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UNIVERSITY OF GONDAR
INSTITUTE OF BIOTECHNOLOGY
DEPARTMENT OF BIOTECHNOLOGY
Probiotics as an alternative potential therapeutic measure against selected foodborne
pathogens and mastitis causing bacteria
A thesis submitted to the department of biotechnology, university of gondar in partial
fulfillment of the requirements for degree of master of science in biotechnology
By
Awel Seid
Advisor: Professor Nega Berhane
Co-Advisor: Getachew Gugsa (DVM, MSc, Associate Professor)
September, 2020
Gondar, Ethiopia
APPROVAL SHEET
This thesis proposal entitled ‘Probiotic as an alternative potential therapeutic measures
against foodborne pathogens and mastitis causing bacteria’ has been Submitted by Awel
Seid for Presentation with My Approval Institute of Biotechnology.
Advisor
Advisor Name: prof. Nega Berhane, Signature: _______, Submission date / /2020
Co-Advisor Name: Dr. Getachew Gugsa, Signature: _______, Submission date: / /2020
Examiners
External Examiner Name: _____________, Signature: ______, Submission date: / /2020
Internal Examiner Name: _____________, Signature: ______, Submission date: / /2020
Chairperson Name: _____________, Signature: _______, Submission date: / /2020
Table of Contents
Acknowledgements ........................................................................................................................ I
List of tables................................................................................................................................... II
List of figures ............................................................................................................................... III
List of annexes ............................................................................................................................. IV
List of abbreviations and acroniums .........................................................................................V
Abstract ........................................................................................................................................ VI
1. Background and justification .................................................................................................. 1
1.1. Statement of the problem .................................................................................................... 4
1.2. Significance of the study ...................................................................................................... 6
1.3. Objectives ............................................................................................................................ 7
1.3.1. General Objective -----------------------------------------------------------------------------------------7
1.3.2. Specific Objectives---------------------------------------------------------------------------------------7
2. Literature review ...................................................................................................................... 8
2.1. Lactic acid bacteria ............................................................................................................. 8
2.2. Taxonomical classification of lactic acid bacteia ............................................................... 9
2.3. Lactic acid bacteria as probiotics ...................................................................................... 10
2.4. Origin and aafety of probiotics ......................................................................................... 12
2.5. Antimicrobial compounds of lactic acid bacteria ............................................................. 12
2.5.1. Organic Acids ............................................................................................................. 13
2.5.2. Reuterin ....................................................................................................................... 14
2.5.3. Bacteriocin .................................................................................................................. 14
2.5.4. Hydrogen peroxide...................................................................................................... 15
2.6. Safety of probiotics ........................................................................................................... 15
2.7. Selection criteria of probiotics .......................................................................................... 16
3. Materials and methods ........................................................................................................... 17
3.1. Description of study sreas ................................................................................................. 17
3.2. Sample collection and handling ........................................................................................ 18
3.3. Standard bacterial strains and clinical isolates .................................................................. 18
3.4. Isolation and identification of Lactpbacillus species ....................................................... 20
3.4.1. Isolation and morphological characterization of Lactobacillus species ................... 20
3.4.2. Identification of Lactobacillus by biochemical tests .................................................. 21
3.5. Antibacterial activity of lactobacillus species................................................................... 23
3.6. Optimization of growth parameters .................................................................................. 23
3.7. Antibiotic sensitivity pattern of lactobacillus ................................................................... 24
3.8. Determination of minimum inhibitory concentration (Mic) ............................................. 25
3.9. Determination of minimum bactericidal concentration (Mbc) ......................................... 27
3.10. Data Management And Analysis ..................................................................................... 27
4. Results and discussion ............................................................................................................ 28
4.1. Isolation and identification of Lactobacillus species ........................................................ 28
4.2. Antibacterial activity assay of Lactobacillus species ....................................................... 29
4.3. Optimization of growth parameters .................................................................................. 33
4.4. Antibiotic sensitivity pattern of Lactobacillus species ..................................................... 37
4.5. Determination of minimum inhibitory concentration (Mic) ............................................. 39
4.6. Determination of minimum Bactericidal concentration (Mbc) ........................................ 41
5. Conclusion and recommendations ........................................................................................ 42
6. References ............................................................................................................................... 44
7. List of annexes ........................................................................................................................ 59
I
Acknowledgements
First and foremost, I would like to sincerely thank my advisor, Professor Nega Birhane, for his
intellectual guidance, constructive comments and devotion of time in appraising this paper. It has
been a great pleasure and honor to have him as my advisor.
I would like forward my warmest thank to my Co-advisor, Dr. Getachew Gugsa, for giving me a
chance to join his research group in my thesis work, it is truly an honor. Successful
accomplishment of this project would never have been possible without his support, from early
design of proposal development up to the final thesis write-up.
I am also deeply thankful to Mr. Demsew Bekele, the laboratory Technician at Wollo University,
for his unreserved assistance and technical support throughout my work and to Dr. Engidaw
Abebe for his wise and professional contribution during the analysis of the present experimental
data.
I would like to extend my gratitude to Amhara National Regional Veterinary Laboratory Institute
Kombolcha Branch, for providing us with some laboratory chemicals and equipment. I also
acknowledge Amhara Public Health Institute Dessie Branch (A.P.H.I) for supplying all standard
test microorganisms and consumable equipment for this project.
I extend my sincere gratitude to the School of Veterinary Medicine, College of Health Sciences,
and School of Bio-Sciences and Technology of Wollo University as well as Dessie Tissue
Culture Center for providing the necessary materials, chemicals, reagents, and facilities required
for the research work.
II
List of tables
Table 1: Physiological and biochemical characteristics of lactobacilli strains. ............................ 27
Table 2:Carbohydrates fermentation profile of Lactobacillus species. ........................................ 28
Table 3: Antimicrobial activity assay of Lactobacillus isolates against Standard bacterial strains
of foodborne pathogens and mastitis-causing bacteria. ................................................... 32
Table 4: Antimicrobial activity assay of Lactobacillus isolates against Drug resistance bacterial
strains of foodborne pathogens and mastitis-causing bacteria. ....................................... 32
Table 5: The growth performance of the Lactobacillus isolates at different pH. ........................ 33
Table 6: Multiple linear regression results of the absorbance values of different isolates among
the two explanatory variables. ......................................................................................... 36
Table 7: Drug sensitivity pattern of Lactobacillus species. .......................................................... 38
Table 8: The MIC and MBC values of the Lactobacillus isolates against the selected standard
bacterial foodborne pathogens and mastitis-causing bacteria in percentage. .................. 40
Table 9: The MIC and MBC values of the Lactobacillus isolates against the selected clinical
isolate bacterial foodborne pathogens and mastitis-causing bacteria in percentage. ...... 41
III
List of figures
Figure 1: Map of the study areas................................................................................................... 17
IV
List of annexes
Annex 1: pH optimization of Lactobacillus isolates..................................................................... 59
Annex 2: Absorbance value of Lactobacillus isolates . ................................................................ 59
Annex 3: Colony morphology and Gram staining of Lactobacilli. .............................................. 65
Annex 4: Antibacterial activity of Lactobacillus against S.aureus American Type Culture
Collection (ATCC25923)/Drug sensitivity of Lactobacillus (D5). .............................. 65
Annex 5: MIC values of Lactobacillus against the standard and clinical pathogens. .................. 66
V
List of abbreviations and acronyms
CDC Center for Disease Control and prevention
CFE Cell-Free Extract
CFU Colony Forming Unit
CLSI Clinical and Laboratory Standards Institute
CSA Central Statistical Authority
EFSA European Food Safety Authority
FAO Food and Agricultural Organization
FBD Food Borne Disease
GRAS Generally Regard As Save
ISAPP International Scientific Association for Probiotics and Prebiotics
LAB Lactic Acid Bacteria
MBC Minimum bactericidal Concentration
MHA Mueller Hinton Agar
MIC Minimum Inhibitory Concentration
MRS de Man Ragosa and Sharpe
OD Optical Density
OF Oxidation Fermentation
WHO World Health Organization
ZDIs Zone Diameter Inhibitions
VI
Abstract
In recent years, the concept of biological control has emerged as one interesting sustainable
alternative to fight against pathogens. Thus, the present study aims to isolate, characterize, and
evaluate indigenous beneficial lactic acid bacterial (LAB) strains as an alternative potential for
the therapeutic and prevention of selected mastitis and foodborne bacterial pathogens. A total of
48 milk samples were aseptically collected. Nine standard bacterial strains and four clinical
isolates were used in this experiment. Antibiotic sensitivity test was performed for both bacterial
pathogens and Lactobacillus isolates. Isolation Lactobacillus species were done using de Man
Ragosa and Sharpe (MRS) agar plates by streak plate method, and primary and secondary
biochemical tests were conducted to phenotypic identification of lactobacillus species. Assay of
antibacterial activity of Lactobacillus species was performed by agar well diffusion method in
triplicates. A volume of 0.1ml of bacterial extract was used. Optimization of growth parameters
for pH, temperature, and the incubation period was conducted. Besides, determination of the
Minimum inhibitory concentration (MIC) and Minimum bactericidal concentration (MBC) were
performed. The obtained results were analyzed by both descriptive and analytic statistics. A total
of 11 isolates were identified as species of genus Lactobacillus. All isolates had varying degrees
of inhibition towards the test pathogens. Lb. acidophilus, Lb. rhamnosus and
Lb. plantarum subsp. plantarum had the most potent antibacterial activity against the standard
bacterial species S. aureus, S. agalactiae and S. pyogenes. All isolated Lactobacillus spp. had
showed maximum growth between pH 5.0 to 7.0. Except B7 and PK2 all isolates had shown the
optimal growth performance at 37°C and 48hr.of incubation period. The highest level of
sensitivity of LAB isolates had shown towards Penicillin G, Ciprofloxacin, Gentamycin,
Chloramphenicol, and Erythromycin. The lowest MIC value (3.125%) was observed in L.brevis
against S. aureus and S. agalactiae and Lb. rhamnosus against S. aureus. The MBC values
revealed that L.brevis (D5), L.rahmanousus (D71) and Lb. acidophilus (K91) had shown the
lowest MBC value with 6.25% against standard and 12.5% Clinical bacteria. It could be
conclude that all isolated LAB showed remarkable inhibitory effect against all the tested
pathogens. These results suggest that some of these isolates could be used as potential probiotic
candidates.
Keywords: Antibacterial activity; Lactic acid bacteria; indigenous; inhibition; Probiotic
1
1. Background and justification
Among the bovine infections, mastitis is one of the most prevalent, important, and expensive
production diseases affecting the dairy cattle industry worldwide (Alert, 1995; Petrovski et al.,
2006 and Kumar, 2017). Mastitis remains the most economically destructive and serious
zoonotic imminent disease for consumers irrespective of many years of research worldwide with
different levels of economic losses identified by different countries (Seegers et al., 2003).
Production losses due to subclinical mastitis in Ethiopia crossbreed dairy cows have been
estimated to be 38 US$ per lactation per cow. Subclinical mastitis accounts for over 90% of the
total loss in milk production. However, most dairy farmers in the country normally do not
recognize subclinical mastitis, which incidentally occurs at a much higher frequency than clinical
mastitis (Mungube, 2005).
Mastitis is caused by a wide spectrum of pathogens and, epidemiologically categorized into
contagious and environmental mastitis (González and Wilson, 2003). More than 140 different
types of organisms may cause mastitis, and these etiological agents are classified into contagious
pathogens and environmental (Radostits et al., 2002 and Langoni et al., 2011). The most
common mastitis pathogens occur either in the cow’s udder, known as contagious pathogens or
in the cow’s surroundings, known as environmental pathogens (Jones and Bailey, 1998). The
main contagious pathogens are Streptococcus dysgalactiae, Streptococcus agalactiae and
Staphylococcus aureus. The environmental pathogens are mainly Streptococcus uberis,
Klebsiella spp. and Escherichia coli (Jones and Swisher, 2009). About 90% of pathogens
responsible for udder inflammations are environmental pathogens (Lassa et al., 2013) which
presence is common in cow-barn environment. Increase number of animal, humidity and
pollutions in cow environment increase bacteria and other pathogens prevalence on animals.
Staphylococcus aureus, E. coli and Klebsiella sp. causes the greatest losses of milk in cows that
suffered from mastitis (Aleksandra et al., 2017).
Several studies conducted in Ethiopia also have supported these findings and documented
prevalence ranging from 1.8 to 21.1 % for clinical and 22.3 to 46.6 % for subclinical mastitis
with significant economic losses associated with the disease (Workineh et al., 2002; Kerro and
Tareke, 2003; Biffa et al., 2005; Hunderra et al., 2005; Mungube et al., 2005; Almaw et al.,
2008; Getahun et al., 2008; Bitew et al., 2010 and Rgbe et al., 2012). Moreover, some of these
2
studies also have shown that the majority of these organisms have developed drug resistance for
the commonly used antimicrobial drugs. Despite intensive research and the implementation of
various mastitis control strategies over the decades, bovine mastitis has not been controlled yet
and the reduction in the prevalence of mastitis has been minimal (Barlow, 2011 and Nickerson,
2009). Although antibiotic therapy to control bovine mastitis is effective in most cases, it can be
detrimental too, because of the emergence of multidrug resistance among gram positive and
gram negative bacteria (Soleimani et al., 2010; Laverty et al., 2011) and occurrence of antibiotic
residues in the milk and meat (Prescott, 2008). Moreover, antibiotic treatments have a low cure
rate during lactation for many mastitis pathogens and frequently resulting in chronic and regular
infections. Therefore looking for an effective biological treatment by other substances than
antibiotics becomes an urgent need (Soleimani et al., 2010).
On the other hand, despite advances in food science and technology, foodborne diseases (FDB)
remain one of the major public health and economic problems all over the world (Legnani et al.,
2004). The risk of foodborne illness has increased markedly, each year 40 million people get sick
from FBD, 128,000 are hospitalized, and 3000 die according to the Center for Disease Control
and Prevention (CDC, 2020). There have been described approximately 250 producing agents of
FBD’s which includes bacteria, virus, fungi, prion parasites, toxins, and heavy metals (Olea
et al., 2012). Some of the principal pathogenic microorganisms transmitted by food that can
affect seriously any individual are E.coli, Listeria monocytogenes, Salmonella enterica, Salmonel
la typhimurium, Shigella species, and S. aureus where even some vulnerable groups such as
pregnant women and babies are the most affected, some of these pathogens can be seriously
harmful and even produce fatal consequences according to Food and
Drug Administration (FDA, 2003). Members of the genus Shigella and Salmonella were being
mentioned microorganisms responsible for the major number of infections caused by
contaminated foods. Few studies conducted in the different parts of Ethiopia showed the poor
sanitary conditions of food preparation establishments and presence of pathogenic organisms like
Campylobacter, Salmonella, S. aureus, Bacillus cereus and E. coli (Bayleyegn et al., 2003;
Abera et al., 2006; Knife and Abera, 2007 and Mekonnen et al., 2013).
