Probiotic characterization of lactic acid bacteria

isolated from chicken meat

A Thesis
For The Fulfillment Of B.S Degree
By
Sidra Zaman

DEPARTMENT OF MICROBIOLOGY
JINNAH UNIVERSITY FOR WOMEN
KARACHI-74600
2018
 APPROVAL SHEET

Name of Candidate: Sidra zaman

Research Supervisor: Miss Erum Mazhar

Lecturer
Department of Microbiology

Program Head of Microbiology: Dr.Taqdees Mallik

Chairperson
Department of Microbiology

External Examiner: Miss Tooba batool

Lecturer
Department of Microbiology
 CERTIFICATE
This is to certify that research work embodied in this thesis entitled isolation and identification
of lactic acid bacteria from chicken meat samples and also its probiotic characterization carried
out by Sidra zaman at Jinnah University for Women Karachi for the fulfillment of BS degree.
The research work has been carried out under my supervision and to my satisfaction. I consider
this work is suitable for the award of BS degree in Microbiology.

Miss Erum Mazhar

Lecturer

Department of Microbiology
Acknowledgment
Foremost, thanks to Merciful Allah for the wisdom he bestowed upon me, the strength, peace
of my mind and good health in order to finish this work.
It is great pleasure to acknowledge my deeper thanks and gratitude to my head of department
Dr. Taqdees Mallik who provide all the facilities, necessary to carry out my project work and
gave me the opportunity to perform my thesis work.
I am highly indebted to my project supervisor Miss Erum Mazhar for her guidance and constant
supervision as well as providing necessary information regarding the project and also for her
support in completing the project. It is a great honor to work under her supervision.
Moreover, I would like to express my deepest thanks to laboratory staff of Jinnah University
for Women for corporative attitude and for providing all the necessary materials required to
complete my research work.
Lastly, I would like to express my gratitude toward my family, friends and all my teachers for
their encouragement which helped me in completion of this project
Abstract
This study is conducted in order to evaluate the probiotic properties of lactic acid bacteria
isolated from chicken meat. As the lactic acid bacteria are health beneficial bacteria and are
use in the production of dairy products, in therapeutics, and as food bio preservative.In this
study lactobacillus was isolated from chicken meat and was exposed to different pH,
temperature, bile salt, NaCl concentrations. Organism was able to tolerate the acidic pH,
different temperatures and was also able to grow in the presence of different bile salt and NaCl
concentrations.the study reveals that the isolated Lactobacillus have good probiotic properties
and have great potential to be use in therapeutics, as substitute of antibiotics in animals feed
and for many other purposes.

Key words:probiotic,therapeutics
Introduction
Gastrointestinal infections are a major cause of morbidity and mortality worldwide. Studies
conducted in 2006 found that, globally, severe diarrhea and dehydration are responsible each
year for the death of 1,575,000 children under the age of five. This represents 15% of the 10.5
million deaths per year of children in this age group [1]. According to recent estimates, acute
gastroenteritis causes as many as 770,000 hospitalizations per year in the United States [2].
Enteric pathogens include viruses (rotaviruses, noroviruses) and bacteria such as different
strains of pathogenic Escherichia coli, toxigenic Clostridium difficile, Campylobacter jejuni,
and Vibrio cholerae. These pathogens produce different types of toxins that can cause severe
or life-threatening dehydration and diarrhea. Despite medical advances in diagnosis and
treatment, the percent and number of hospitalized pediatric patients less than 5 years of age
with severe rotavirus infection significantly increased when a recent time period (2001– 2003)
was compared to an earlier time period (1993– 1995) [3]. In addition to the typical pattern of
acute gastroenteritis, infectious agents such as enteropathogenic E. coli (EPEC) may cause
persistent, chronic diarrhea in children lasting longer than 1 week [4]. Such persistent infections
may increase the risk of dehydration and long-term morbidities.

