Functional efficacy of Enterococcus faecium HN4 and Lactobacillus delbrueckii HN5 strains isolated from human milk

ABSTRACT

Aims

The microbiota of human milk is described at length; however, variation in different bacterial genera in milk has been reported. Here, breast milk samples from Indian mothers were used to target bacterial species from the pool of microbial communities in human milk with probiotic potential.

Materials and methods

A culture-dependent technique was used to isolate bacterial cultures, and their physiological and functional properties were appraised.

Results

Two bacterial cultures, E. faecium HN4 and L delbrueckii HN5, were identified as showing considerable tolerance to acid and bile conditions with 73.0–87.0% survival. The cultures showed other in vitro beneficial properties.

Conclusion

The study highlights the presence of potential probiotics in human milk that could be investigated for further use.

Plain Language Summary

Human milk is made of nutrients as well as bacteria that can be beneficial to baby’s health and the development of the bacteria within their gut. This study looked at bacteria from milk samples from Indian mothers to see whether they had probiotic (promote the growth of beneficial bacteria) properties. The study successfully identified two bacterial species, namely E. faecium HN4 and L. delbrueckii HN5, which show very early potential as probiotics but need further testing to confirm their benefits, if any.

1. Introduction

Human milk is considered as the best food for developing infants as it harbors several bioactive peptides and nutritional compounds such as carbohydrates, fatty acids, minerals, vitamins, immunoglobulins, lactoferrin, lysozyme, and antimicrobial proteins. It also comprises a distinct microbiome containing beneficial and commensal microorganisms, which play a crucial role in establishing an infant’s gut microbiota. Breast milk differs in composition from colostrum to late lactation stage, where colostrum is the initial fluid secreted immediately after the newborn's birth and lasts for approximately 2–4 days. It is an amalgam of proteins, immunoglobulins, fat-soluble vitamins, minerals, and growth factors and is considered highly beneficial for infants. Transition (next 4–14 days) and mature milk (follows 2 weeks) are produced after that, including fat and lactose to fulfill the nutritional needs of the developing infant [1]. An infant consumes approximately 800 ml of milk daily, thus ingesting nearly 10⁶-10⁷ microbial cells [2]. The genera predominating in human milk include Enterococcus, Streptococcus, Staphylococcus, Lactobacillus, and Bifidobacterium. The specific bacterial species originating from human milk include Lactobacillus gasseri, Lactobacillus oris, Lactobacillus fermentum, Streptococcus salivarius, Pediococcus pentosaceus, Enterococcus faecalis, Enterococcus durans, and Bifidobacterium brevis, and their characterization has been well documented [3,4]. The probiotic microbiota in breast milk can benefit both maternal and infant health by preventing or treating mastitis, preventing diarrhea, treating allergic diseases, and promoting normal gut bacterial colonization in premature infants. Recently, the novel probiotic strain of human milk origin gained interest for being employed as a safe treatment to control various human diseases. For instance, Rajoka et al. [5] reported Lactobacillus rhamnosus SHA111, SHA112, and SHA113 in human milk that proved to have apoptotic activity against the cervix cancer cells (HeLa) lines. Similarly, L. fermentum CECT5716 and Lactobacillus salivarius CECT5713 originated from breast milk boost the immune system through the augmentation of cytokine and IgA production [6]. Likewise, human milk-derived species, viz. L. gasseri BNR17, L. fermentum MA-7, and L. fermentum MA-8 displayed cholesterol-lowering abilities [7,8]. Furthermore, human breast milk (HBM) produces a variety of compounds that are responsible for antioxidant activity, such as catalase, superoxide dismutase, glutathione peroxidase, and other non-enzymatic substances like lactoferrin, glutathione (GSH), and vitamins A, C, and E. All these compounds protect infants from the oxidative stress caused by free radicals and reactive oxygen species [9–11]. Interestingly, a clinical trial demonstrated that the supplementation of Lacticaseibacillus casei CRL-431 reduced oxidative stress after 15 and 30 days [12].

