Bioscience Biotechnology Research Communications

An International  Peer Reviewed Refereed Open Access Journal

P-ISSN: 0974-6455 E-ISSN: 2321-4007

Bioscience Biotechnology Research Communications

An Open Access International Journal

Riteshkumar Arya1, Jaspreet Singh2 and Amar P. Garg*3

1Research Scholar, School of Life Sciences, Jaipur National University, Jaipur, 302017, India.

2Associate Professor, School of Life Sciences, Jaipur National University, Jaipur, 302017, India.

3Vice Chancellor, Shobhit Institute of Engineering & Technology (Deemed to be University), Meerut, 250110, India.

Corresponding author email: amarprakashgarg@yahoo.com

Article Publishing History

Received: 10/02/2020

Accepted After Revision: 20/03/2020

ABSTRACT:

The first thick milk produced immediately after the delivery is called human colostrum (HC). Its composition and functions are quite different than mature milk. It contains high levels of proteins, vitamins, immunoglobulins, carbohydrates, amino acids and many other nutrients. Apart from its nutritional aspects, HC also contains large number of Lactic Acid Bacteria (LAB) with huge probiotic potential. These LAB helps in nourishment, proper growth and development of infants in the early stages of life. The main objective of the study was to characterize and evaluate the probiotic potential of LAB from HC. The study showed several LAB with probiotic potential. The isolated LAB fulfilled all the necessary criteria of a standard probiotics such as growth at low pH, different temperatures, tolerance against bile salts, resistance against antibiotics and antimicrobial activities against common human pathogens. Four isolates of the study were found to be very promising in showing resistance against antibiotics and antimicrobial response against common pathogens such as Escheria coli ATCC 25922, Proteus vulgaris ATCC 33420, Staphylococcus aureus ATCC 25922, Salmonella typhi ATCC 733 and Pseudomonas aeruginosa ATCC 27853. On the basis of biochemical characterization, the isolates were identified as Lactobacillus brevis, L. acetotolerans, L. casei and Pediococcus acidilactici. The present paper deals with the isolation, characterization and  evaluation of probiotic potentials of LAB isolated from HC.

KEYWORDS:

Antagonistic activity, Human Colostrum, Infant Gut,  Lactic Acid Bacteria, Probiotics.

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Arya R, Singh J, Garg A. P. Isolation, Characterization and Evaluation of Probiotic Potential of Lactic Acid Bacteria Isolated from Human Colostrum. Biosc.Biotech.Res.Comm. 2020;13(1).


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Arya R, Singh J, Garg A. P. Isolation, Characterization and Evaluation of Probiotic Potential of Lactic Acid Bacteria Isolated from Human Colostrum. Biosc.Biotech.Res.Comm. 2020;13(1). Available from: https://bit.ly/36qMxrd

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INTRODUCTION

For many years, HC was considered to be a sterile fluid, but recent studies have revised this dogma (Fernandez et al., 2013). The period of flow of HC is from 1st  to 6th day of lactation. The milk produced after the 6th day is mature milk (Castellote et al., 2011). HC is a thick fluid rich in nutrients and contains vitamins, proteins, amino acids, carbohydrates and lipids along with several immune cells which provide immunity to infants in early stages of growth and development (Ballard et al., 2013). Recent studies reveals that apart from all the nutritional aspects of HC, it also contains large number of probiotic bacteria which helps in digestion and protection against infections (Marchesi et al., 2016). The study on milk of Rheses monkey (Mucaca mulatta) first showed that milk contains 19 different species of bacteria belonging to 8 genera in its constituents (Jin et al., 2011). HC also contains large number of other bacteria (Hunt et al., 2011). These bacteria also play a very important role in the development of immune system of infant (Wang et al., 2018).

The number of bacteria in HC are about thrice more than mature milk. The number of bacteria in mature milk lower downs with the continuous regular flow of milk (McGuire et al., 2015). From the studies carried out in the past, majority of the bacteria isolated from human milk  were generally Lactic Acid Bacteria (LAB) (Jost et al., 2015). LAB is a large group of bacteria used worldwide as a probiotic. This group of bacteria involves the microorganisms of  genera Lactobacillus, Lactococcus, Aerococcus, Enterococcus, Pediococcus, Leuconostoc, Streptococcus, Sporolactobacillus, Vagococcus and Carnobacterium (Pavli  et al., 2018 & Neha  2019).

