IDENTIFICATION AND CHARACTERIZATION OF LACTOBACILLUS

ISOLATES, RECOVERED FROM LOCALLY FERMENTED MILK (NUNU)

WITHIN BAUCHI METROPOLIS.

BY

ISMAILA ABDULLAHI

17/46480U/1

A PROJECT PROPOSAL SUBMITTED TO THE DEPARTMENT OF

MICROBIOLOGY ABUBAKAR TAFAWA BALEWA UNIVERSITY,
BAUCHI, NIGERIA IN PARTIAL FULFILMENT OF THE REQUIRMENT FOR
THE AWARD OF THE BACHELOR OF TECHNOLOGY (B.Tech.) DEGREE
IN APPLIED MICROBIOLOGY

SURPERVISED BY:

MRS AGBO VERONICA

 MARCH, 2024

CHAPTER ONE

1.1 Background of the study

Milk is an excellent source of vitamins and minerals, particularly calcium. Milk

proteins increase the value of poorer quality cereal and vegetable proteins in the
diet by providing the amino acids these proteins lack, protects against tooth decay,
reduces oral acidity (which causes decay), stimulates saliva flow, and decreases
plaque formation. (Silva et al.2016) Raw milk has low keeping quality and at room
temperature, spontaneous microbial spoilage occurs turning the product sour few
some days. This is brought about by the activity of lactic acid bacteria. (Rodríguez
et al. 2019) Locally fermented milk is a form of food processing where microbes,
for example, lactic acid bacteria (LAB) are utilized for food production via the
process known as fermentation. Fermentation in food processing is the conversion
of carbohydrates to alcohol and carbon dioxide or organic acids using yeast and/or
bacteria, under anaerobic conditions. (franc,et.al2014)Fermentation is one of the
classic methods to preserve foods. The fermentation techniques are often a small
scale and household basis, characterized by the use of simple non-sterile
equipment, chance or natural inoculums, unregulated conditions, sensory
fluctuations, poor durability and unattractive packing of the processed products
resulting in food of unpredictable quality [4]. Locally fermented milk is processed
by collecting fresh cow milk and allowing it to ferment for a day or two. The
Fulanis ferment the milk in calabashes, or rubber buckets. Locally fermented milk
(nunu) is yoghurt-like in taste (a sharp acid taste) and can be consumed alone or
with sugar and fura. Even when the milk is fermented, the fermentation process
with the attendant drop in pH may not rid the product of these organisms and may
be carried to consumers. Salmonellae and other microorganisms have been known
to underscore the importance of milk and milk products as vehicle for human
infections.

Lactobacillus is considered as part of the indigenous microflora of the mammalian

gastrointestinal tract and of many other niches and fermented foods. (Naeem et al.
2016) Locally fermented milk contains a good balance of protein, fat and
carbohydrate and is a very important source of essential nutrients including:
Calcium, Riboflavin, Phosphorous, Vitamins A and D, Pantothenic acid. There are
claims that the digestibility of the milk proteins is improved by fermentation The
Lactobacillus genus consists of a genetically and physiologically diverse group of
rod-shaped, Gram-positive, non-spore forming, none pigmented catalase negative
and microaerophilic to strictly anaerobic. Mainly, the ability to reduce serum
cholesterol levels, antimicrobial substrate production and immune modulation are
considered as effective properties, in which, Lactobacillus (Lactic Acid Bacteria,
LAB) is a commercially important bacterium with wide variety of application, both
in the food industry and as a probiotic agent for the improvement of human health
From the health point of view, ingestion of live cells of certain species and strains,
the probiotic concept of lactobacilli in adequate amounts is believed to confer
several beneficial physiological effects on the host such as maintaining a healthy
and equilibrated intestinal microbiota and reducing incidence of intestinal
infection. The criteria for the in vitro selection of lactobacilli to be used as health-
promoting, probiotic ingredients, in food and pharmaceutical preparations include
antibiotic tolerance as well as the production of lactic acid that inhibits the growth
of other microorganisms, which allow them to be established in the intestinal tract.
Most Lactobacillus strains regardless of their source harbour at least one
indigenous plasmid and often more in which, some lactic acid bacteria may carry
potentially transmissible plasmid encoded antibiotic resistance genes and any
strains harbouring antibiotic resistance plasmids are considered unsuitable for use
as human or animal probiotics. However, the importance of intrinsic antibiotic
resistant strains which may benefit patients whose normal intestinal micro biota
has become unbalanced or greatly reduced in numbers due to administration of
various antimicrobial agents have also been reported [13], [14]. These health
benefits have been attributed to an array of antimicrobial substances produced by
lactobacillus especially bacteriocin.“Nunu”, as a locally fermented milk is being
produced in limited daily consumable quantities due to its poor keeping quality.
Deterioration of this locally fermented milk starts quickly during storage in terms
of growth of microorganisms (lactic and non-lactic) present naturally or as
contaminant. Poor hygiene, practiced by handlers of the products, may lead to
introduction of pathogenic microorganisms into the products and since they do not
undergo further processing before consumption, this food may pose risk to the
consumer. Therefore, screening of locally fermented milk products for
lactobacillus composition by species, cell density, and probiotic functions remain
the standard measures of quality and marketability in human population to promote
public health. Knowledge of the biochemical and microbial changes that are
associated with its processing will obviously enhance the production and proper
utilization on a larger scale.

