“Isolation and characterization of lactic acid bacteria from

raw cow milk”

APROJECTREPORTS
UBMITTED
INPARTIALFULFILLMENT OF
REQUIREMENTSFORTHEDEGREEOF

MASTEROF
SCIENCEIN
BIOTECHNOLOGY

Submittedby:
Anshika Tyagi

Underthesupervisionof:

Mrs. MEGHA SIROHI

(Asst.Professor)

DepartmentofBiotechnology,M.I.E.T.,Meerut

N.H.58, Delhi-
RoorkeeHighway,BaghpatRoadBypassCrossingMeerut-
250002,U.P.Ph:-0121-2439019,Fax:-0121-2439057
 ACKNOWLEGDGEMENTS

Research is an ever evolving discipline which needs the close co-operation of friends, colleague
andthe guidance of experts in the field to achieve something worthwhile and substantial to reach
thepinnacleofsuccessin scientificattributes.

I feel pleasure in expressing my profound sense of gratitude and indebtedness to the

Management,Meerut Institute of Engineering and Technology N.H. 58, Delhi-Roorkee Highway,
BaghpatRoadBypassCrossing,Meerut,whoprovidedmetheopportunitytoutilizethefacilitiesavailablein
labs.

IamhighlyobligedtoDr.ShaliniSharma(Principal,DepartmentofBiotechnologyandMicrobiology),M
eerutInstitute ofEngineeringAnd Technology,
Meeruttoarrangeformyprojectintheiresteemedorganization.

I express my heart-leftgratitude tomy guide Mrs. Megha Sirohi,Asst. Professor (project

mentor)whose unique guidance,all embracing help,valuable suggestions and encouragement have
enabledme tocomplete this task which wouldnot have been a success without them.His constant co-
operation,wholeheartedinterest,involvementateachstepofmyworkwithtimelyhelpandcommunicationofr
esearchpapersandfaithinmehasenabledmetocompletethisworksuccessfully. I believe that, hisguidance
will surely formthe basisformy research activitiesinfutureaswell.

Last but not theleast I wish to express my appreciation and regards toMy Parents and AlmightyGod
whohas been the source of motivation, strength andsupport,which ledme toattain success.No amount
of thanks would express the gratitude towards them, as their unconditional love
andaffectionandunfailingsupportwhichhavedirectedmetoachievemygoal.
I wish tothank each andevery individual whom I amfailedtomention personally
forhisorherhelpwithouttheirsupporttheworkwouldnothavebeenpossible.

ANSHIKA TYAGI
 Table of Contents

ABSTRACT ............................................................................................................................................ 4

REVIEW OF LITERATURE................................................................................................................ 8

MATERIALS AND METHODS ......................................................................................................... 15

RESULTS .............................................................................................................................................. 17

CONCLUSION ..................................................................................................................................... 19

ACKNOWLEDGMENTS .................................................................................................................... 20

REFERENCES ..................................................................................................................................... 21
 Abstract

This study aimed to isolate and determine the characteristics of lactic acid bacteria (LAB) from raw cow

milk using serial dilutions and colony & cell morphology.

Lactic acid bacteria are found naturally in raw cow milk as indigenous microbiota and are

utilized as a starter culture to make fermented dairy products. Lactic acid bacteria were recovered from

raw cow milk samples in this study. The serial dilutions of raw milk were made and plated onto M17

agar containing thallium acetate and adjusted to pH 5.5 with lactic acid. The isolates wereidentified

based on cell morphology.

The spotted LAB isolates were characterized as Lactobacillus, Lactococcus, and Leuconostoc.