The development of legal procedures for the prevention and microbiological quality control of
foods can contribute to FBD’s reduction due to bacterial organisms, although taking into
3
consideration the consumers protection through the employment of vaccines, probiotics, and
functional foods, in which some of them should present certain molecules in their intrinsic
composition showing proven biological activity as a potential preventative measure against
FBD’s pathogenic microorganisms (Hernandez, 2010 and Quevedo, 2014). The ingestion of
certain bacteria allows the maintenance of a certain type of microorganisms, in this context the
concept of probiotic is born, which is defined as those microorganisms (bacteria and yeasts) pure
or in mixed active cultures that when consumed in adequate quantities by human and animals
can exert a beneficial effect in the guest health (Lorente et al., 2001; Quera et al., 2005; Anadon
et al., 2006; Ramirez et al., 2011 and Diosma et al., 2013). Also, the concept of “ideal probiotic”
has been established, which is the one that would present the majority of the following
particularities: skill to adhere to cells, to multiply, and the ability to generate antibacterial
compounds against the growth of pathogens (organic acids, bacteriocins, among others),
generally regard as safe (GRAS), non-invasive, non-carcinogenic, non-pathogenic, and be
capable to co-aggregate to form part of normal well-balanced flora (Zamora-Vega et al., 2014).
In recent years, the concept of biological control has emerged as one of interesting sustainable
alternative to fight against pathogens. The range of applications of probiotic bacteria thus has
broadened, and they are now considered a possibility for alternative treatments against mastitis
as well as they are favorable choice to treat many infectious diseases of human and animal
(Soleimani et al., 2010; Klostermann et al., 2010 and Espeche et al., 2012). Currently, dairy and
dairy-related products (both fermented and non-fermented), and human milk are a good source of
probiotics (Liong, 2011 and Yu et al., 2011). Probiotics particularly lactic acid bacteria (LAB)
have long been used in fermentation to preserve the nutritive qualities of various foods. The main
antimicrobial effect exerted by LAB is that they ferment different sugars to lactic acid, thereby
reducing the pH to a level that harmful bacteria cannot tolerate (Frola et al., 2012). In addition, a
variety of antimicrobial compounds are produced by LAB, which a detrimental impact on
harmful bacteria and inhibit their growth (Ramirez et al., 2011 and Serna and Enriquez, 2013).
There are a knowledge of the importance of probiotics and wide range of applications of Lactic
acid bacteria and their health benefits for both animal and human, their antagonistic properties
and antimicrobial activity has getting more attention to investigate against mastitis bacterial
pathogens. However, there is a limitation on research finding on LAB as potential mammary
4
probiotic isolated from cow milk against bovine mastitis-causing pathogens in the study area as
well as at the national level. Therefore, this research problem will invite the medical world to
bring probiotic therapeutic measures against mastitis and foodborne pathogens alternative to
antibiotics (Matios et al., 2009; Bekele et al., 2010 and Geberyohannes et al., 2010).
1.1. Sstatements of the problem
Ethiopia has the largest cattle population in Africa with an estimated population of 56.7 million,
of this, around 11.8 million of the total cattle heads are milking cows (CSA, 2017). However,
milk production does not satisfy the country’s requirements; bovine mastitis is one of the major
problems. Medical therapy involving antibiotics and still a key tool in the scheme of mastitis
treatment and control (Mekonnen, 2013). The long time use of antibiotics in the treatment of
mastitis has directed further problem of emergency of antibiotic resistant strains, therefore there
is continual worry about treatment failure and the resistant strains entering the food chain
(Espeche et al., 2012). Moreover, while antibiotics have had a major impact on dairy cow health
and consequently on milk quality, their use is questioned because of traces of antibiotics in milk
for human consumption (Klostermann et al., 2010).
On the other hand, many control measures in the food industry are provided to prevent or
minimize bacterial contamination, including the appearance or growth of food-borne pathogens.
Good manufacturing practices, sanitation, and hygiene measurements for raw material, the food
industry environment, and so forth do not avoid the occurrence of food-borne outbreaks (Crispie
et al., 2008). Traditionally, in veterinary areas, medicine treatment therapies and prophylaxis
have been based primarily on the use of antibiotics. It is known that the indiscriminate use of
antibiotics for the treatment of bovine mastitis is a main public concern, as cited before. Some
pathogenic organisms might become resistant and they can often spread the resistance genes to
other related microorganism. In addition, the presence of antibiotic residues in milk and other
dairy products may not be detected in time and get to the market chain to the consumer
producing various disorders. It is also important to note that the presence of residues of antibiotic
in milk is also a serious problem for the dairy industry because it can lead to the failure of the
fermentation processes (Espeche et al., 2012).
5
Intramammary inflammation is the main cause of antimicrobial usage on dairy farms and herd-
level associations between the use of antimicrobial agents and antimicrobial resistance in some
mastitis pathogens have been demonstrated (Pol and Ruegg, 2007). The potential public health
risks related to milk may result from the presence of pathogens which are resistant to
antimicrobials or possess genes encoding resistance to such antibiotics, that may transfer their
resistance determinants to pathogenic bacteria, which leads the emergency of multi-drug resistant
food-borne pathogens (Saini et al., 2013). Even though, many considerable researches on the
disease treatment for bovine mastitis, alternative biological therapy has not been developed yet.
During the last decades, world tendency to limit the use of antibiotics in dairy cattle, has lead
researchers toward the study of cows natural defense mechanisms in order to ensure their
absence in dairy products with the aim of satisfy consumers demand for “organic products”. It
has also been recognized that LAB are capable of producing inhibitory substances other than
organic acids (lactic and acetic) that are antagonistic toward other microorganisms (Quevedo,
2014).
In recent years, there is an increasing tendency on the need to apply preventive strategies in all
human and animal areas. In this way, the use of different and novel preventive approaches are
being suggested and assayed, which includes the use of probiotic microorganisms (Besler and
Essack, 2010). These probiotics improve the health status, increase the weight gain and inhibit
pathogens (Herstad et al., 2010). Therefore, scientific research towards developing the cow’s
natural defense mechanisms against mastitis is crucial. Developing indigenous lactic acid
bacteria probiotics has a novel preventive approach for the treatment of mastitis and food borne
pathogens, through legal procedures, thereby decreasing drug resistant bacteria and its infection.
In 2010, Klotermann et al. compared an antibiotic therapy and a potentially probiotic containing
L. lactis DPC3147 in the treatment of chronic subclinical mastitis and clinical mastitis. After the
intramammmary infusion probiotic, the application of the organism was as effective as the
antibiotic in the treatment of clinical mastitis. The development of legal procedures for the
prevention and microbiological quality control of foods can contribute to FBD’s reduction due to
bacterial organisms, although taking into consideration the consumers protection by means of the
employment of probiotics and functional foods, in which some of them should present certain
molecules in their intrinsic composition showing proven biological activity as a potential
6
prevention measure against FBD’s pathogenic microorganisms (Hernandez, 2010). In particular,
bacteriocin producing lactic acid bacteria probiotics have received much of interest due to the
great potential for their use in food to control food-borne pathogens and to increase the shelf life
of food products, as well as for the development of products for human and veterinary medicine
by pharmaceutical industries (Cotter et al., 2005). In order to avoid the frequent use of antibiotics
and to control potentially pathogenic bacteria, probiotics could be successfully employed (Arias
et al., 2013). LAB produced antimicrobials have been successfully used to prevent mastitis and
food borne pathogens (Abo-Amer, 2013).
1.2. Significance of the study
Developing potent and cost effective indigenous probiotic drug, as alternative means to control
and prevent selected bacterial foodborne and mastitis causing pathogens is the major outcome of
this project at the end of in-vitro and in-vivo tests. Moreover, application of these probiotics may
reduce antibiotic residues and emergency of drug resistance pathogens, which is in agreement
with global pressure to limit their use in dairy cattle. On the other hand, the current research has
also demonstrated the role of antimicrobial compounds as protective mechanism against
intestinal pathogens and therefore certain strains could have a probiotic potential on both in food
matrix and in gastrointestinal tract.
Thus, the policy makers are given the opportunity to take part in future as an outcome of this
proposed project for the implementation of the use of probiotics as alternative potential
therapeutics and preventive measures against selected bacterial foodborne and mastitis causing
pathogens. Hence, the humble end users will be benefited since from the efforts to be made in
response to the expected outcome (their livelihood will be improved). The control and prevention
of selected bacterial foodborne and mastitis causing pathogens which have a negative economic
and public health impact is a major goal for programmed aimed at poverty alleviation. These
project will be addresses some beneficiaries and stockholders like; Ministry of Health, Livestock
and Fishery Minister, different pharmaceuticals , food processing factories, consumers of food of
animal origin, farm owners and individuals who are working at slaughter houses, butcher shops,
restaurants, wholesalers, cafeterias, super markets, and farms.
7
1.3. Objectives
1.3.1. General objective
The general objective of the current study was:-
To isolate, characterize, and evaluate indigenous beneficial LAB strains as a potential
probiotic for the therapeutic and prevention of the selected mastitis-causing and
foodborne bacterial pathogens.
1.3.2. Specific objectives
The specific objectives were:
To isolate, characterize, and identify Lactobacillus species.
To evaluate the antibacterial activity of the isolated Lactobacillus species against the
selected mastitis-causing and foodborne bacterial pathogens.
To optimize the growth parameters: pH, temperature, and incubation period.
Assay of antibiotic resistance patterns of Lactobacillus species.
To determine the minimum inhibitory concentration and minimum bactericidal
concentration of the potential probiotic Lactobacilli strains.
8
2. Literature review
2.1. Lactic acid bacteria
The Lactic acid bacteria (LAB) is a group of Gram-positive microorganisms which mostly grows
at a pH of 4-4.5, the temperature is a key factor of their growth, being mesophiles (20-25°C) or
thermophiles (40-45°C). Furthermore, they share morphological, physiological, and biochemical
characteristics (bacillus, width from 0.5 to 0.8 μm, non-spore, non-mobile, without cytochromes;
non-respiring but aerotolerant, fastidious, anaerobic, and microaerophilic, oxidase, catalase, and
cytochrome oxidase negatives and they cannot reduce the nitrate to nitrite). They are generally
used in the food industry in fermentation processes such as yogurt, cheese, pickles, sausage
production, and are also involved in beer and wine elaboration and used as food supplements.
Additionally, they are widely used in the livestock and farming industry to improve animal
production. Lactic acid bacteria are present in natural form in fruits, vegetables, milk products,
meat, and in fact in the digestive tract and reproductive systems of mammals
(Rattanachaikunsopon and Phumkhachorn, 2010).
In this way LAB are responsible for the fermentation of different foods as they facilitate their
preservation/ shelf-life by the production of different antimicrobial compounds as CO2, H2O2,
bacteriocins, exopolysaccharides, and lactic acid improving sensorial characteristics (odor,
flavor, and texture), nutritional quality, shelf-life and the safety of the final product (Parada et
al., 2007; Rattanachaikunsopon and Phumkhachorn, 2010 and Serna and Enriquez, 2013). The
present interest in LAB, besides their fermentative and preservative effects in food, is more
focused on human and animal health aspects due to their antimicrobial effects against pathogenic
microorganisms which contaminate foods (Serna and Enriquez, 2013) peruse promoting a variety
of illnesses when they are consumed (Emiliano, 2014). After the 1980s researchers observed the
widespread application of LAB in the field of biomedicine, food preservative, food processing,
and fermentation, and animal husbandry (Pessione et al., 2012).
The Lactic acid bacteria are classified into homo-fermentative and hetero-fermentative
organisms based on their ability to ferment carbohydrates (Kuipers et al., 2000). The homo-
fermentative LAB such as Lactococcus, some Lactobacilli, and Streptococcus; mainly produce
lactic acid from two molecules of lactates from one glucose molecule whereas hetero-
9
fermentative LAB such as Leuconostoc, Wiessella, and some Lactobacilli generates lactate,
ethanol, and CO2 from one molecule of glucose (Salminen et al., 1998 and Smith, 2017). LAB
produces lactic acid and some other organic acid during sugar fermentation, which results in the
reduction of pH of the environment and thereby inhibiting the growth of spoilage and pathogenic
bacteria (Smith, 2017). As it was reported by Chow (2002), the concept that food could serve as
medicine was first conceived thousands of years ago by the Greek philosopher and father of
medicine, Hippocrates, who once wrote: 'Let food be thy medicine, and let medicine be thy food'.
During recent times, the concept of food having medicinal value has been reborn as 'functional
foods'.
One of the most promising areas for the development of functional food components lies in the
use of probiotics and prebiotics which scientific researchers have demonstrated therapeutic
evidence (Smith, 2017). Besides the nutritional values, the ingestion of LAB and their fermented
foods have been suggested to confer a range of health benefits (Soccol et al., 2010). LAB was
first isolated from milk. They can be found in fermented products as meat, milk products,
vegetables, beverages, and bakery products. In the food industry, LAB is widely used as starters
to achieve favorable changes in texture, aroma, flavor, and acidity (Leory and De Vuyst, 2004).
However, there has been an important interest in using bacteriocin and/or other inhibitory
substances producing LAB for non-fermentative biopreservation applications (Parada et al.,
2007). Trillions of microorganisms (“microbiota/microflora”) are colonized in the intestine and
the gut of the mammalian system, which are vital for the human and animal health.
Lactobacillus, Pediococcus, Bifidobacterium, Lactococcus, Streptococcus, and Leuconostoc are
the most extensively isolated organisms from the fermented foods, beverages and also from the
human and animal gut (Rahul et al., 2018). Numerous health-promoting LAB strains (such as
Bifidobacterium sp., Lactococcus sp., and Lactobacillus sp.) have been found that have shown
optimistic consequences upon human health (Rahul et al., 2018).
2.2. Taxonomical classification of lactic acid bacteria
In recent taxonomic classification, LAB come under the Phylum of Firmicutes, class Bacilli, and
order Lactobacillales. They are a heterogeneous group of bacteria comprising about 20 genera
includes; Lactobacillus, Lactococcus, Pediococcus, Enterococcus, Streptococcus, Melissococcus,
10
Leuconostoc, and Bifidobacterium are the main LAB genera involved (Leroy et al, 2008).
However, the largest genus in this group is the Lactobacillus and it consists of more than 140
recognized species and 30 subspecies (Paul et al., 2009).
2.3. Lactic acid bacteria as probiotics
The most tried and tested manner in which the gut microbiota composition may be influenced is
through the use of live microbial dietary additions, as probiotics (Tunick and van Hekken, 2015).