Recent studies have highlighted long-term morbidities associated with gastroenteritis. Early
childhood diarrhea predisposes children to lasting disabilities, including impaired fitness,
stunted growth,and school performance [6]. Along with this data, new research on maternal
and child under nutrition reported in The Lancet in January 2008 links poor nutrition with an
increased risk for enteric infections in children. Furthermore, irritable bowel syndrome (IBS),
a costly and difficult to treat condition that affects 20% of the United States population [7], has
medical costs of up to $30 billion per year, excluding prescription and over-the-counter drug
costs [8]. IBS is precipitated by an episode of acute gastroenteritis in up to 30% of all cases in
prior studies [9]. Therefore, preventing or treating acute gastroenteritis before long-term sequel
develop would drastically reduce hospitalizations, disability adjusted life years, and both direct
and indirect medical costs. Accurate diagnosis of acute gastroenteritis is an ongoing challenge
even in sophisticated academic medical centers. In a pediatric patient population exceeding
4,700 children, less than 50% of stool samples that underwent complete microbiologic
evaluation yielded a specific diagnosis [10]. Enteric viruses represented the predominant
etiologic agents in acute gastroenteritis in children less than 3 years of age, and bacteria caused
the majority of cases of acute gastroenteritis in children older than 3 years of age [10]. The
diagnostic challenges with enteric viruses include the relative paucity of stool-based molecular
or viral antigen tests and the inability to readily culture most enteric viruses. Bacterial
pathogens may be difficult to identify (such as most strains of disease causing E. coli) because
of the lack of specific assays for these infections. The relative insensitivity of stool based toxin
assays for the detection of toxigenic C. difficile precludes accurate diagnosis. In a children’s
hospital setting, combination toxin antigen testing yielded sensitivity below 40% in pediatric
patients (J. Versalovic, unpublished data). The introduction of new molecular assays for
realtime PCR detection of toxin genes directly in stool has markedly improved the ability to
diagnose antimicrobial associated diarrhea and colitis due to toxigenic C. difficile [11]. In
addition, approximately 15–25% of cases of antimicrobial-associated diarrhea are caused by
C. difficile. The prevalence of antimicrobial-associated diarrhea and gastrointestinal disease
highlights the importance of alternatives to antibiotic strategies for treatment. Furthermore,
antibiotics have limited utility for the treatment of gastroenteritis in general. Antimicrobial
agents are not generally recommended as prevention strategies because of the problems of
antibiotic resistance and antimicrobial associated disease. Thus, instead of suppressing
bacterial populations with antibiotics, probiotics can be used to remodel or shift microbial
communities to a healthy state [12]

Probiotics

The term probiotic is derived from Greek and literally means “for life.” It was first coined in 1965 by
Lilley and Stillwell to describe substances secreted by one microorganism that stimulate the growth
of another [1, 2]. In 1974, Parker modified this definition to “…organisms and substances which
contribute to intestinal microbial balance” [1, 3]. The current definition of probiotics by Food and
Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) is
“live microorganisms which when administered in adequate amounts confer a health benefit to the
host” [4–6]. Probiotic organisms require certain characteristics to enable them to exert maximum
therapeutic effects. Of these characteristics, there are some that are considered almost essential for
a probiotic to have therapeutic effects, including gastric acid and bile salt stability, ability to adhere to
the intestinal mucosa, and ability to colonize the intestinal tract [1, 7].

Classification

There are many different microorganisms currently used as probiotics [1, 20, 25]. To better
understand how bacteria are named and classified, the following discussion may be helpful. Genus is
the first name of a bacterium (e.g., Lactobacillus). It is somewhat general and refers to a grouping of
organisms based on similarity of qualities, such as physical characteristics, metabolic needs, and
metabolic end products. Species is a bacterium’s second name (e.g., acidophilus). It is a much more
narrow classification based on shared common characteristics that distinguish them from other
species. Strain is an even more specific classification that divides members of the same species into
subgroups based on several properties that these bacteria have in common that are distinct from
other members of the species (e.g., strain LA5) [1, 26]

Bifidobacterium species

Bifidobacterium is an anaerobic, Gram-positive, nonspore-forming, pleomorphic rod. Bacteria in the

Bifidobacterium genus produce lactic and acetic acids as by-products of glucose utiliza‐ tion. BB536 is
a type of probiotic bacteria, which, according to secondary sources, was first isolated from the
intestinal tract of healthy infants. Bifidobacteria, in combination with Lactobacillus species and the
probiotic yeast Saccharomyces boulardii, seem to reduce the adverse effects of Helicobacter therapy,
but do not seem to improve compliance [46]. In addition, Bifidobacterium infantis in combination with
Lactobacillus acidophilus seems to reduce the incidence of NEC and NEC-associated mortality in
critically ill neonates [47].

Bacillus species

Bacillus coagulans is a Gram-positive rod, which produces lactic acid, and therefore is often
misclassified as lactic acid bacteria, such as lactobacillus. In fact, some commercial products containing
B. coagulans are marketed as Lactobacillus sporogenes or "spore-forming lactic acid bacterium." It
forms spores, which is an important factor in differentiating these species. B. coagulans is used
therapeutically in a similar manner as other probiotics such as lactobacillus and bifidobacterium;
however, B. coagulans is not a component of the normal human flora. In order to be effective for
restoring normal flora and prevent pathogenic colonization, probiotics must have the ability to persist
and colonize in the intestinal mucosa. When the Bacillus spore is ingested by humans, it is unknown
what happens to the spore. It is unknown if the Bacillus spore is capable of germinating in the intestinal
tract or if colonization occurs [48].