Keywords: Raw cow milk. Lactic acid bacteria. Isolation. Morphology.Serial dilution
Figure 1. Bacterial structure and morphology. From Hart and Shears, 1997. (Source)
 Introduction

Lactic acid bacteria (LAB) are an industrially important group of bacteria and used as starter cultures for

the production of fermented milk products (e.g. yoghurt and some cheeses) in the dairy industry. LAB contain the

following genera: Aerococcus, Alloicoccus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus,

Leuconostoc, Oenococcus, Pediococcus, Streptococcus, Symbiobacterium, Tetragenococcus, Vagococcus and

Weissella (HORVATH et al., 2009). These groups of bacteria have been isolated from the raw materials of food,

such as raw milk (CORROLER et al., 1998; RODRIGUEZ et al., 2000; TULINI et al., 2016; ASPRI et al., 2017;

PERIN et al., 2017), plants (ALEMAYEHU et al., 2014), meat (EGAN, 1983), vegetables (KELLY et al., 1998),

and fruits (CHEN et al., 2017). Natural habitats, including the indigenous flora of raw milk, can be good source

of novel LAB strains with the potential desirable properties for use in the production of novel fermented dairy

products (CORROLER et al., 1998; RODRIGUEZ et al., 2000; WOUTERS et al., 2002; DELAVENNE et al.,

2012; TULINI et al., 2016; PERIN; NERO, 2014). The most frequently isolated LAB genera from raw milk and

dairy products that were made from raw milk were Enteroccoccus, Lactococcus, Lactobacillus, Leuconostoc, and

Streptococcus (PERRY; SHARPE, 1960; FRANCIOSI et al., 2009). The major sources of bacteria in raw milk

are from within the udder, the exterior of the teats and the udder, the milking machine, the storage equipments,

the housing, bedding, feed, air and water (QUIGLEY et al., 2013).
Cow Milk is a pale liquid produced by the mammary glands of cow. It is the primary source of
nutrition for infant mammals before they are able to digest other types of food. It contains many other
nutrients including protein and lactose (Pehrsson et al., 2000). Moreover, Milk itself is known as one
of the natural habitats of lactic acid bacteria (LAB) (Delavenne et al., 2012), (Wouters et al.,2002).

Lactic acid bacteria (LAB) are a group of Gram- positive, non-sporulating, anaerobic or facultative
aerobic cocci or rods, which produce lactic acid as one of the main fermentation products of the
metabolism of carbohydrates (Axelsson., 2004), (Hayek and Ibrahim., 2013). Lactic acid bacteria
(LAB) are naturally present in milk and milk products. LAB is generally associated with habitat rich
in nutrients such as milk, cheese, meat, beverages and vegetables. In addition, (Tserovska et al.,
2002), (Chen et al.,2005)
showed that lactic acid bacteria could be also isolated from soil, lakes, intestinal tract of animals and
humans. Lactic acid bacteria (LAB) have been used for the fermentation of food and feed products
since ancient days and today their major applications are still in the food and feed industry as starter
cultures (Desmons et al., 1998), (Boonmee et al.,2003).Lactic acid bacteria are the most important
bacteria in desirable food fermentations, being responsible for the fermentation of sour dough bread,
fermented foods and beverages, all fermented milks and fermented vegetables. It plays an essential
role in the production of all dairy products and is involved in the production of many other fermented
foods and beverages, sausages, pickles, boza etc. Based on the end product of glucose fermentation
lactic acid bacteria are grouped as either Homofermenters or Heterofermenters. The Homofermenters
producelactic acid as the major product of fermentation of glucose. The Heterofermenters produce
lactic acid, carbon dioxide, acetic acid, and ethanol from the fermentation of glucose. According to
(Sharma et al., 2012), (Steele et al., 2013) LAB are recognized for their fermentative ability and thus
enhancing food safety, improving organoleptic attributes, enriching nutrients and increasing health
benefits Many LAB species play an important role in the ripening process of cheese, especially to
improve the consistency, aroma and flavor (Hannon et al., 2003), (Duan et al., 2008). Certain LAB
strain characterized by their ability to transform lactose and improves the digestibility of fermented
dairy products (Weinberg et al., 2007) as well as their preservation (Abdelbasset and Djamila., 2008).
They also employed for improvement of the taste, texture and viscosity in the manufacture of dairy
products (Soukoulis et al., 2007). Lactic acid bacteria can be recovered from fermented foods and
beverages, vegetables, milk and milk products. The objective of the study was to isolate and identify
naturally occurring lactic acid bacteria from raw cow milk.
.
 Review of Literature
Lactic acid bacteria are Gram-positive, non-spore-forming, non-respiring but aerotolerant, which

