A RESEARCH PROPOSAL

ON

THE PROBIOTICS POTENTIALS OF SOME LACTIC ACID

BACTERIA ISOLATED FROM SOY MILK SOLD IN DUTSIN-

MA METROPOLIS.

BY

DUNIYA DANIEL DAVIDSON

LSC/2021/20116

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 CHAPTER ONE

INTRODUCTION

1.1 BACKROUND OF THE STUDY

The term probiotic was defined as “a live microbial feed supplement

which beneficially affects the host animal by improving its microbial

balance’’ (Aslam and Qazi, 2010). Probiotic bacteria may produce various

compounds, which are inhibitory to the pathogen’s growth, which

include organic acids (lactic and acetic acids), bacteriocins, and reuterin.

The organic acids not only lower the pH, thereby affecting the growth of

the pathogen, but they can also be toxic to the microbes (Tambekar and

Bhutada, 2010). There is increasing evidence that probiotics are

beneficial in gastrointestinal disturbances, such as diarrhoea, dysentery,

typhoid etc (Tambekar and Bhutada, 2010). It is important to underline

when considering the effectiveness and biological activity of probiotics,

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prebiotics or their combination (synbiotics) that they are food products

and not drugs.

Furthermore, in many cases, their effects are mainly prophylactic in

nature, rather than therapeutic, i.e. preventive rather than curative

(Suskovic et al., 2001). Lactic acid bacteria were referred to as probiotics

in scientific literature by Lilley and Stillwell. Lactic acid bacteria (LAB) are

a group of Gram positive, non-spore forming, cocci or rods which

produce lactic acid as major end product from fermentation of

carbohydrates. Majority of microorganisms used as probiotics belong to

the LAB and bifidobacteria. Within the group of LAB, Lactobacillus

species are most commonly utilized group of microorganisms for their

potential beneficiary properties as probiotics. The antagonistic activity of

such bacteria is known to inhibit a large number of enteric and urinary

pathogenic bacteria (Hutt et al., 2006). Lactic acid bacteria including

Lactobacillus, leuconostoc, Lactococcus, pediococcus and

Bifidobacterium are found throughout the gastrointestinal tract.

Lactobacillus and Bifidobacterium spp. are prominent members of the

intestinal flora and are the commonly studied probiotics bacteria. They

cause reduced lactose intolerance alleviation of some diarrhoeas,

lowered blood cholesterol, increased immune response and prevention

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of cancer. The selection criteria for probiotic LAB include: safety,

viability/activity in delivery vehicles, resistance to acid and bile,

adherence to gut epithelial tissue ability to colonise the gastro intestinal

tract, production of antimicrobial substances, ability to stimulate a host

immuneresponse and the ability to influence metabolic activities such as

vitamin production, cholesterol assimulation and lactose activity

(Savodago et al., 2006). Therefore an attempt was made to isolate LAB

from food samples and to evaluate their potential as probiotics. 3

1.2 STATEMENT OF PROBLEM

The increasing demand for plant-based dietary alternatives has led to

the widespread consumption of soymilk, which is often favored for its

nutritional content, including proteins, vitamins, and minerals. While

soymilk has been recognized as a source of beneficial microorganisms,

the probiotic potential of lactic acid bacteria (LAB) isolated from soymilk

remains insufficiently studied. LAB are known for their health benefits,

such as improving gut health, enhancing immune responses, and

preventing pathogen growth (Ouwehand et al., 2016). However,

research into the probiotic properties of LAB isolated specifically from

soymilk is limited, and the mechanisms through which these bacteria

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could contribute to human health have not been thoroughly

investigated.

Probiotics derived from dairy sources are well-documented, but the lack

of comprehensive studies on the probiotic potential of LAB from plant-

based alternatives like soymilk presents a critical research gap (Shah,

2012). Moreover, there is insufficient evidence regarding the survival of

LAB strains isolated from soymilk under gastrointestinal conditions, their

antimicrobial activity, and their ability to adhere to intestinal cells, which

are key factors in determining their effectiveness as probiotics (Mangal

et al., 2015). This gap in knowledge limits the potential development of

functional, dairy-free probiotic foods that could cater to individuals with

lactose intolerance, vegans, or those seeking plant-based probiotic

options.

1.3 JUSTIFICATION OF THE RESEARCH

The research on the probiotic potentials of some lactic acid bacteria

(LAB) in soy milk is crucial for several reasons, considering the increasing

interest in plant-based foods and the therapeutic benefits of probiotics.

Studies have shown that probiotics in soy-based foods can aid in the

management of lactose intolerance, improve gut microbiota diversity,

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and contribute to the reduction of inflammation. Additionally, LAB in soy

milk can help in the prevention of gastrointestinal disorders, such as

irritable bowel syndrome (IBS) and inflammatory bowel diseases (IBD)

(Nami et al., 2018).

1.4 AIM AND OBJECTIVES

AIM

The aim of this study research is to investigate the probiotic potentials of

some lactic acid bacteria in soy milk sold in Dustin-metropolis.

OBJECTIVE

 To enumerate bacteria associated with soy milk sold in Dustin-Ma

metropolis.

 Isolation and identification of lactic acid bacteria associated with

soy milk sold in Dustin-Ma metropolis.