Therefore, seeking new bacterial isolates with a probiotic perspective among the microbiota of human milk may provide clues about a novel microbial species of an individual. Breast milk microbiota is significantly influenced by the geographic location, antibiotic treatment, dietary habits, and genetic factors [13,14]. Moreover, the route of delivery, ethnicity, lactation stage, and maternal health also play important roles in the microbial diversity of human milk [15,16]. So, the study aimed to isolate and characterize lactic acid bacteria from human milk of healthy Indian mothers, and their probiotic potential was explored using a series of in vitro tests, such as survival under simulated gastrointestinal tract conditions, antibiotic profiling, adhesion potential, antioxidant capacity, β-galactosidase assay, hemolytic activity, and gelatinase activity.

2. Materials and methods

2.1. Sample collection

The human milk samples (n = 14) were collected from healthy lactating women belonging to different districts of Haryana, India. The age of volunteers was not considered in this study. Before collection, the mammary areola and the surrounding skin were washed with sterile water, followed by chlorhexidine. The process involves gently squeezing the breast without using a pump and drop-wise collection of milk samples in a sterile container. Prior to the collection, the first few drops of milk were discarded, and then, 2–5 ml were collected. The samples were transported to the laboratory for bacterial enumeration in an icebox with a cooling bag in it and kept at −20°C until use.

2.2. Isolation and characterization of lactic acid bacteria (LAB)

The milk samples were gently vortexed, and serial dilutions were made using a sterile saline solution (0.85% NaCl). Appropriate dilutions were used, spread onto the de Man, Rogosa Sharpe (MRS) agar (Himedia, Mumbai, India), and incubated aerobically at 37°C for 24–48 h to enumerate LAB. The randomly selected colonies were purified in MRS medium using the streak plate method. Various biochemical and morphological tests such as Gram staining, catalase test (reaction with 3.0% H₂O₂), nitrate reduction (ring formation in nitrate broth after addition of sulfanilic acid solution and α-Naphthylamine solution), citrate utilization (utilization of citrate in media), arginine hydrolysis (reaction with 0.5% arginine in broth), growth in litmus milk (10.0% skim milk containing litmus dye), and analysis of sugar discs (carbohydrate fermentation pattern) including xylose, lactose, glucose, trehalose, mannitol, and arabinose (Himedia, Mumbai, India) were performed as per Bergey’s Manual of Systematic Bacteriology to identify the isolates [17].

2.3. Molecular characterization of isolates

Genomic DNA was extracted from overnight MRS-grown cultures (5 ml) using a QIAamp DNA kit (QIAGEN, Hilden, Germany) as per the instruction manual, and the quality was assessed by 0.8% agarose gel electrophoresis. The 16S rRNA gene was amplified using 27F (5’-AGAGTTTGATCC TGGCTCAG-3’) and 1492 R (5’-GGTTACCTTGTTACGACTT-3’) universal primers. The reaction was carried out in 0.2 ml PCR tubes with a total volume of 50 μl containing 25 μl of PCR master mix, 0.25 μl of 27’F, 0.25 μl of 1492’R, 23.5 μl of molecular-grade water, and 1 μl of DNA template. The reaction was performed in a thermocycler (Bio-Rad T100, USA) under the following conditions: denaturation at 94°C for 30 s, annealing at 52°C for 30 s, and extension at 72°C for 90 s, with a final extension at 72°C for 10 min for 32 cycles. Further, the amplified products were purified using a Qiaex gel extraction kit (Qiagen Hilden, Germany) and sequenced (Applied Biosystems^TM Model no. ABI3130, Thermo Scientific, Germany) by BioKart Pvt. Ltd., New Delhi. Then, the forward and reverse sequences were evaluated using BioEdit 7.2.5 software [18] and curated to form a consensus sequence. The query sequences were then compared using the BLASTn tool (www.ncbi.nlm.nih.gov.in, NCBI, USA) to find sequence homology between previously deposited data in the NCBI database.