produce lactic acid as one of the key fermentation products by utilizing carbohydrates during

fermentation. These bacteria produce lactic acid as an end product of carbohydrate catabolism and also

make organic substances that contribute to the flavor, texture, and aroma that result in unique

organoleptic characteristics (SADISHKUMAR et al., 1998). Orla Jensen (1919) first published a

monograph that laid the foundation for classifying lactic acid bacteria. This system of classification was

linked to certain factors that entailed the following; glucose fermentation characteristics, cell

morphology, capacity to utilize sugars, and optimum growth temperature range. This classification

system thus recognized only four lactic acid bacteria genera: Lactobacillus, Pediococcus, Leucononstoc,

and Streptococcus (QUINTO et al., 2014).

Figure 2. Schematic representation of the worldwide occurrence (left) and multiple health applications (right) of lactic acid
bacteria. (Source)
 Lactic acid bacteria has also been classified into different genera/species based on their acid

production characteristics by fermenting sugars and its growth at specific temperatures (PARVEZ et al.,

2006). Additionally, the lactic acid bacteria can be classified as homofermentative or heterofermentative

organisms based on their ability to ferment carbohydrates (MOKOENA et al., 2017). The

homofermentative lactic acid bacteria such as Lactococcus and Streptococcus yield two molecules of

lactates from one glucose molecule whereas heterofermentative lactic acid bacteria such as

Leuconostoc, Wiessella, and some lactobacilli generate lactate, ethanol, and carbon dioxide from one

molecule of glucose (SALMINEN et al., 1998). The conventional approach to lactic acid bacteria

classification was based on physiological and biochemical characteristics; however, more recently,

molecular characterization has become an important tool for classification and identification of lactic

acid bacteria. Molecular characterization includes random amplified polymorphic DNA profiling, 16S

rRNA gene sequencing, PCR-based fingerprinting, and soluble protein patterns (SHARMA et al., 2020)

and differentiation of species by multiplex PCR assay by using specific recA derived primers (NI et al.,

2015).

The genus Lactobacillus has recently been reclassified by scientists into 25 genera. This

reclassification was necessitated due to the extent of how diverse the original genus was, which made it

very challenging to classify, name, and distinguish between different lactobacilli (ZHENG et al., 2020).

The new genera are Lactobacillus, Paralactobacillus and the 23 novel genera. The twenty three (23)

novel genera includes: Amylolactobacillus, Acetilactobacillus, Agrilactobacillus, Apilactobacillus,

Bombilactobacillus, Companilactobacillus, Dellaglioa, Fructilactobacillus, Furfurilactobacillus,

Holzapfelia, Lacticaseibacillus, Lactiplantibacillus, Lapidilactobacillus, Latilactobacillus,

Lentilactobacillus, Levilactobacillus, Ligilactobacillus, Limosilactobacillus,Liquorilactobacillus,

Loigolactobacilus, Paucilactobacillus, Schleiferilactobacillus, and Secundilactobacillus (ZHENG et al.,

2020).
 Figure 3. Some probiotic strains giving a brief idea of LAB’s cell morphology. (Source)

Lactic acid bacteria constitute a ubiquitous bacterial group that is widespread in nature in niches

of dairy (fermented), meat and vegetable origin, the gastrointestinal and urogenital tracts of humans and

animals, and soil and water (LIU et al., 2014). The ecology of lactic acid bacteria has transitioned over

time from theirsoil and plant habitats to the gut of mammals. The mammalian intestine is a repository of

100 trillion microorganisms generally called microbiota (HOOPER et al., 2010). The microbiota

colonizes the gastrointestinal tract and is essential for health by enhancing metabolism, digestion and

boosts the immune system (HOOPER et al., 2010). The microbiota is well adapted to the mammalian

gut, based mainly on three factors which include adhesion to intestinal cells, resistance to host barriers,

and substrate fermentation in the gut (LEBEER et al., 2008). Bile salts and low pH also affect the lipid

membrane composition of the microbiota (RUIZ et al., 2013). The adhesion of lactic acid bacteria to the

intestinal cells is facilitated by the action of peristalsis which is coupled with lubrication from mucins

that protect and line the epithelial intestinal cells. This coordination thus ensures an increased adherence

capacity of lactic acid bacteria to the intestinal cells (CORFIELD et al., 2000). Intestinal mucins are
thus very important as their continuous production impedes and prevents pathogenic bacteria from

adhering to the intestinal epithelial cells, thus promoting the activity of resident intestinal bacteria.

