequencing techniques of bioinformatics have been used in
420 Food Microbial and Molecular Biology

food-related pathogens and their production (Alkema et al., 2016). Using the
computational algorithms and bioinformatics software the molecular modeling
and dynamic simulation can be possibly performed to the taste receptors and
also such simulations will possibly help to develop more efficient tasting
components as additives for the food.

FIGURE 13.6 Applied bioinformatics in food microbiology.

13.6.3 MICROBIAL INFORMATICS

Bioinformatics plays a major role in determining desirable and undesirable

effects of microbes on food. Food nutrition has growing importance in
the growth of various chronic diseases; a lot of efforts have been taken to
increase the nutritional composition and food quality (Alkema et al., 2015).
The omic techniques have been enumerated in the area of food sciences.
The advancement of bioinformatics in food microbiology is providing
several ways for the researchers to accept new area or discipline. Microbial
bioinformatics is used to optimize the biomass yield to improve the food
safety, quality, and production (Lemay et al., 2007). To do this, sequencing
has been carried at the genome level of the microorganism and metabolic
models are designed for many microbes including food-associated microor-
ganisms (Bukin et al., 2019).
The bioinformatics tools, comparative metabolomics and software can
be applied to understand the genetic constitution or the organization of the
Sequencing, Bioinformatics Tools, and Databases 421

pathogenic microbes. This genomic information can be further sequenced to

know the protein function, gene elucidation and it also enriches the knowl-
edge about the microbe and its disease mechanism. Microbial genomic infor-
mation thus obtained forms the basis to study the microbe related protein
and genes and synthesized toxins. Thus, bioinformatics approaches quicken
and facilitate the analysis of cellular processes in order to control or treat the
microbes (Brul et al., 2006).

13.6.4 BIOINFORMATICS INVOLVED IN SECURING THE SAFETY OF

FOOD

Bioinformatics databases, tools, and software are playing a major role

in securing the quality of food taste flavor and also maintaining food
safety. The molecular evolution of the food taste receptors can also be
tested using the bioinformatics tools (Chibuike et al., 2014). Many other
studies have been carried out to focus primarily on the food taste receptors
that links between the receptors that taste bitter and glucose regulation.
Recently the chemical properties of many compounds associated with the
food flavor and taste were included in the electronic database (Lemay
et al., 2007). Because of the importance of bioinformatics in the field of
food microbiology, various projects related to the genomic sequencing
are concentrating on the foodborne pathogens, due to which now
bioinformatics has started an innovative way to determine the cause of the
foodborne illness (Bukin et al., 2019).

13.7 FOOD SCIENCE DATABASES

13.7.1 FOODOMICS DATABASE

It is the database that involves molecular profiles of the food. Twenty-eight

standard references of USDA National Nutrient databases were released
that contain the data for about 150 food-related molecules. The concurrent
nutrient knowledge of less than 15 nutrients is provided by the nutrition
facts labels (NFLs) (Mayo et al., 2019). The Foodomic profiles help us in
implementing the precise medicine for every person; this can be done only
when there is integration between NFLs and SR28. This is the precise way to
give food molecules in the right amount at the right time to the right person
(Gomes et al., 2015).
422 Food Microbial and Molecular Biology

13.7.2 FOODWIKI DATABASE

It is the source of nutritive value and food information (Kumar et al., 2017).
The immense amount of information or the data stored in this database
possibly utilizes the bioinformatics strategies and protocols. Such source
of information about the food and nutrition helps in enhancing the nutritive
value, safety, and quality of the food (Mayo et al., 2019). This database
plays a crucial role in the field of food sciences, which in turn leads to the
progression of bioinformatics in the area of food microbiology.

13.7.3 EUROFIR-BASIS

This database mainly contains the information about the plant-based food
compounds and uniquely pools biological activity and composition of the
food. It comprises of 330 main edible parts of the food plants and various
compound classes (Mochida et al., 2010). This database plays a very major
role in providing a valuable source of information in the field of development
of products in the food industry and diet and health-related research activities.

13.7.4 FOODB

This database is the major resource that provides the knowledge about the
biology, chemistry related to the food constituents (Chibuike et al., 2014). It
contains the information about macro and micronutrients of the compounds
involved in food taste, color, aroma, texture, and flavor (Desiere et al., 2001).
Every chemical entry of this database consists of more than 100 different data
sets associated with precise physiological, compositional, and biochemical
information (Mochida et al., 2010).

