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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