2.4. Acid and bile tolerance

The survivability of the isolates was assessed by exposing the cultures to low-pH and high bile salt concentration solutions as described by Jacobsen et al. [19], with slight changes. The overnight-grown cultures in MRS broth (5 ml) were harvested by centrifugation (2500 × g, 10 min) using a centrifuge (Sorvall ST8R, Thermo Scientific, Germany) and washed twice using a phosphate buffer saline (PBS) solution (g/l: NaCl, 7.65; Na₂PO₄, 0.72; K₂HPO₄, 0.21; pH 7.0) procured from Himedia, Mumbai, India. The cells were then resuspended into a PBS solution containing different bile salts (Cat. No. RM008-500 G) concentrations, i.e., 0.5%, 1.0%, and 1.5% (w/v) with an initial optical density (OD) of 0.9–1.0. Similarly, the isolates were resuspended in different solutions with pH 1.5, 2.5, and 3.5 (adjusted with 1 M hydrochloric acid (HCl) solution), having the desired OD. The PBS solution with pH 7.0 and without bile salt was taken as a control. One ml aliquot from each pH solution was taken immediately (0 h) and after 1, 2, and 3 h of incubation to determine the viability of cultures using the plate count method. Similarly, the aliquot was collected from different bile salt solutions after 0, 2, 6, and 8 h of incubation to assess the viability.

2.5. Cell surface hydrophobicity

The adhesion potential of the isolates was assessed as per the method of Tiago et al. [20] with slight modifications. In brief, overnight-grown cultures in MRS broth were harvested by centrifugation (9500 × g for 5 min) at 4°C and washed with PBS solution. The cell pellets were resuspended in the same buffer, and the initial OD at 580 nm (A₀) was determined using a spectrophotometer (Eppendorf AG22331, Germany). Then, the bacterial suspension was mixed with the solvent used for hydrophobicity, i.e., n-hexadecane and xylene individually in a 1:1 ratio. The mixture was then vortexed vigorously for 2 min and incubated for 30 min at 37°C for phase separation. The aqueous phase was separated in a clean test tube, and the final OD₅₈₀ (A) was measured. The percentage of hydrophobicity (H%) was calculated using the following equation:

$$\mathit{H}\mathrm{\%}=\left(\mathit{Ao}-\mathit{A}\right)/\mathit{Ao}\times 100$$

where A₀ = Initial OD

A = Final OD

2.6. Auto-aggregation assay

The assay was performed as per the procedure of Shobharani and Halami [21], with certain modifications. In brief, overnight grown cultures were centrifuged (9500 × g for 10 min at 4°C), washed the resultant pellets, and resuspended in PBS buffer (pH 7.0). Then, the initial OD was measured at 600 nm (A₀), and the cell suspension was left undisturbed at 37°C temperature for 1 h and 2 h. The upper portion of the suspension was separated carefully into a fresh tube to measure the final OD (A_t). The auto-aggregation percent of the isolates was measured using the following equation.

$$\mathit{Auto}-\mathit{aggregation}\left(\mathrm{\%}\right)=\left(\mathit{ODA}\mathrm{o}-\mathit{ODA}\mathrm{t}/\mathit{Ao}\right)\times 100$$

2.7. β-galactosidase assay

For the enzymatic assay, the cell-free extract (CFE) was prepared using the glass bead method. The method involves an overnight MRS-grown culture (5 ml) harvested by centrifugation (2500 × g 10 min, 4°C) and resuspended in 2.5 ml of PBS solution after washing in the same buffer. The sterile glass beads (0.2 mm diameter) were then added to the cell suspension and vigorously vortexed for up to 5 min with constant cooling on ice. The lysate was then centrifuged (3700 × g 10 min, 4°C), and the CFE thus obtained was collected in a fresh tube for the assay. The enzymatic assay was performed as per the method of Kara [22] with minor changes. In short, 100 μl of CFE was mixed with 500 μl of a 10 mm O-nitrophenol-D galactopyranoside (ONPG) (Cat. no: N1127, Sigma Aldrich, St Louis, MO, USA) solution (prepared in 0.05 M sodium phosphate buffer pH 7.0) and incubated for 15 min at 40°C in a water bath. The reaction was stopped by adding 600 μl of 0.05 M sodium carbonate (Na₂CO₃) solution to the reaction mixture and incubating for 5 min at room temperature. The absorbance was then measured at 420 nm, and the unit of the enzyme was calculated using a standard curve. One unit of enzyme is defined as the quantity of enzyme liberating 1 μmol of o-nitrophenol (ONP) from ONPG per unit of time under the assay conditions.