The bacteria of these genera have high probiotic potential and have been proven safe for human consumption (Guesh et al., 2019). The first bacteria that enters the infant gut is from HC. These bacteria enters infant gut through HC and remains in the gut for entire life (Pang et al., 2007). The gut microflora get involves in various biochemical processes and serves several functions in the welfare of human gut (Dunlop et al., 2015). LAB have innumerable health benefits such as blood pressure lowering (Robles-Vera et al., 2017), prevention of colon cancer (Rafter 2003), reduction of allergic symptoms (Cuello-Garcia et al., 2017), reduction of cholesterol (Agerholm-Larsen et al., 2002), boosting of immune system (King et al., 2014), prevention of urinogenital infections (Shortliffe et al., 2013), reduction of Helicobacter pylori infections (Hamilton 2003), Intestinal Inflammation (Jin-Sil  et al., 2018), antimicrobial effects on pathogens (Tankoano  et al., 2019) and many more. Therefore, it can also be said that LAB are boon to infant gut. The present study deals with isolation, characterization and evaluation of probiotic potential of HC.

MATERIAL AND METHODS

Sample Collection: Total 60 different HC samples were collected from lactating mothers who voluntarily gave their consent for our study. All the samples were collected immediately after the delivery from the maternity ward of Jaipur National University Hospital, Jaipur (India). The tubes used for sample collection were autoclaved using standard protocols. The nipples of lactating mothers were cleaned properly with cotton dipped in alcohol to avoid any contaminations of breast skin microflora. The mid flow of HC was carefully aseptically collected in the tubes with the help of experts.

Isolation of LAB:The isolation of LAB from HC was quickly processed after completion of sample collection. The HC samples were serially diluted upto 10-6 using sterile peptone water. The last three dilutions were inoculated on MRS agar plates using Spread Plate Technique. The inoculated plates were incubated at 37℃  for 48 h under anaerobic conditions using anaerobic gas jar.

Enumeration of LAB:After incubation, the bacterial colonies were counted using digital colony counter and Colony Forming Unit (CFU) per mL of HC were calculated using standard method.

Biochemical Characterization of Isolates:Isolated bacteria were sub-cultured to get pure form of colony. The colony characteristics of each isolate were recorded carefully. The pure colonies were further chosen for biochemical characterization. Gram’s staining, Catalase test, Oxidase Test, Arginine Hydrolysis Test and Sugar Fermentation tests were performed to characterize the isolates to be LAB as per recommendation of Bergey’s Manual of Determination Bacteriology.

Gram’s Staining:A single drop of sterile water was dropped on a clean glass slide and a pure colony of isolate was picked from the plate and was mixed gently to prepare a smear. The smear was heat fixed carefully. The standard procedure of Gram’s staining was followed and the slide was observed under oil emulsion lens (10x X 100x) of compound microscope. As LAB are Gram positive in nature, all the isolates which showed Gram positive nature were further processed for other biochemical tests.

Catalase Test:Catalase test was performed for all the isolates which were Gram’s positive. Catalase is a type of enzyme which is produced by several microorganisms that breaks down hydrogen peroxide into water and oxygen and forms bubbles of gas. The 3% hydrogen peroxide solution was mixed gently on the surface of clean glass slide and was observed for bubble formation. As LAB are catalase negative, all the isolates that showed negative results of catalase test were further tested for oxidase test.

Oxidase Test:All the isolates which showed catalase activity negative were further tested for oxidase test. Cytochrome c oxidase is an enzyme found in several bacterial electron transport chain. Presence of cytochrome c oxidase  oxidizes the reagent called tetramethyl-phenylenediamine into indophenols (purple color) end product. As LAB are oxidase negative, all the isolates which showed negative results were further tested for its arginine hydrolysis.