The history of probiotics is as old as human history, as it is firmly related to the utilization of
fermented food. Metchnikoff is known as the father of probiotics at the beginning of the
20thcentury, and he put the first scientific basis and conceptualized probiotics. In 1907, he
suggested that there are some kinds of bacteria present in the fermented milk products that
produce acids if consumed habitually, lead to a healthier and long life. He hypothesized that the
normal gut microflora could exert adverse effects on the host and that consumption of ‘soured
milks’ reversed this effect (Vasiljevic and Shah, 2008).
The probiotic (Lactobacillus bulgaricus) discovered by Metchnikoff was involved in the
combination of fermented milk. Based on its Greek etymology, probiotic is the combination of
the words “pro bios” literary meaning “for life”. The origin of the first use can be traced back to
Kollath (1953), who used it to describe the restoration of the health of malnourished patients by
different organic and inorganic supplements. Later, Vergin in1954 proposed that the microbial
imbalance in the body caused by the antibiotic treatment could have been restored by a probiotic-
rich diet; a suggestion cited by many as the first reference to probiotics as they are defined
nowadays (Okuro et al., 2013). Similarly, Kolb recognized the detrimental effects of antibiotic
therapy and proposed prevention by probiotics (Vasiljevic and Shah, 2008).
In 2002, a Working Group of a Food and Agriculture Organisation of the United Nations and
World Health Organisation (FAO/WHO) Expert Consultation proposed the following definition:
‘Live micro‐organisms which when administered in adequate amounts confer a health benefit on
the host’ (FAO/WHO, 2002). The 2002 definition, although widely accepted at least in the
scientific community, has not been adopted into any international standard (at least to date). In
2014, a similar panel of scientific experts organised by the International Scientific Association
for Probiotics and Prebiotics (ISAPP) agreed that the FAO/WHO (2002) definition for probiotics
11
was still relevant, but advised a minor grammatical correction as follows: ‘Live micro‐organisms
that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al.,
2014). The idea of health-promoting effects of LAB is by no means new, as Metchnikoff
proposed that lactobacilli may fight against intestinal putrefaction and contribute to long life
(Holzapfel et al., 2001 and Belhadj et al., 2010). Probiotics interact with the potential of
pathogenic microbes by producing metabolic compounds and other products (Holowacz et al.,
2016).
Today, around 25 Lactobacillus species and 5 Bifidobacterium strains represent the great
majority of marketed probiotics. Other probiotic bacteria include Pediococcusacidi lactici,
Lactococcus lactis subsp. lactis, Leuconostoc mesenteroides, Enterococcus faecium,
Streptococcus thermophiles (Santiago‐Lopez et al., 2015). Species from other bacterial genera
such as E. coli Nissle, Bacillus subtilis, and Enterococcus have also been used, but there are
concerns surrounding the safety of such probiotics as these genera contain opportunistic
pathogenic species (FAO/WHO, 2002). Few non-bacterial microorganisms such as probiotic
yeasts are non-pathogenic strains generally belonging to species of Saccharomyces cerevisiae
and Saccharomyces boulardii strains, studied and commercialized as probiotics (Zorica et al.,
2016).
A number of different strategies can be applied to modify microbial intestinal populations
(Newburg and Grave, 2014). Antibiotics can be effective in eliminating pathogenic organisms
within the intestinal microbiota. However, they carry the risk of side effects and cannot be
routinely used for longer periods or prophylactically. The consumption of probiotics aims to
directly supplement the intestinal microbiota with live beneficial organisms. Prebiotics represent
a third strategy to manipulate the intestinal microbiota (Barczynska et al., 2015). Prebiotics are
nondigestible food ingredients generally oligosaccharides, that selectively stimulate the
proliferation and/or activity of desirable bacterial populations, Lactobacilli and Bifidobacteria
already resident in the consumer’s intestinal tract (Pandey et al., 2015). There is an obvious
potential to use prebiotics and probiotics together in a complementary and synergistic manner
Therefore, foods containing both probiotic and prebiotic ingredients have been termed synbiotics
(Legette et al., 2012).
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2.4. Origin and safety of probiotics
Nowadays many foods are considered as a probiotics source and are commercial authorized
mainly as nutritional supplements specifically in countries like the USA (Oyetayo et al., 2003
and Forssten et al., 2011). Many of them containing viable cells of LAB being the dairy products
the conventional vehicle to commercialize them such as yogurt, pickles, sausages, cheese, ice
cream or butter, also it has been identified other food products as soy milk, mayonnaise, juices,
peanuts, and soups (Ramirez et al., 2011). Otherwise, species from Lactobacillus, Leuconostoc,
Pediococcus, Enterococcus, and Bifidobacterium genras have been isolated from a wide variety
of different infectious lesions, questioning their safety as probiotics. There are many assessments
which have been considered in the probiotics evaluation safety (in vitro, animal and/or clinical
studies), such as pathogenicity, noninfectious behavior, and virulence, even though other factors
such as toxin production, metabolic and hemolytic activities, screenings for virulence factors and
resistance to a host defense mechanism, metabolic activity and intrinsic properties of these
microorganisms have to be taken into account (FAO/WHO, 2002 and Forssten et al., 2011).
Lactic acid bacteria traditionally used in fermented dairy products have a long history of safe
use. However, as interest grows in using new strains, safety testing will become important.
Probiotic strains such as Lactobacillus species and Bifidobacterium species have a long history
of safe use and are GRAS (Millis et al., 2011). Another key aspect of safety is the specification
of the strain origin. Potential probiotic cultures have been isolated from a variety of sources
including animal, human, and food sources. However, there is now growing evidence that strains
are host specific and for that reason, it is generally accepted that strains to be used for human
applications should be human isolates. Of course, whatever the origin or the taxonomic identity,
the candidate probiotic strains need a series of in-vitro tests and animal trials to verify safety
issues (Millis et al., 2011).
2.5. Antimicrobial compounds of lactic acid bacteria
The antimicrobial activity of starter cultures and probiotic lactic acid bacteria has been attributed
to the production of metabolites such as organic acids (lactic and acetic acid), hydrogen
peroxide, ethanol, diacetyl, acetaldehyde, other low molecular mass compounds with
antimicrobial activity and bacteriocins (Ammor et al., 2006 and Ratsep et al., 2014). The
13
carbohydrates fermentation products from most of LAB are organic acids as lactic, formic,
propionic, butyric and acetic, furthermore other compounds such as H2O2, acetaldehyde,
diacetyl, CO2, and bacteriocins, which inhibit the growth of microorganisms responsible of the
spoilage and development of pathogens in food against L. monocytogenes, S. aureus, B. cereus.,
Enterococcus spp., Cl. botulinum through a wide variety of action mechanisms (Reis et al.,
2012).
The LAB antimicrobial activity generated against Gram-positive bacteria mainly comes from
the action of bacteriocin, while the antimicrobial activity against Gram-negative bacteria is due
to organic acid’s action and also to other compounds as H2O2. Different authors acknowledge
that the chemical composition of the cell wall of Gram-negative bacteria is considered as a
protection factor against Bacteriocins since the external membrane of these microorganisms
functions as a permeability barrier (Serna and Enriquez, 2013). The application of antimicrobial
agents produced by Lactobacillus spp. has been demonstrated in many food systems, which in
many cases demonstrates the effectiveness of these potent inhibitors to control undesirable
bacteria. Several in vitro studies have also examined the antimicrobial potential of Lactobacillus.
From clinical studies, the role of Lactobacillus antimicrobial agents as one of the desirable
properties of a probiotic is becoming apparent (Mobolaji and Wuraola, 2011).
2.5.1. Organic acids
The primary antimicrobial effect exerted by LAB is due to the production of organic acids
(Olaoye and Onilude, 2011). The most important and best characterized antimicrobials produced
by LAB are lactic and acetic acid. During growth, the sugars are mostly converted to lactic acid,
which exerts most of the inhibitory capacity against microorganisms. Lactic acid is preferred for
food and pharmaceutical applications and as starting material in the production of biopolymers
(Papagianni, 2012). The accumulation of both lactic and acetic acid end-products and associated
with low pH as well as H2O2 formation resulting in the strongest antimicrobial activity and wide
inhibitory spectrum including both Gram-positive and Gram-negative bacteria (Ammor et al.,
2006). These organic acids exert their antimicrobial effect by penetrating the microbial cell, and
breakdown essential metabolic functions, cellular growth inhibition, and reducing the
intracellular pH of the pathogens (Reid, 2006). Aciduric organisms such as yeasts, molds, and
most acid-producing bacteria are tolerant to acids and a low pH (Rattanachaikunsopon and
14
Phumkhachorn, 2010). The production of lactic acid and reduction of pH are dependent on
species or strain, culture composition, and growth conditions (Olaoye and Onilude, 2011).
2.5.2. Reuterin
Reuterin is glycerol derived antimicrobial compound produced under anaerobic conditions, and
production is enhanced by the presence of glycerol. Reuterin is a potent, broad-spectrum
antimicrobial agent effective against Gram-negative (e.g. Salmonella and Shigella) and Gram-
positive (e.g. Clostridium, Staphylococcus, and Listeria) bacteria, yeasts, fungi, and protozoa
(Chow, 2002). It has been proposed that reuterin and/or reuterin-producing Lactobacilli may
have an application in the preservation of food and feed by reducing pathogenic and spoilage
microorganisms (Yang, 2000). Reuterin was initially reported to be produced by Lactobacillus
reuteri, which is part of the endogenous bacterial flora in both humans and animals (Nes et al.,
2012).
2.5.3. Bacteriocin
Bacteriocins are ribosomally synthesized antimicrobial peptides produced by one bacterium that
are active against other bacteria. It has been suggested that one of the desirable properties of a
probiotic strain is the ability to produce antimicrobial substances, such as bacteriocins, which
potentially offers a competitive advantage in colonization and competition in the GI tract
(Hegarty et al., 2016). Bacteriocins are peptides with antimicrobial activities targeting bacteria
closely related to the producer. Whereas most bacteriocins produced by Gram-negative bacteria
only act on very closely related (Gram-negative) species, most bacteriocins of Gram-positive
bacteria exhibit a broader inhibitory spectrum towards a wide range of Gram-positive species
(Yang et al., 2012).
Bacteriocins have been reported to be produced by strains of Lactococcus, Lactobacillus,
Pediococcus, Leuconostoc, Carnobacterium, Streptococcus, Enterococcus, and Bifidobacterium
(Zacharof and Lovitt, 2012). Bacteriocins produced by LAB can be defined as biologically active
proteins or protein complexes displaying a bactericidal mode of action exclusively towards
Gram-positive bacteria and particularly towards closely related species. As an example, ninsin
forms ion-permeable channels in the cytoplasmic membrane of susceptible cells, resulting in an
increase in the membrane permeability (linkage), which causes dissipation of the membrane
15
potential and efflux of ATP, amino acids and essential ions such as potassium and magnesium.
Ultimately, energy production and biosynthesis of macromolecules including cell wall
biosynthesis are inhibited resulting in cell death (Zacharof and Lovitt, 2012). Associated with
the discovery of new bacteriocins, several probiotic strains have been shown to display the
ability to produce inhibitory peptides. In this respect, most of the probiotic bacteriocins
characterised to date are of Lactobacillus origin. Knowledge on bacteriocin producers in situ and
their function in the gut of healthy animals is still limited due to a scarcity of in vivo studies
(Umu et al., 2016). There are two major classes of bacteriocins in LAB, were known; lantibiotics
(Class I) and non-lantibiotics (Class II). Lactobacilli are most often cited for the production of
bacteriocins and produce all classes of bacteriocins (Juodeikiene et al., 2012).
2.5.4. Hydrogen peroxide
Hydrogen peroxide (H2O2) is produced by LAB in the presence of oxygen (Abbas et al., 2010).
The antimicrobial effect of H2O2 may result from peroxidation of membrane lipids which would
explain the increased membrane permeability caused by hydrogen peroxide. The resulting
bactericidal effect of these oxygen metabolites has been attributed not only to their strong
oxidizing effect on the bacterial cell but also damage basic molecular structures of nucleic acids
and cell proteins ( Zalan et al., 2005).
2.6. Safety of probiotics
As viable, probiotic bacteria have to be consumed in large quantities, over an extended period, to
exert beneficial effects; the issue of the safety of these microorganisms is of primary concern
(Leroy et al., 2008). Until now, reports of a harmful effect of these microbes to the host are rare.
However, many species of the genera Lactobacillus, Leuconostoc, Pediococcus, Enterococcus,
and Bifidobacterium were isolated frequently from various types of infective lesions like
bacterial endocarditis, bloodstream infections and local infections (Gasser, 1994). Although
minor side effects of the use of probiotics have been reported, infections with probiotic bacteria
occur and invariably only in immunocompromised patients or those with intestinal bleeding
(Leroy et al., 2008).
According to FAO/WHO guidelines and recommendations, candidate probiotic
strains/microorganisms should not harbor transmissible drug resistance genes encoding
16
resistance to clinically used drugs (FAO/WHO, 2002). In addition, probiotic strains should be
evaluated for several parameters, including antibiotic susceptibility patterns, toxin production,
metabolic and hemolytic activities, and infectivity in immunocompromised animals (FAO/WHO,
2002). In vitro safety screenings of probiotics may include, among others, antibiotic resistance
assays, screenings for virulence factors, resistance to host defense mechanisms, and induction of
hemolysis. The efficiency, side effects of (if any) and safety of probiotic strains should be
assessed on animal models; like models of immunodeficiency, endocarditis, colitis, and liver
injury (Forssten et al., 2011).
2.7. Selection criteria of probiotics
Many in-vitro tests are used for screening and pre-selection of potential probiotic strains
(Morelli, 2007). In general, the initial screening and selection of probiotics include testing of
their phenotype and genotype stability, carbohydrate and protein utilization patterns, acid and
bile tolerance, survival and growth, intestinal epithelial adhesion properties, ability to produce
antimicrobial substances and inhibit known pathogens, antibiotic resistance patterns and
immunogenicity (Desai, 2008). The competitiveness of the most promising strains selected by in
vitro assays is furthered need to be evaluated using various in vivo tests for monitoring of their
host specificity, safeness, adsorption to the gut surface and persistence in the gut, antimicrobial
activity, and beneficial effects (e.g., enhanced nutrition and increased immune response) in the
host (Tuomola et al., 1999). Finally, the probiotic must be viable under normal storage
conditions and technologically suitable for industrial processes (e.g., lyophilized) (Kabir, 2009).
