MAINTENANCE AND ASSESSMENT OF MICROBIAL CELL VIABILITY IN SELECTED CARRIERS (CRYOPROTECTANTS)

ABSTRACT

This study investigated the maintenance and assessment of microbial cell viability in selected carriers (cryoprotectants). Drinking yogurt and pap samples were collected for the laboratory isolation of lactic acid bacteria. Identification of the suspected colonies and probiotic characterization tests were carried out. The colonies were found to be lactic acid bacteria and they showed probiotic potentials. In the maintenance of the viability of this lactic acid bacteria cell, Freeze drying technique was selected and a cryoprotective carrier, combination of skim milk and sucrose was incorporated in the culture medium prior to freeze drying. A standard plate count was carried out on the culture before and after freeze drying in cryoprotectants. A plate count was also carried out on a control culture for freeze drying without cryoprotectants. Result showed that there was improvement of viability of the cells in culture freeze dried with cryoprotectants with increase in growth from 0.72×105 to 0.74×105 CFU after 7 days of storage at room temperature. While viability of cells freeze dried without cryoprotectants was partly contained but cells suffered from sub lethal injury during freezing. This study thus concludes that the incorporation of a cryoprotectants medium in microbial cell culture while freeze drying is very effective for the maintenance of the cell viability.

TABLE OF CONTENTS

Title Page                                                                                                                      i

Certification                                                                                                                  ii

Dedication                                                                                                                    iii

Acknowledgement                                                                                                       iv

Table of Content                                                                                                           v

List of Figures                                                                                                              viii

List of Tables                                                                                                                ix

Abstract                                                                                                                         x

 CHAPTER ONE: INTRODUCTION

1.1        Background of Study                                                                                        1

1.2        Aims of the Study                                                                                             3

 CHAPTER TWO: REVIEW OF LITERATURE  

2.1        Microbial cell viability                                                                                     4

2.1.1     Viability in Contrast to Culturability                                                                5

2.2        Factors affecting Viability                                                                                8

2.2.1    Low Temperature                                                                                             8

2.2.2    High Osmotic Gradient                                                                                   9

2.2.3    Ozone                                                                                                                9

2.2.4    High Temperature                                                                                             9

2.2.5    High Pressure                                                                                                  10

2.2.6    High Acceleration                                                                                           12

2.2.7    Other Factors Affecting Viability                                                                   12

2.3       Measurement of Viability; Colony Count                                                       12

2.3.1    Total Viable Count                                                                                         12

2.3.2    Total Count                                                                                                     12

2.3.3    Viable Count                                                                                                   14

2.4       Viability of Lactic Acid Bacteria (LAB)                                                        16

2.4.1    Industrial benefits of LAB                                                                              17

2.4.2    Growth media and growth condition of LAB                                                 18

2.4.3    Sweet Potato Base Medium                                                                            19

 CHAPTER THREE: MATERIALS AND METHODS

3.1       Samples Collection                                                                                          21

3.2       Isolation of Lactic Acid Bacteria                                                                    21

3.3       Identification of LAB Isolates                                                                         21

3.4       Morphological and Physiological Tests                                                          22

3.4.1    Cell Morphology                                                                                            22

3.4.2    Spore Staining                                                                                                22

3.5       Biochemical Tests                                                                                           22

3.5.1    Catalase Test                                                                                                   22

3.5.2    Starch Hydrolysis Test                                                                                   22

3.5.3    Sugar Fermentation Test                                                                                23

3.5.4    Gas Production from Glucose                                                                        23

3.6       In Vitro Characterization of Probiotic Properties                                           23

3.6.1    Determination of Optimum pH and Temperature for Growth                     23

3.6.2    Tolerance to NaCl and Phenol                                                                        24

3.6.3    Antibiotic Susceptibility Test                                                                         24

3.6.4    Antimicrobial Activity                                                                                   24

3.7       Cryoprotective Medium and Preparation of Suspension                                25

3.7.1    Sample preparation                                                                                          25

3.7.2    Lyophilization                                                                                                  25

3.8       Stabilization                                                                                                     26

3.9       Determining the Conversation of the Viability Over Time                                      26

CHAPTER FOUR: RESULTS

4.1       Morphological and physiological tests                                                           27

4.2       Biochemical tests                                                                                            27

4.3       In Vitro Characterization of Probiotic properties                                           27

4.3.1    Effect of optimum pH                                                                                     27

4.3.2    Effect of optimum temperature for growth                                                    28

4.3.3    Assay for NaCl and phenol tolerance                                                             28

4.4       Antibiotic susceptibility test                                                                           28

4.5       Detection of antimicrobial activity                                                                 28

4.6       Microbial Cell Viability after Treatment with Selected Carries (Cryoprotectants)

and stored for some period of time.                                                                             29

 CHAPTER FIVE: DISCUSSION AND CONCLUSION

5.1       Discussion                                                                                                       36

5.2       Conclusion                                                                                                       38