Consequently, these gastrointestinal bacteria serve as a barrier system that acts against pathogens

(MOAL et al., 2006). Antimicrobial substances that are produced by Lactobacillus and Bifidobacterium

spp. have been confirmed to possess antimicrobial properties that are exerted against enteropathogenic

bacteria linked to causing diarrhea against (SERVIN et al., 2004), and both genera can exert an

inhibitory effect on the action of pathogenic enteric bacteria (MOAL et al., 2006).

Lactic acid bacteria are among the most important groups of microorganisms used in food

fermentations. They contribute to the taste and texture of fermented products and inhibit food spoilage

bacteria by producing growth-inhibiting substances and large amounts of lactic acid. As agents of

fermentation LAB are involved in making yogurt, cheese, cultured butter, sour cream, sausage,

cucumber pickles, olives and sauerkraut, but some species may spoil beer, wine and processed meats.

It can be challenging to isolate lactic acid bacteria (LAB) from raw milk that has been

refrigerated without pre-incubation since the flora tends to be dominated by Gram-negative bacteria and

the LAB are present in low numbers.

There is no single agar medium that is suitable for the selective isolation of strains all genera of

LAB present in raw milk.

While selective media can be used for some LAB including lactobacilli, some streptococci

including faecal streptococci, leuconstocs and pediococci general purpose growth media and incubation

conditions generally need to be adapted for projects. M17 (Terzaghi and Sandine, 1975) which is a good

general purpose growth medium is generally not selective for LAB. While some strains of Lactobacillus

delbrueckii subsp. bulgaricus are inhibited by the β-glycerophosphate present in M17, caution should be

used in using M17 to enumerate Streptococcus thermophilus in environments containing L. delbrueckii

subsp. bulgaricus.
 MRS MRS + GP

0 9
No Growth

1 29
Poor Growth

0 1
Medium Growth

57 13
Good Growth

*No. of strains (58 tested). From: Shankar and Davies (1977) (Source)

Effect of β-glycerophosphate (1.9 %) on growth of L. delbrueckii subsp. bulgaricus strains in MRS broth. Incubation at 37°C

for 48 h

Note that 23% of the L. delbrueckii subsp. bulgaricus strains tested grew well on M17 indicating

the need for authors using this medium to enumerate Str. thermophilus in an environment containing L.

delbrueckii subsp. bulgaricus to confirm the absence of L. delbrueckii subsp. bulgaricus (Gram stain

and catalase reaction as a minimum).

The simplest method for isolating LAB from raw milk is probably to use M17 or preferably

PLGYG agar (Mullan et al., 1981) containing thallium acetate and adjusted to pH 5.5 with lactic acid.

As discussed M17 is inhibitory to many strains of L. bulgaricus. The thallium acetate inhibits the

growth of Gram-negative bacteria, the usual concentration is 1 part in 2000 parts of media (Harrigan

and McCance, 1998). The lactic acid and the lower pH provide a more selective environment for LAB.

The lactic acid is added aseptically to the agar media after sterilisation and prior to use. The selective

pressure can be increased further by e.g. lowering the pH below 5.5, addition of salts e.g. sodium

chloride, sodium or calcium lactate, sodium acetate, replacing the glucose with sucrose or other sugars

and using antibiotics (Billlie et al, 1992). Incubation temperature can be varied (e.g. 30°C for

mesophiles) as required as can gaseous environment.