Arginine Hydrolysis Test:Nessler’s reagent and arginine MRS medium were used to check the production of ammonia from arginine. 5 mL of MRS broth was transferred to empty test tube and 100 μl of test culture (O. D 1.0 at 600 nm) was inoculated and the tubes were incubated at 37±1°C for 24h. After incubation, an equal volume of Nessler’s reagent was added to each tube. The immediate appearance of dark orange color was interpreted as positive (presence of ammonia) while indication of yellowish color was interpreted as negative reaction (absence of ammonia) (Kavitha and Devasena 2013).

Sugar Fermentation Test:Carbohydrate when fermented by microorganisms form an acid or acid with gas at the end. Depending on the microorganisms involved, the end products may vary. All the isolates which were Gram positive and catalase and oxidase negative were tested for their sugar fermentation activity. Sugars were prepared using standard protocol (HiMedia) and each tube of sugar contained Durham’s tube in inverted position. Each isolate was inoculated in all different sugars (Glucose, Lactose, Maltose, Fructose, Mannitol, Galactose and Sucrose) to note down the breakdown of sugars into acid and/or acid + gas. Incubation for 48 h at 37 ℃ were given to all the sugars. Results were recorded after completion of incubation period. On the basis of Sugar fermentation activity, the isolates were identified using Bergey’s Manual of Systematic Bacteriology (Hammes P et al., 2009).

Determination of Probiotic Potential.After biochemical characterization, all the isolates were tested for their probiotic potential by testing their growth at low pH, different temperatures, tolerance against bile salts, resistance against common human pathogens and resistance to antibiotics.

Growth at low pH:The pH of human stomach ranges between 2 to 3. It is also believed that food eaten by us stays in stomach for at least 4 h (Bistha N et al., 2019). Therefore, it is the necessary for the isolate to survive at low pH for more than 4 h. To check the growth of isolates at low pH, all the isolates were inoculated in peptone water prepared with different pH (6, 5, 4, 3, 2) for a period of 6 h. After incubation period, the isolates were inoculated on MRS agar plates and were incubated under anaerobic conditions to check their survival at different pH. All the isolates were further checked for their tolerance against bile salts.

Tolerance against Bile Salts:The concentration of bile salts in the intestine is believed to be 0.3% (w/v) and the food eaten stays in small intestine is suggested to be 4 h (Kumari A et al., 2019). Therefore, all the isolates were examined for their growth at different bile salts concentrations. Peptone water with different bile salts concentration was prepared using Oxoid and active cultures of isolates were inoculated in the medium for 6 h. After incubation, the isolates were inoculated on MRS agar plates for its viable count. The isolates which showed good growth on plates were further tested for their growth at different temperatures.

Growth at Different Temperatures: To examine the growth of isolates at different temperature, the active cultures of isolates were inoculated on MRS agar plates and were incubated at different temperatures (25, 30, 35, 40 ℃) in anaerobic conditions. The isolates which showed best growth at both high as well as low temperatures were further screened for their resistance against antibiotics.

Resistance to Antibiotics:The isolates which gave best growth at high as well as low temperature were tested for their resistance against common antibiotics using Kirby Bauer method. The isolates were spreaded on the entire surface of MH agar plates and the discs of antibiotics with different concentrations were placed on the surface of agar and gently pressed. The plates were allowed to incubate at room temperature for 24-48 h. The isolates which did not give appropriate zone of inhibition  around the discs of antibiotics according to standard chart were further examined for their antimicrobial activities against common human pathogens.

Antimicrobial activity:All the isolates which fulfilled the above mentioned criteria were further tested for their antimicrobial activities against common human pathogens using agar well diffusion method. The indicator pathogenic microorganisms were spreaded on the entire surface of Muller Hilton (MH) agar plates and using a sterile core borer of 7 mm diameter. 5 different wells of same size were made by puncturing the MH agar plates. Using micropipettes, 80 μL of overnight grown culture of isolate were inoculated carefully in the wells. The plates were incubated for 24 h in upright position. Thereafter, the zone of inhibition were measured. The isolates which showed greater zones of inhibition were considered having good probiotic potential.