B. coagulans might reduce pathogenic bacteria colonization through several mechanisms. B. coagulans
produces coagulin and lactic acid, which have antibacterial activity and might reduce pathogenic
bacteria growth through this mechanism [29, 49, 50]. Animal model research also suggests that
ingesting bacillus spores increases immune response [48]. Proponents of B. coagulans suggest that
this species of probiotics offers advantages over others such as lactoba‐ cillus because Bacillus species
can be stored indefinitely in desiccated forms [48]. Bacillus spores are also resistant to high
temperatures and to acid.
Saccharomyces species

S. boulardii, also known as Saccharomyces cerevisiae, is a nonpathogenic yeast strain that has been
used for the treatment and prevention of diarrhea resulting from multiple etiologies. S. boulardii has
been isolated from the skins of tropical fruits found in Indochina. The indigenous population of
Indochina has long used these fruit skins to prevent and treat diarrhea [51].

S. boulardii is prepared by lyophylization (freeze drying) of live yeast organisms and encap‐ sulation
using lactose in the preparation. S. boulardii cannot be distinguished from other S. cerevisiae strains
by phenotypic criteria, so identification of these infections requires molecular typing. Comparative
molecular studies show that S. boulardii is genetically very close or nearly identical to S. cerevisiae
[52]. Results suggest that microsatellite polymorphism analysis of the YKL139w and YLR177w genes
and the analysis by Ty917 hybridization are the most useful tools for the correct identification of S.
boulardii strains [53]. However, metabolically and physiologically, S. boulardii shows a very different
behavior than S. cerevisiae, particularly in relation to growth yield and resistance to temperature and
acidic stresses, which are important characteristics for a microorganism to be used as a probiotic.

Lactobacillus species

Lactobacillus refers to a group of lactic acid–producing Gram-positive rods that are obligate and
facultative anaerobes in the human gastrointestinal and genitourinary tracts [27, 29–32]. The name
lactobacillus refers to the bacterium's ability to produce lactic acid, not to the ability to digest lactose
[28]. Lactobacilli are used therapeutically as probiotics, the opposite of antibiotics. They are
considered "friendly" bacteria and are taken for the purpose of recolo‐ nizing areas of the body to
provide nutritional benefits including inducing growth factors and increasing the bioavailability of
minerals [32]. Lactobacilli also stabilize the mucosal barrier and decrease intestinal permeability [33].

Altering the normal flora allows for potential colonization by pathogenic organisms [34], which can
result in side effects, such as diarrhea, cramping, and less commonly pseudomem‐ branous colitis
(PMC), caused by C. difficile. The theory is that taking lactobacillus probiotics during antibiotic
treatment can prevent or minimize normal flora depletion and pathogenic bacteria colonization. There
is some evidence to support this theory [35, 36]. Hydrogen peroxide–producing lactobacilli are
bactericidal to the vaginal pathogen Gardnerella vaginalis, and their presence in the vagina has been
associated with decreased frequencies of bacterial vaginosis and trichomoniasis [37]. In the vagina,
lactic acid from lactobacilli lowers vaginal pH, which can prevent pathogen growth.

There is some preliminary evidence that lactobacilli and other probiotics might help protect against
cancer. In animal models, lactobacillus has been shown to bind dietary carcinogens [38] and decrease
development of tumors in the colon after carcinogen challenge [39, 40]. Preliminary research also
suggests that lactobacilli, especially L. plantarum, can reduce the severity of chemotherapy-induced
enterocolitis [41]. According to other research studies, Lactobacillus bulgaricus and Lactobacillus
sporogenes might have hypolipidemic and antiathero‐ sclerotic effects. Limited clinical evidence
suggests that it can reduce total and low-density lipoprotein (LDL) cholesterol with no effect on high-
density lipoprotein (HDL) [42, 43]. Fermented dairy products, such as yogurt and acidophilus milk, also
seem to have a beneficial effect on cholesterol. Lactobacilli and other probiotic bacteria seem to bind
bile acids to cholesterol. They also seem to increase fatty acid production in the intestine, which
decreases circulatory fatty acid concentrations either by inhibiting hepatic cholesterol synthesis or
redistributing cholesterol from the plasma to the liver.

Most researchers agree that the effectiveness of lactobacilli and other probiotics for all indications
depends on their ability to colonize an area of tissue. To do this, lactobacillus preparations must
contain live and viable organisms. Products stored for long periods of time or stored improperly may
contain few live and active organisms. For oral preparations, bacteria must also remain viable after
passing through the gut, and then they must be able to latch on to the intestinal epithelium.
Lactobacilli strains might vary in their effectiveness due to differences in their ability to adhere to the
epithelial cells by host factors such as hormone levels [30, 44, 45]. This ability can change during a
woman's menstrual cycle in response to changing hormone levels. In postmenopausal women,
correcting low estrogen levels can help restore lactobacillus colonization without supplementation
[29, 30].