 Cell Lactate DNA (mole Growth on Rogosa
Genus
Morphology* Fermentation isomer % G+C)** agar

Cocci in
Lactococcus chains Homo L 33-37 -

D/L, D,
Lactobacillus Rods Homo/hetero L 32-53 ±

Leuconostoc Cocci Hetero D 38-41 ±

±, Most strains are

Oenococcus Cocci Hetero D positive

Cocci in
Streptococcus chains Homo L 40 -

Pediococcus Cocci, tetrads Homo D/L 34-42 +

Tetragenococcus Cocci, tetrads Homo D/L +

*Distinguishing between a short rod and a coccus can be difficult. (Source)

Characteristics of genera of lactic acid bacteria used as starter cultures

The differential characteristics listed in the table are based on phenotypic properties and despite

their limited validity in current microbial classification they are still used and provided the limitations are

understood have some utility.

Molecular and chemotaxonomic methods are being used to assign genus and species designations. The

extent of DNA–DNA and DNA–rRNA hybridization, similarity between profiles produced by restriction

mapping of chromosomal DNA, and the nucleotide sequence of the 16S and 32S RNAs have been found to be

particularly useful in the creation of the genus. Additional methods, including serology, also have provided

further evidence for the validity of genus designation (e.g., antisera) against purified superoxide dismutase, which

has been used to demonstrate a similarity between lactococci but not streptococci or enterococci (Mullan,2014).
 Materials and Methods
Instruments

The instruments used in this study are as follows: sterile bottles, wash bottles, pipette, M17 agar

plate, petri dish, measuring cylinder, beaker glass, stirring rod, test tubes, test tube rack, microscope,

refrigerator, incubator, and pencil.

Materials

The materials used in this study are as follows:raw cow milk, distilled water,and thallium

acetate.

Instruments and Materials Sterilization

All instruments washed until clean and dried. Then the instruments and materials were sterilized

using an autoclave at a temperature of 121 °C and a pressure of 15 psi for 20 minutes.

Sample collection

The study was conducted to isolate and characterize the naturally occurring lactic acid bacteria from

raw cow milk. Several raw cow milk samples were aseptically taken into the sterile bottles from bulk

tanks at different dairy farms located in Meerut. Samples were collected using sterilized sample bottles

and brought to laboratory with icebox for microbiological investigation. Samples were kept in a

refrigerator (around 4°C) till the analysis began.

Isolation of lactic acid bacteria

Serial dilutions of the milk samples were immediately made indistilled water by adding one drop

of raw cow milk in 10mL of distilled water.These dilutions were spread ontoM17 agarcontaining

thallium acetate and adjusted to pH 5.5 with lactic acid. The lactic acid was added aseptically to the agar

media after sterilization and prior to use. The plates were incubated for 48 h at 30ºC and 45ºC anaerobic

conditions. After incubation, the colonies displaying the general characteristics of lactic acid bacteria

were chosen from each plate and streaked on M17 agar plates for characterization.

Morphological Observation of Lactic Acid Bacteria Colonies and Cells

Observations of colony and cell morphology was done after getting a pure culture in a petri

dish.These observations were made visually include shape, color, edges, and the elevation ofbacterial

colonies and cells. The surface of colonies and cells can be seen from the side and the edge of the

colony can be seen from the above.

 Results
Isolates of raw cow milk were obtained from the dilution method. Dilutions of 10-1 were grown

on selective media for lactic acid bacteria, de M17 agar.

Characterization is the basis for bacterial identification. Characterization can be done based on

colony morphology, characteristic of cytology (cell type and Gram staining) and physiological

characteristics. Observed morphology in this study was colony morphology and cell morphology.

Colony morphology includes color types, edges, and elevation of colonies that observed visually, while

the cell morphology was observed using a microscope.

Colony Morphology

Colony Morphology Colony morphology of isolates bacteria are round (circle) shape (100%), has slippery

(smooth) edge (100%), the form of convex elevation (100%), and milky white (75%) and cream (25%) of colony

color.

Cell Morphology

Cell morphology was done to determine the colored smear culture under a microscope and see how the shape of

the cell. The results showed that some isolates are with rod shape of cell and some with cocci shape of cell. These

results are supported by researches (Syahrurachman, 1994) which states that the lactic acid bacteria (LAB) are in

the group of Grampositive with the shape of cell cocci or bacilli.