2.8. Antagonistic activity against pathogens

The antagonistic activity of the cultures against various pathogens was evaluated using an agar well diffusion method described previously by Tagg et al. [23]. For this, the cultures were grown in MRS medium at 37°C for 24–48 h, and the cell-free supernatant (CFS) was obtained by centrifugation at 9500 × g for 15 min. The CFS was carefully collected in a clean tube and 30 µl poured in a well of Brain Heart Infusion (Himedia Pvt. Ltd. Mumbai, India) agar plates previously seeded with 500 µl of pathogenic bacterial species, i.e., Escherichia coli NCDC249, Klebsiella pneumoniae MTCC 530, Pseudomonas aeruginosa MTCC1035, Bacillus subtilis MTCC 1335, Bacillus cereus MTCC 1305, Salmonella typhimurium MTCC 1253, and Mammiliococcus lentus MKS (human isolate with accession number ON035499). The plates were then incubated horizontally (plate lids to the upright position) for 18–24 h at 37°C for the inhibition zone.

2.9. Hemolytic and gelatinase activity

The hemolytic activity was determined by streaking the isolates in the MRS medium supplemented with defibrinated mammalian blood (5.0%, v/v) and incubated at 37°C for 48 h for lysis of red blood cells. Similarly, the gelatinase activity of the cultures was examined by streaking on MRS agar plates enriched with 0.3% gelatin (Himedia Pvt. Ltd., Mumbai, India). The plates were initially incubated at 37°C for 48 h and then refrigerated at 4°C for 4 h. The plates were then flooded with a mercuric chloride (HgCl₂) solution (15 g HgCl₂ and 20 ml 4 N HCl solution) to observe any halo around the colonies.

2.10. Mucin degradation assay

The isolates were assessed for their ability to degrade gastric mucin under in vitro conditions as per the method of Miller and Hoskin [24]. To assess mucin degradation ability, the cultures were grown on tryptone agar (a glucose-free medium) supplemented with 0.3% (w/v) hog gastric mucin (Sigma, St Louis, USA) for 72 h at 37°C. The plates were then stained with an amido black solution prepared by dissolving 0.1% (w/v) amido black (Cat. no. MB165-25 G, HiMedia Laboratories Pvt. Ltd., Mumbai (India)) in 3.5 M acetic acid solution, followed by destaining using 1.2 M acetic acid solution.

2.11. Antibiotic susceptibility profile

The antibiotic susceptibility pattern of the cultures was determined by the disc diffusion method. Briefly, the overnight-grown cultures were spread on MRS agar plates which provide a nutrient-rich environment for the growth of isolates. The selected panel of antibiotics with specific concentrations was chosen to evaluate the resistance effect on the tested isolates. The antibiotic discs were then placed on it and incubated uprightly at 37°C for 24–48 h. The inhibition zones were measured using a ruler to determine the diameter of the clear area surrounding the antibiotic disc on the agar plate. The edges of the zones were defined by the point at which bacterial growth became visible. The results are expressed in terms of resistance, moderate susceptibility, or susceptibility as described in Clinical and Laboratory Standards Institute (CLSI) guidelines [25]. Various antibiotic discs used were ampicillin (Cat. no. SD002A), azithromycin (Cat. no. SD204), cefixime (Cat. no. SD211), ceftazidime (Cat. no. SD207), ciprofloxacin (Cat. no. SD060), clindamycin (Cat. no. SD051), doxycycline hydrochloride (Cat. no. SD012), erythromycin (Cat. no. SD013), linezolid (Cat. no. SD215), methicillin (Cat. no. SD019), nafcillin (Cat. no. SD246), penicillin (Cat. no. SD028), rifampicin (Cat. no. SD030), trimethoprim (Cat. no. SD039), vancomycin (Cat. no. SD045), gentamicin (Cat. no. SD016) procured from Himedia Pvt Ltd., Mumbai, India).