RESULTS AND DISCUSSION

A total of 130 LAB were isolated from the HC of 60 different lactating mothers. The isolates were identified on the basis of physiological and biochemical characteristics. On the basis of Bergey’s Manual of Systematic Bacteriology, 72 different species of LAB were identified. Of these, 4 isolates of LAB were found to be very promising with the potential of probiotics. These isolates were selected on the basis of their antimicrobial activities and their resistance against antibiotics. The average number of LAB count per ml of HC of a health lactating mothers were found to be 108 to 109. The LAB count was measured on the basis Standard Plate Count (Total Viable Count). The isolates were initially confirmed  by using biochemical test such as Catalase, Oxidase, Grams Staining, Arginine Hydrolysis test and Sugar Fermentation test. All the isolates in the present study were found Gram’s positive, Catalase and Oxidase negative and also had the capacity to breakdown sugars into acids and gas [Table 1]. On the basis of their Sugar Fermentation activity and Gram’s morphology [Figure 1], the isolates were identified using Bergey’s Manual of Systematic Bacteriology (Hammes et al., 2009).

Figure 1: Microscopic observation of Gram’s Staining

Figure 1 Microscopic observation of Gram’s Staining

Determination of LAB to be potentially probiotic: All the isolates identified as LAB through biochemical tests were further screened for determining their probiotic potential. Firstly, the growth of isolates were checked at low pH. Out of 130 isolates, 79 showed its positive growth at pH 2 which were further screened for their tolerance against different bile salts concentrations. Out of 130 total isolates, 96 were found to be prominent against tolerating the 0.3% (w/v) bile salts concentrations. These isolates were further examined for their growth at different temperatures. Out of 130 isolates, 77 showed a good growth at 40 ℃ and even at 25 ℃. On basis of these three criteria’s, 34 best isolates were selected for checking their resistance against antibiotics from which 16 best isolates were screened for testing their antimicrobial activity against common human pathogens. Out of 16 isolates, only 4 showed very high degree of zone of inhibition against pathogenic bacteria. The details of these for isolates are mentioned below in Table I, II, III and IV.

Table 1 :Fermentation of different sugars for identification of isolates as per the recommendations of Bergey’s Manual