Characteriistics of colo
ony morphollogy and celll morphology
gy of isolatess and identifi
fied bacteria

Isolate C
Colony C
Cell Lactic Acid
A
Code M
Morphology
y M
Morphology Bacteriaa

T
Type Color Eddge Elevation C Shape
Cell

ISL-1 C
Circle Milky White
W Sm
mooth Coonvex B
Bacilli (rod) Lactobaacillus
ISL-2 C
Circle Milky White
W Sm
mooth Coonvex B
Bacilli (rod) Lactobaacillus
ISL-3 C
Circle Milky White
W Sm
mooth Coonvex C
Cocci (in Lactocooccus
c
chains)
ISL-4 C
Circle Milky White
W Sm
mooth Coonvex C
Cocci Leuconoostoc
(
(spherical)
ISL-5 C
Circle Milky White
W Sm
mooth Coonvex B
Bacilli (rod) Lactobaacillus
ISL-6 C
Circle Milky White
W Sm
mooth Coonvex B
Bacilli (rod) Lactobaacillus
ISL-7 C
Circle Milky White
W Sm
mooth Coonvex C
Cocci (in Lactocooccus
c
chains)
ISL-8 C
Circle Beige/C
Cream Sm
mooth Coonvex B
Bacilli (rod) Lactobaacillus

Ideentified Baccteria in Iso

olates

Lactobacillus Lacctococcus Leuconosstoc

 Conclusion
The study was conducted to isolate and identify the naturally occurring lactic acid bacteria from raw

cow milk collected from different dairy farms in Meerut. With lactic acid bacteria belonging to the

genus Lactobacillus, Lactococcus, and Lecunostoc, were identified from eight raw cow milk samples.

The results obtained from the present study demonstrated that there is a diversity of lactic acid bacteria

in raw cow's milk. The presence of LAB in milk and milk products enhances bioavailability of nutrients

and act as a preservative. From this study, we can conclude that raw can milk is a rich source of LAB.

The local raw milk could serve as source for beneficial lactic acid bacteria in future researches.
 Acknowledgments

The authors would like to acknowledge MIET and faculties of Biotechnology department for their willingness

and providing laboratory facility.

 References
Abdelbasset M, Djamila K, Antimicrobial activity of autochthonous lactic acidbacteria isolated

from Algerian traditional fermented milk “Raïb”. Afr. J. Biotechnol. 2008:7:2908-2914.

Bille, P.G., Espie, W.E. and Mullan, W.M.A. (1992). Evaluation of media for the isolation of

leuconstocs from fermented products. Milchwissenschaft. 47:637-640. This can be downloaded from

www.researchgate.net .

Corfield, A.P.; Myerscough, N.; Longman, R.; Sylvester, P.; Arul, S.; Pignatelli, M.Mucins and

Mucosal Protection in the Gastrointestinal Tract: New Prospects for Mucins inthe Pathology of

Gastrointestinal Disease. Gut 2000, 47, 589–594.

Delavenne E, Mounier J, Déniel F, Barbier G, Le Blay G “Biodiversity of AntifungalLactic Acid

Bacteria Isolated from Raw Milk Samples from Cow, Ewe and Goat over One-Year Period,”

International Journal of Food Microbiology, 2012:Vol. 155, No. 3, , pp. 185-190.

Mullan, W.M.A. (2015). Isolating lactic acid bacteria from milk. [On-line]. Available from:

https://www.dairyscience.info/index.php/cheese-starters/250-isolating-lab.html?jjj=1627495268523 .

Accessed: 28 July, 2021. Updated June, 2016; September, 2019.

Ghosh, T.; Beniwal, A.; Semwal, A.; Navani, N.K. Mechanistic insights into probiotic properties

of lactic acid bacteria associated with ethnic fermented dairy products. Front. Microbiol. 2019, 10, 1–

19.

Harrigan W. F, McCance M. E, Laboratory Methods in Food and Dairy Microbiology.1st Edn.,

Academic Press, London, 1976:pp: 25-29.

Hayek S. A, Ibrahim S. A, Current Limitations and Challenges with Lactic AcidBacteria: A

Review. Food and Nutrition Sciences, 2013:4, 73-87.

 Hooper, L.V.; Macpherson, A.J. Immune Adaptations That Maintain Homeostasiswith the

Intestinal Microbiota. Nat. Rev. Immunol. 2010, 10, 159–169.