Mechanism of action

The exact mechanisms by which probiotics accomplish their beneficial actions have not been well
documented. However, there are several postulated mechanisms that explain many of their favorable
effects [8] (Figure 1). One of such mechanisms is a competition for adhesion sites, which means
probiotics fight for cellular attachments. Many pathogenic organisms must associate with the GI tract
epithelium to colonize effectively [9]. However, some strains of bifidobacteria and lactobacilli can
adhere to the epithelium and act as “colonization barriers” by preventing pathogens from adhering to
the mucosa [1, 10]. This effect was demonstrated with the Lactobacillus rhamnosus strain GG and
Lactobacillus plantarum 299v. Both of these organisms showed the ability to inhibit attachment of
Escherichia coli to human colon cells [1, 11]. Another possible mechanism of action is the modification
of the microbial flora through the synthesis of antimicrobial compounds [12]. Many types of
lactobacilli and bifidobacteria produce bacteriocinsor and other antimicrobial compounds.
Bacteriocins are defined as “compounds produced by bacteria that have a biologically active protein
moiety and a bactericidal action” [1, 13]. Other biologically active compounds produced by lactic acid
bacteria include hydrogen peroxide, diacetyl, and short-chain fatty acids. The release of these
compounds by probiotic organisms results in a beneficial modification of the microflora [1, 14].
However, not all strains of lactobacilli or bifidobacteria produce antimicrobial compounds, and some
produce compounds that are fairly nonspecific in their activity, so that beneficial bacteria, as well as
pathogenic organisms, may be negatively affected [1]. It has also been observed that probiotics can
stimulate the immune response [15]. This immune response may take the form of increased secretion
of immunoglobulin-A (IgA) [1, 16], elevated numbers of natural killer cells, or enhanced phagocytic
activity of macrophages [1, 17]. Increased secretion of IgA may decrease numbers of pathogenic
organisms in the gut, thus improving the composition of the microflora [1, 10]. Due to these
immunomodulating effects, some researchers think probiotics might not only fight intestinal and
urogenital pathogens, but might also be helpful for conditions, such as inflammatory bowel disease
(IBD), pouchitis, food allergy, and for use as an adjuvant to vaccination [18–22]. Probiotics may also
compete for nutrients that would otherwise be utilized by pathogens [1, 23]. This situation occurs with
Clostridium difficile, a potentially pathogenic organism that is dependent upon monosacchar‐ 20
Probiotics and Prebiotics in Human Nutrition and Health ides for its growth. Probiotic organisms in
sufficient numbers can utilize most of the available monosaccharides, which results in the inhibition
of C. difficile [1, 24].

Mechanistic details of probiotic action

The use of probiotics to prevent and treat a wide variety of conditions has gained favor in the past
decade. This is in part due to a need to find alternatives to traditional therapies such as antibiotics as
well as the lack of good treatments for GI ailments. While there are increasing reports of the efficacy
of probiotics in the treatment of diseases such as pouchitis [13, 14], diarrhea [15–17], and irritable
bowel syndrome [18].The concept of using probiotic microorganisms to prevent and treat a variety of
human ailments has been around for more than 100 years [19]. With the rise in the number of
multidrug resistant pathogens and the recognition of the role that the human microbiota plays in
health and disease, a recent expansion in the interest in probiotics has been generated. This
phenomenon is apparent in both the numbers of probiotic products being marketed to consumers as
well as the increased amount of scientific research occurring in probiotics. Although many of the
mechanisms by which probiotics benefit human beings remain unclear, probiotic bacteria are being
utilized more commonly to treat specific diseases.(20)
 Stimulation of host antimicrobial defenses

Many probiotics have been shown to produce antipathogenic compounds ranging from small
molecules to bioactive antimicrobial peptides. Conceptually, an antimicrobial compound produced by
an organism would need to be produced at a high enough level and in the right location in the
intestinal tract to exert a strong effect on a pathogen in vivo. An elegant proof of principle for direct
action of a probiotic-produced antimicrobial against a pathogen was recently reported by Corr et al.
who demonstrated that production of the bacteriocin Abp118 by Lactobacillus salivarius was sufficient
to protect mice from disease by infection with Listeria monocytogenes [21]. To prove the action of the
bacteriocin was directly responsible for the protection of the mice, they generated a L. salivarius strain
that was unable to produce Abp118 and showed that this mutant was incapable of protecting against
L. monocytogenes infection. Notably, they were able to express a gene that confers immunity to the
Abp118 bacteriocin within L. monocytogenes and showed that this strain was now resistant to the
probiotic effect of L. salivarius within the mouse. This study provided clear evidence that a probiotic-
derived bacteriocin could function directly on a pathogen in vivo.