References                                                                                                                              38

LIST OF FIGURES

Figures                                                                    Pages

1          Typical Microbial Growth Curve                                                           4

LIST OF TABLES

Tables            Pages

1.               Morphological and Physiological Test of The Isolated Bacterial Strains            30

2.               Biochemical Test                                                                                           31

3.               In Vitro Characterization of Probiotic Properties                                          32

4.               Antibiotic Susceptibility Test                                                                        33

5.               Antimicrobial activity                                                                                    34

6.               TVC of the Lactobacillus plantarum at day 0 and day 7 storage                        35

 CHAPTER ONE

INTRODUCTION

1.1       BACKGROUND OF THE STUDY

Ever since the discovery of microorganisms, humans have for variety of reasons tried to assess, control, utilize or even restrict their growth and viability. Sometimes the presence of living cells is essential, thus the ability to measure the viability of bacterial cells has an important role in microbiological analyses. The definition of viability, however, is a challenging task, and there is no simple answer how to define it. First it should be established how to separate viable micro-organism from nonviable ones. This question is of particular importance since one has to know a prerequisite of viability before it can be stated if bacteria are viable or not. The growth of bacteria is a fast and dynamic process, and mostly bacterial culture consists both of living and dead cells. The formation of new bacterial cells occurs typically within minutes to some hours and all environmental factors which bacteria are confronted have a distinct effect on bacterial growth and death thus making the exact definition of bacterial viability sometimes quite sophisticated.

Lactic acid bacteria (LAB) play a critical role in food, agricultural, and clinical applications. The fast growing characteristics of LAB and their metabolic activity have been the key in most applications including food production, agricultural industry, and probiotics. However, the biochemical and biophysical environments have significant effect on the growth and metabolic activity of LAB.

According to the definition, probiotic is: live microorganisms which administered in adequate concentration confer a health benefit. The probiotic bacteria should be present in a viable form at a suitable level during production until consumption and maintain high viability throughout the gastrointestinal tract.

Lactic acid bacteria (LAB) represent a major group of microorganisms used as starter cultures and probiotics in the food industry.

The industrial application of LAB depends on concentration and conservation technologies that are required to ensure the long term stability of cultures in terms of viability and functional activity. It is essential, both technologically and economically, maximizing the viability of laboratory cultures during drying and subsequent storage for long periods. There are several mechanisms that allow us to preserve the viability of the bacteria over time, being cryopreservation and lyophilization the most prominent. In the production of compound feed, LAB are subjected to various stressful technological procedures. It is essential to maintain the viability of probiotics to remain effective. Although, the freeze-drying and subsequent storage produce cell viability decrease due to the drying exposes cells to an additional stage of stress processing. Different species show different degrees of survival to freeze drying. The degree of viability loss depends on inherent microorganism factors (strain properties, growing conditions and the state of growth) and other factors inherent in the process (technical parameters such as cooling rate and temperature, the presence of cryoprotectants, and the type of rehydration buffer). These factors may cause osmotic shock and membrane injury during recrystallization by the formation of intracellular crystals.

To minimize this damage, cryoprotectant substances are commonly used. Some sugars are recognized as protectors and used for the preparation of freeze-dried cultures. These sugars stabilize the cell membrane by a mechanism of replacement of water and a series of interactions between membrane phospholipids and sugars. Skim milk and sucrose are commonly used as cryoprotectants. Skimmed milk is considered to be able to prevent cell damage by stabilizing the cell membrane, providing a protective layer for the cells, while the protective activity of sucrose suggests that is due to its ability to prevent harmful eutectic fluid cell freezing. Other polysaccharides such as maltose, lactose and trehalose, as well as maltodextrin also have use as cryoprotectants, increasing the viability of lactic acid bacteria during freezing and freeze-drying processes. When the water content of the samples is low, the sugars form a glassy matrix that is characterized by high viscosity and low mobility. Under ideal conditions for drying and storage, trehalose is probably more effective than other oligosaccharides in the preservation of biomaterials. This increased tolerance to desiccation appears to be result of the ability of certain disaccharides to lower the temperature of the membrane during the transition to the drying and maintain the structure of the proteins in the dried state.

1.2       AIM OF THE STUDY

The aim of this study was to evaluate the effect of different cryoprotectants (skim milk powder, sucrose and trehalose) on the viability of a Lactobacillus plantarum probiotic strain when it is subjected to a stressful process of freezing and subsequent lyophilization, determining the optimal combination of cryoprotectants to improve the viability of the preparations before its incorporation into a premix feed. The specific objectives are to:

i.             assess the viability of lactic acid bacteria as a probiotic in a premix feed or as a starter culture.

ii.           evaluate the carriers suitable for protecting the probiotic bacteria during freezedrying, storage processes and application processes.

iii.         determine the optimal temperature (storage and freezing) required to maintain the live probiotics.

iv.          determine the viability range of the probiotic bacteria.

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