17
3. Materials and methods
3.1. Description of study areas
The study was conducted in Dessie and Kombolcha towns. Dessie is located in the Northern
parts of Ethiopia, Amhara national regional state Southen Wollo zone at 11°8'N-11°46’N degree
latitude and 39°38'E 41°13’E longitude at 400 km distance from Addis Ababa. It is relatively
bounded by Kutaber Woreda in the North, Dessie Zuriya Woreda in the East, and by Kombolcha
town in the South. The topography of Dessie is a highland type surrounded by ‘Tossa’ mountain
(Dawit, 2013). Its elevation ranges between 2,470 and 2,550 meters above sea level. Annual
maximum and minimum temperatures of Dessie are 23.7°C and 9°C, respectively. Dessie is one
of the reform towns in the region and has a city administration consisting of a municipality, 10
urban and 6 peri-urban kebeles.
Source: Prepared by Dr. Engdaw Abebe
Figure 1: Map of the study areas.
Kombolcha is an industrial town found in the North-central part of Ethiopia in South Wollo Zone
of the Amhara Regional State of Ethiopia. It is situated at a distance of 377 km from North of
Addis Ababa, 505 km from the Regional capital city, Bahirdar, 23 km from the zonal town
18
Dessie and 533 km from port Djibouti. Astronomically, the town is located at about 11° 6’ N
latitude and 39° 45’ E longitudes. The delimitation of the town is bounded by Dessie Zuria
Woreda in the North East and North West, KaluWoreda in the South, and Albuko Woreda in the
South West (Muluwork, 2014). Mean annual rainfall is 1046 mm while annual maximum and
minimum temperatures are 28.1°C and 12.9°C, respectively. The town is located in a range of
altitudes between 1, 500, and 1,840 meters above sea level. Kombolcha is one of the reform
towns in the region and has a town administration municipality, 5 urban, and 6 peri-urban
kebeles (Eskinder et al., 2010). The reason why milk samples were collected from these local
areas was to increase the chance of getting potent probiotic candidates.
3.2. Sample collection and handling
A total of 48 cow milk samples were collected aseptically from the local households of Dessie
and Kombolcha Towns in their environs, and 24 samples from each. Purposive sampling method
was followed to selected households for milk sample collection. Approximately 250 ml of cow
milk samples were collected from half litter of each cow and transported to the Veterinary
Microbiology Laboratory, School of Veterinary Medicine, at Wollo University on the day of
collection through icebox containing ice packs. Aseptic sampling was followed as described by
the Health Protection Agency HPA (2004) and the Food and Drug Administration FDA (2003).
All the milk samples stayed for 48 hour at room temperature for yogurt making. Bacterial culture
was started immediately after yogurt has getting ready.
3.3. Standard bacterial strains and clinical isolates
These indicator organisms were selected based on their public health importance. The standard
bacterial strains or the American Type Culture Collection including; S. aureus (ATCC25923), S.
agalactiae (ATCC13813), S.pyogenes (ATCC19615), L.monocytogenes (ATCC7644), E.coli (A
TCC25922), K.pneumoniae (ATCC700603), S.Typhimurium now Salmonella enterica subsp.
enterica serotype typhimurium (ATCC14028), S. enterica subsp. enterica (ATCC 13076), and
Shigella flexneri (ATCC12022) were obtained from Amhara Public Health Institute Dessie
Branch (A.P.H.I.) and multidrug resistant clinical isolates including: S.Typhimurium (Salmonella
enterica subsp. enterica serotype Typhimurium), E. coli, S. aureus and S. agalactiae were
obtained from School of Veterinary Medicine, Wollo University. These test organisms were used
19
for determination of antimicrobial activity and minimum inhibitory concentration of the Lactic
acid bacteria isolates and for the control of in vitro tests.
3.4. Antibiotic sensitivity patterns of the test bacterial pathogens
To assess the antibiotic sensitivity pattern, in vitro antimicrobial susceptibility was done using
the agar disk diffusion method described by Bauer et al. (1996). All the 13 indicator bacteria
were tested against to clinically important antimicrobials. This test was aimed to know the actual
antibacterial sensitivity of test organism before used. For standard bacterial species; E. coli,
S. enterica, and S. Typhymurium tested for Gentamicin (GM) (10µg), Nalidlixic acid (NA)
(30µg), Kanamycine (K) (30µg), Tetracycline (TE) (30µg), Trimethoprim (T) (5µl), and
Sulphamethaxaloze (SxT) (23.75µg); S.aureus tested for Vancomycin (VA) (30µg), Oxacilline
(OX) (1µl), Amoxicillin (A) (10µg), Streptomycin (S) (10µg), Kanamycine (K) (30µg), and
Nalidlixic acid (NA) (30µg); L. monocytogenes for Gentamicin (GM) (10µg), Erythromycin (E)
(15µg), Tetracycline (TE) (30µg), Penicillin G (PG) (10µg), Streptomycin (S) (10µg),
Chloroapimnicol (C) (30µg), and Vancomycin (VA) (30µg); K. pneumonia for Gentamicin
(GM) (10µg), Erythromycin (E) (15µg), Tetracycline (TE) (30µg), Nalidlixic acid (NA) (30µg),
and Amoxicillin (A) (10µg); S. pyogenes for Gentamicin (GM) (10 µg), Tetracycline (TE)
(30µg), Penicillin G (PG) (10µg), and Chloroapimnicol (C) (30µg) were tested; for Shigella
Sulphamethaxaloze (SxT) (23.75µg), Gentamicin (GM) (10µg), Amoxicillin (A) (10µg),
Tetracycline (TE) (30µg), Ampicillin (25µg), and Kanamycine (K) (30µg); and for S. agalactiae
tested for Gentamicin (GM) (10µg), Erythromycin (E) (15µg), Tetracycline (TE) (30µg),
Penicillin G (PG) (10µg) and Amoxicillin (A) (10µg).
The clinical isolates such as S. agalactiae tested for against Gentamicin (GM) (10µg),
Erythromycin (E) (15µg), Tetracycline (TE) (30µg), Chloroapimnicol (C) (30µg), Clindamycin
(DA) (2µg), and Sulphamethaxaloze (SxT) (23.75µg); E. coli for Erythromycin (E) (15µg),
Penicillin G (PG) (10µg), Amoxicillin (A) (10 µg), Nalidlixic acid (NA) (30µg), and Cefazolin
(C) (30µg); S. aureus for Gentamicin (GM) (10µg), Chloroapimnicol (C) (30µg), Nalidlixic acid
(NA) (30µg), Penicillin G (PG) (10µg), Ampicillin (25 µg), and Kanamycine (K) (30µg); and
S.Typhimurium for Gentamicin (GM) (10 µg), Erythromycin (E) (15 µg), Nalidlixic acid (NA)
(30µg) and Chloroapimnicol (C) (30µg) were used. All indicator organisms were tested against
20
different class of antibiotics and more emphasis was given to; cell-wall synthesis inhibitor
antibiotics for the gram positive bacteria and protein synthesis inhibitors for the gram negative.
For inoculum preparation, 4-5 well-isolated colonies of each test pathogens from nutrient agar
plates were taken into tubes containing 5 ml of a normal saline solution until it achieved the 0.5
McFarland turbidity standards, and then a sterile cotton swab was dipped into the adjusted
suspension and the excess broth was purged by pressing and rotating the swab firmly against the
inside of the tube above the fluid level. The cotton swab was then spread evenly over the entire
surface of the plate of Mueller-Hinton agar to obtain uniform inoculums. The plates were then
allowed to dry for 3 to 5 minutes. Antibiotics impregnated disks were then applied to the surface
of the inoculated plates with sterile forceps. Each disk was gently pressed down onto the
Mueller-Hinton agar to ensure complete contact with the agar surface. Even distribution of disks
and minimum distance of 24 mm from center to center was ensured and from the edge of the
plates to prevent overlapping of the inhibition zones. Five antibiotic disks were placed in each
petri-dish. Within 15 minutes of the application of the disks, the plates were inverted and
incubated at 37°C. After 18 to 24 hours of incubation, the plates were examined, and the
diameters of the zones of complete inhibition to the nearest whole millimeter were measured by
a digital caliper. The clear zone (inhibition zones of bacterial growth around the antibiotic disc
(including the disc diameter)) for individual antimicrobial agents was interpreted and categorized
as per the table of the Clinical Laboratory Standard Institute into Sensitive (S), Intermediate (I),
and Resistant (R) (CLSI, 2015 and CLSI, 2017).
3.4. Isolation and identification of lactic acid bacteria species
3.4.1. Morphological characterization of Lactobacillus species
The species of Lactobacillus were isolated from yogurt samples using de-Man Ragosa and
Sharpe (MRS) agar according to the method described by Harrigan and McCance (1976) by the
spread plate method. 1 ml of each yogurt sample was homogenized in to 9 ml sterile saline
solution (0.85%, w/v NaCl) to make initial dilution. Serial dilution up to 10-4 was made using a
sterile pipette by transferring 1ml from 10 ml of suspension into 9 ml of sterile NaCl solution.
After proper homogenization, 0.1 ml of 10-4 serially diluted sample was spread on MRS agar
21
medium and uniformly swabbed by a sterile cotton swab. The plates were incubated
anaerobically for 24-48 hr at 37°C using anaerobic jar.
After 48 hour when the colonies become predominant, morphologically distinct and well-isolated
colonies were picked and transferred to new MRS agar using streak plate method. Colonies
showing typical characteristics of LAB on agar surface were picked up randomly and transferred
into MRS broth for further enrichment. Further, their purity was checked on MRS agar. The pure
isolates were subjected to identification: the macroscopic appearance of all the colonies was
examined for cultural and morphological characteristics. The size, shape and color of the
colonies were recorded. The isolates were stained by Gram's Method by using S. aureus (purple
cocci) and E. coli (pink rods) as gram stain control slides. All isolates were examined for catalase
reaction by mixing a drop of 3% H2O2 to a loopful of fresh culture on a slide to observe bubble
formation by using S. aureus and S. agalactiae as a positive and negative control organisms,
respectively. Those isolates readily identified as Gram-positive rods and catalase-negative were
included for further characterization (Dhanasekaran et.al. 2010).
3.4.2. Identification of lactic acid bacteria by biochemical tests
After performing the preliminary isolation, those Gram-positive, catalase-negative and rod
shaped isolates were considered as presumptive Lactobacillus species according to Seifu et al.
(2012). According to the method described by Harrigan and McCance (1976) pure presumptive
lactobacillus species were sub-cultured on MRS agar and maintain colonies on MRS broth at
4°C for further use by supplement with 20% (v/v) glycerol. Identification to species level was
conducted by subjecting isolates to various carbohydrates fermentation, from (1% w/v): D-
Glucose, L-Arabinose, D-fructose, Cellobiose, Esculin, Lactose, Maltose, D-galactose, Mannitol,
Starch, Raffinose, Rhamnose, D-sorbitol, Sucrose, Trehalose, Inulin and Sorbose in 5ml MRS
broth. Phenol red used as an indicator of acid production after 48 hr anaerobic incubation at 37°C
by inoculating a loopful of lactobacillus isolated colonies.
Methyl red and voges-proskauer test, indole production test, and citrate utilization test were
performed. Presumptive isolates that showed the general characteristics of lactobacillus bacteria
were selected randomly and subjected to different biochemical tests according to the method
described by Harrigan and McCance (1976) and that included; Growth at different
22
temperatures: Overnight cultures of isolates were inoculated in to 5ml of MRS broth and
incubated at 15°C and 45ºC for 48 hr. Growth was determined by observing the turbidity. Gas
production from glucose: Overnight isolated cultures were inoculated at 10% (w/v) in to 5ml of
MRS broth containing inverted Durham tubes and incubated at 37ºC. The gas production from
glucose was observed after 48 hr. Indole test: Isolates were inoculated in to 5ml of Trypton
broth and incubated at 37°C for 48 hr, the formation of red-ring at the top of broth in the test tube
was determined the ability of an organism to produce indole and indicated by Kovac’s reagent.
E. coli used as positive control organisms. Methyl red and voges-proskauer test: A loop full of
fresh culture was inoculated into 5ml of sterile MR-VP broth medium and incubated 48hr at
37°C anaerobically. For the MR test, methyl red was used as a reagent whereas for VP test 5%
alpha naphtha and 40% KOH were used as reagents E. coli was used as positive control
organisms for MR test (Bettache et al., 2012).
Citrate utilization test: The isolates were inoculated in Simon’s Citrate agar and incubated at
37°C for 48 hr. The appearance of blue coloration indicated the positive for the test. Salmonella
spp. was used as positive control organisms. Oxidation fermentation test (OF test): All isolates
were determined their oxidative or fermentative ability to sucrose, glucose and lactose in OF
broth medium containing trypton and bromothymol blue. 10g per 100ml distilled water of the
tested sugars were prepared and sterilized. 4.5 ml of oxidation fermentation media and 0.5ml of
each respective sugar was added into a test tube. The overnight cultures of Lactobacillus isolates
in MRS broth were centrifuged at 6000 rpm for 15 min to obtain free cells (Estifanos et al.,
2016). The cell pellets were washed with saline solution for at least two times to avoid false
positive result. The supernatant was discarded and sediment (cells) was used for sugar
fermentation. For each test LAB isolate; tubes were inoculated by stabbing the cell pellets with a
sterile loop in duplicate. Oil immersion was overlaid and tightens to one of each duplicate tube to
made anaerobic condition, and loosen overlaid tube. Incubate tubes at 37°C. The sugar
fermentation pattern of each isolate was followed for seven days by checking the color change
from green to yellow (Dhanasekaran et.al., 2010).
23
3.5. Antibacterial activity of Lactobacillus species by agar well -diffusion method
Antimicrobial activity of LAB is one of the most important selection criteria and traits. Inoculum
densities of all test organisms were adjusted to McFarland 0.5 turbidity standard, made by
barium sulphate solution. For this purpose, the isolates were inoculated in to 10 ml of sterile
MRS broth and incubated at 37°C for 48 hrs for the production of antibacterial substances. After
48 hr. of incubation, a volume of 1 ml of broth cultured was transferred into 2ml capacity
Eppendorf tube and a cell-free supernatant was obtained by centrifuging the bacterial culture at
6000 rpm for 15 min followed by filtration of the supernatant through 0.20 μm pore size
Whitman filter paper and the filtrate transferred into new Eppendorf tube. The antagonistic
activities of the Lactobacillus isolates against pathogens were determined by the agar well diffusion assay
performed in triplicate according to Estifanos et al. (2016) and Vinderola et al. (2008).