13.8 FOOD BIOINFORMATICS CONCEPTS AND TECHNIQUES

13.8.1 GENOME SEQUENCING

When considered a microbe of interest its complete genome sequence

can be determined (Solieri et al., 2013). The high-quality and high-
throughput sequencing data can be obtained by using NGS technique
(Mochida et al., 2010).
Sequencing, Bioinformatics Tools, and Databases 423

13.8.2 COMPARATIVE GENOMICS

As the name indicates, the genome content or the sequences of various organ-
isms are compared (Solieri et al., 2013). This genomic content must include
nucleic acid sequences (DNA or RNA), gene order, genes, and regulatory
sequences.

13.8.3 METABOLIC MODELING

By using the genome sequencing method, the metabolic pathways requirement

as well as the growth prediction of the microbes can be carried out (Mochida
et al., 2010). The construction of metabolic models in Genome-scale is done
by using comparative or automated genomics (Solieri et al., 2013). With
the help of these constructed metabolic models the growth simulation can
be carried out. The bioinformatics tools used for the modeling are Vanted
(https://omicstools.com/vanted-tool), PySces (https://omictools.com/pysces-
tool) and Seed (https://pubseed.theseed.org/).

13.8.4 MICROBIOME ANALYSIS

The overall microbe content in a specific niche are said to be microbiome. This
microbiome content is usually analyzed using sequence-based techniques like
next-generation sequencing (Solieri et al., 2013).

13.8.5 16S rRNA SEQUENCING

Sequence generation from the 16S gene is termed as 16S rRNA sequencing
(Ronholm et al., 2018). This technique is well established and cheap when
compared to other sequencing techniques and plays a very major role in
the characterization of compound cultures which has a limited prior data is
available.

13.8.6 FUNCTIONAL PREDICTION

The result of the data generated from the 16S rRNA sequencing can be further
used to determine the gene functions of a particular taxa using bioinformatics
424 Food Microbial and Molecular Biology

tool like PICRUSt (https://omictools.com/picrust-tool). This tool utilizes the

already known data of the taxa where the genomes are already sequenced
and generates the gene functions (Ronholm et al., 2018).

13.8.7 SHOTGUN METAGENOMICS AND META-TRANSCRIPTOMICS

These techniques are very powerful as the circumventing of culturing

microbes is allowed while still determining their gene expression and gene
content (Yi et al., 2015). Due to which it provides the knowledge about
the functions of the encoded DNA at the molecular level and also for the
expressed mRNAs.

13.8.8 ASSEMBLY

The sequence derived from the DNA/RNA can be arranged into large contigs,
for these obtained large fragments the functional annotations can be effec-
tively carried out.

13.8.9 ANNOTATION

The metagenome sequences can be taxonomically and functionally annotated

by predicted or compared gene sequences using the known functions (Yi et al.,
2015). Bioinformatics tools such as PhymmBL, MG-RAST, and MetaPHlAn.

13.8.10 PREDICTING PHENOTYPES

Statistical techniques, machine learning, and gene-matching methods are

used to determine the bacterial strain phenotype based on the pathways, gene
particulars or depending upon the classification (Wingender et al., 1996).

13.8.11 METABOLOMICS

Several metabolites in the biological samples are simultaneously measured.

The reaction products of various microorganisms in the food samples and
in the culture media can be determined using metabolomics technique (Yi
et al., 2015).
Sequencing, Bioinformatics Tools, and Databases 425

13.9 CONCLUSION

NGS techniques will eventually reform food microbiology. Detecting the

pathogen, profiling the microbial populations, data mining, phenotype, and
genotype linking, determining the pathogens over food production and shelf
life of the food products are the realm of this NGS techniques. There is
an increasing success rate of bioinformatics approaches related to the food
sciences, not only food sciences but it is also expanding the research by
becoming a very essential tool in the field of life sciences. In the area of
food sciences, it helps in protein sequence analysis of the food products
to determine the function and allergenicity of it. Comparative genomic
sequencing techniques of bioinformatics have been used in food related
pathogens and their production. In concern to secure the FSQ, using bioin-
formatics, a lot of advancement has been made which allows automatic
food property detection.

KEYWORDS

• bioinformatics
• fermentation
• genomics
• microorganisms
• next-generation sequencing
• proteomics

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