ABSTRACT
This study was aimed at analyzing the antimicrobial activities of Lactic Acid Bacteria (LAB) on some food spoilage microorganisms. Changes in pH and titratable acidity (TA) of the samples were investigated for a period of four days (96 hours) and a decrease in pH was associated with an increase in titratable acidity. The isolation and characterization of eleven tentative LAB from fermented maize and cassava (Ogi and Fufu respectively) as well as identification of the spoilage organisms from fish were aseptically performed and the antimicrobial activity was determined by agar well diffusion method against three isolated food spoilage organisms (Pseudomonas spp, Enterobacter spp and Bacillus spp). Out of eleven isolates, six best strains designated as OG1, W22, W12,Y11, F12 and F6 showed good inhibitory activities (11.25 ± 1.06 - 22.00 ± 0.71mm) against all the food spoilage organisms with F12 having the highest inhibitory diameter of 22.00±0.71mm against Bacillus spp. These species of the isolates were selected and further identified as Lactobacillus amylolyticus strain L6, Lactobacillus plantarum strain ci-4wand Lactobacillus sakei strain MLS1by the aide of genotypic characteristics (16S rRNA gene sequences). These strains were screened for their exopolysaccharide (EPS) producing activity, resistance to low pH and bile salts as well as bacteriocin activity. The investigation in the screening for EPS synthesis showed that Lactobacillus amylolyticus strain L6 is an active producer of exopolysaccharide. These three isolated strains showed good tolerance to pH 3.0 after 3 hours and 6 hours with a range of 52.50±6.36 –90.00±4.24log CFU/ml; however, all the isolates were able to survive in the presence of bile salts (0.1, 0.3 and 0.5 %) where Lactobacillus plantarum strain ci-4w and Lactobacillus sakei strain MLS1 showed the highest survival rates. The bacteriocins produced by these strains were prominent antibacterial, tolerated acidic pH 4 although recorded the lowest inhibitory activities at pH 9 but indicate highest inhibitory activities between pH 6.0 and 7.0. The effects of different temperature on the bacteriocin activity of these Lactobacillus strains exhibits full bacteriocin stability at 40°C, 60°C and 80°C. This present study revealed that the activity of bacteriocin was gradually decreased after 48 hours storage period. There was a significant difference (P < 0.05) between the antimicrobial activities of the isolated Lactic acid bacteria (LAB) species. Therefore, this present study suggested that Lactobacillus amylolyticus strain L6, Lactobacillus plantarum strain ci-4wand Lactobacillus sakei strain MLS1have an excellent probiotic potential and the possibility of using their bacteriocins as a food preservative against food spoilage organisms.
TABLE
OF CONTENTS
Page
Title page i
Certification ii
Declaration iii
Dedication iv
Acknowledgement v
Table of Contents vi
List of Tables x
List of Figures xi
Abstract xii
CHAPTER 1:
INTRODUCTION 1
1.1 Background
of the Study 1
1.2 Statement of Research Problem 3
1.3 Justification 4
1.4
Significance of Study 5
1.5 Aim 5
1.6 Specific
Objectives 5
CHAPTER 2: LITERATURE REVIEW 6
2.1 Description of Lactic Acid Bacteria 6
2.1.1 Sources of lactic acid bacteria 7
2.1.2 Significance of antimicrobial metabolites of lactic acid bacteria 7
2.1.2.1
Organic acids 8
2.1.2.2
Hydrogen peroxide 8
2.1.2.3
Carbon dioxide 9
2.1.2.4
Diacetyl 9
2.1.2.5
Reuterin 9
2.1.2.6
Bacteriocins 10
2.2 Role of Lactic Acid Bacteria 11
2.2.1 Lactic acid bacteria as probiotics 12
2.2.1.1
Definition of probiotics 12
2.2.1.2 Features of probiotics 12
2.2.1.3 Probiotic mechanisms of action 13
2.2.1.4 Benefits of probiotics 13
2.2.2 Lactic
acid bacteria as safe microbiota
in biopreservation 15
2.2.2.1
Definition of biopreservation 15
2.2.2.2 LAB Bacteriocins as
potential food biopreservatives 15
2.2.2.3 Features of
safe bacteriocins 16
2.2.2.4 Classification
of bacteriocins 16