Therefore, this work investigates the bacteriocin-like activity and probiotic value
of locally fermented milk consumed in Bauchi, Bauchi,Nigeria. The objectives are
to identify Lactobacillus isolates present in a selected sample of locally fermented
milk consumed within Bauchi metropolis in Bauchi State of Nigeria. To determine
the total Lactobacillus bacteria,count in the locally fermented milk product and
their percentage resistance to antibiotics by the isolated Lactobacillus strains. To
determine the bacteriocinogenic activity of the Lactobacillus isolates against multi
drug resistance E. coli (FAO/WHO, 2015).

1.2 Statement of problem

Locally fermented milk products play a significant role in many cultures

worldwide, offering nutritional benefits and contributing to the preservation of
traditional food practices. However, despite their importance, there is often limited
knowledge regarding the microbial composition and probiotic potential of these
indigenous fermented milk products. The identification and characterization of
Lactobacillus isolates recovered from locally fermented milk present several key
challenges and gaps in current understanding

Microbial Diversity: Locally fermented milk products are known to harbor a

diverse microbial community, including various lactic acid bacteria such as
Lactobacillus species. However, the specific composition and abundance of
Lactobacillus isolates within these products remain largely unexplored.
Probiotic Potential: While Lactobacillus species are widely recognized for their
probiotic properties, including gastrointestinal health benefits, the probiotic
potential of isolates from locally fermented milk is poorly understood. It is
essential to assess the viability, functionality, and safety of these isolates to
determine their suitability as probiotic candidates.

Characterization Challenges: Traditional methods of phenotypic characterization

may not provide sufficient resolution to accurately differentiate between closely
related Lactobacillus species. Molecular techniques, such as genomic sequencing,
are necessary to elucidate the genetic diversity and functional attributes of these
isolates.

Impact on Food Quality: The presence and activity of Lactobacillus strains in

locally fermented milk products can influence the sensory attributes, shelf-life, and
safety of these foods. Understanding the role of specific Lactobacillus isolates in
the fermentation process is crucial for optimizing product quality and ensuring
consumer acceptance. Addressing these challenges requires a comprehensive
approach that integrates molecular biology, microbiology, food science, and
probiotic research. By elucidating the microbial composition and probiotic
potential of Lactobacillus isolates recovered from locally fermented milk, this
study aims to fill critical knowledge gaps and provide insights that can inform the
development of nutritious, safe, and culturally relevant fermented dairy products.

1.3 Aim of the study

The aim of this study is to understand the microbial diversity present in the locally
fermented milk (Nunu), specifically focusing on the Lactobacillus genus. This
involves isolating individual strains of Lactobacillus bacteria from the fermented
milk, identifying them through various molecular and biochemical techniques, and
characterizing their phenotypic and genotypic traits. By doing so, researchers can
gain insights into the potential probiotic properties, technological characteristics,
and safety aspects of these Lactobacillus strains, which can have implications for
improving the quality and health benefits of locally fermented milk products.
1.4 Objectives of the study

The specific objectives of this study are;

i. To Isolate and purify Lactobacillus strains from locally fermented milk

samples.