Pathogen exclusion via indirect mechanisms

In addition to producing antimicrobial compounds that act directly on pathogens, probiotics may
stimulate host antimicrobial defense pathways. The intestinal tract has a number of mechanisms for
resisting the effects of pathogens including the production of defensins [22]. Defensins are cationic
antimicrobial peptides that are produced in a number of cell types including Paneth cells in the crypts
of the small intestine and intestinal epithelial cells. Probiotics may act to stimulate defensin activity
via at least two mechanisms. First, probiotics may stimulate the synthesis of defensin expression. This
has been demonstrated for human beta defensin 2 (hBD-2), whose expression is upregulated by the
presence of several probiotic bacteria via the transcription factor NF-κB [26, 27]. The implication is
that probiotic strains with this capability would strengthen intestinal defenses by increasing defensin
levels. Second, many defensins are produced in a propeptide form that must be activated via the
action of proteases.

Immunomodulation
Rather than directly inhibiting the growth or viability of the pathogen, probiotics may compete for an
ecological niche or, otherwise, create conditions that are unfavorable for the pathogen to take hold
in the intestinal tract. There are many possible mechanisms for how pathogen exclusion may take
place. First, several probiotics have been demonstrated to alter the ability of pathogens to adhere to
or invade colonic epithelial cells in vitro, for example, see [30, 31]. Second, probiotics could sequester
essential nutrients from invading pathogens and impair their colonization ability. Third, probiotics may
alter the gene expression program of pathogens in such a way as to inhibit the expression of virulence
functions [32]. Lastly, probiotics may create an unfavorable environment for pathogen colonization
by altering pH, the mucus layer, and other factors in the local surroundings. It is important to note
that although many of these possible effects have been demonstrated in vitro, the ability of probiotics
to exclude pathogens in vivo remains to be proven.

Enhancing intestinal barrier function

Probiotics may have strain-dependent effects on the immune system. Different strains representing
different Lactobacillus species demonstrated contrasting effects with respect to proinflammatory
cytokine production by murine bone marrow-derived dendritic cells [33]. Specific probiotic strains
counteracted the immunostimulatory effects of other strains so that probiotics have the potential to
yield additive or antagonistic results Different probiotic Lactobacillus strains of the same species may
also yield contrasting effects with respect to immunomodulation. Human breast milkderived
Lactobacillus reuteri strains either stimulated the key proinflammatory cytokine, human tumor
necrosis factor (TNF), or suppressed its production by human myeloid cells [34]. The mechanisms of
action may be due, not surprisingly, to contrasting effects on key signaling pathways in mammalian
cells. Probiotic strains such as Lactobacillus rhamnosus GG (LGG) may activate NF-κB and the signal
transducer and activator of transcription (STAT) signaling pathways in human macrophages [35]. In
contrast, probiotic Lactobacillus strains may suppress NF-κB signaling [36, 37] or MAP kinase-/c-Jun-
mediated signaling [34]. Stimulation of key signaling pathways and enhancement of proinflammatory
cytokine production may be important to “prime” the immune system for defense against
gastrointestinal infections. Conversely, suppression of immune signaling may be an important
mechanism to promote homeostasis and tolerance to microbial communities with many potential
antigens, and these immunosuppressive functions may promote healing or resolution of infections.

Probiotics and the prevention and treatment of gastroenteritis

probiotic bacteria have been implicated as therapy for a range of digestive diseases, including
antibiotic-associated colitis, Helicobacter pylori gastritis, and traveler’s diarrhea [46]. Probiotic
formulations may include single strains or combinations of strains. L. reuteri is indigenous to the
human gastrointestinal tract, is widely present in mammals, and has never been shown to cause
disease.15

Probiotics may be effective for the prevention or treatment of infectious gastroenteritis. In the context
of disease prevention, several studies with different probiotic strains have documented that these
bacteria may reduce the incidence of acute diarrhea by 15–75% depending on the study [17, 47–50].
Although the relative impacts on disease incidence vary depending on the specific probiotic strain and
patient population, consistent benefits for disease prevention have been demonstrated in multiple
clinical studies.49

The potential role of probiotics in treating Clostridium difficile-associated

diarrhea (CDAD)