The inhibitory activity was performed against all the test pathogenic bacteria by dipping sterile
cotton swap into the standardized suspension of the test bacteria and then streaking the swap
over the entire Muller-Hinton Agar to obtain uniform inoculation. Around 6mm diameter of 6
wells was made at equidistance on pre-inoculated Muller-Hinton Agar. Then, each well was
filled with 100µl of cell-free supernatant of lactobacillus isolates till its fullness (Patel et al.,
2011). Plates were allowed to dry, inverted, and incubated at 37°C for 24hr. Antibacterial
activity of the isolates was determined by measuring the “zone of inhibition (clear zone of
diameter)” expressed in millimeter (mm) in diameter using a digital caliper. This experiment was
performed in triplicates and each data was recorded as mean zones of inhibition. The antimicrobial
activity of the LAB strains was determined by the development of inhibition zones in millimeters around
the wells (Estifanos et al., 2016).
3.6. Optimization of growth parameters
According to Sarkar and Paul (2019) production of antimicrobial compounds by Lactobacillus
species depends on many parameters like pH, temperature, and incubation period. Optimum
growth performance of all isolates were determined by incubating colonies in MRS broth at
different pH (2, 3, 5, 6.5, 7, and 9), temperatures (10, 27, 37, and 45°C), and incubation period
(18, 24, 48, 72, 96, and 120 hr) ranges. This experiment was conducted in triplicates. 24hr of
24
fresh single colony from MRS agar plate was inoculated in to sterile MRS broth, after overnight
incubation, vortex and properly mixed to maintain similar inoculum size. For all growth
parameters, 100 µL of fresh Lactobacillus culture was inoculated into another 10ml MRS broth
containing test tubes. Four incubators were adjusted at 10, 27, 37 and 45°C ranges. All isolates
were incubated for18-120hr at each temperature. Regarding pH optimization: MRS broth was
first maintained its pH 6.5 ±0.2 (control pH) at 25°C by using a digital pH meter and water bath
before being adjusted to the required pH range. Respective pH ranges were adjusted by using
hydrochloric acid and 40% NaOH solution inside sterile beaker container and homogenized with
electric-stirrer followed by pouring in to test tubes. All isolates were inoculated into all pH
ranges and incubated anaerobically for 48hr. at 37°C. The optimum parameters for the highest
growth of the identified Lactobacillus spp. were determined by measuring and comparing the
optical density (OD) at 600 nm (OD600).
3.7. Antibiotic sensitivity pattern of Lactobacillus
As part of the European Food Safety Authority (EFSA) and Food and Drug Administration
(FDA) requirements for the safety assessment of bacteria intended for a probiotic purpose, such
organisms should not possess acquired resistance determinants to antibiotics of medical
importance. However, the recent detection of antibiotic-resistant LAB and the continuous
exposure to environmental conditions may promote that LAB became as intrinsic or extrinsic
reservoirs for antibiotic resistance (AR) genes, which can be horizontally transfer to the
pathogenic bacteria through the food chain (Fraqueza, 2015 and Mermelstein, 2018).
To assess the antibiotic sensitivity pattern, in vitro antimicrobial susceptibility was done using
the agar disk diffusion as a method described by (CLSI, 2015). The method was originally
standardized as per ISO 10932/IDF 233 standards with minor modifications (Hobbs, 2000). The
following ten different antibiotic disks with their concentrations given in parentheses were used
in the antibiograms: Penicillin G (10µg), Ampicillin (25µg), Amoxicillin (10µg), Ciprofloxacin
(5µg), Gentamycin (10µg), Chloramphenicol (30µg), Erythromycin (15µg), Oxacilline (1µg),
Tetracycline (30mg) and Vancomycin (30mg). For this, four to five well-isolated colonies of
each isolate from MRS agar plates were taken into tubes containing 5 ml of a normal saline
solution until it achieved the 0.5 McFarland turbidity standards, and then a sterile cotton swab
was dipped into the adjusted suspension within 15 minutes and the excess broth was purged by
25
pressing and rotating the swab firmly against the inside of the tube above the fluid level. The
swab was then spread evenly over the entire surface of the plate of MRS agar to obtain uniform
inoculums. The plates were then allowed to dry for 5 minutes to avoid excess moisture.
Antibiotics impregnated disks were then applied to the surface of the inoculated plates with
sterile forceps. Each disk was gently pressed down onto the MRS agar to ensure complete
contact with the agar surface. Even distribution of disks and minimum distance of 24 mm from
center to center was ensured and from the edge of the plates to prevent overlapping of the
inhibition zones. Five antibiotic disks were placed in each petri-dish. Within 15 minutes of the
application of the disks, the plates were inverted and incubated at 37°C.
After 24 hours of incubation, the plates were examined, and the diameters of the zones of
complete inhibition to the nearest whole millimeter were measured by a digital caliper. The
interpretation was done based on the table of the Clinical Laboratory Standard Institute (CLSI,
2015). The inhibition zone diameter of isolates less than or equal to 14 mm was considered as
resistant, zone diameter more than 20 mm as sensitive and zone diameter in between 14 and 20
mm as intermediate.
3.8. Determination of minimum inhibitory concentration
The lowest concentration of an antimicrobial substance that prevents the visible growth of
bacteria are used to evaluate the antimicrobial efficacy of various compounds by measuring the
effect of decreasing concentrations of antibiotics over a defined period in terms of inhibition of
microbial population growth. The Clinical and Laboratory Standards Institute (CLSI) has
established protocols and standards for MIC in products. The antibacterial potential of each
Lactobacillus extract and their synergistic effects were evaluated by minimum inhibitory
concentration (MIC) using resazurin based 96-well microdilutions method. Production of the
antibacterial extract was performed according to Estifanos et al. (2016). Each isolate was
incubated in MRS broth at 37°C for 48 hr. After incubation, cell-free supernatant was obtained
by centrifuge the LAB culture at 6000 rpm for 20 minutes in Eppendorf tubes, followed by the
filtration of the supernatant through 0.20μm pore size Whatman filter paper. Inoculum
suspensions were made by a loop full of discrete bacterial colonies with similar morphology
were inoculated into 5ml of sterile saline solution and properly homogenized by vortex mixer
before being used and adjusted to 0.5 McFarland Standard. Broth microdilutions were performed
26
according to the Clinical and Laboratory Standards Institute (CLSI, 2015) with some
modification.
It was performed in a 96-U-shaped well (round-bottom) microtitre plate composed of 12 rows (1-
12) and 8 columns (A-H). One lactobacillus extract was tested for two pathogenic organisms 4
columns for each bacteria used as 4 replicas from A-D and E-H in one plate. The assay was
composed of 10 rows of extract and suspension of test organisms; the 11th row was used as
growth control line, broth having test bacteria without extract, and the 12th row was used as broth
sterility control, without extract and test pathogen. All the 96 wells were dispensed with 100µl of
sterile trypton soya broth. Equal volume (100µl) of bacterial extract was added in first row of
eight wells. Titration was made by a multichannel pipette starting form the first 1st horizontal
row wells and mixed thoroughly by proper pipetting up to 5-10 times. Two-fold serial dilution
was made and continued up to the 10th row. In each row 100μl diluted solution was transferred
from the 1st to 10th row.
Lastly, 100μl was removed and discarded from the 10th row. The final concentration of bacterial
extract was now one-half of the original concentration in each row. Then, a separate and sterile
pipette was used to dispense 50μl of bacterial suspension into the wells of 4 columns for each
test organism including the 11th row except the last broth sterility control row. One LAB extract
was tested against two pathogens in a single plate. Plates were sealed with a plastic cover to
protect cross-contamination and evaporation during incubation (Ehsani et al., 2016). After 24 hr
incubation at 37°C, resazurin solutions was prepared by dissolving 337.5mg of resazurin tablet in
50ml distilled water in a sterile flask container and mix the solution to ensure homogeneity.
Around 30µl of resazurin dye was added to all wells and incubated at 37°C for another 2-4hours.
Changes of color were observed and recorded. The lowest concentration prior to color change
into pink/reddish was considered as the MIC. The lowest LAB concentration that prevented
bacterial growth (no visible bacterial growth) was considered as MIC value (Abdollahzadeh
et al., 2014).
27
3.9. Determination of minimum bactericidal concentration
To determine minimum bactericidal concentration (MBC) value, a Loop-full of sample was
taken from wells of the plate with no visible growth in the MIC experiment and sub-cultured on
the freshly prepared Mueller Hinton agar plates and incubated at 37°C for 24 hr. After
incubation, the lowest concentration of the LAB with bactericidal effects (the well of extract that
did not permit bacterial colony growth on the agar plate) was considered as MBC value (Ehsani
et al., 2016).
3.10. Data management and analysis
The data were summarized and compiled by sum up different laboratory findings. All raw data
were stored in Microsoft Excel 2010 spreadsheet and transferred to STATA Version 12 for
statistical analysis. Both descriptive and analytic statistics were used. In addition to descriptive
analysis, multiple linear regression analysis was also computed to determine how the average
absorbance values of each of the isolates vary with (depend on) the values of temperature and
incubation period. The assumptions of multiple linear regression analysis were checked using
graphical checking methods by plotting residuals in different ways. Collinearity analysis was
carried out to ensure whether there are correlations among the explanatory variables (temperature
and incubation period) or not, and the better the model fits the data were examined by observing
both R2 and adjusted R2 values. Statistical significance was considered at p-value less than 0.05.
28
4. Results and discussion
4.1. Isolation and identification of Lactobacillus species
In the current experiment, a total of 11(eleven) isolates were identified out of the 48 samples as
genus Lactobacillus and lactobacillus species based on their morphological, cultural,
physiological, biochemical and sugar fermentation patterns. The isolates grown on MRS agar
plates were white creamy, rods shape with long or rounded ends. They appeared mostly as a
chain of 3-4 cells or single or in pairs and similar finding were observed as research conducted
by Mamta et al., (2017). Gram-positive and non-catalase producing isolates were for further
characterization as shown. According to the primary biochemical tests, all isolates were found to
be negative for catalase production, citrate utilization, indole production and voges-proskauer
whereas positive in methyl red and oxidation fermentation tests. Identification of Lactobacillus
species carried out through procedures of Bergey’s manual of systemic Bacteriology and by
comparing the result with previously published scientific research work of Bettache et al. (2012)
and Estifanos et al. (2016).
The identified Lactobacillus spp. were Lactobacillus delbrueckii subsp. delbrueckii (B11),
Lactobacillus delbrueckii subsp. indicus (B5), Lactobacillus acidophilus (K91 and B6),
Lactobacillusplantarum subsp. Plantarum (W31, PK2 and K1), Lactobacillus rhamnosus (D71),
Lactobacillus delbrueckii subsp. Bulgarikus (B7), Lactobacillus brevis (D5), Lactobacillus delb
rueckii subsp. lactis (B1). Of these eight species, Lactobacillus plantarum subsp. plantarum and
Lactobacillu brevis grew at 15°C whereas Lactobacillus delbrueckii subsp. delbrueckii,
Lactobacillus delbrueckii subsp. indicus, Lactobacillus acidophilus, Lactobacillus delbrueckii
subsp. bulgarikus, and Lactobacillus delbrueckii subsp. lactis were able to grow at 45°C
incubation temperature. On the other hand, Lactobacillus rhamnosus (D71) was grown both at
15°C and 45°C. Regarding gas production from glucose, all isolates, except Lactobacillus
plantarum subsp.plantarum (B7&Pk2) and L.delbruskii subsp. Lactis (B5), were unable to
produced gas. Physiological and biochemical characteristics and carbohydrate utilization profile
of Lactobacillus isolates were demonstrated in Tables 1 and 2.
27
Table 1: Physiological and biochemical characteristics of Lactobacillus Species.
Isolates
Colony morphology
Gram Stain and Biochemical Test Results
Growth
Size Color Cell-
Shape
Gram
stain
Aerobicity Catalase Indole MR VP Citrate OF Gas from
Glu.
15ᴼC 45ᴼC
B5 Large White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + + ‒ +
B1 Large White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ ‒ +
B6 Medium White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ ‒ +
B11 Medium White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ ‒ +
B7 Medium White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + + ‒ +
D71 Large White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ + +
D5 Medium White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ + ‒
K1 Small White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ + ‒
K91 Medium White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ ‒ +
pk2 Large White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + + + ‒
W31 Large White
creamy
Rod + F.a ‒ ‒ + ‒ ‒ + ‒ + ‒
Note: F.a= facultative anaerobic; + = positive reaction; - = negative reaction.
28
Table 2: Carbohydrates fermentation profile of Lactobacillus species. Isolates Carbohydrates Species Identification
Tre Sor Lac Man Gul Gal Cel Rha Suc Fru Ara Mal Sta Raf Esc Sorbo Inu
B5 ‒ + + ‒ + + + ‒ + + + ‒ + + + + + L.delbruskii subsp. Lactis
B11 ‒ + + + + + + ‒ + + + + + + + + +
L.delbruskii subsp.
Bulgaricus
B6 + + + + + + ‒ + + + + + + ‒ + + + L.acidophilus
B1 ‒ + + + + + + + + + + + + ‒ v + v
L.delbruskii sub
sp.Bulgaricus
K1 + v + + + + v + + + + v + + + + +
L.plantarum sub. Sp.
plantarum
W31 + + + + + + + + + + + + + + + + +
L.plantarum sub. Sp.
plantarum
D71 ‒ + + + + + + + + + ‒ + + ‒ + + ‒ L.rahmanosus
D5 ‒ + + + + + ‒ + + + + + + + + + + L.brevis
Pk2 + + + + + + + + + + + + + + v + +
L.plantarum sub. Sp.
plantarum
B7 + + + + ‒ + + + + + + + v + + v +
L.plantarum sub. Sp.
plantarum
K91 + + + + + + + + ‒ + + + + + + + ‒ L.acidophilus
Note: Tre = Trealose; Suc = sucrose; Sorb = Sorbose; Str = Starch; Inu = Inulin; Sor = Sorbitol; Ara = Arabinose; Gal = Galactose;
Rha = Rhamnose; Esc = Esculin; Mal = Maltose; Cel = cellubiose; Man = Mannitol; Mos = Mannose; Fru = Fructose; Glu = Glucose;
Lac = Lactose; Raf = Raffinose; + = positive ; ‒ = negative; V= variable.
29
4.2. Antibacterial activity assay of Lactobacillus species
The eleven Lactobacillus isolates were evaluated for their antimicrobial activity against all
ATCC strains of nine (9) standard and 4 (four) drug resistant clinical isolates of mastitis-causing
and foodborne pathogenic bacteria. The results revealed that all isolates had varying degrees of
inhibition towards the test pathogens. According to Nigam et al. (2012) ˃1 mm inhibition zone
around the colonies of the producer strain was scored positive for inhibition. It was assumed that
the greater the diameter of the inhibition zone, the greater the antibacterial activity of the
isolates. As described by Handa (2012), isolates having clear zones ≤9mm and ≥12mm diameter
against the test pathogens indicated poor and strong antimicrobial activity, respectively. The
inhibition zone diameter was interpreted as described earlier by Nigam et al., (2012); as less
activity (≤10 mm), moderate activity (11-15 mm), and strong activity (˃15mm). Surprisingly, all
of the current LAB isolates in this study have exhibited antimicrobial activity with inhibition
zone of ≥12.2 mm against all the test organisms as the data presented in Tables 3 and 4. The
obtained results of present study showed that Lactobacillus were the most important probiotic
organism which they have growth inhibitory effects against different isolates of Gram-positive
and Gram-negative bacteria.