2.2.2.5 Factors limiting bacteriocin efficiency in food systems 18
2.3 Locally Fermented Foods in Nigeria 18
2.3.1 Ogi 19
2.3.1.1 Production processes of Ogi 19
2.3.2 Fufu 21
2.3.2.1 Production
processes of Fufu 22
2.4 Microorganisms
Involved in Fermented Food Production 22
2.5 Significance of Food Fermentation and its Benefits 24
2.5.1. Enhancement of organoleptic properties 24
2.5.2 Preservative Properties 25
2.5.3 Provision of nutritional quality 26
2.5.4 Detoxification during Fermentation 27
2.5.5 Improvement of health 27
2.5.6 Decreased cooking time 28
2.6 Factors Influencing the Development of Fermented Foods 28
2.6.1 Salt Concentration 29
2.6.2 Temperature 29
2.6.3 Hydrogen ion concentration (pH) 29
2.6.4 Water activity 29
2.6.5 Nutrients 29
2.6.6 Oxygen availability 30
2.7 Constrains
Associated with Traditional Fermented Foods 30
2.8 Improvement and
Industrial Development of Traditional Fermented Foods 31
2.8.1 Control of processing environment 32
2.8.2 Raw material development 32
2.8.3 Starter development 32
2.8.4 Development of fermentation processes 34
2.8.5 Finished product
development 35
CHAPTER 3:
MATERIALS AND METHODS 36
3.1 Source
and Collection of Samples 36
3.1.1 Sample preparation 36
3.2
Physicochemical Analysis 37
3.2.1 Determination
of pH 37
3.2.2 Determination of Titratable Acidity (T.A) 37 3.3 Microbiological
Analysis 38
3.3.1 Isolation and Characterization of Lactic Acid Bacteria 38
3.3.2 Isolation and Identification of Test Organism 39
3.4
Antimicrobial Assay of Lactic Acid
Bacteria 39
3.5 Evaluation of In
Vitro Probiotic Potentials of the Selected LAB Isolates 40
3.5.1 Screening of the LAB isolates for Exopolysaccharide Production 40
3.5.2 Acid tolerance test 40
3.5.3 Bile salt tolerance test 40
3.6
Evaluation of biopreservation
potential of lactic acid bacteria 41
3.6.1 Extraction
of bacteriocin 41
3.6.2 Detection of inhibitory activity of bacteriocin from selected isolates 41
3.6.3 Effect of pH on crude bacteriocin 41
3.6.4 Effect of temperature on crude bacteriocin 41
3.7 Molecular
Identification of Lactic Acid Bacteria 42
3.7.1 DNA
extraction 42
3.7.2 PCR analysis 42
3.7.3 Sequencing 43
3.7.4 BLAST
analysis 43
3.7.5 Phylogenetic Analysis 43
3.8
Statistical Analysis 44
CHAPTER
4 45
4.1 Results 45
4.2 Discussion 68
CHAPTER 5 77
5.1 Conclusion 77
References 79 Appendix 101
LIST
OF TABLES
Table Title Page
4.1: Morphological
and Biochemical Characterization and Identification of Test Organisms 50
4.2: Morphological
and Biochemical Characterization and Identification
of Lactic Acid Bacteria Isolates 51
4.3: Physiological
characterization of Isolates 52
4.4: Inhibitory activity of the LAB isolates
against different test organisms 53
4.5: Molecular
Identification of Lactobacillus amylolyticus strain L6 55
4.6: Molecular
Identification of Lactobacillus plantarum strain ci-4w 56
4.7:
Molecular
Identification of Lactobacillus sakei strain MLS1 57
4.8: Exopolysaccharide
(EPS) production by different LAB isolates 59
4.9:
Survival of probiotic LAB at acidic
pH levels 60
4.10:
Inhibitory effect of crude bacteriocin 62
4.11:
Stability of Crude Bacteriocin to
Temperature 66
4.12:
Effect of Storage time on bacteriocin
activity 67
LIST
OF FIGURES
Figure Title Page
3.1: Flow chart
for traditional
method of Ogi processing 36
3.2: Flow chart
for traditional
method of fufu processing 37
4.1: Changes in pH of Fermented
food products (Ogi and Fufu) 47
4.2: Changes in Titratable
Acidity (T.A) of LAB isolates during 96h growth. 48
4.3: Agarose
gel electrophoresis of the 16S rRNA gene of the Lactobacillus
Amylolyticus
strain L6 (OG1), Lactobacillus Plantarum strain
ci-4w (Y11)
and Lactobacillus sakei strain
MLS1 (F12). 53
4.4: Evolutionary relationship
of the isolated LAB strains 57
4.5: Growth of Lactic Acid Bacteria at different
bile salt 60
4.6: Effect of pH on crude bacteriocin activity of
Lactobacillus amylolyticus
strain
L6 62
4.7:
Effect of pH on crude bacteriocin
activity of Lactobacillus Plantarum strain
