USING BIOCONTROL AGENTS TO CONTROL SALMONELLA SPECIES IN DAIRY PRODUCTS

ABSTRACT

Essential oil produced from turmeric and  Bacteriocin produced from Lactobacillus plantarum  were used to control Salmonella species found in dairy products. Six dairy products inculding  milk powdered,cheese,icecream,yoghourt, tin milk, fresh milk were screenced for Salmonella species, only three isolates were identified which inculde Salmonella typhi, Salmonella  enteric, Salmonella chloeraceau. Results showed the presene of  Salmonella species  in three  products yoghourt, fresh milk and milk powdered. Lactobacillus was  isolated from “ogi”(fermented maize starch) and it’s yield  of bacteriocin was 0.36% while essential oil yield from turmeric was  4.94%. The highest zone of inhibition was 24.33mm were recorded for bacteriocin activity aganist Salmonella species while the highest zone of inhibition for essential oil  showed antibacteria activity with  zone of inhibition in diameter of 14.33mm aganist the isolates .The test of biocontrol agents more also are found to be active  at low concentraction of 15mg/ml and 21mg/ml for bacteriocin and essential respectively. Comparatively the bacteriocin extract was found to have higher activity than the essential oil extracts and should be incoperated in strater culture in production of dairy products.

TABLE OF CONTENTS

Title page i

Certification ii

Acknowledgments iii

Dedication iv

Table of Contents v

List of tables           vi

Abstract vii

CHAPTER ONE

1.0 Introduction

1.1        Introduction 1

1.2 Aim and Objectives 2

CHAPTER TWO

2.0 Literatue Review 4

2.1 Latic acid bacteria 4

2.2 Latic acid bacteria and bacteriocin 6

2.3 Bacteriocin 7

2.4. Classification of bacteriocin 8

2.4.1 Class 1 (Lantibiotics) 8

2.4.2 Class 11 (Small heat stable peptides) 9

2.4.3 Class 111(Large heat labile bacteria) 9

2.4.4 Class iv 9

2.5.0 Essential oil from turmeric 10

2.5.1 Scientific classification of turmeric 10

2.5.2  origin and  uses of turmeric 11

CHAPTER THREE

3.0 Materials And Methods 15

3.1 Materials 15

3.2 Methods 15

3.2.1 Sample Collection 15

3.2.2 Media Prearation 15

3.3.0 Isolation Of Salmonella From Dairy Products Determination of Micriobiral

Load (cfu/g) 15

3.3.1 Confirmation of  Salmonella 16

3.3.2 Microscopic Examination/Gram Staining 17

3.4.0 Sources of Lactic Acid Bacteria 17

3.5.0 Biochemical  Tests 18

3.5.1 Indole Test 18

3.5.2 Cirtrate Utilization Test 18

3.5.3 Nitrate Reduction Test 18

3.5.4    Catalase Test 19

3.5.5   Cogulase Test 19

3.5.6 Oxidase Test 19

3.5.7 Sugar Fermentaion Tests 19

3.7.0  occurance  of Salmonella  in  Dairy Products 20

3.8.0 Extracton Of Biocontorl Extracts (Bacteriocin From Lactobacillus plantarum  and Essental Oil From Turmeric/Determination Yield 20

3.8.1 Extracion of Essential Oil from Turmeric/Determination Yield 20

3.8.2 Extraction of  Bacteriocin /Detremination Yield 21

3.9 Antibacterial Activity Test 22

3.10 Antibiotics Senstivity Test 23

3.11   Minimum  Inhibtion Concentraction (MIC) 23

3.12 Statistical Method 23

CHAPTER FOUR

4.0 Results 24

CHAPTER FIVE

5.0 Discussion, Conclusion And Recommendation 29

5.1 Discussion 29

5.2. Conclusion 30

5.3 Recommendation 31

References                                                                                                               32

Appendix

List of Tables

Tables                                                     Title Pages

1.           Occurance of Salmonella Species in Dairy Products                                    25

      2.             Precentage Yield Of Esseential oil And Baceteriocin 26

       3.             Zone of Inhibition of Bacteriocin and Essential Oil Aganist 27

Salomonella Species in Diameter (mm)  

      4.              Minimum Inhibition  Concentration of Essential Oil and 28

Bacteriocin of  Salmonella Species.

CHAPTER ONE

1.0 INTRODUCTION

Inspite of morden improvement in food production techiques, food safety is still an improtant  aspect  of  public health .It has been estimated  that as many as 30% of people in industrised  countries has most  occuring bacterial food borne  disease each year (WHO, 2002) defined food disease as disease infection or toxification caused by the consumption of containmated  food or water. Salmonellosis was the most occuring  bacterial  food borne disease with more than 325,000 cases per year (Baeulmer et al, 2005) .Food borne  outbreak of Salmonella assoicated with consumption of contaminated dairy products and other ready-to- eat products has been reported to occur with increased  frequancy Doyle (1992), Baeulmer et al 2005, Bredhoit et al, 2005) also Staphylococus anerus, Eschericha coli  cause food borne infection. Salmonella belongs to the  family of enterobacterioaceae which inculdes the genera of Salmonella ,Proteus, Klesbsiella,Shigella, Enterococus, Eschericha coli, there up to 200 serotypes and 70 sub-species of Salmonella (Jawetz et al, 2005).