An estimated 500,000–3,000,000 cases of Clostridium difficile-associated diarrhea (CDAD) occur

annually with related health care costs exceeding $1 billion per year [58– 60]. CDAD occurs primarily
in patients that have undergone antibiotic therapy in a health care setting, indicating that alterations
in the intestinal microbiota are important for the initiation of CDAD. In a small but increasing number
of cases, more severe complications will occur including pseudomembranous colitis and toxic
megacolon. Moreover, the emergence of metronidazole-resistant strains of C. difficile has diminished
the efficacy of metronidazole, and vancomycin- and metronidazole-induced cecitis reinforces the
need for new therapies for the treatment and prevention of CDAD [61, 62]. Approximately 10–40% of
patients treated for an initial bout of CDAD will show recurrent disease, often with multiple episodes
[63]. Such recurrences are often refractory to existing therapies including antibiotic therapy. Patients
with recurrent CDAD had a marked decrease in the diversity of organisms in their fecal microbiota
while patients that were free of recurrent disease had a normal microbiota [64]. Thus, therapies that
restore a normal microbiota or suppress C. difficile growth while allowing the repopulation of the
intestine with a favorable microbiota may be important to resolve infections and maintain intestinal
health.
Material and Method
Requirements
Lactic acid bacteria (LAB) sourced from non-dairy product(meat sample).
Media
MRS was used for selective growth, enrichment culture and indication of specific properties of
lactobacillus.
De Man, Rogosa and Sharpe (MRS) Agar and Broth
De Man, Rogosa and Sharpe Agar and broth were designed to encourage the growth of the
“lactic acid bacteria” which includes species of the following genera: Lactobacillus,
Streptococcus, Pediococcus and Leuconostoc. It typically contains 1.0 % peptone, 1.0 % beef
extract, 0.4 % yeast extract, 2.0 % glucose, 0.5 % sodium acetate trihydrate, 0.1 % polysorbate
80 (also known as Tween 80), 0.2 % dipotassium hydrogen phosphate, 0.2 % triammonium
citrate, 0.02 % magnesium sulfate heptahydrate, 0.005 % manganese sulfate tetrahydrate and
the desired amount of distilled water. It can be used as a solid medium by adding 1.0% agar.
Sample Collection
Meat sample was collected from Karachi.Then sample was stored at 4°C until use for further
microbiological analysis.
Isolation of Lactobacillus
Ten gram meat sample was immediately processed under aseptic conditions by suspending in
90 mL of peptone water and was vortexed for proper mixing. Then appropriate serial dilution
(10-1to10-3) was prepared for sample using 1mL of well mixed sample then one milliliter of
appropriate dilution was enriched in 9 mL of sterile Lactobacillus selection MRS broth for 24
h at 37 °C. Before inoculation of sample, the pH of MRS broth was adjusted to 6.5 ± 0.2. The
enriched samples were streaked on the petri plates containing Lactobacillus selection MRS
agar with the help of calibrated inoculating loop and incubated anaerobically at 37 °C for 48 h
and observed for the growth of colonies. After the incubation, colonies were restreaked on the
MRS agar Petri plate for the formation of isolated colonies.
Identification of Selected Lactobacilli
The identification and further characterization of Lactobacilli isolates grown on MRS agar was
done mainly with the help of the following tests: microscopic examination (Gram staining),
catalase test, fermentation of different carbohydrates, etc. Conformation of the Lactobacillus
 isolated from meat by testing for the absence of catalase enzyme and the presence of acid
produced by fermentation of glucose.
Gram staining
1. Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. ..
2. The addition of iodide, which binds to crystal violet and traps it in the cell.
3. Rapid decolorization with ethanol or acetone.
4. Counterstaining with safranin.
Microscopic Examination
The purity morphological identification of the isolates as Lactobacilli was confirmed
microscopically by performing Gram staining, for which single colony of isolate was picked
up and stained as per the standard protocol and viewed under oil immersion lens.
Biochemical Characterization of Isolates
a) Catalase Test: The test was performed in order to determine the ability of the isolated
cultures to degrade the hydrogen peroxide by producing the enzyme catalase. The test
was carried out as the slide method, using an inoculating needle. For this, culture from
a typical colony was placed onto a clean grease-free glass slide and drop of 3%
hydrogen peroxide solution was added onto the culture and closely observed for the
evolution of bubbles. The production of bubbles indicated positive catalase reaction
and was recorded accordingly for the presence or absence of enzyme.
b) Nitrate Reduction Test: Nitrate reduction is an important criterion for differentiating
and characterizing different types of bacteria. Therefore, the isolates were incubated at
37 °C for 24 h in trypticase nitrate broth. After incubation, 0.5 mL each of sulphanilic
acid (0.8%, in 5N Acetic acid) and -naphthylamine (0.5%, in 5N Acetic acid) were
added into the tubes. The appearance of red or pink color indicated the positive test for
nitrate reduction and was recorded accordingly for the isolate tested in the present
study.
c) Citrate Utilization Test: The isolates were inoculated in Simmons citrate agar
incubated at 37 °C for 24 h. After incubation, the appearance of blue coloration
indicated the positive test for citrate utilization and was recorded accordingly for the
isolate tested.
Carbohydrate Utilization Test
Phenol red dextrose, sucrose, fructose, lactose, manitol and glucose broths of 6 ml were
prepared by autoclaving at 15 psi 121°C for 15 minutes in separate test tubes. Using sterile
technique, a small amount of the experimental bacteria from 24 hour old pure culture was
inoculated into the broths by means of loop inoculation. All the tubes were incubated for 24
hours at 37°C.