Lebeer, S.; Vanderleyden, J.; De Keersmaecker, S.C. Genes and Molecules ofLactobacilli

Supporting Probiotic Action. Microbiol. Mol. Biol. Rev. 2008, 72, 728–764.

Moal VL, L.; Servin, A.L. The Front Line on Enteric Host Defence againstUnwelcome Intrusion

of Harmful Microorganisms: Mucins, Antimicrobial Peptides andMicrobiota.Clin. Microbiol. Rev.

2006, 19, 315–337

Mokoena, M.P. Lactic Acid Bacteria and Their Bacteriocins: Classification,Biosynthesis and

Applications against Uropathogens: A Mini-Review. Molecules 2017, 22,1255.

Mullan, W.M.A., Daly, C. and Fox, P.F. (1981). Effect of cheesemaking temperatures on the

interactions of lactic streptococci and their phages. J. Dairy Res. 48, 465-471.

Ni, K.; Wang, Y.; Li, D.; Cai, Y.; Pang, H. Characterization, identification andapplication of

lactic acid bacteria isolated from forage paddy rice silage. PLoS ONE 2015,10, e0121967.

Parvez, S.; Malik, K.A.; Ah Kang, S.; Kim, H.Y. Probiotics and their fermented foodproducts are

beneficial for health. J. Appl. Microbiol. 2006, 100, 1171–1185.

Quinto, E.J.; Jimenez, P.; Caro, I.; Tejero, J.; Mateo, J.; Girbes, T. Probiotic LacticAcid Bacteria:

A Review. Food Nutr. Sci. 2014, 5, 1765–1775.

Ruiz, L.; Margolles, A.; Sánchez, B. Bile resistance mechanisms in Lactobacillus

andBifidobacterium. Front. Microbiol. 2013, 4, 1–8.

Sadishkumar, V.; Jeevaratnam, K. In vitro probiotic evaluation of potential antioxidantlactic acid

bacteria isolated from Idli batter fermented with Piper betle leaves. Int. J. FoodSci. Technol. 2017, 52,

329–340.

Salminen, S.; Bouley, C.; Boutron-Ruault, M.C.; Cummings, J.H.; Franck, A.;Gibson, G.R.;

Isolauri, E.; Moreau, M.C.; Roberfroid, M.; Rowland, I. Functional foodscience and gastrointestinal

physiology and function. Br. J. Nutr. 1998, 80 (Suppl. 1), S147-S171.

 Servin, A.L. Antagonistic Activities of Lactobacilli and Bifidobacteria againstMicrobial

Pathogens. FEMS Microbiol. Rev. 2004, 28, 405–440.

Sharma, A.; Kaur, J.; Lee, S.; Park, Y.S. Tracking of Intentionally Inoculated LacticAcid

Bacteria Strains in Yogurt and Probiotic Powder. Microorganisms 2020, 8, 5.

Sawsan A. A, Maymouna M. O, Isolation and Identification of Lactic Acid Bacteriafrom Raw

Cow Milk, White Cheese and Rob in Sudan Pakistan Journal of Nutrition 2010: 9(12): 1203- 1206.

Terzaghi, B.E. and Sandine, W.E. (1975). Improved medium for lactic streptococci and their

bacteriophages. Appl. Microbiol. 29, 807-813.

Tserovska L, Stefanova S, Yordanovu T, Identification of lactic acid bacteria isolatedfrom katyk,

goat. Milk and cheese. J. Culture Collections, 2002: 3: 48-52.

Wouters J. T, Ayad E. H, Hugenholtz J, Smit G, “Microbes from Raw Milk forFermented Dairy

Products,” International Dairy Journal, 2002:Vol. 12, No. 2-3,pp. 91-109.

Zheng, J.; Wittouck, S.; Salvetti, E.; Franz, C.M.; Harris, H.M.; Mattarelli, P.;Watanabe, K. A
taxonomic note on the genus Lactobacillus: Description of 23 novel genera,emended description of the
genus Lactobacillus Beijerinck 1901, and union ofLactobacillaceae and Leuconostocaceae. Int. J. Syst.
Evol. Microbiol. 2020