Sample No. Isolate no. Glucose Maltose Lactose Mannitol Galactose Fructose Sucrose Identification Based on Bergey’s Manual
A G A G A G A G A G A G A G
HC1 I1001 + + + + + + + + + + Lactobacillus acidophilus
I1002 + + + + + + + + Lactobacillus sakei
HC2 I2001 + + + + + + + +` Lactobacillus paracasei
I2002 + + + + + + + + + Lactobacillus fermentum
I2003 + + + + + + + + + Streptococcus oralis
HC3 I3001 + + + + + + + + + + Lactobacillus gasseri
I3002 + + + + + + + Lactobacillus agilis
HC4 I4001 + + + + + + + + Bifidobacterium longum
I4002 + + + + + + +` Lactobacillus rhamnosus
HC5 I5001 + + + + + + + + + + + Bifidobacterium breve
I5002 + + + + + + + + + + Pediococcus demnosus
HC6 I6001 + + + + + + + + + Lactobacillus oris
I6002 + + + + + + Lactobacillus curtus
HC7 I7001 + + + + + + + + + + + Bifidobacterium magnum
I7002 + + + + + + + + + + Lactococcus garvieae
HC8 I8001 + + + + + + + + + Bifidobacterium dentium
I8002 + + + + + + + + + Lactobacillus johnsonii
HC9 I9001 + + + + + + + + + + Lactobacillus gasseri
I9002 + + + + + Lactobacillus helviticus
I9003 + + + + + + + +` Lactobacillus backii
HC10 I0101 + + + + + + + + + Lactobacillus parakefiri
I0102 + + + + + + + Stretococcus bovis
HC11 I1101 + + + + + + + Lactobacillus silage
I1102 + + + + + + + + Lactobacillus rennini
HC12 I1201 + + + + + + + + + Bifidobacterium bifidum
I1202 + + + + + + + + + Lactobacillus rapi
HC13 I1301 + + + + + + + + + + + + + Pediococcus cellicola
I1302 + + + + + + Lactobacillus ozensis
HC14 I1401 + + + + + + + Lactobacillus helviticus
I1402 + + + + + + + Bifidobacterium hapal
HC15 I1501 + + + + + + Bifidobacterium merycicum
I1502 + + + + + + + + + + Lactobacillus acidipiscis
I1503 + + + + + + + + + Lactococcus piscium
HC16 I1601 + + + + + + + + + + + + Lactobacillus acetotolerens
I1602 + + + + + + + +   + Lactobacillus florum
HC17 I1701 + + + + + + + + + + + Bifidobacterium breve
I1702 + + + + + + + + + Lactococcus plantarum
HC18 I1801 + + + + + + + + + Bifidobacterium reuteri
I1802 + + + + + + + + + Bifidobacterium bifidum
HC19 I1901 + + + + + + + + + Lactobacillus plantarum
I1902 + + + + + + + + + + + Lactobacillus acidophilus
HC20 I0201 + + + + + + + + Lactobacillus agalis
I0202 + + + + + + + + + Bifidobacterium dentium
HC21 I2101 + + + + + + + + Pediococcus inopinatus
I2102 + + + + + + + + + Lactobacillus casei
I2103 + + + + + + + + + + Lactobacillus larvae
HC22 I2201 + + + + + + Lactobacillus nagelii
I2202 + + + + + + + + + + Lactococcus formosensis
HC23 I2301 + + + + + + Pediococcus stilesii
I2302 + + + + + Lactobacillus helviticus
HC24 I2401 + + + + + + + + + + Lactobacillus brevis
I2402 + + + + + + + +` Lactobacillus paracasei
HC25 I2501 + + + + + + + + + Pediococcus ethanoliduran
I2502 + + + + + + + + Lactobacillus pontis
I2503 + + + + + Lactococcus raffinolactis
HC26 I2601 + + + + + + + + + + Lactobacillus nuruki
I2602 + + + + + + + + + Bifidobacterium reuteri
HC27 I2701 + + + + + + + Bifidobacterium bombi
I2702 + + + + + + + Lactobacillus perolens
HC28 I2801 + + + + + + + + Pediococcus pentosaceus
I2802 + + + + + + + + + Lactobacillus raoutii
HC29 I2901 + + + + + Lactobacillus helviticus
I2902 + + + + + + + + + + + + + Lactobacillus mobilis
HC30 I0301 + + + + + + Streptococcus ferus