Standard strain of Streptococcus pyogenes was the most susceptible bacteria. Gram-positive
bacteria are more sensitive than gram-negative groups in this experiment. Among these isolated
LAB species, Lactobacillus acidophilus, Lactobacillus rhamnosus, and Lactobacillus
plantarum subsp. plantarum had the most potent probiotic effect which showed strong
antibacterial activity against the standard bacterial such as; S. aureus, S. agalactiae, and
S. pyogenes, with inhibition zone ranged from 17.8 to 20.2mm in diameters. Similarly,
Lactobacillus delbrueckii subsp. delbrueckii had shown a promising effect against S. aureus and
S. typhimurium; Lactobacillus acidophilus and Lactobacillus rhamnosus also had strong activity
against clinical bacteria isolates except for E. coli. Lactobacillus acidophilus had shown the
maximum value of the zone of inhibition (20.2±0.3mm) against standard strain of S. pyogenes
(ATCC19615).
The minimum inhibition zone diameter was observed against drug resistant Salmonella
Typhymurium by Lactobacillus plantarum subsp. Plantarum with the value of 12.2±0.3mm. This
is due to the fact that LAB mostly the Lactobacilli possessing the capacity to alienate the
30
bacterial pathogens through the production of some antimicrobial metabolites, such as hydrogen
peroxide, organic acids (mainly, lactic acid), diacetyl and bacteriocin (Nigam et al., 2012). The
variation in antibacterial activities as illustrated by different authors might be due to the amount
of culture supernatant and quality of metabolites produced by LAB isolates. All isolates were
displayed a broad-spectrum antibacterial activity against both mastitis-causing and foodborne
bacterial pathogens. Several studies have been carried out to evaluate antagonistic properties and
effect of probiotic microorganisms. Similar results were obtained by Hoque et al. (2010) and
Mashak (2016) in an experiment carried out with Lactobacillus spp. isolated from yogurt,
indicated that Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus rhamnosus
isolated from traditional fermented dairy products had shown excellent antibacterial activity
against E. coli with zone diameter of inhibitions (ZDIs) 17, 21 & 14 mm, respectively. Rahman
(2015) also indicated that Lactobacillus acidophilus isolated from buffalo milk showed
inhibitory activity against S. typhimurium, E. coli, and Shigella species with ZDIs 10-22 mm.
The production capability of a wide range of antimicrobial substance by lactobacilli had a
greater advantage over other LAB species against the growth of many spoilage and pathogenic
microorganisms, such as species of Salmonella, Shigella species, E. coli, S.aureus and Listeria
(Abbas et al., 2010 and Mamo, et al., 2015). Tigu et al., 2016 also reported that two
Lactobacillus spp. isolated from traditional Ethiopian fermented condiments, namely, Datta and
Awaze, inhibited the growth of S. Typhimurium and E. coli with inhibition zones ranging from
10.3 to 14.3mm. In line with this, Haghshenas et al. (2017) and Jose et al. (2015 ) reported that
among the selected eight LAB isolated from fermented dairy products, Lactobacillus species,
particularly Lactobacillus plantarum had showed the most efficient antagonistic activity against
S. aureus, L. monocytogenes, S. typhimurium, and E. coli with inhibition zones of 11.7, 13.7,
12.3, and 12.3mm diameters, respectively. Likewise, Rajoka et al. (2017) and Grosu-Tudor et
al. (2014) had verified that all the Lactobacillus rhamnosus isolated from human milk inhibited
the growth of S.aureus, S. Typhimurium, and E. coli using agar-well diffusion method with
variable diameters (6 to14mm).
31
In general, the present isolated Lactobacilli strains showed strong activity against standard
bacteria and moderate activity against drug-resistant bacterial pathogens and exhibited a broad-
spectrum of antimicrobial activity. The broad spectrum of antagonistic activity against both
gram-negative and gram-positive pathogens exhibited by the Lactobacillus strains examined in
this study is in agreement with the findings of many other researchers (Liasi et al., 2009;
Setyawardani et al., 2014 and Jose et al., 2015).
32
Table 3: Antimicrobial activity assay of Lactobacillus isolates against standard bacterial foodborne pathogens and mastitis-causing bacteria.
LAB Isolates Standard Bacterial Strains
S. aureus S. agalactiae S. pyogenes L. monocytogenes E. coli S. typhimurium S. enterica K. pneumoniae Shigella
B11 15.4±0.56 15.04±1.4 16±0.28 15±0.56 15.5±0.70 15±0.84 16.23±1.4 17±0.07 15±0.70
B5 17.6±1.3 16±1.40 15.3±0.45 17±1.40 15±0.70 16.5±0.70 14.9±0.9 16.1±0.28 15±0.20
K91 19±0.21 18.1±0.5 20.2±0.3 16.3±0.08 14.3±0.03 17.6±0.2 16.5±0.4 15.4±0.2 14.5±0.38
W31 18.1±0.09 18.3±0.07 17.8±0.07 13.5±0.14 14.7±0.04 13.6±1.4 15.2±0.3 15.6±0.5 14.7±0.3
D5 16.6±0.22 16.7±0.35 15.8±0.35 15.9±0.04 14.6±0.4 15±0.31 16.3±0.3 14.4±0.01 14.8±1.9
D71 18.4±0.5 18.8± 0.2 18.06± 0.5 17.2± 0.4 14.6± 0.31 15.8± 0.33 16.6 ±0.38 14.3± 0.03 15±0.21
K1 13.9 ±0.69 14.5± 0.55 13.5± 0.54 13.7± 0.26 14 ±0.9 13.8 ±0.69 13.9 ±0.5 13.8± 0.55 14.9± 0.3
PK2 16.5± 0.7 15.6± 0.4 15.8 0.3 15.7 0.14 14.5± 0.01 15.1± 0.17 15.4± 0.44 15.7 0.43 15.4 0.21
B7 14.4±0.2 15.1±0.04 15.24 ±0.2 13.7 ±0.2 15.3±0.1 14.1±0.1 14.4 ±00 15.3±0.4 14.9 ±0.2
B1 15.5±0.1 15.7±0.4 15.4±0.04 14.6±1.0 15.8±0.2 15.6±0.7 15±0.6 15.2±0.3 14.3±0.5
B6 19±0.24 18.1±9.4 18.1±0.1 14.3±0.3 15.1±0.14 15.2±0.28 16.4±0.14 15.1±0.3 13.6±0.2
Table 4: Antimicrobial activity assay of Lactobacillus isolates against clinical bacterial foodborne pathogens and mastitis-causing bacteria.
LAB Isolates Clinical Isolates
S. aureus S. agalactiae E. coli S. typhimurium
B11 15.4±0.70 14.9±0.9 14.7±0.45 15 ±1.13
B5 14.5±0.14 14.3±0.56 14±0.28 14.5±1.4
K91 15.8±0.03 15.6±0.24 14.8±1.5 15.6±0.1
w31 13.7±0.2 14.3±0.4 14.2±1.1 14.3±0.1
D5 14.2±0.24 14.3±0.2 13±0.2 13.4±0.4
D71 15.9± 0.6 15.1± 0.7 14.9± 0.2 15.2 ± 0.4
K1 12.4± 0.1 12.3 ±0.04 13.1± 0.03 12.2 ±0.6
Pk2 13.3 ±0.3 14.2±0.4 13.8 ±0.12 13.6±0.31
B7 12.8±0.02 13.4 ±0.2 13.3±0.7 13.6±0.2
B1 14±0.14 13.08±0.5 13.7±0.7 13.2±0.1
B6 14±0.28 14.2±0.8 13.2±0.2 13.3±0.07
NB: The value indicated is means± SD; in triplicate determination.
33
4.3. Optimization of growth parameters
All isolated Lactobacillus species have showed maximum growth between pH5.0 to 7.0 (the
optimum pH range). Growth was dramatically decreased as pH below 5.0. The optical density
(OD) reading was the average value of the three replicas as it is described in Table 5.
Table 5: The growth performance of the Lactobacillus spp. isolates at different pH
LAB Isolates pH optimization
pH=2 pH=3 pH=5 6.5 ±.2 (control) pH=7 pH=9
B5 0.13± 0.24 0.2 ± 0.12 0.48 ± .24 0.52 ± .34 0.48 ± .24 0.31 ± .23
B1 0.12 ± 0.21 0.13 ± 0.05 0.5 ± .26 0.51 ± .24 0.48 ± .50 0.32 ± .06
B6 0.20± 0.12 0.2 ± 0.34 0.5 ± .15 0.51 ± .06 0.5 ± .00 0.3 ± .23
B11 0.21± 0.11 0.21 ± 0.23 0.5 ± .09 0.51 ± .32 0.5 ± .40 0.42 ± .34
B7 0.11 ± 1.02 0.2 ± .42 0.5 ± .02 0.501 ± .51 0.5 ± .03 0.30 ± .04
D71 0.20 ± 0.05 0.2 ± .22 0.5 ± .41 0.51 ± .02 0.5 ± .21 0.31 ± .22
D5 0.10 ± 0.13 0.11 ± .21 0.5 ± .22 0.51 ± 0.3 0.50 ± .20 0.48 ± .23
K1 0.12 ± 0.22 0.14 ± 0.07 0.5 ± .21 0.511 ± .06 0.5 ± .22 0.48 ± .67
K91 0.10 ± 0.30 0.12 ± .61 0.5 ± .04 0.501 ±.11 0.51 ± .30 0.46 ±.07
PK2 0.11 ± .21 0.11 ± .42 0.49 ± .31 0.51 ± .041 0.52 ± .21 0.39 ± .28
W31 0.06 ± 0.4 0.13 ± .28 0.5 ± .5 0.52 ± .08 0.5 ± 0.33 0.37 ± .34
NB: The indicated value is mean ± SD; n=3.
34
The growth performances of the eleven (11) Lactobacillus isolates were observed at different pH,
incubation temperature, and incubation time ranges. The multiple linear regression results
indicated that six of the eleven isolates (B5, B6, B1, B11, B7, and K91) showed significant
variation in absorbance values with the different temperature ranges. However, except for isolate
B11, the remaining ten isolates did not show statistically significant dependency with the
different incubation periods (18, 24, 48, 72, 96, and 120hr) as shown in Table 5. All isolates had
shown optimal growth performance at 37°C. Regarding incubation time, B7 & Pk2 isolates
shown optimum growth performance at 72 hr., whereas the rest of the isolates at 48hr. of
incubation period as it is described in Annex 2.
The maximum lactic acid production was obtained at pH 6.5 on 24h of incubation. From pH 4.0
to 6.5 the fermentative products drastically increase, whereas after optimum pH 6.5, the lactic
acid production sharply decreased as illustrated by Sarkar and Paul (2019). Krischke et al. (1991)
also reported that for Lb. casei strain a pH range of 6.0-6.5 has been optimal for lactic acid
production. Ha et al. (2013) also suggested that, pH 5.5 has been optimum for lactic acid
production by using the strain L. helveticus. The lactic acid production increased sharply with
increase in the temperature from 25°C up to 37°C; and best production was found at 37°C.
However, lower lactic acid production was found at 45°C as shown by Sarkar and Paul (2019).
According to Ha et al. (2013) the optimal temperature for growth of lactic acid bacteria varies
between the 20 to 45°C and obviously it varies on species to species. Ilmen et al. (2007)
maximum lactic acid production was reported by Lb. casei at 37°C. Technologically important
parameters like pH, temperature and incubation period for industrially used probiotic strains are
well known. Cachon and Divie`s (2003) found that maximum growth and lactic acid production
of L.lactis subsp. Lactis, Lactobacillus delbrueckii subsp.bulgaricus, Lactobacillus acidophilus,
Lactobacillus paracasei and L. bulgaricus optimal pH range of 6.3 to 6.9 with temperature
ranging from 27 to 40ᴼC.
The present experimental study results of the ideal conditions were in line with the reports of
Tomas et al. (2002) and Talluri et al. (2017) who reported as the best temperature 37°C and pH
6.5 for the growth performance of the isolated Lactobacillus. According to Bergey’s Manual of
systemic bacteriology, for lactobacillus spp. Growth temperature range 2–53°C, best generally
30–40°C. Aciduric, optimal pH usually 5.5–6.2; growth generally occurs at pH 5.0 or less; the
35
growth rate is often reduced in alkaline conditions. Regarding incubation period, an increase in
lactic acid production was found increased till 120 h and thereafter sharply decrease as reported
by Talluri et al. (2017) and Sarkar and Paul ( 2019). This is due to the growth of the culture
entered to the stationary phase and as a consequence of slow down the metabolic activity.
However, many researcher reported that incubation period of 48 h has been generally used for
lactic acid production using different lactobacilli cultures( Garg et al., 1995; Fu et al., 1995;
Gavrilescu, 2005 and Gonzalez et al., 2006. All of the above findings were supported to our
results. As multiple linear regression results in following table indicate that; β1 and β2
Unstandardized Coefficients values indicated that the slope by how much temperature and
incubation period affects the growth performance of isolates; standard error value indicate that
the probability of doing an error during this specific test. P-value is the strong evidence towards
null hypothesis or against alternative hypothesis.
36
Table 6: Multiple linear regression results of the absorbance values of different isolates
among the two explanatory variables.
LAB
Isolates
Explanatory Variables Unstandardized
Coefficients
Standard Error p-value
B5 Constant α 0.124 0.057 0.043
Temperature β1 0.009 0.001 0.001
Incubation period β2 0.00008 0.0005 0.874
B6 Constant α 0.22305 0.039312 0.000
Temperature β1 0.00657 0.000998 0.000
Incubation period β2 0.00027 0.000353 0.458
B1 Constant α 0.2689499 0.0409871 0.001
Temperature β1 0.0055858 0.001040 0.001
Incubation period β2 0.000018 0.000368 0.961
B11 Constant α 0.2377 0.0453 0.001
Temperature β1 0.0046 0.0011 0.001
Incubation period β2 0.0010 0.0004 0.023
B7 Constant α 0.295354 0.038166 0.001
Temperature β1 0.004982 0.000969 0.001
Incubation period β2 -0.000023 0.000343 0.947
D5 Constant α 0.53166 0.07593 0.001
Temperature β1 -0.00212 0.00193 0.285
Incubation period β2 -0.00084 0.00068 0.232
K1 Constant α 0.4779 0.0440 0.001
Temperature β1 0.0001 0.0011 0.920
Incubation period β2 -0.0005 0.0004 0.177
W31 Constant α 0.57152 0.07181 0.001
Temperature β1 -0.00274 0.00182 0.147
Incubation period β2 -0.00103 0.00064 0.126
D71 Constant α 0.42046 0.03230 0.001
Temperature β1 0.00168 0.00082 0.053
Incubation period β2 -0.00017 0.00029 0.567
PK2 Constant α 0.44476 0.02995 0.001
Temperature β1 0.00092 0.00076 0.241
Incubation period β2 -0.000038 0.00027 0.890
K91 Constant α 0.205644 0.046092 0.001
Temperature β1 0.006996 0.00117 0.001
Incubation period β2 0.000390 0.000414 0.357
NB: Statistical significance was considered at P-value (p < 0.05).