Probiotic characterization
pH tolerance
The isolated bacterial cultures were inoculated into sterile MRS of various pHi.e.2,8,10 and
were incubated at 370C anaerobically for 24 to 48hrs.After incubation period MRS broth
cultures were observed for turbidity.
Temperature tolerance
For the determination of growth at various temperatures, MRS broth was inoculated with
colony of fresh overnight culture of LAB and incubated at 25, 37, and 45 °C for 24 h. The
growth was evaluated by spreading on MRS agar and monitors their growth.
Bile salt tolerance
Bacterial strain were inoculated into MRS broth having different concentrations of bile salts
(0.2, 1, 2 and 3.0%), incubated at 370C for 48h. The growth was evaluated by spreading on
MRS agar and monitors their growth.The development of LAB culture on MRS agar plates
were used to consign isolates as bile salt tolerant.
NaCl tolerance
For NaCl tolerance determination of isolated LAB strain, MRS broth adjusted with varying
concentration of NaCl (2%, 8%, and 10%). After sterilization, each test tube was inoculated
with fresh overnight culture of bacterial isolates and was incubated at 370C for almost 24h.
Results
In this study, isolate collected from the non-dairy product(meat), and cultured on MRS agar
anaerobically for 48 hours at 37°C. The isolate further sub cultured to obtain pure colonies and
were further screened for the presence of Lactobacilli based on the morphological and
biochemical characteristics.
Identification of Selected Isolates
Microscopic Examination
The Gram reaction property and cell morphology was examined using standard staining
procedure. The isolate was found to be Gram-positive rods of varying sizes and rods are
arranged as singly, or in chains, under oil-immersion microscope.
Table.01 Microscopy
S/No source Gram Reaction Shape And Arrangement
1 Chicken meat Positive Scattered short rods

Fig-1 fig-2 fig-3

Biochemical Characterization of Isolates

a) Catalase Test: Catalase, an extracellular enzyme secreted by several microorganisms,
helps in degradation of hydrogen peroxide produced during carbohydrates utilization
for energy production, thereby its presence or absence in a microbial cell can be used
as a significant diagnostic tool. In the present study isolate was found to be catalase
negative. These results obtained for catalase further support the identification of isolates
as Lactobacilli.
b) Nitrate Reduction Test: In microbial taxonomy, nitrate reduction is an important
criterion for characterization and identification of different types of bacteria. This is
 due to the fact that certain bacteria have the capability to reduce nitrate to nitrite while
others are capable of further reducing nitrite to ammonia. The formation of ammonia
changes the pH of media to alkaline thus, changing the color of media from yellow to
cherry red. In the nitrate reduction test the isolate tested showed negative reaction, a
characteristic of Lactobacillus group of organism.
c) Citrate Utilization Test: For further characterization and identification, all the isolates
were subjected for their potential to utilize citrate as the sole carbon source. Certain
bacteria utilize citrate with the help of enzyme citrate permease and citrase-producing
diacetyl, a flavoring compound as end product. Following incubation on Simmons
citrate agar, citrate-positive cultures were identified by the presence of growth on the
surface of slant, accompanied by blue coloration whereas negative cultures did not
show any growth and medium remained green. In citrate utilization test the isolate
tested showed negative results
Table.02 Biochemical Characteristics
Catalase Test Negative

Nitrate Reduction Test Negative

Citrate Utilization Test Negative

Carbohydrate Utilization Test

For accounting the sugar utilization pattern of the isolate suspected to be the Lactobacilli after
their growth on Lactobacillus selection MRS agar, the sugar utilization tests were performed
using four different sugars and the isolate ferment these sugars the results indicated by the
production of pink colour.
Table.03 Carbohydrate Utilization
Lactose Sucrose Glucose Manitol
++ + + +
++ + + +
++ + + +
++ + + +
++ = acid and gas production and + = acid production
Probiotic characterization

Temperature

In this study being carried out, isolated LAB were able to grow at 25 ˚C,37 ˚C and 45 ˚C. This
temperature range was used during the study to investigate if bacterial strains can grow within
range of around body temperature or not. If isolated strains failed to survive within the present
temperature range then they would been unable to continue to exist in gut of human, animal
and its most important criteria for selection of probiotic bacteria, the results of this study were
found positive.

Table.04 Temperature tolerance

Temperature Growth

25 ˚C ++
37 ˚C ++
45 ˚C +
++=Good growth, +=Growth

Fig-4:Growth at different temperatures

NaCl

In this study isolated LAB was able to tolerate 2,8 and 10% of NaCl concentration.NaCl having an
inhibitory action can prevent the growth of various types of bacteria. If the lactic acid bacteria are not
tolerant to NaCl then they would be unable to show their action in the availability of NaCl, so it was far
important to test the isolated strains resistance to NaCl.

