ACID-BILE TOLERANCE AND ANTIMICROBIAL PROPERTIES OF SOME LACTOBACILLUS SPECIES ISOLATED FROM FERMENTED MILK PRODUCTS

ABTSRACT

Acid-bile tolerance and antibacterial properties of some Lactobacillus species were studied. Milk products (Nunu, Fan milk and Yoghurt) were plated on De-Mann Rogosa Sharpe (MRS) agar and incubated for 24 hours. The Lactobacillus species isolated were L. acidophilus, L. bulgaricus, L. casei and L. plantarum with the occurrence range of (100%), (66%), (33%) and (33.3%) respectively. The Lactobacillus isolates were analyzed for their antimicrobial activity against some test pathogenic organisms such as Staphylococcus sp, Pseudomonas sp, Escherichia coli, Candida sp and Aspergillus sp. Antimicrobial activity of Lactobacillus isolates against the test organisms mentioned earlier ranged from 12.33 ± 1.15mm to 21.00 ± 2.00mm for Lactobacillus acidophilus, 11.69 ± 1.15mm to 12.67 ± 0.58mm for L. bulgaricus, 10.67 ± 1.15mm to 22.67 ± 1.15mm for L. casei and 9.67 ± 1.15mm to 15.67 ± 1.15mm for L. plantarum. This result showed L. bulgaricus to be least active against Pseudomonas sp with inhibition zone of 8.67 ± 0.58mm while L. casei showed the highest activity against Aspergillus sp. The acid-bile tolerance ability of the different Lactobacillus isolates was tested and the results show varying levels of the ability of the isolates to thrive in acid and bile environment.

TABLE OF CONTENTS

CHAPTER ONE

1.1       INTRODUCTION

1.2       Aim and objectives of the study

CHAPTER TWO

2.1       LITERATURE REVIEW

2.2       Lactobacillus

2.2.1    Species of Lactobacillus

2.2.2    Lactic acid production

2.2.3    Metabolic potentials  of Lactobacillus

2.2.4    Functional properties of Lactobacillus

2.2.5    Clinical uses of Lactiobacillus

2.2.6    Food production

2.3       Probiotics

2.3.1    Effects of probiotics on the immune system

2.3.1.1 Cholesterol reduction

2.3.1.2 Mechanism of probiotics

2.3.1.3 The effects of probiotics on health

CHAPTER THREE

3.1       MATERIALS AND METHODS

3.2       Source of materials

3.3       Samples and media preparation

3.3.1    Sample preparation

3.3.2    Media preparation

3.3.3    Isolation of LAB

3.4       Characterization of isolates

3.4.1    Colony features

3.5      Biochemical Tests

3.5.1   Catalase test

3.5.2    Coagulase Test

3.5.3  Citrate Test

3.5.4    Motility, Indole, Urease Test (MIU)

3.5.5    Sugar utilization tests

3.6       Identification of bacteria isolates

3.7       Acid-bile tolerance test

CHAPTER FOUR

4.1       RESULTS     

CHAPTER FIVE

5.1       DISCUSSION, CONCLUSION AND RECOMMENDATION

5.2       Discussion

5.3       Conclusion

5.4       Recommendation

REFERENCES

APPENDIX I: E. coli

APPENDIX II: Staphylococcus

APPENDIX III: Pseudomonas

APPENDIX IV: Aspergillus

APPENDIX V: Caudida

APPENDIX VI: Morphological characteristics of Lactobacillus species isolated from fermented milk products

APPENDIX VII: Characteristic features of bacterial isolates from fermented milk products

CHAPTER ONE

1.1       INTRODUCTION

Species of Lactic acid bacteria (LAB) belong to numerous genus under the family of Lactobaciliacae. They represent as potential microorganisms and have been widely applied in food fermentation worldwide due to their well known status as generally recognized as safe (GRAS) microorganisms. They are also recognized for their fermentation ability and thus enhancing food safety, improving organoleptic attributes, enriching nutrients and increasing health benefits (Liu et al., 2011). Fermentation is generally considered as a safe and acceptable preservation technology of food and fermentation using LAB can be categorized into two groups based on the raw material used, non-dairy and dairy fermentation. Milk from different mammalian animals can be used in dairy fermentation to produce different products. Milk of cow followed by milk of goat and sheep are the most widely used materials to produce particular economic value fermented milk worldwide. Due the characteristics of milk that is highly perishable, the main purpose of milk fermentation using LAB is to prolong its shelf-life as well as the preserve the nutritious components of milk. It is also recognized that fermentation of milk using LAB will undoubtedly produce good quality of products with highly appreciated organoleptic attributes. Recently, there is a growing interest to develop a variety of fermented milk products for other beneficial purposes, particularly for health purposes and preventing of toxins produced by food borne pathogens and spoilage bacteria that enter human body . The beneficial effects of fermented milk products are produced by a variety of bioactive compounds of LAB (Grifth and Tellez, 2010). Lactic acid bacteria represents as the most extensively studied microorganisms in food milk fermentation. The presence of LAB in milk fermentation can be either as spontaneous or inoculated starter cultures. Milk itself is known as one of the natural habitats of LAB. Although under spontaneous fermentations, the growth of LAB cannot be predicted on controlled, but this procedure has been practiced and carried out traditionally for years. A procedure called as back slopping is often used. There are some examples of fermented milk by LAB produced under this procedure such as those of artisanal cheeseklil (Sun et al., 2010). In general, the technology of milk fermentation is relatively simple and cost effective. On the other hand, standardized fermented milk products are produced and manufactured in large-scale production under controlled conditions and becomes an important industrial application of LAB as starter cultures. There are some important features of LAB starter in fermented milk products. A single potential starter culture will dominate and reduce the diversity of microorganisms in fermented milk products compare to that of products under natural fermentation.

