PRODUCTION OF ANTI-RESPIRATORY BIOACTIVE SUPPLEMENTS FROM CITRUS AURANTIFOLIA

CHAPTER ONE

INTRODUCTION

Bacteriocins are extremely heterogeneous group of substances constituting an active protein moiety alone or in conjugated form (Anil et al., 2007). According to Wikipedia, Bacteriocins are proteinaceous or peptidic toxins produced by bacteria to inhibit the growth of similar or closely related bacterial strain(s). They are similar to yeast and paramecium killing factors, and are structurally, functionally, and ecologically diverse. Narayanapillai et al., (2012) defined Bacteriocin as a proteinaceous substance that exhibit bactericidal activity against closely related organisms. Bacteriocins are ribosomally synthesized antimicrobial peptides produced by microorganisms belonging to different eubacterial taxonomic branches. Most of them are small cationic membrane-active compounds that form pores in the target cells, disrupting membrane potentials and causing cell death (Sabiha, 2017).

Kunun-zaki is a fermented non-alcoholic cereal beverage, which is popular in northern Nigeria. Kunun-zaki production is essentially a home-based industry and at present, there is no large-scale factory production. (Agarry et al., 2010). It is usually made from millet (Efiuvwevwere and Akona, 1995)

Microbes produce an extraordinary array of microbial defence systems. These include broad-spectrum classical antibiotics, metabolic by-products, such as the lactic acids produced by lactobacilli, lytic agents such as lysozymes, numerous types of protein exotoxins, and bacteriocins, which are loosely defined as biologically active protein moieties with a bactericidal mode of action. This biological arsenal is striking not only in its diversity, but also in its natural abundance. (Riley and Chavan, 2007).

Lactococcus is a genus of lactic acid bacteria that were formerly included in the genus Streptococcus Group N1. (Schleifer et al., 1985) They are known as homofermenters meaning that they produce a single product, lactic acid in this case, as the major or only product of glucose fermentation. Their homofermentative character can be altered by adjusting environmental conditions such as pH, glucose concentration, and nutrient limitation. They are Gram positive, catalase-negative, non-motile cocci that are found singly, in pairs, or in chains. The genus contains strains known to grow at or below 7°C (James, 1992).

Lactobacillus is a genus of Gram-positive, facultative anaerobic or microaerophilic, rod-shaped, non-spore-forming bacteria (Makarova et al., 2006).

The indicator organisms, test reference pathogens from American Type Culture Collection (ATCC) such as Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC7090), Escherichia coli (ATCC25922) (Clark and Geary, 1974) are all opportunistic pathogens (Muller et al., 2015) and are coincidentally notorious for causing Bacteraemia (Douglas et al., 2004; Cesar,2015; Luzzaro et al., 2002) which lead to a host of complications especially pneumonia, meningitis (Wendy et al., 2006) and septic arthritis etc. (Rasmussen et al., 2011) and it is known to be particularly prevalent and severe in the very young and very old (Tong et al., 2015).

Pasteur and Joubert in 1877, recorded observation of antagonistic interactions between different bacteria. They noted that B. anthracis was inhibited by the effect of common bacteria present in urine. Later in 1925 a Belgian scientist, Gratia discovered that the filtrates of the cultures of Escherichia coli called principle V strongly inhibited the growth of another strain of same species. Since Gratia’s observations similar substances were found to be produced by numerous strains of family Enterobacteriaceae including Escherichia, Enterobacter, Salmonella, Shigella, and Proteus species. Later on Gratia and Fredricq (1946) coined the term “COLICIN’ for this inhibitory substance. Whereas the term “bacteriocin” was first used by Jacob and his co-workers in 1953. They are called ‘colicins’ because a substance produced by any member of the group may be active on strains belonging to any other species of the family including E. coli. Although the nature of the inhibitory substance was observed previously, but it was suggested that many of the observed interactions were caused due to substances that are now classified as bacteriocins. This bacteriocin is the general and the individual type of bacteriocin are generally named according to the species of the organisms originally produce it. (Anil et al., 2007).

Allelopathy, defined as the suppression or death of one organism due to the toxic chemicals excreted by another organism, is a ubiquitous phenomenon within microbial communities. In bacterial assemblages, the agents of allelopathic interaction are the bacteriocins. Bacteriocins are narrow-spectrum antimicrobial proteins found within nearly every major lineage of Bacteria. Given that bacteriocinogenic (toxin-producing) strains kill closely related non-producing strains, bacteriocins are commonly interpreted to be anti-competitor compounds. Over the past few decades, there has been much interest in exploring the microbial dynamics of toxic consortia.

Some of these studies have shown that Socrates’ insight carries particular salience for communities with bacteriocinogenic members – allelopathy may play a critical role in maintaining diversity in these systems (Riley and Chavan, 2007).

The study of inter-bacterial inhibition, similarly to so many other fundamental facets of microbiology, can trace its origins to Louis Pasteur. In 1877 Pasteur, together with his assistant Joubert, in seeking a way to control the growth of the anthrax bacillus, reported both in vivo and in vitro inhibitory activity associated with co-inoculated “common bacteria” (probably Escherichia coli) isolated from urine. Pasteur’s pioneering studies heralded several decades of investigations, predating the antibiotic era, which focused upon the dosing of patients with relatively harmless bacteria in an attempt to counter the proliferation of pathogens – the so-called bacterial interference strategy – an approach to infection control now experiencing a renaissance after the half century of neglect that followed the discovery of penicillin, and the associated smug dependence of clinicians on the profligate use of therapeutic non-ribosomally synthesized antibiotics to control bacterial infection. Most of the early successes in defining the nature of bacteriocins related to those of Gram-negative bacteria, especially the colicins, and much of this knowledge stemmed from the work of Gratia and Fredericq. It was Gratia

who first described antagonism between strains of E. coli (Gratia, 1925). Interestingly, the first documented inhibitory strain produces colicin V, a bacteriocin of the microcin class that, in many respects, more closely resembles bacteriocins typically produced by Gram-positive bacteria (Havarstein et al., 1994). Fredericq used specific (receptor-deficient) colicin-resistant

mutants to classify the colicins (Fredericq, 1946). General characteristics of the colicins included;

(1) plasmid-encoded, large domain-structured proteins;

(2) bacteriocidal activity via specific receptors and;

(3) lethal SOS-inducible biosynthesis.

The study of bacteriocins of Gram-positive bacteria got off to a relatively faltering start, largely focusing on the staphylococci, and with various attempts to apply similar principles of classification to those that had been established for the colicins. However, relatively few of the protein antibiotics of Gram-positive bacteria fit closely the classical colicin mold. Major differences include their relatively broad activity spectra, less defined specific producer cell self-protection (immunity), and absence of SOS inducibility. In the past three decades, studies of bacteriocins of Gram-positive bacteria, especially those of the lactic acid bacteria (LAB), have come to dominate the bacteriocin-related literature, a change largely driven by

commercial imperatives. (Riley and Chavan, 2007).

1.1 AIM AND OBJECTIVES

The aim of this research work is to compare the bactericidal activity of bacteriocins of Lactococcus species and Lactobacillus species isolated from Kunun-zaki against some very common test reference pathogens. To achieve this, the objectives include;

1.    To isolate Lactococcus species and Lactobacillus species in Kunun-zaki.

2.  To screen them for their ability to produce bacteriocin.

3. To study the antimicrobial activity of their bacteriocin against common human pathogens such as Escherichia coli, Enterococcus faecalis, and Staphylococcus aureus.

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