ii. To Identify the isolated Lactobacillus strains using molecular techniques

such as PCR and sequencing.

iii. To characterize the phenotypic traits of the Lactobacillus isolates, including

morphology, growth characteristics, and biochemical properties.

iv. To valuate the probiotic potential of the isolated Lactobacillus strains,

including acid and bile tolerance, adhesion to intestinal epithelial cells, and
antimicrobial activity.

v. To compare the identified Lactobacillus isolates with known probiotic

strains for potential commercial applications in dairy products,
understanding the microbial diversity and dynamics in locally fermented
milk to improve traditional production methods and product quality.
 CHAPTER TWO

LITERATURE REVIEW

2.1 An Overview Of Lactobacillus

Lactobacillus is a genus of bacteria commonly found in the digestive, urinary, and

genital systems of humans and animals. They are considered beneficial or
"friendly" bacteria because they can help maintain a healthy microbial balance.
Lactobacillus species are used in various fermented food production, such as
yogurt, cheese and pickles (George et al 2018)Additionally, they are often used as
probiotics to promote digestive health and boost the immune system. Lactobacillus
strains are diverse, with different species and strains offering various health
benefits. (Zheng et, al 2020)

Lactobacillus is a genus of Gram-positive, facultative anaerobic bacteria that

belongs to the phylum Firmicutes. It encompasses a diverse group of species, many
of which are naturally found in the gastrointestinal tract, oral cavity, and female
reproductive system of humans and animals. These bacteria play significant roles
in maintaining the health of their host organisms through various mechanisms,
particularly in the context of the gut microbiota.

2.2 Taxonomy and Classification:The genus Lactobacillus is taxonomically

classified within the order Lactobacillales, family Lactobacillaceae. As of the latest
taxonomic updates, there are over 260 recognized species within the genus, with
ongoing research leading to the discovery of new strains and variants. ( Salam,
2016)

2.3 Morphology and Physiology: Lactobacillus species are typically rod-

shaped or bacilli, although some may appear coccoid or filamentous under certain
conditions. They are non-spore forming and have a simple cell structure with a
thick cell wall composed of peptidoglycan. Like other Firmicutes, they lack an
outer membrane.In terms of metabolism, Lactobacillus species are facultative
anaerobes, meaning they can grow in both the presence and absence of oxygen.
They primarily ferment sugars to produce lactic acid, which contributes to their
acidophilic nature and ability to thrive in acidic environments.

2.4 Ecology and Habitat: Lactobacillus species are widespread in nature and
can be found in various ecological niches, including the human body, plants, soil,
water, and fermented foods. In humans, they are particularly abundant in the
gastrointestinal tract, where they form a significant portion of the gut microbiota,
contributing to its stability and functionality.

2.5 Characteristics of Lactobacillus: Lactobacillus is a genus of Gram-

positive, facultatively anaerobic bacteria belonging to the phylum Firmicutes. They
are rod-shaped, non-spore-forming, and typically appear singly, in pairs, or short
chains under a microscope. Lactobacilli are widely distributed in nature,
particularly in dairy products, plant materials, and the gastrointestinal tracts of
animals and humans. Here are some extensive characteristics of lactobacilli:

2.5.1 Metabolism: Lactobacillus species are heterofermentative or

homofermentative. Homofermentative lactobacilli primarily produce lactic acid as
the major end product during fermentation, while heterofermentative lactobacilli
produce lactic acid, acetic acid, ethanol, and carbon dioxide. This metabolic
versatility allows them to thrive in diverse environments.
2.5.2 Acid tolerance: Lactobacilli are acid-tolerant bacteria, capable of
surviving and proliferating in acidic conditions. This characteristic is crucial for
their survival in the acidic environment of the stomach and the fermentation of
acidic foods such as yogurt, pickles, and sauerkraut.

2.5.3 Probiotic properties: Many lactobacillus species exhibit probiotic

properties, promoting gastrointestinal health and offering various health benefits.
They can inhibit the growth of pathogenic bacteria by competing for nutrients and
adhesion sites in the gut, modulate the immune response, and enhance the barrier
function of the intestinal epithelium.