Table.05 NaCl tolerance

NaCl concentrations Growth

2% ++

8% ++

10% +

Bile salt
Isolated LAB survived in 0.2, 1, 2 and 3.0% of bile salt concentrations The isolates not only
survived in above mentioned concentration of bile salts but also multiplied well. In this study
design, 0.2,1,2 and 3.0% of bile concentration were used, as it is equalent to that found in the
human intestine tract and in healthy men almost 0.3% of bile is present.

Table.06 Bile salt tolerance

Bile salt Growth

0.2% ++
1% ++
2% ++
3% ++
 Fig-5:Growth at different bile salt concentrations

pH
pH is the most important factor that can influence bacterial growth. In this study design the
growth of isolated LAB strains were observed in various pH value 2,8 and 10.The results of
pH tolerance indicated that the isolated LAB strain tolerated and survived in both acidic as
well as alkaline ph. The probiotic bacteria for human use have to survive during the passage
through the stomach where the pH is 1.5-3.0, before they arrive at intestinal tract and must
remain viable for almost 4 hr or even more.

Table.07 ph tolerance

ph Growth

2 ++

8 ++

10 ++
 Discussion
Bacteria were gram-positive,rod shaped and occurred as separate rods and in chain. The
catalase test is the most important test being useful for the identification of bacteria because
this test is quite simple. In catalase test results, no bubble were observed thus indicated that the
isolated bacterial strain was catalase negative and were unable to result in the decomposition
of hydrogen peroxide to produce oxygen.
It is a long time that scientists are trying to substitute synthetic drugs with natural products.
Nowadays, various natural materials and methods are used to prevent or treat diseases.The use
of probiotics is one of these methods. Lactobacilli and Bifidobacterium are normal intestinal
flora which by preventing intestinal infection, lowering cholesterol, stimulating the immune
system, and reducing the risk of colon cancer play an important role in human health. Probiotic
bacteria produce lactic acid and organic acids, reduce the pH environment, and try to prevent
the growth of many bacteria. These bacteria produce antimicrobial compounds such as
bacteriocin which can be used as natural preservatives.
Probiotics pass through the stomach before entering the gut, so the ability to fight gastric juice
is the primary condition for probiotic screening. Usually, the pH of gastric juice in human body
is about 3, and the digestion time is between 1-3 h. the isolated LAB in this study was able to
grow in acidic and alkaline ph. When probiotics enter the intestinal tract, the bile salts in the
small intestine inhibit the probiotics. Therefore, the tolerance of the strains to bile salts is often
one of the important indicators for screening probiotics. Generally, the concentration of bile
salts in human body fluctuates within the range of 0.03% to 0.3% Studies have shown that the
survival rate of lactic acid bacteria more than 70% can grow in different concentrations of bile
salt, and the growth efficiency decreases with the increase of bile salt concentration. The reason
is that high concentration bile salt can make the membrane permeability change, membrane
protein dissociation, eventually lead to intracellular material flow, some cell death. In the
screening of probiotics in vitro, the surface hydrophobicity of bacteria reflects the ability of
bacteria colonization in vivo, as with strong hydrophobic ability, lactic acid bacteria have good
adhesion and strong colonization ability.In this study isolated LAB was also able to survive in
range of 0.03% to 0.3% bile salt concentrations.

.
 Conclusion
In this study lactic acid bacteria was isolated from non-dairy product chicken meat. For lactic
acid bacteria as the main probiotics, its probiotic properties have been an important basis for
scholars to study probiotics. Studies have shown that probiotic lactic acid bacteria can regulate
intestinal flora balance, inhibit cholesterol absorption, antioxidant, anti-tumor and cancer
prevention and so on. In this study, the isolation and identification of a strain of lactic acid
bacteria through physiological and biochemical tests and the ability of isolated LAB to tolerate
different ph,temperature bile salt and NaCl concentrations proved that this lactic acid bacteria
has good probiotic potential with good value in food and bio pharmaceutical development.
 Appendices
Appendix A
List of material
 Pipette
 Microscope
 Petri plates
 Microscopic slides
 Test tubes
 Flask
 Marker
 Dropper
 Wire loop
 Match stick
 Burner

Appendix B
Composition of agar
o Peptone proteose................................10,00
o Meat extract.......................................8,00
o Yeast extract......................................4,00
o D(+)-Glucose...................................... 20,00
o Sodium acetate...................................5,00
o Triammonium citrate.............................2,00
o Magnesium sulfate...............................0,20
o Manganese sulfate.............................. 0,05
o Dipotassium phosphate........................ 2,00
o Polysorbate 80....................................1,00
o Agar..................................................14,00
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