I0302 + + + + + + + +` Lactobacillus buchnerii
HC31 I3101 + + + + + + + + + Lactobacillus parakefiri
I3102 + + + + + + Lactococcus hircilactis
I3103 + + + + + + + + Lactobacillus agalis
HC32 I3201 + + + + + + + + + Lactobacillus casei
I3202 + + + + + + + + + Bifidobacterium reuteri
HC33 I3301 + + + + + + + + + Bifidobacterium bifidum
I3302 + + + + + + + Pediococcus claussenii
HC34 I3401 + + + + + + + + + Aerococcus suis
I3402 + + + + + + + + Bifidobacterium boum
HC35 I3501 + + + + + + + + + Lactobacillus pasteurii
I3502 + + + + + + + + + Lactobacillus raoutii
HC36 I3601 + + + + + + + + + Lactococcus laundensis
I3602 + + + + + + + + + Lactobacillus saniviri
HC37 I3701 + + + + + + + + + Bifidobacterium reuteri
I3702 + + + + + + + + Lactobacillus rogosae
HC38 I3801 + + + + + + + + + Bifidobacterium dentium
I3802 + + + + + + +` Lactobacillus sunkii
I3803 + + + + + + + + + Streptococcus downei
HC39 I3901 + + + + + + + +   + Lactobacillus florum
I3902 + + + + + + + +` Bifidobacterium myosotis
HC40 I0401 + + + + + + + + + Lactobacillus sakei
I0402 + + + + + + + + + + Pediococcus acidilactici
HC41 I4101 + + + + + + + + + Lactobacillus casei
I4102 + + + + + + + + + + + Lactobacillus gasseri
HC42 I4201 + + + + + + + + + Bifidobacterium bifidum
I4202 + + + + + + Lactobacillus acetotoleren
HC43 I4301 + + + + + + + + Lactobacillus rennini
I4302 + + + + + + + + + Lactococcus piscium
HC44 I4401 + + + + + + + + + Lactobacillus plantarum
I4402 + + + + + + + + + Lactobacillus ozensis
HC45 I4501 + + + + + + + + + + + + + Pediococcus cellicola
I4502 + + + + + + + + + + + Lactobacillus acidophilus
I4503 + + + + + + + + + Lactobacillus fructivorans
HC46 I4601 + + + + + + + + + Pediococcus parvulus
I4602 + + + + + + + + + Lactobacillus pasteurii
HC47 I4701 + + + + + + Lactococcus hircilactis
I4702 + + + + + + + + + + Pediococcus demnosus
HC48 I4801 + + + + + + + + + Lactobacillus oris
I4802 + + + + + + Lactobacillus acetotoleren
HC49 I4901 + + + + + + + + + + Pediococcus acidilactici
I4902 + + + + + + + + + Bifidobacterium reuteri
HC50 I0501 + + + + + + + + + + Lactococcus garvieae
I0502 + + + + + + + Lactobacillus perolens
HC51 I5101 + + + + + + + + + Lactobacillus pasteurii
I5102 + + + + + + + + + Bifidobacterium bifidum
HC52 I5201 + + + + + + + Lactobacillus perolens
I5202 + + + + + + + + + Aerococcus sanguinicola
I5203 + + + + + + + + + Lactobacillus plantarum
HC53 I5301 + + + + + + + + + + + Lactobacillus gasseri
I5302 + + + + + + + + Lactobacillus vini
HC54 I5401 + + + + + + + + + + Lactobacillus larvae
I5402 + + + + + + + +` Lactobacillus paracasei
HC55 I5501 + + + + + + Lactococcus hircilactis
I5502 + + + + + + + + + + Lactobacillus gasseri
HC56 I5601 + + + + + + Lactobacillus acetotoleren
I5602 + + + + + + + + + + + + + Lactobacillus mobilis
HC57 I5701 + + + + + + + + Lactobacillus agalis
I5702 + + + + + + + + + Lactococcus lactis
I5703 + + + + + + + + + Bifidobacterium minimum
HC58 I5801 + + + + + + + + + Lactobacillus oris
I5802 + + + + + + + + + Lactobacillus casei
HC59 I5901 + + + + + + Pediococcus stilesii
I5902 + + + + + + + + Bifidobacterium longum
HC60 I0601 + + + + + + + + + + + + + Lactobacillus mobilis
I0602 + + + + + + + + + + Lactococcus garvieae