2.5.4 Fermentation: Lactobacilli play a vital role in the fermentation of

various foods, including dairy products, vegetables, and meats. They contribute to
the development of flavor, texture, and preservation of fermented foods through
acidification, production of antimicrobial compounds, and metabolic activities.

2.5.5 Genetic diversity: The genus Lactobacillus encompasses a wide

range of species with diverse genetic characteristics. This diversity allows
lactobacilli to adapt to different ecological niches and perform various metabolic
functions, contributing to their versatility and importance in industrial and
biomedical applications.

2.5.6 Health benefits: Beyond their role in food fermentation, lactobacilli have
been extensively studied for their potential health benefits. These include
improving digestive health, alleviating symptoms of irritable bowel syndrome,
preventing antibiotic-associated diarrhea, enhancing immune function, and
reducing the risk of certain infections.

2.5.7 Safety: Lactobacilli are generally regarded as safe (GRAS) by regulatory

authorities and have a long history of safe use in food fermentation and probiotic
applications. However, some strains may pose a risk to individuals with
compromised immune systems or underlying health conditions, highlighting the
importance of strain-specific safety assessments.

2.5.8 Industrial applications: Due to their metabolic activities and

probiotic properties, lactobacilli have numerous industrial applications. They are
used in the production of fermented dairy products, sourdough bread, pickles,
kimchi, and other fermented foods. Additionally, they are employed as probiotic
supplements in dairy products, capsules, and functional foods.

In summary, lactobacilli are versatile bacteria with diverse metabolic capabilities,

probiotic properties, and industrial applications. Their ability to ferment sugars into
lactic acid, tolerate acidic conditions, and promote gastrointestinal health make
them invaluable in food production, human health, and biotechnology.

2.7 Fermentation Process Of Lactobacillus

Lactobacillus fermentation is a fundamental biological process used in various

industries, particularly in food production, biotechnology, and pharmaceuticals.
Lactobacillus is a genus of Gram-positive, facultative anaerobic bacteria that
belong to the lactic acid bacteria group. These bacteria are renowned for their
ability to convert sugars into lactic acid through fermentation, a process that not
only preserves food but also imparts unique flavors and textures. Here, we will
delve extensively into the fermentation process of Lactobacillus, exploring its
biochemical pathways, regulatory mechanisms, and industrial applications. The
fermentation process in Lactobacillus begins with the uptake of sugars such as
glucose, fructose, sucrose, or lactose. These sugars are transported into the
bacterial cell and metabolized through the glycolytic pathway. During glycolysis,
glucose is enzymatically converted into pyruvate in a series of sequential reactions,
generating a small amount of ATP and reducing equivalents (NADH).Lactic Acid
Production: In the absence of oxygen, pyruvate is further metabolized to lactic acid
through the process of homolactic fermentation. This conversion is catalyzed by
the enzyme lactate dehydrogenase, which reduces pyruvate to lactic acid while
oxidizing NADH to NAD+. The regeneration of NAD+ is crucial for sustaining
glycolysis, allowing Lactobacillus to continue metabolizing sugars
anaerobically.Regulation of pH: Lactic acid production by Lactobacillus leads to a
decrease in pH, creating an acidic environment in the fermentation medium. This
decrease in pH inhibits the growth of spoilage microorganisms and pathogens,
contributing to the preservation of the fermented product. pH regulation is essential
for maintaining the stability and safety of fermented foods. Lactobacillus employs
carbon catabolite repression to regulate the expression of genes involved in sugar
metabolism. When multiple sugars are present in the environment, Lactobacillus
preferentially utilizes the most easily metabolizable sugar while repressing the
utilization of others. This regulatory mechanism ensures efficient energy utilization
and substrate preference. Lactobacillus maintains intracellular pH homeostasis to
survive and thrive in acidic environments. This involves the expression of proton
pumps and ion transporters that regulate the influx and efflux of ions across the
cell membrane, balancing the intracellular and extracellular pH levels.
Lactobacillus fermentation is extensively used in the production of various
fermented foods and beverages, including yogurt, cheese, sauerkraut, kimchi,
sourdough bread, pickles, and fermented meats. The fermentation process not only
enhances the flavor, texture, and nutritional quality of these products but also
extends their shelf life through acidification and preservation. Certain strains of
Lactobacillus are utilized as probiotics, which are live microorganisms that confer
health benefits when consumed in adequate amounts. Probiotic Lactobacillus
strains are commonly found in fermented dairy products, dietary supplements, and
functional foods, where they promote digestive health, boost the immune system,
and may alleviate gastrointestinal disorders. Lactobacillus fermentation is
employed in various biotechnological applications, including the production of
lactic acid, which serves as a precursor for the synthesis of biodegradable plastics,
pharmaceuticals, cosmetics, and food additives. Additionally, Lactobacillus-based
fermentation processes are used in the production of enzymes, vitamins, and other
bioactive compounds. (Philip et al., 2017)
 CHAPTER THREE