ci-4w 63
4.8: Effect of pH on crude bacteriocin activity
of Lactobacillus sakei
strain
MLS1 64
CHAPTER
1
INTRODUCTION
1.1
BACKGROUND OF THE STUDY
Over
the years, lactic acid bacteria (LAB) have received much attention due to the
health-promoting properties of certain strains, called probiotics. They are
normal inhabitants of the healthy gut microbiota as they improve the balance of
the microbial community in the intestine, confer protection against potential
pathogenic bacteria, prevent or cure intestinal diseases and present in
numerous fermented food products (Rijkers et
al. 2011; Brown and Valiere 2004; Adak et
al. 2002). LAB are used in a wide range of fermented food, they play a
critical role in food processing and spontaneous fermentation (Elayaraja et al. 2014) also, they have shown a
major potential for use in biopreservation due to their GRAS (generally
recognized as safe) status (Salem 2012:
Vignolo et al. 2008: Radha
and Padmavathi 2015).
They exert a strong antagonistic activity against many food contaminating
microorganisms and these effects are mediated by production of antimicrobial
metabolites such as organic acids (for example lactate, acetate, and butyrate),
hydrogen peroxide, bacteriocins, and competition with harmful bacteria for
nutrients or adhesion receptors (Maurya and Thakur 2012; Wilson et al. 2011). LAB are
among the most important microbes which are used in food fermentations, as well
as in enhancing taste and texture in fermented food products (Van
Geel-Schuttená et al.1998; Hati et al. 2013).
However, fermented foods are of great
significance as they provide and preserve vast quantities of nutritious foods
in a wide diversity of flavours, aromas and textures which enrich the human
diet and they are consumed throughout the world (Oyedeji et al. 2013: Steinkraus
1997). Fermented foods are associated with a unique group of microflora which
increases the level of proteins, vitamins, essential amino acids and fatty
acids, thereby, helping in solving malnutrition problems in population (Bali et al. 2011). The most common
microorganisms found in fermented foods are yeasts and lactic acid bacteria
(known as probiotics). These organisms form stable mixed populations and the
species composition depends on the raw materials used, geographical factors,
preparation methods and production hygiene (Abriouel et al. 2006; Schoustra et al.
2013). Therefore, non-pathogenic microorganisms in fermented foods or rather
probiotics are increasingly being employed by medical experts in the treatment
of diseases. They are the reason why the water from fermented products (Ogi, kununzaki,
and burukutu) stop diarrhea (Ukwu
2011) and also offers the possibility of extending storage life of high quality
foodstuffs without the use of artificial chemicals (Oguntoyinbo et al. 2007). Fermented
foods are largely consumed in Africa where they constitute a bulk of the diet
among the many African traditionally fermented food stuffs like Ogi and fufu
(Ageni et al. 2017; Opeifa et al. 2015). Although fermented foods
are derived from substrates like roots, legumes, cereals, oilseeds, nuts, meat,
fish, milk, palm tree, sap, etc. (Uzogara et
al.1990;
Akobundu and Iwuoha 1992) but in Nigeria, the most common substrates for
fermentation are cassava and cereal grains such as maize, sorghum and millet
(Adesokan et al., 2010).
Several studies have revealed that LAB have been isolated
from locally fermented foods such as Ogiand fufu with the ability to retard spoilage, preserve food as well as improve
food safety (Oyedeji et al. 2013; Izah et al. 2016; Oyinlola et al.
2016). Therefore, Ogi and fufu are
rich sources of LAB. Fermented foods have a role beyond provision of energy and
body maintenance (Achi and Ukwuru 2015) as some fermentation microorganisms are known to produce
antimicrobial substances which lead to safe and long shelf life of food
products (Corgan et al. 2007; Kalui et al. 2009; Kalui et al.