Table 2: Determination of probiotic potential based on growth at low pH, bile salt tolerance and growth at variable temperatures

Sample No. Isolate no. Growth at different pH Bile Salt Tolerance (%) Growth at different Temperatures (℃)
pH6 pH5 pH4 pH3 pH2 0.2 0.3 0.4 0.5 25 30 35 40
HC1 I1001 + + + + + + + + + +
I1002 + + + + +          + + + + + + +
HC2 I2001 + + + + + + + + + +
I2002 + + + + + + + + + +
I2003 + + + + + + + + + +
HC3 I3001 + + + + + + + + +
I3002 + + + + + + + + +
HC4 I4001 + + + + + + + + + +
I4002 + + + + + + + + + + +
HC5 I5001 + + + + + + + + + + + +
I5002 + + + + + + +
HC6 I6001 + + + + + + + + +
I6002 + + + + + + + + + +
HC7 I7001 + + + + + + + + + + +
I7002 + + + + + + + + + + + + +
HC8 I8001 + + + + + + + + + +
I8002 + + + + + + + + + + + +
HC9 I9001 + + + + + + + + + + + + +
I9002 + + + + + + + + + +
I9003 + + + + + + + +
HC10 I0101 + + + + + + + + + + +
I0102 + + + + + + + + + + + + +
HC11 I1101 + + + + + + + + + + + +
I1102 + + + + + + + + + +
HC12 I1201 + + + + + + + + + + +
I1202 + + + + + + + + +
HC13 I1301 + + + + + + + + + + + + +
I1302 + + + + + + + + + + + +
HC14 I1401 + + + + + + + + +
I1402 + + + + + + + +
HC15 I1501 + + + + + + + + + + + + +
I1502 + + + + + + + + + + +
I1503 + + + + + + + + + + +
HC16 I1601 + + + + + + + + + + + + +
I1602 + + + + + + + +
HC17 I1701 + + + + + + + + + + + + +
I1702 + + + + + + + + + + + +
HC18 I1801 + + + + + + + + + +
I1802 + + + + + + + + +
HC19 I1901 + + + + + + + + + + + +
I1902 + + + + + + + + + + + +
HC20 I0201 + + + + + + + + + +
I0202 + + + + + + + + + + +
HC21 I2101 + + + + + + + + +
I2102 + + + + + + + + + + +
I2103 + + + + + + + + + + +
HC22 I2201 + + + + + + + + + + + + +
I2202 + + + + + + + + + + +
HC23 I2301 + + + + + + + + + + +
I2302 + + + + + + + + + + + + +
HC24 I2401 + + + + + + + + + + +
I2402 + + + + + + + + +
HC25 I2501 + + + + + + + + + + + + +
I2502 + + + + + + + + + +
I2503 + + + + + + + + + +
HC26 I2601 + + + + + + + + + + + +
I2602 + + + + + + + + + + + + +
HC27 I2701 + + + + + + + +
I2702 + + + + + + + + + + + + +
HC28 I2801 + + + + + + + + + + + + +
I2802 + + + + + + + + +
HC29 I2901 + + + + + + + + + + +
I2902 + + + + + + + + + + + +
HC30 I0301 + + + + + + + + + +
I0302 + + + + + + + + + + +
HC31 I3101 + + + + + + + + +
I3102 + + + + + + + + + + +
I3103 + + + + + + + + +
HC32 I3201 + + + + + + + + + + + + +
I3202 + + + + + + + + + +
HC33 I3301 + + + + + + + + + + + + +
I3302 + + + + + + + + + +
HC34 I3401 + + + + + + + + +
I3402 + + + + + + + + + +
HC35 I3501 + + + + + + + + + + + +
I3502 + + + + + + + + + + + + +
HC36 I3601 + + + + + + + + + + + + +
I3602 + + + + + + + + + + +
HC37 I3701 + + + + + + + + + + + + +
I3702 + + + + + + + + + +
HC38 I3801 + + + + + + + + + + +
I3802 + + + + + + + + +
I3803 + + + + + + + +
HC39 I3901 + + + + + + + + + + + + +
I3902 + + + + + + + + + + +
HC40 I0401 + + + + + + + + + +
I0402 + + + + + + + + + + + +
HC41 I4101 + + + + + + + + + + + + +
I4102 + + + + + + + + +
HC42 I4201 + + + + + + + + + +
I4202 + + + + + + + + + + + + +
HC43 I4301 + + + + + + + + + + + + +
I4302 + + + + + + + +
HC44 I4401 + + + + + + + + + +
I4402 + + + + + + + + + + +
HC45 I4501 + + + + + + + + +
I4502 + + + + + + + + + +
I4503 + + + + + + + + + + + + +
HC46 I4601 + + + + + + + + + + + + +
I4602 + + + + + + + +
HC47 I4701 + + + + + + + + + + +
I4702 + + + + + + + + +
HC48 I4801 + + + + + + + + + + + + +
I4802 + + + + + + + + + + + + +
HC49 I4901 + + + + + + + + +
I4902 + + + + + + + + + +
HC50 I0501 + + + + + + + + +
I0502 + + + + + + + + + + + + +
HC51 I5101 + + + + + + + + + + +
I5102 + + + + + +
HC52 I5201 + + + + + + + + + + + + +
I5202 + + + + + + + + + + + + +
I5203 + + + + + + + + + + +
HC53 I5301 + + + + + + + + + + + +
I5302 + + + + + + + + +
HC54 I5401 + + + + + + + + + + + +
I5402 + + + + + + + + + + + + +
HC55 I5501 + + + + + + + +
I5502 + + + + + + + + + + + + +
HC56 I5601 + + + + + + + + + + + + +
I5602 + + + + + + + + + + + +
HC57 I5701 + + + + + + + + + + +
I5702 + + + + + + + + + + + +


Table 3: Evaluation of Resistance of isolates against common antibiotics using disc diffusion method.