MATERIALS AND METHODS

3.0 Study Area

This study will be carried out in Abubakar Tafawa Balewa University Bauchi,
Yelwa Campus. The geographic location of Bauchi state located between latitudes
9° 3´ and 12° 3´ North and longitudes 8° 50´ and 11° 0´ in the North-Eastern part
of Nigeria has a total land area of 49,119 km2 representing about 5.3% of the
country’s total land mass and extents two distinct vegetation zones, namely the
Sudan savannah and the Sahel savannah. The Sudan savannah type of vegetation
covers the southern part of the state with the vegetation getting richer and richer
towards the South. The Sahel type of savannah becomes manifest from the mid of
the state as one moves from south to northern part of the State.

3.1.1 Study Design

3.1.2 Collection Of Sample

Thirty (30) non-replicate samples of locally made fermented milk will be collected
from six different locations at different times over one month in Bauchi metropolis,
of Bauchi State. They will be bought randomly and collected aseptically from
sellers at the following locations: Yelwa market , ATBU yelwa campus, ATBU
Gubi campus, Awala round about, Wunti market and Yankari motor park.

3.1.3 Materials and Reagents Use

They include wire loop, autoclave, beaker, Conical flask, Incubator, Distilled
water, Test tubes, Hot air oven, Bunsen burner, Petri dishes, Cotton wool, Hand
gloves, Microscope, sterile glass ware, 70% alcohol, samples of Locally Fermented
Milk (Nunu)MRS agar, Forcep, Gram staining kit and biochemical testing
reagents.

3.2 Serial Dilution

9 ml of sterile distilled water will be dispensed into six (6) test tubes. After which 1
ml of the sample (Nunu) will be transferred into the first test tube and then mixed
properly after which 1 ml again will be removed from the first test tube and then to
be transferred to the second test tube and then repeat for the third, fourth, fifth and
sixth tube in other to obtain a serial dilution of (10-1, 10-2, 10-3, 10-4, 10-5 and
10-6). The procedure will be repeated for all the samples to be collected.

3.3 Preparation of Media

3.3.1 Preparation of Man Rogosa De-Sharpe (MRS) Agar

The MRS agar will be prepared according to the manufacturer’s instruction. 62.2 g
of MRS agar will be dissolved in distilled water (1 L) and then heat to dissolve.
The solution will be sterilized by autoclaving at 121 °C for 15 min then allow to
cool at 45 °C before dispensing into petri-dish. The MRS Agar will be used for
isolation of the lactobacillus

3.4 Isolation of lactobacillus from nunu (Pour Plate Method)

Each sample will be allowed to stand for two minutes, and the last test tubes with
dilution 10-6 is to be shaken in order to have a homogenous mixture and
homogenous distribution of the microorganism with the aid of a sterile pipette, 0.1
ml of each solution will be transferred into different sterile Petri dishes, before the
media are poured into the Petri dishes using a pour plate technique. The inoculated
plates will be incubated at 37°C for 24 hours for bacteria and at 37°C for 72 hours
for fungi. The plates will therefore be observed for colonies and other
morphological appearances.

3.5 Subculturing of lactobacillus Isolates

Pure colonies will be obtained by subculturing presumed isolates of lactobacillus

on Nutrient agar slant. The nutrient agar slant will be kept in the fridge until they
are used for morphological and biochemical test.