 To determine the probiotic potential of the isolated Lactic Acid

Bacteria (Antibiotic resistance assay, acid tolerance test and

biofilm formation).

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CHAPTER THREE

MATERIALS AND METHOD

3.1 STUDY AREA

This study was conducted in Dutsin-Ma, Dutsin-Ma is a Local

Government Area (LGA) in Katsina State Nigeria. It is located on latitude

and longitude 12°27'18''N, 7°29'29''E respectively. The LGA has an area

of 527 km2 and population of 169,671 as of 2006 census, with Zobe Dam

lying to the south of the town [Wikipedia. Dutsin-Ma. 2025. Accessed on

5th febuary 2025]. The inhabitants of the local government are

predominantly Hausa and Fulani by tribe. Their main occupation is

farming and animal rearing.

3.2 COLLECTION OF SAMPLES

Soy milk samples will be collected from different locations in Dutsin-Ma

Metropolis. Two(2) samples each will be collected from three different

locations namely: Wednesday market, Hospital road, and Hayin gada

local market. Samples will be collected in a sterile container and will be

transported to the Department of Microbiology Laboratory, Federal

University, Dutsin-Ma for further analysis.

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3.3 Isolation of Lactic Acid bacteria

De Man Rogosa Sharpe (MRS) agar will be prepared according to the

manufacturer’s instruction. The isolation of Lactic acid bacteria will be

carried out using the pour plate method. A serial dilution of each sample

will be made by diluting 10-fold in sterile water and 1ml of 10µl aliquots

of 10-3, 10-5 dilutions will be poured into MRS agar and plates will be

incubated at 37oC for 24-48 hours.

3.4 Selection of Lactic Acid Bacteria (LAB)

After incubation, the plates will be examined for colonies with LAB

morphology. The selected colonies will then be sub-cultured on MRS

agar to obtain pure cultures by employing streak plate method. After

streaking on the MRS plate, it was incubated anaerobically at 37oC for

24 – 48hrs. The pure culture obtained will be stored in MRS broth in

McCartney bottle and kept in the refrigerator at 4oC until when

required.

3.5 Identification and Characterization of Isolated Organisms

Suspected colonies of Lactobacillus will be identified based on the

following standard tests: morphology on MRS plate, gram staining,

biochemical tests such as; oxidase test, catalase test, methly red Voges

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Proskauer test, sugar fermentation including fructose, sucrose, maltose,

mannose, sorbitol and cellulose at 1% concentration as substrates using

methly red as indicator

3.5.1 Gram Reaction

Microscopy of isolates will be carried out using Gram staining technique

for all isolates. Under aseptic condition, a smear of fresh culture at 18-

24hrs of the isolates will be mixed with a drop of sterile water on a clean

slide using a well flamed wire loop. The smears will be air dried and heat

fixed by passing the slides over flame. The slides will then be flooded

with crystal violet for 1 – 2mins each. They will be rinsed with sterile

water. The resulting smear will be flooded with gram’s iodine and left to

stand for a minute and rinsed off with water. The smear will then be

decolorized with 75% alcohol. It will be counterstained with safranin for

30 seconds. The slides will finally be dried and examined under an oil

immersion lens.

3.6 Antimicrobial Susceptibility Testing (AST)

The Antibiotic susceptibility testing will be carryout using Agar well

diffusion method. Using a sterile loop, few colonies of 18-24 hours old

test isolate on Mueller-Hinton agar (MHA) will be dislodged into normal

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saline and standardized to 0.5 McFarland standard. A pathogenic

organism (Escherichia coli) will be inoculated onto the MHA plate by

streaking using a sterile loop. Thereafter, wells will be made in MHA

plate uniformly using a sterile cork bore. 100μl of the LAB culture will be

added to the wells. The antibiotic discs will be aseptically dispense on

the surface of the MHA using forceps. The antibiotics to be used include:

Amoxicillin/clavulanic acid (AMC) 30μg, gentamicin (GEN) 10μg,

ciprofloxacin (CIP) 5μg, tetracycline (TET) 30 μg, and ampicillin (AMP) 10

μg. The plates will be incubated at 37 ℃ 24 hours after which it will be

observed for the presence of clear zone. The clear zones will be

measured in millimeters and the results will be interpreted using the

guidelines of Clinical Laboratory Standard Institute (CLSI, 2024).

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REFERENCES

Mangal, M., Mendiratta, S., Sharma, B. (2015). Probiotics and their role

in gut health. International Journal of Food Science and Technology,

50(1), 25-35.

Nami, Y., Ghazali, H. M., & Ramasamy, K. (2018). "Probiotic potential of

lactic acid bacteria in soy milk: Functional properties and health

benefits." Food Research International, 108, 469-479.

Ouwehand, A., Salminen, S., von Wright, M. (2016). Lactic Acid Bacteria:

Microbiological and Functional Aspects. Springer.

Ram Kumar Pundir, Satish Rana Neha kashyap and Amandeep Kaur.

Probiotic Potential of Lactic acid Bacteria Isolated from food Samples –

an in Vitro study. J App Pharm Sci. 2013; 3 (03): 085-093.

Shah, N. P. (2012). Probiotics and prebiotics: A brief overview. Food

Science and Technology Bulletin: Functional Foods, 8(1), 1-6.

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