37
4.4. Antibiotic sensitivity pattern of Lactobacillus species
The antibiotic susceptibility pattern of lactic acid bacteria is important because bacteria used as
probiotics may serve as host for antibiotic resistant genes which can horizontally transfer to the
pathogenic bacteria (Akalu et al., 2017). The zone of inhibition was measured after 24 hours of
incubation period and interpretations were done according to CLSI (2015). All our isolates were
susceptible to the various antibiotics tested in this study. The highest level of sensitivity was
observed towards Penicillin G, Ciprofloxacin, Gentamycin, Chloramphenicol and Erythromycin;
and resistance was observed against Vancomycin and Oxacilline as it is shown below in Table 7.
Susceptibility may be of a disadvantage, if the host takes orally administered antibiotics which
may eventually eliminate established probiotic LAB (Tigu et al., 2016).
Susceptibility of Lactobacilli to both Erythromycin and Chloramphenicol has also been indicated
by previous studies (Akalu et al., 2017). Strains of lactobacilli (Lactobacillus plantarum, L.
acidophilus, L.brevis, L. casei) resistant to penicillin G, Cloxacillin, Streptomycin, Gentamycin,
Tetracycline, erythromycin and chloramphenicol were isolated from “home-made” Spanish
cheeses (Herrero et al., 1996 and Erginkaya et al. 2017). The current finding was also in
agreement with the reports of Pan et al. (2015) who reported that among 12 Lactobacillus
species, of which 9 species were resistant to Ampicillin, and 8 isolates resistant to Tetracycline.
On the contrary, the findings of Amraii et al. (2014), Abriouel et al. (2015), Tigu et al. (2016),
and Zheng et al. (2017) revealed that the LAB isolates were sensitive towards Ampicillin,
Oxacilline, Amoxicillin and Tetracycline. Moreover, Zheng et al. (2017) reported that some
strains of Lactobacillus rhamnosus and Lactobacillus plantarum were resistance to Penicillin G.
There is an intrinsic resistance to Kanamycin, Gentamycin, Streptomycin and Vancomycin by
LAB (Danielson and Wind, 2003; Franz et al., 2015; Erginkaya et al., 2017 and Wolupeck et al.,
2017)
38
Table 7: Drug Sensitivity Pattern of Lactobacillus Species
LAB Isolates Used Antimicrobial Disks
E C Cip Amox AP TE OX VA GN PG
B5 S S S R I R R R S S
K91 S S S I R S R R S S
B11 S S S R R S R R S S
D71 S S S R S I R R I I
PK2 S S S I I I R R S S
B7 S S S R S S R R S S
B1 S S S R S S R R S S
W31 S S S S S R R R S S
B6 S S S R I I R R S S
D5 S S S R R R R R S S
K1 S S S I I S R R S S
Note: E: Erythromycin; C: Chloramphenicol; VA: Vancomycin; Cip: Ciprofloxacin; TE: Tetracycline; A:
Ampicillin; OX: Oxacilline; Amox: Amoxicillin; PG: Penicillin G and GN: Gentamycin; R: resistant, I:
Intermediate and S: sensitive.
Intrinsic resistance of LAB against many antibiotics may be considered as advantageous for
those isolates with probiotic potential. Such resistance could be helpful for the sustainable
utilization of the strains in the human intestine to maintain the natural balance of intestinal
microflora during antibiotic therapy (Ketema Bacha et al., 2010). However, there is the danger of
transferring multiple drug resistance genes to pathogens in the intestinal environment. The
susceptibility of LAB isolates to the clinically important antimicrobials, on the other hand, is
beneficial as it minimizes the chances of horizontal genes transfer to pathogens both in the food
matrix and/or in the gastrointestinal tract (Ketema Bacha et al., 2010 and Federici et al., 2016).
The previous report is strictly to convince the current finding which indicates these isolates are
wild strains that are not more exposed to antibiotics.
39
4.5. Determination of minimum inhibitory concentration
The lowest minimum inhibitory concentration (MIC) value (3.125) was observed in
Lactobacillus brevis against S. aureus (ATCC25923) and S. agalactiae (ATCC13813) and
Lactobacillus rhamnosus against S. aureus (ATCC25923), whereas pooled-1 and 2 showed the
highest MIC value (25%) against the clinical isolates E. coli, S.Typhimurium, and S. aureus. All
Gram-positive standard bacterial pathogens, except L. monocytogenes, were found to be sensitive
and inhibited by a most antibacterial substance with MIC value between 3.125-12.5%, it is said
to be the lowest MIC value in this observation, while Gram-negative standard microorganisms;
E. coli, S. enterica, and K. pneumoniae were shown more resistance and value ranges from 6.25-
12.5%.
Among the different LAB isolates, B1, B6, K1, B11, PK2, and W31 display higher MIC value
against all tested pathogens and less potent as compared to other remaining isolates. Both pool-1
and pool-2 had shown 6.25% MIC value against all Gram-positive, while the value had shifted to
12.5% when demonstrated against Gram-negative standard bacteria. On the other hand, the
pooled LAB strains had shown a highest MIC value of 25% against all clinical pathogens except
for S. agalactiae (12.5%). In this observation all isolates were exhibited wide range of
antibacterial activity, in fact resistance in clinical pathogens were more prevalent as shown in
Table 8 and 9.
40
Table 8: The MIC and MBC values of the Lactobacillus isolates against the selected standard bacterial foodborne pathogens and mastitis-
causing bacteria in percentage.
LAB
Isolates
Standard Bacterial Strains
S. aureus S. agalactiae S. pyogenes L. monocytogenes E. coli Shigella K. pneumoniae S. typhimurium S. enterica
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
B1 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 12.5 12.5 25
B5 6.25 12.5 6.25 12.5 12.5 25 12.5 25 6.25 12.5 6.25 12.5 12.5 25 6.25 12.5 12.5 25
B6 12.5 25 6.25 12.5 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 12.5 12.5 25
B7 12.5 25 6.25 12.5 6.25 12.5 12.5 25 12.5 25 12.5 25 6.25 12.5 6.25 25 12.5 25
B11 12.5 25 6.25 12.5 6.25 12.5 6.25 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25
D5 3.125 6.25 3.125 6.25 6.25 12.5 12.5 25 6.25 12.5 6.25 12.5 6.25 12.5 6.25 25 6.25 12.5
K1 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 12.5 6.25 12.5
D71 3.125 6.25 6.25 12.5 6.25 6.25 6.25 12.5 12.5 25 6.25 12.5 12.5 25 6.25 12.5 12.5 25
W31 12.5 25 12.5 25 6.25 12.5 12.5 25 12.5 25 6.25 12.5 12.5 25 12.5 25 12.5 25
K91 6.25 6.25 6.25 12.5 6.25 12.5 12.5 25 12.5 25 12.5 25 6.25 12.5 6.25 12.5 12.5 25
Pk2 12.5 12.5 12.5 12.5 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 25 12.5 12.5
Pool-1 6.25 12.5 6.25 12.5 6.25 12.5 6.25 12.5 12.5 25 12.5 25 12.5 25 12.5 25 12.5 12.5
Pool-2 6.25 12.5 6.25 12.5 6.25 12.5 6.25 12.5 6.25 25 6.25 12.5 12.5 25 12.5 25 12.5 12.5
41
Table 9: The MIC and MBC values of the LAB isolates against the selected clinical isolate
bacterial foodborne pathogens and mastitis-causing bacteria in percentage.
LAB Isolates Clinical Isolates
S. aureus S. agalactiae E. coli S. typhimurium
MIC MBC MIC MBC MIC MBC MIC MBC
B1 12.5 25 12.5 25 12.5 25 12.5 25
B5 12.5 25 12.5 25 12.5 25 12.5 25
B6 12.5 25 12.5 25 12.5 25 12.5 25
B7 12.5 25 12.5 25 12.5 25 12.5 25
B11 12.5 25 12.5 25 12.5 25 12.5 25
D5 6.25 12.5 6.25 12.5 12.5 25 12.5 25
K1 12.5 25 12.5 25 12.5 25 12.5 25
D71 6.25 12.5 6.25 12.5 12.5 25 12.5 12.5
W31 12.5 12.5 12.5 25 12.5 25 12.5 25
K91 12.5 12.5 6.25 25 12.5 25 12.5 12.5
Pk2 12.5 25 12.5 25 12.5 25 12.5 12.5
Pool-1 25 25 12.5 25 25 25 25 25
Pool-2 25 25 12.5 25 25 25 25 25
4.6. Determination of minimum bactericidal concentration
The minimum bactericidal concentration (MBC) determination result revealed that Lactobacillus
rhamnosus (D71) against of S. pyogenes (ATCC19615) had shown 6.25%, both
Lactobacillus delbrueckii subsp.lactis (B1) and Lactobacillus plantarum subsp. plantarum (K1)
against S. typhimurium (ATCC14028) had shown 12.5%, and Lactobacillus plantarum subsp.
plantarum (PK2) against S. aureus, S. agalactiae also had shown 12.5% MBC values which are
equivalent to their MIC whereas, all other isolates had shown doubled their MIC value against all
standard and clinical indicator bacteria. The MBC value for the standard bacterial strains ranges
from 6.25-25%, and for the clinical isolates from 12.5-25%. The lowest MBC values (6.25%)
against the standard bacteria were recorded by Lactobacillus brevis (D5) against S. aureus
(ATCC25923) and S. agalactiae (ATCC13813), Lactobacillus rhamnosus (D71) against S. aureus
(ATCC25923) and S. pyogenes (ATCC19615), and Lactobacillus acidophilus (K91) against S.
aureus (ATCC25923). Whereas the lower MBC values (12.5%) against clinical isolates were
observed by Lactobacillus brevis (D5) against S. aureus and S. agalactiae; Lactobacillus
rhamnosus (D71) against S. aureus, S. agalactiae, and S.Typhimurium; Lactobacillus
42
plantarum subsp. plantarum (PK2) and Lactobacillus acidophilus (K91) against S. Typhimurium.
Higher MIC and MBC values were observed in clinical bacteria.
5. Conclusion and recommendations
In the current experimental study, a total of 11 possible indigenous probiotic Lactobacillus
species were identified. These eleven isolates were tested for their antibacterial activity against
the standard bacterial strains: S. aureus (ATCC25923), S. agalactiae (ATCC13813), S. pyogenes
(ATCC19615), L. monocytogenes (ATCC7644), E. coli (ATCC25922), K. pneumoniae
(ATCC700603), S.Typhimurium (ATCC14028), S. enterica (13076), and Shigella Flexneri
(ATCC12022), and drug resistant clinical isolates: S.Typhimurium, E. coli, S. aureus, and S.
agalactiae and had shown varying degree of inhibition. In this regard L.acidophilus, Lb.
rhamnosus, and Lb. plantarum subsp. plantarum were high in antimicrobial activity across the
tested pathogens. All Lactobacillus isolates had shown sensitive to Erythromycin, Chloramphenicol,
and Ciprofloxacin, whereas resistant to Vancomycin and Oxacilline. However, these isolates
were only characterized phenotypically and the optimization of growth parameters were only
determined for pH, temperature, and incubation period. 5.0-7.0 pH, 37ᴼC, and 48hr. were
identified as optimum pH, temperature and incubation period respectively. Though the MIC and
MBC of the isolates were determined, the concentration of the metabolites that have
antimicrobial activity was not yet determined. L.brevis and Lb. rhamnosus had shown the lowest
MIC and MBC in this observation. These results suggest that some of these isolates could be
used as potential probiotic candidates.
Therefore, based on the above conclusion the following recommendations are forwarded:
The isolates should also be characterized for their autoaggregation, coaggregation, cell-surface
hydrophobicity, hemolytic activity, acid and bile tolerance, phenol tolerance, NaCl tolerance,
resistance to lysozyme, and milk coagulation activities.
Quantification of organic acid and determination of pH value should be done.
Determination and characterization of metabolites (antimicrobial substances) should be done.
Biolog based characterization of the isolates should be performed
Molecular identification of Lactobacillus isolates using16s rRNA should be conducted.
43
Future research should focus on the genetic mechanisms underlying the phenotypic resistance
by analyzing antibiotic resistance genes in the potential probiotic Lactobacillus strains.
Further in vitro and in vivo tests should be done for the approval and use of the potent
indigenous candidate probiotics as an alternative therapeutics.
44
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7. List of annexes
Annex 1: pH optimization of Lactobacillus isolates.
LAB Isolates pH Optimization
pH=2 pH=3 pH=5 6.5 ±.2 (control) pH=7 pH=9
B5 0.13± 0.24 0.2 ± 0.12 0.48 ± .24 0.52 ± .34 0.48 ± .24 0.31 ± .23
B1 0.12 ± 0.21 0.13 ± 0.05 0.5 ± .26 0.51 ± .24 0.48 ± .50 0.32 ± .06
B6 0.20± 0.12 0.2 ± 0.34 0.5 ± .15 0.51 ± .06 0.5 ± .00 0.3 ± .23
B11 0.21± 0.11 0.21 ± 0.23 0.5 ± .09 0.51 ± .32 0.5 ± .40 0.42 ± .34
B7 0.11 ± 1.02 0.2 ± .42 0.5 ± .02 0.501 ± .51 0.5 ± .03 0.30 ± .04
D71 0.20 ± 0.05 0.2 ± .22 0.5 ± .41 0.51 ± .02 0.5 ± .21 0.31 ± .22
D5 0.10 ± 0.13 0.11 ± .21 0.5 ± .22 0.51 ± 0.3 0.50 ± .20 0.48 ± .23
K1 0.12 ± 0.22 0.14 ± 0.07 0.5 ± .21 0.511 ± .06 0.5 ± .22 0.48 ± .67
K91 0.10 ± 0.30 0.12 ± .61 0.5 ± .04 0.501 ±.11 0.51 ± .30 0.46 ±.07
pk2 0.11 ± .21 0.11 ± .42 0.49 ± .31 0.51 ± .041 0.52 ± .21 0.39 ± .28
W31 0.06 ± 0.4 0.13 ± .28 0.5 ± .5 0.52 ± .08 0.5 ± 0.33 0.37 ± .34
NB: The indicated value is mean ± SD; n=3.
