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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.



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 Organic acids                                                                                                  8 Hydrogen peroxide                                                                                         8 Carbon dioxide                                                                                               9 Diacetyl                                                                                                           9 Reuterin                                                                                                          9 Bacteriocins                                                                                                    10

2.2       Role of Lactic Acid Bacteria                                                                          11

2.2.1    Lactic acid bacteria as probiotics                                                                    12 Definition of probiotics                                                                                  12 Features of probiotics                                                                                     12 Probiotic mechanisms of action                                                                      13 Benefits of probiotics                                                                                     13

2.2.2    Lactic acid bacteria as safe microbiota in biopreservation                              15 Definition of biopreservation                                                                          15 LAB Bacteriocins as potential food biopreservatives                                    15 Features of safe bacteriocins                                                                           16 Classification of bacteriocins                                                                          16 Factors limiting bacteriocin efficiency in food systems                                 18

2.3       Locally Fermented Foods in Nigeria                                                              18

2.3.1    Ogi                                                                                                                  19 Production processes of Ogi                                                                           19

2.3.2    Fufu                                                                                                                 21 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


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                               









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











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).



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).



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).




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.      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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