2008; Parvez et al. 2006)
and they
have emerged as not only the source of nutrition but also as functional and
probiotic foods, which besides nutritional value have health effects or provide
protection against food-borne diseases (Mulaw et al. 2019). LAB made it
possible for human to increase the shelf life of food and food products by
utilizing their antimicrobial activities without damaging food contents (Tamang
et al. 2016).Many researchers have
investigated the antimicrobial activity of lactic acid bacteria against
undesirable microorganisms, e.g. Escherichia
coli, Salmonella, Staphylococci, Yersiniae, Bacilli, and Pseudomonads
(Garriga et al. 1993; Pazakova et al. 1997; Pepe et al. 2003). Substantiating the antimicrobial activities of LAB
will affirm their use in the development of functional foods for the betterment
of the health of the consuming public (Chuayana et al. 2003).
1.2
STATEMENT OF RESEARCH PROBLEM
One
of the concerns in food industry is the contamination by food spoilage
microorganisms and pathogens, which are frequent cause of food spoilage and
food borne diseases. Due to food spoilage, one-third of the worlds' food
produced for the consumption of humans is lost every year. An
important aspect of food contamination by microorganisms is the presence of
potentially pathogenic species, which pose a great risk for the human and
animal health (Broberg et al. 2007).
Bacteria and various fungi are the cause of spoilage and can create serious
consequences for the consumers. Some troublesome spoilage microorganisms
include aerobic psychrotrophic Gram-negative bacteria, yeasts, molds,
heterofermentative lactobacilli, and spore-forming bacteria. They can
cause extensive damage of the food such as unpleasant smell, taste or
appearance as well as formation of harmful substances for the consumer’s health
(Dinev et al. 2017).
In spite of modern technologies, good manufacturing practices, quality control
and hygiene and safety concepts such as risk assessment and HACCP, the reported
numbers of food borne illnesses and intoxications still increased over the past
decade (Garcia et al. 2010; Garcha
2018).
In order to achieve improved food safety against such
spoilage microorganisms, food industry makes use of chemical preservatives or
physical treatments (e.g. high temperatures). These preservation techniques
have many drawbacks which includes the proven toxicity of the chemical
preservatives (e.g. nitrites), the alteration of the organoleptic and
nutritional properties of foods, and especially recent consumer demands for
safe but minimally processed products without additives (Ananou et al., 2007; Sharma et al., 2006).
The biggest challenge in the food industry now is the
effort to reduce economic losses caused by food spoilage, reduce the price of
the food production process, reduce the possibility of pathogen transfer, and
satisfy the growing consumer need for ready-to-use food that tastes fresh, has
a high nutritional and vitamin value, and has been minimally processed and
treated with preservatives (Nath et al.,
2013).
1.3
JUSTIFICATION
However, the increasing resistance of food spoilage
microorganisms to current preservatives, the consumer’s high demand for safe,
minimally processed foods, the alteration of the organoleptic and nutritional
properties of foods and the hazards associated with the use of high doses of
chemical preservatives has led to the need for finding safer alternatives in
food preservation and disease prevention (Garcia et al., 2010).Therefore, the need for alternatives to extend the
shelf life of foods without changing their sensory properties and use in the
treatment or prevention of gastrointestinal disease, has launched research on
probiotics and biopreservation technologies, which are based on the use of non-pathogenic
microorganisms (Lactic Acid Bacteria) or their metabolites to retard food
spoilage or to improve food safety and confer health benefit (De Martinis et al., 2001; Ross et al., 2002).
1.4
SIGNIFICANCE OF STUDY
This research is designed to
alleviate the rate of malnutrition, to provide health benefits such as reducing
gastrointestinal diseases, allergies and improving immune health as it
evaluates probiotics properties, also, to enhance the shelf-life of fermented
food through assessing the biopreservation potency of lactic acid bacteria with
the aim of developing starter/protective culture with predictable
characteristics, for use in industrial application.
1.5
AIM
The aim of this research is to
analyze antimicrobial activities of lactic acid bacteria isolated from
fermented food.
1.6
SPECIFIC OBJECTIVES
1. To
isolate and identify lactic acid bacteria associated with traditional
fermentation of Ogi and Fufu.
2. To
evaluate the antimicrobial activity of the isolated lactic acid bacteria
against food spoilage microorganisms.
3. To
determine the in-vitro probiotic potentials of lactic acid bacteria isolate.
4. To
evaluate biopreservation potentials of the lactic acid bacteria.
5. To
carryout molecular (genotypic) identification of selected LAB isolates
with high probiotic and antimicrobial activities.
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