According to cheesbrough 2006,Salmonella are gram negtive bacteria with rods,areobes, facuitves they grow between the temperature of 15-45⁰C. They are motile withe pertriochas  flagella , they  ferment  maltose, manitole .They produce hydorgen suphides, non –sporing ,non-capsulated ,biochemical test of Salmonella is urease, indole negtive and cirtates postive. The natural habitat is intestinal tract of  human’s and animals ,the main infective dose  is 10⁵-10⁸(CDCP,1993). Some Enteric organism are part of normal  microbiota and incidently cause disease, but Salmonella-Shigella are pathogenic to humans. salmonella grows in simple media and selective media  such as Salmonella-Shigella ager (SSA). They survive freezing and can stay for longer  period, they are isolated  from food and other poducts such as  dairy  products there symptoms inculdes vomitting ,fever,diarrohea and abdomial pains after ingestion of the infected foods between  12-36hours (Baeulmer et al 2005). Colony morophololgy , identification of Salmonella on SSA are in cluster and has black pigment.

According to Reidtke et al (1993) biocontrol agents refers to the suppression, reduction or inactivation of food borne pathogen  bacteria  by other bacteria or micobiral agents. Progress in research and advanced in techonology has led to various practical ways for microbiral control of food borne . Biocontrol agents have anti microbiral activites aganist Staphylococus auerus, Escheriha coli, Pseudomonas  and other  pathogenic oraganism (Parish et al 2003, Liao et al 2010) .Biocontrol are also known as biological control or bioactive microbes may inhibit or suppress food borne   pathogen. Latic acid bacteria (LAB) can be used as biocontrol agent  or its  extract like Bacteriocin. LAB are group bacteria gram negtive bacteria  that can be used as biopreservative ,bicontrol  .They are used  to prolong life shelf of food by producing  a number  of organic  compounds that  that are antagonistic to other microorganism (Brink et al, 1994). Among these compounds proteinaeous bacteriocin has grained much attention especially regarding  to much  reserach  focused on utilization of bacteriocin as Novel food  biopreservatives ,also the interest  in using them as biocontrol  of  bacteria in human because of the  probiotics potenial of these bacteria is also vastly investiagated ( Redtke  et al, 1993, Onyeagba et al 2004 , Diez-Gonzaales , 2007). Alot of Lactobacillus has been noted to have nutitional benfits, imprived lactose utilization have anti-cholesterol and carcinogenic activites and protection agenist other disease. Bacteriocin has received attention for use as natural or Biopreservatives in food in recent years (Savadogo et al, 2004).

Accoring to Dimick et al (1947) using solvent extraction method to extract curde bacteriocin from Lacto-bacillus Plantarum. In recent year use of essential oil (EOs) as biocontrol agent has recevied  attention both in pharamcetical, comestic,agrcultural and food indusrty because of the biological activity, notably antibacterial, antifungal, activity and efficeney of  EOs to be used as antimicrobiral agents in food system depends on the factors as temperaure, _PH, apperance, load of microbiral flora and favourable evironment. In recent years, serveral research  have shown that many spices and herbs who exert  antimicrobial activity due to their essential oil fraction can be used in food system for inhibiting   the growth of pathogenic microorganism this way ensuring the microbiological saftey of food products (Bankkali et al, 2008, Burt, 2004,Yardugul et al, 2002), and that antimicrobiral effects of EOs exhibit strctural or functional damges of membrane of pathogenic bacteria by various antimicrobiral mechanism (Cotter et al 2000, Burt 2007) the plant EOs is gotten from Turmeric (Curcuma longa) the EOs is extracted by solvent extraction method  according to (Prasad et al 2001). Turmeric is a pereninal plant that is cultivated throughout tropices of Asia,india and chian,the plant grows at the height of 0.9-1.5m and bears large oblong leaves and funnel-shaped, dull yellow flowers. recently ,The EOs  strated to used because of their antimicrobiral activity in food package,to edible films provide incoprating of compounds into edible films provides a noval way to improve the safety and shelf life of ready-to-eat food (Diez-Gonzale et al, 2007, cutter 2000).

1.2 Aim And Objectives

 To evaluate inhibitory activities of bacteriocin and essential  oil aganist  salmonella species on dairy products.

i. To determine the antibacterial activites  and Minimum Inhibitory Concentraction of bacteriocin and essential oil

ii. Isolation and  identification of Salmonella from different dairy products .

iii. Extraction of biocontrol agents (bacteriocin from Lactobacillus planatrum  and essential oil from turmeric).

iv. To determine the effects of these bicontorl agents aganist Salmonella in dairy products to reduce foodborne infection.

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