Isolate No. Names of Antibiotics
Erythromicin Tetracycline Pencillin Gentamicin Streptomicin Amoxicillin Ciprofloxacin
Measurement on Zone of Inhibition in (mm) and its Resistance (R) or Sensitivity (S) against Antibiotics
L. casei 13 R 9 R 10 R 11 R 9 R 12 R 13 R
L. brevis 12 R 10 R 12 R 12 R 8 R 9 R 14 R
P. acidilactici 9 R 11 R 8 R 9 R 10 R 10 R 10 R
L. acetotoleren 14 R 9 R 9 R 13 R 9 R 11 R 12 R

Table 4: Antimicrobial activity of isolated LAB against common pathogens

Isolate No. Names of Pathogens
E.coli

ATCC-25922

P. vulgaris ATCC-33420 S. aureus ATCC -25922 S. typhi

ATCC-733

P. aeruginosa

ATCC-27853

L. casei 18 mm 17 mm 18 mm 17 mm 18 mm
L. brevis 21 mm 14 mm 16 mm 19 mm 20 mm
P. acidilactici 19 mm 18 mm 18 mm 20 mm 18 mm
L. acetotoleren 20 mm 16 mm 15 mm 16 mm 16 mm

4 best species of LAB were screened out of 130 isolates. Identification of LAB was made on the basis of colony morphology, physiological and biochemical tests as per the guidelines mentioned in Bergey’s Manual of Systematic Bacteriology (Hammes et al., 2009). Similar tests performed by earlier researchers found 8 species of LAB that were Gram positive, catalase and oxidase negative and also showed active hydrolysis of arginine (Kang et al., 2019).

The acidic pH of stomach and antimicrobial actions of pepsin provide an effective barrier for LAB to survive in gastrointestinal tract (Kang et al, 2019). For exerting beneficial effects on host, probiotic should be able to maintain its viability along the gastrointestinal transit by surviving under harsh conditions (Tongwa et al., 2019). The survival rate of the isolates of our study were found to be best even at the pH of 2. Traditional techniques of microbiology were used in the study rather than modern molecular techniques because it is more reliable. Modern techniques have some limitations such as the viability of milk microbes cannot be analyzed, total bacteria counts may be over- or underestimated because of cell-wall composition, DNA extraction methods and the number of microbial 16S gene copies which may lead to the over- or underestimation of bacteria counts. Contamination in DNA extraction kit and reagents was also reported in the past studies (Mc Guire, 2015).

CONCLUSION: Human Colostrum contains of large number of bacteria with probiotic potential which greatly helps the infant in boosting up its immunity and in maintaining the gut microbiome. The number of LAB below this count can be a cause of worry for infant. We also found that LAB have the great potentials of fighting against common human pathogens. In our study, we have found that some LAB have great efficiency to resist against antibiotics. Such species of LAB should be commercialized and marketed at a global stage so that problems related to imbalance in gut microbiome can be solved. Through our studies, we also came to know that unnecessary consumption of antibiotics during the time of pregnancy may reduce the LAB count in HC. Therefore, use of antibiotics used be minimized. There are several other facts which are still not known till date such as existence of LAB in HC is still a mystery. LAB in HC is a wide area of research and still needs lots of genuine studies to be carried out to solve the unknown.

ACKNOWLEDGMENTS

Our team is greatly thankful to the entire staff members of Institute for Medical Sciences and Research Center, Jaipur National University (JNU) for directly and indirectly contributing to our studies. A special thanks to the members of Institutional Ethics Committee of JNU for approving our work. We are also thankful to the nursing staff of JNU Hospital especially Mrs. Mamta for helping us in sample collection throughout our entire study.

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