Probiotics are live microorganisms which are defined by the World Health Organization/Food and Agricultural Organization (2001) as live microorganisms whose administration in adequate amount of the body is able to confer a health beneficial effect on the host. The most common types of microbes which are used as probiotics are lactic acid bacteria (LAB) and Bifido bacteria. A number of genera within formicutes phylum like Lactobacillus, Lactoccocus, Lactosphaera, Carnobacterium, Enteroccocus, Streptoccocus Tetragenoccocus, Oenoccocus, Pediccocus, Meisella, Melissoccocus, Vagoccocus constitute lactic acid bacteria (Jay, 2000, Holzaptel et al., 2002). LAB are Gram positive bacteria  able to fermentation carbohydrates into lactic acid and energy (Jay,2000). Some LAB differ in their metabolic pathway for example homofermentative bacteria like Lactoccocus and Streptococcus produce two lactate molecules from one glucose into ethanol, lactate and carbon dioxide (Fitzgerald, 2006; Jay, 2000; Kuipers et al., 2000). Furthermore, lactic acid bacteria yield some organic compounds that contribute to the aroma as well as flavor of the fermented products (Fitzgerald et al., 1999). Human milk is a complex biological fluid that is species specific and completely fulfills both nutritional and microbiological requirements of the new born. Breast milk boots up immune system and builds body defense against various infectious diseases which makes it superior to other supplements for infants. Various bioactive compounds like Immunoglobulins. Lyzozyme, antimicrobial acids, Oligosaccharide, glycoproteins for example Lactoferrin, polyamines, immune cells and bioactive peptides present in breast milk that are responsible for its anti-infective effect (Saavedra, 2002, Isaac, 2005). These bioactive compounds of human milk play a major role in the regulation of the anti inflammatory system. Due to Immunomolulatory action of human milk, the incidence as well as severity of various infectious diseases like tetanus, poliomyelitis and diphtheria is lesser in breast-fed infants than those fed with other food formulae. The addition of breast milk probiotics to infant formulae could be a new alternative to mimic some of the functional effect of human milk in children who are not breastfed. That is why breast milk was selected as source of probiotic bacteria in this study. In human milk, most frequently occurring LAB are Lactobacillus, Lactococcus, and Enterococcus. Probiotics are a sub-group of microorganisms will positive effects on the host health through improving the gut bacterial balance. These bacteria were first discovered by Mechnoof in 1907 (Setyawardani et al., 2011. Probiotic bacteria should be resistant to gastric acidity and bile salts, so that they can reach to colon and create their desirable effects (Setyawaardani et al., 2011, Both et al., 2010). Lactic acid bacteria (LAB) are the most common types of probiotics. These bacteria have a long-term survival in fermented products (Barakat et al., 2011).

Lactobacillus is a Gram-positive, non-spore forming, rarely motile bacteria, while Lactoccocus is a Gram-positive spherical and rarely motile bacteria both of which are present in considerable amounts in dairy products. Some beneficial effects of probiotics include enhancing the immune system function, reducing the symptoms of lactose intolerance and growth in acidic foods like free amino acids as well as in compounds such as Nisinin which they can have antibacterial activites (Tharmaraj et al., 2009). LAB makes an acidic condition and present the growth of pathogens by converting the milk sugar (lactose) into lactic acid (Simora et al., 2008). Some pathogens such as Staphylococcus aureus are important in food hygiene, because they produce toxins and cause food poisoning (Charlier et al., 2009). Salmonella typhimurium is another pathogens that causes gastroenteristis. Therefore, controlling these bacteria in food products is important for human health (Monads et al., 2010). Antibiotics have been used for treating bacterial diseases for long time. However, the continued usage of antibiotics has caused innumerous problems including microbial resistance. As a result, scientists are determine to find substitute solutions and probiotics are on the top of their list (Wysong, 2006, Sanders et al., 2010). Some probiotics have chronic effects on health and can improve the digest tract function, their consumption is advised (Shirazi et al., 2011). Although a lot of studies have been performed on traditional dairy products of sabzevar, Iran, not enough research has been performed to isolate LAB from these products.

1.2       Aim and objectives of the study

The aim of this work is to isolate some Lactobacillus species from fermented milk products.

Objectives of the study

1.     To isolate and identify some Lactobacillus species from fermented milk (products).

2.     To determine the bile and acid tolerance of isolates

3.     To determine the antimicrobial activities of isolates against selected bacterial and fungal species.

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