Annex 2: Absorbance value of Lactobacillus isolates at different temperature ranges and
incubation periods.
LAB Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
B5 B5-1 10°C 0.071 0.084 0.187 0.191 0.198 0.194
B5-2 10°C 0.067 0.086 0.186 0.191 0.194 0.188
B5-3 10°C 0.065 0.081 0.187 0.191 0.199 0.197
0.067667 0.083667 0.186667 0.191 0.197 0.193
B5-1 27°C 0.497 0.501 0.509 0.503 0.501 0.487
B5-1 27°C 0.488 0.495 0.511 0.506 0.501 0.487
B5-1 27°C 0.468 0.489 0.508 0.508 0.502 0.485
0.484333 0.495 0.50933 0.505667 0.501333 0.486333
B5-1 37°C 0.499 0.505 0.512 0.508 0.493 0.483
B5-1 37°C 0.498 0.504 0.514 0.507 0.491 0.487
B5-1 37°C 0.498 0.504 0.514 0.511 0.493 0.483
0.49833 0.504333 0.5133 0.508667 0.492333 0.484333
B5-1 45°C 0.498 0.488 0.461 0.442 0.433 0.421
B5-1 45°C 0.483 0.488 0.469 0.452 0.445 0.432
B5-1 45°C 0.483 0.489 0.481 0.471 0.462 0.443
0.488 0.488333 0.470333 0.455 0.446667 0.432
60
LAB Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
B6 B6-1 10°C 0.172 0.188 0.298 0.302 0.301 0.301
B6-2 10°C 0.173 0.189 0.293 0.302 0.301 0.308
B6-3 10°C 0.179 0.189 0.284 0.311 0.301 0.304
0.174667 0.188667 0.291667 0.305 0.301 0.304333
B6-1 27°C 0.488 0.506 0.505 0.501 0.481 0.481
B6-2 27°C 0.489 0.506 0.509 0.501 0.499 0.485
B6-3 27°C 0.487 0.504 0.511 0.501 0.499 0.486
0.488 0.505333 0.508333 0.501 0.493 0.484
B6-1 37°C 0.496 0.499 0.505 0.501 0.501 0.498
B6-2 37°C 0.488 0.506 0.512 0.503 0.501 0.491
B6-3 37°C 0.491 0.501 0.511 0.502 0.501 0.493
0.491667 0.502 0.509333 0.502 0.501 0.494
B6-1 45°C 0.465 0.476 0.491 0.481 0.478 0.471
B6-2 45°C 0.461 0.498 0.503 0.495 0.486 0.471
B6-3 45°C 0.479 0.503 0.511 0.488 0.482 0.472
0.468333 0.492333 0.501667 0.488 0.482 0.471333
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
B1 B1-1 10°C 0.178 0.293 0.305 0.302 0.301 0.287
B1-2 10°C 0.177 0.294 0.305 0.305 0.305 0.287
B1-3 10°C 0.178 0.294 0.306 0.307 0.302 0.297
0.177667 0.293667 0.305333 0.304667 0.302667 0.290333
B1-1 27°C 0.491 0.498 0.508 0.501 0.501 0.491
B1-2 27°C 0.487 0.499 0.509 0.511 0.504 0.491
B1-3 27°C 0.491 0.495 0.507 0.511 0.507 0.491
0.489667 0.497333 0.508 0.5076 0.504 0.491
B1-1 37°C 0.508 0.508 0.513 0.513 0.495 0.489
B1-2 37°C 0.499 0.509 0.516 0.513 0.492 0.487
B1-3 37°C 0.499 0.502 0.516 0.513 0.498 0.491
0.502 0.506333 0.515 0.513 0.495 0.489
B1-1 45°C 0.462 0.464 0.507 0.468 0.438 0.421
B1-2 45°C 0.462 0.479 0.508 0.475 0.447 0.429
B1-3 45°C 0.461 0.464 0.507 0.474 0.445 0.425
0.461667 0.469 0.507333 0.472333 0.443333 0.425
61
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
B11 B11-1 10°C 0.183 0.288 0.288 0.396 0.395 0.392
B11-2 10°C 0.195 0.298 0.298 0.396 0.396 0.496
B11-3 10°C 0.193 0.293 0.299 0.396 0.397 0.396
0.190333 0.293 0.295 0.396 0.396 0.428
B11-1 27°C 0.161 0.471 0.511 0.505 0.502 0.488
B11-2 27°C 0.172 0.472 0.511 0.505 0.499 0.486
B11-3 27°C 0.167 0.474 0.512 0.506 0.496 0.485
0.166667 0.472333 0.511333 0.505333 0.499 0.486333
B11-1 37°C 0.474 0.508 0.513 0.505 0.497 0.491
B11-2 37°C 0.476 0.508 0.512 0.508 0.501 0.492
B11-3 37°C 0.484 0.508 0.514 0.503 0.499 0.489
0.478 0.508 0.521667 0.505333 0.499 0.490667
B11-1 45°C 0.449 0.493 0.501 0.489 0.481 0.478
B11-2 45°C 0.401 0.496 0.501 0.491 0.486 0.474
B11-3 45°C 0.453 0.501 0.501 0.489 0.483 0.475
0.434333 0.496667 0.501 0.489667 0.483333 0.475667
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
B7 B7-1 10°C 0.294 0.301 0.307 0.308 0.304 0.299
B7-2 10°C 0.295 0.301 0.309 0.311 0.301 0.297
B7-3 10°C 0.195 0.301 0.311 0.307 0.303 0.298
0.261333 0.301 0.309 0.308667 0.302667 0.298
B7-1 27°C 0.502 0.503 0.511 0.513 0.506 0.501
B7-2 27°C 0.496 0.504 0.511 0.512 0.506 0.501
B7-3 27°C 0.501 0.505 0.511 0.513 0.503 0.495
0.499667 0.504 0.511 0.512667 0.505 0.499
B7-1 37°C 0.508 0.511 0.514 0.516 0.511 0.503
B7-2 37°C 0.512 0.511 0.515 0.515 0.512 0.501
B7-3 37°C 0.511 0.512 0.513 0.516 0.513 0.501
0.510333 0.511333 0.514 0.515667 0.512 0.501667
B7-1 45°C 0.452 0.477 0.466 0.461 0.455 0.449
B7-2 45°C 0.453 0.462 0.457 0.451 0.444 0.435
B7-3 45°C 0.469 0.469 0.461 0.455 0.449 0.437
0.458 0.469333 0.461333 0.455667 0.449333 0.440333
62
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
D5 D5-1 10°C 0.351 0.388 0.411 0.409 0.401 0.391
D5-2 10°C 0.365 0.386 0.409 0.408 0.408 0.391
D5-3 10°C 0.364 0.389 0.411 0.411 0.407 0.401
0.36 0.387667 0.410333 0.409333 0.405333 0.394333
D5-1 27°C 0.472 0.501 0.509 0.508 0.508 0.497
D5-2 27°C 0.455 0.502 0.509 0.506 0.507 0.492
D5-3 27°C 0.466 0.497 0.506 0.501 0.508 0.497
0.464333 0.5 0.508 0.505 0.507667 0.495333
D5-1 37°C 0.505 0.509 0.514 0.511 0.505 0.501
D5-2 37°C 0.508 0.508 0.514 0.512 0.507 0.499
D5-3 37°C 0.504 0.508 0.513 0.511 0.508 0.501
0.505667 0.508333 0.513667 0.511333 0.506667 0.500333
D5-1 45°C 0.491 0.395 0.292 0.287 0.081 0.078
D5-2 45°C 0.489 0.392 0.291 0.285 0.081 0.078
D5-3 45°C 0.472 0.301 0.299 0.287 0.083 0.077
0.484 0.362667 0.294 0.286333 0.081667 0.077667
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
K1 K1-1 10°C 0.391 0.395 0.406 0.411 0.408 0.398
K1-2 10°C 0.388 0.394 0.406 0.411 0.407 0.391
K1-3 10°C 0.391 0.398 0.406 0.411 0.409 0.399
0.39 0.395667 0.406 0.411 0.408 0.396
K1-1 27°C 0.495 0.501 0.512 0.509 0.501 0.495
K1-2 27°C 0.495 0.509 0.511 0.504 0.501 0.489
K1-3 27°C 0.495 0.505 0.513 0.507 0.508 0.496
0.495 0.505 0.512 0.5066 0.503333 0.493333
K1-1 37°C 0.511 0.513 0.513 0.511 0.493 0.488
K1-2 37°C 0.513 0.511 0.514 0.511 0.494 0.485
K1-3 37°C 0.505 0.511 0.515 0.512 0.494 0.483
0.509667 0.511667 0.514 0.5113 0.493667 0.485333
K1-1 45°C 0.491 0.494 0.395 0.391 0.282 0.272
K1-2 45°C 0.486 0.483 0.399 0.381 0.281 0.275
K1-3 45°C 0.401 0.409 0.399 0.382 0.28 0.275
0.459333 0.462 0.397667 0.384667 0.281 0.274
63
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
W31 W31-1 10°C 0.387 0.499 0.411 0.413 0.408 0.397
W31-2 10°C 0.377 0.498 0.411 0.411 0.409 0.394
W31-3 10°C 0.383 0.496 0.412 0.411 0.409 0.399
0.382333 0.497667 0.411333 0.411667 0.408667 0.396667
W31-1 27°C 0.497 0.509 0.511 0.512 0.511 0.503
W31-2 27°C 0.498 0.506 0.511 0.511 0.511 0.502
W31-3 27°C 0.497 0.505 0.509 0.513 0.511 0.502
0.497333 0.506667 0.510333 0.512 0.511 0.502333
W31-1 37°C 0.506 0.515 0.515 0.506 0.505 0.499
W31-2 37°C 0.507 0.515 0.515 0.506 0.501 0.492
W31-3 37°C 0.509 0.513 0.516 0.507 0.502 0.495
0.507333 0.514333 0.515333 0.506333 0.502667 0.495333
W31-1 45°C 0.489 0.392 0.291 0.191 0.181 0.071
W31-2 45°C 0.485 0.392 0.291 0.191 0.181 0.075
W31-3 45°C 0.489 0.393 0.289 0.186 0.181 0.073
0.487667 0.392333 0.290333 0.189333 0.181 0.073
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
D71 D71-1 10°C 0.353 0.399 0.402 0.405 0.402 0.397
D71-2 10°C 0.341 0.399 0.402 0.412 0.401 0.396
D71-3 10°C 0.348 0.399 0.402 0.408 0.401 0.389
0.347333 0.399 0.402 0.408333 0.401333 0.394
D71-1 27°C 0.502 0.505 0.515 0.511 0.511 0.505
D71-2 27°C 0.495 0.499 0.516 0.511 0.512 0.505
D71-3 27°C 0.501 0.501 0.516 0.512 0.511 0.505
0.499333 0.501667 0.515667 0.5113 0.511333 0.505
D71-1 37°C 0.498 0.504 0.515 0.511 0.506 0.495
D71-2 37°C 0.495 0.511 0.518 0.511 0.502 0.495
D71-3 37°C 0.497 0.511 0.515 0.512 0.506 0.495
0.496667 0.508667 0.516 0.511333 0.504667 0.495
D71-1 45°C 0.481 0.501 0.412 0.422 0.411 0.383
D71-2 45°C 0.482 0.494 0.405 0.426 0.401 0.388
D71-3 45°C 0.481 0.492 0.407 0.436 0.421 0.381
0.481333 0.495667 0.408 0.428 0.411 0.384
64
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
Pk2 Pk2-1 10°C 0.389 0.397 0.399 0.409 0.491 0.482
Pk2-2 10°C 0.386 0.395 0.395 0.409 0.493 0.481
Pk2-3 10°C 0.389 0.397 0.398 0.409 0.491 0.481
0.388 0.396333 0.397333 0.409 0.491667 0.481333
Pk2-1 27°C 0.499 0.501 0.503 0.501 0.504 0.501
Pk2-2 27°C 0.495 0.499 0.501 0.506 0.504 0.501
Pk2-3 27°C 0.499 0.501 0.502 0.503 0.504 0.501
0.497667 0.500333 0.502 0.503333 0.504 0.501
Pk2-1 37°C 0.494 0.511 0.512 0.513 0.502 0.498
Pk2-2 37°C 0.496 0.511 0.511 0.512 0.505 0.501
Pk2-3 37°C 0.498 0.512 0.511 0.512 0.504 0.499
0.496 0.511333 0.511333 0.51233 0.503667 0.499333
Pk2-1 45°C 0.481 0.491 0.496 0.408 0.394 0.385
Pk2-2 45°C 0.489 0.494 0.499 0.409 0.399 0.385
Pk2-3 45°C 0.486 0.491 0.499 0.409 0.401 0.385
0.485333 0.492 0.498 0.408667 0.398 0.385
LAB
Isolate
Replicas
Temperature
Incubation Period
18hr 24hr 48hr 72hr 96hr 120hr
K91 K91-1 10°C 0.086 0.188 0.292 0.304 0.312 0.311
K91-2 10°C 0.089 0.197 0.299 0.303 0.312 0.311
K91-3 10°C 0.097 0.199 0.291 0.304 0.314 0.313
0.090667 0.194667 0.294 0.303667 0.312667 0.311667
K91-1 27°C 0.501 0.507 0.511 0.509 0.509 0.501
K91-2 27°C 0.495 0.507 0.511 0.511 0.514 0.501
K91-3 27°C 0.495 0.502 0.511 0.511 0.513 0.501
0.497 0.505333 0.511 0.51033 0.512 0.501
K91-1 37°C 0.501 0.514 0.516 0.504 0.501 0.496
K91-2 37°C 0.499 0.513 0.518 0.511 0.504 0.494
K91-3 37°C 0.505 0.514 0.518 0.507 0.501 0.492
0.501667 0.513667 0.51733 0.507333 0.503 0.494
K91-1 45°C 0.486 0.505 0.501 0.491 0.484 0.481
K91-2 45°C 0.481 0.505 0.501 0.493 0.485 0.479
K91-3 45°C 0.484 0.501 0.501 0.491 0.481 0.471
0.483667 0.503667 0.501 0.491667 0.483333 0.477
65
Annex 3: Colony morphology and Gram staining of Lactobacilli.
Annex 4: Antibacterial activity of LAB against S.aureus (ATCC25923)/Drug sensitivity of
Lactobacillus (D5)
66
Annex 5: MIC values of LAB against the standard and clinical pathogens.