3.6 Morphological Characteristics of Isolates

3.6.1 Gram Staining Techniques

Gram staining will be carried out on the isolates. A smear of the culture will be
made on a clean grease free slide labeled with each isolate code and heat fixed to
dry. The smear will then be stained with crystal violet for 60 seconds after which
it will be rinsed in water. Few drops of Lugol's iodine solution (Gram’s iodine) will
be added and allowed for 60 seconds. The smear will be decolourized with 95%
ethanol for 30 s and immediately rinse with tap water. The slide will be counter
stained with Carbon Fuchsin for 1 min and rinse with water and then dried with
Whatman filter paper. Gram-positive cells will appear purple while Gram-negative
cells are red (Ochei et al., 2000).

3.7 Biochemical Characteristic of the Isolates

3.7.1 Catalase Test

A microscopic slide will be placed inside a petri dish. Using a sterile inoculating
loop, a small amount of microorganism from 24-hour pure culture will be placed
onto the microscopic slide. 3% H2O2 solution will be added to each of the slides
and a portion of the bacterial colony will be mixed with it. Production of bubble
will indicate the presence of catalase enzyme in the bacteria (Reiner, 2010).

3.7.2 Citrate utilization

This test will be used to determine if an organism can utilize citrate as its sole
source of carbon and energy. The citrate test uses a medium in which sodium
citrate is the source of carbon and energy. In the Simon's citrate agar, the pH
indicator is bromothymol blue, which is green neutral pH and becomes blue when
the medium become alkaline.
Slopes slant of Simon’s citrate agar will be prepared in bijou bottles and the test
organisms will be inoculated by streaking the surface and stabbing the butt with a
sterilized inoculating needle and to be incubated at 35 ℃ for 48 hours and will be
observed for a bright blue colour in the medium which indicates a positive result
(Chessbrought 1991).

3.7.3 Methyl Red Test

Methyl red test should be performed to determine the ability of the bacteria to
oxidize glucose with the production of high concentration of acidic end products.
Glucose phosphate broth will be inoculated with the isolates and incubated for 2-5
days at 37°C. Then 5 drops of methyl red indicator (0.2%) will be added and
carefully observed for immediate development of a red color which indicates a
positive test for acidic products (Ferdous et al., 2013).

3.7.4 Voges Proskauer test

The Voges Proskauer test indicates the production of acetoin from the fermentation
of glucose. The MR-VP broth (5ml) will be inoculated with the test organisms and
incubated for 48 hours. 0.6ml of alpha-napthol and 0.2ml of 40% potassium
hydroxide will be added to the second test tube. The broth will be left to stand for
15 min for colour development after thorough agitation. If acetoin is produced,
there will be a red colour change. A yellow to brown colour indicates a negative
result (Chesbrough 2006).

REFERENCES

1. International Journal of Probiotics & Prebiotics, 2017

2. Albert Wu G. (2014). Sequencing of diverse mandarin, pomelo and orange

genomes reveals complex history of admixture diary food domestication.
Nature Biotechnology. 32 (7): 656–662.
3. Albuquerque, T.M.R., Garcia, E.F., de Oliveira Araújo, A., Magnani, M.
(2018). In Vitro Characterization of Lactobacillus Strains Isolated from food
products as Potential Probiotics. Probiotics and Antimicrobial Proteins. 10,
704-716.

4. Asian Food Science Journal, 2018

5. Araújo, K.L.G.V., Magnani, M., Nascimento,J.A., Souza, A.L.,

Epaminondas, P.S., Souza, A.L. (2017). Antioxidant activity of co-products
from , cow milk produced in the Brazilian Northeast. Molecules 19, 3110–
3119.

6. Ayala-Zavala, J.F., Rosas-Domínguez, C., Vega-Vega, V., and González-

Aguilar, G.A. (2015). Antioxidant enrichment and antimicrobial protection
of fresh-cut fruits using their own byproducts: looking for integral
exploitation. Journal of Food Science. 25,175-181.

7. Aziz SA, Olival KJ, Bumrungsri S, Richards GC, Racey PA (2016). The
Conflict Between Pteropodid cows Species, Legislation and Mitigation. In
Voigt C, Kingston T (eds.). cows in the Anthropocene: Conservation of
cows in a Changing World. Springer. pp. 377–426.

8. Identification and Characterization of Lactobacillus Isolates Recovered from

Locally Fermented Milk (Nunu) consumed within Lagos Metropolis".
Afribary, Afribary, 05 Nov. 2021. Web. 11