2.12. Free radical scavenging ability

The DPPH (2,2-diphenyl-1-picrylhydrazyl) hydrate assay was performed as per the method of Hamad et al. [26]. The isolates were grown overnight in the MRS medium and harvested by centrifugation at 2500 × g for 10 min at 4°C. The supernatant obtained was filtered with a micro-syringe filter (pore size 0.2 µm) (Advanced Microdevices Pvt. Ltd., Ambala, India) to obtain CFS. The cell pellets, on the other hand, were suspended in PBS (pH 7.0) buffer with the OD at 530 nm adjusted in the range of 1.0–1.2 and termed as intact cells. Then, 800 µl freshly prepared DPPH (Himedia Pvt. Ltd., Mumbai, India) (7.8 mg in 100 ml methanol) solution was added to CFS and intact cells separately and mixed well for 30 s. The mixture was then incubated for 30 min in dark conditions at room temperature. The OD was measured at 517 nm (OD_sample) against the control (DPPH in methanol only). The DPPH scavenging ability percentage was calculated as follows:

$$\mathrm{DPPH}\mathrm{scavenging}\mathrm{ability}\left(\mathrm{\%}\right)=\mathit{\hspace{0.17em}}\left(\mathrm{ODcontrol}-\mathrm{ODsample}\right)/\mathrm{ODcontrol}\times \mathit{\hspace{0.17em}}100$$

2.13. Titratable acidity (TA)

TA of lactic cultures was determined as per the method of Baba et al. [27] with minor changes. The isolates were grown overnight at 37°C in skim milk (10.0% nonfat milk solids). 10 ml of the milk sample was taken into the beaker, and an equal amount of distilled water was added. 1 ml of 0.5% phenolphthalein indicator was added, and titration was carried out with a 0.1N NaOH solution. The results were reported as % titratable acidity (expressed as lactic acid). The titratable acidity of fermented milk samples was measured as follows:

$$\%\mathrm{TA}(\mathrm{Titratable}\mathrm{acidity})=\frac{9\mathrm{x}\mathrm{normality}\mathrm{of}\mathrm{NaOH}\mathrm{xVolume}\mathrm{of}\mathrm{NaOH}\mathrm{used}}{\mathrm{Volume}\mathrm{of}\mathrm{sample}}\mathit{\hspace{0.17em}}$$

2.14. Bile salt deconjugation assay

The bile salt hydrolase (BSH) ability of the cultures was determined as per the method of Du Toit et al. and Shehata et al. [28,29] with few modifications. For this, MRS agar was supplemented with 0.5% (w/v) sodium salts of glycodeoxycholic acid (Cat. no. 361311, Sigma Aldrich, USA) and 0.037% (w/v) calcium chloride (Himedia Pvt. Ltd., Mumbai, India) to examine the BSH activity of the cultures. The plates were inoculated in the form of zig-zag streaking with overnight-grown isolates and incubated at 37°C for 72 h. The plates were observed to form a deoxycholic acid precipitation zone around the colonies.

2.15. Statistical analysis

The experiments were carried out in triplicate, and the mean values were presented. The SPSS 16.0 version (SPSS Inc., Chicago, IL, United States) was used to analyze the data using one-way analysis of variance (ANOVA) and to compare the variances between the means of the acid and bile salt test results; the post hoc Tukey test was used. When the probability value of the means was p < 0.05, the differences were statistically significant.

3. Results

3.1. Isolation and biochemical characterization of isolates

Twenty-two bacterial colonies were shortlisted based on typical traits displayed by LAB cultures using a culture-dependent technique. Two out of them were further selected based on their ability to curdle milk within 16–24 h, negative catalase reactions, and named as HN4 and HN5. These isolates displayed white or creamy colored colonies, viscid, buttery, brittle, or mucoid in texture, and other characteristics are tabulated in Table 1. The physiological and biochemical tests indicated that the isolates tentatively belonged to the Lactobacillus and Enterococcus genus.

Table 1.

Morphological and biochemical characterization of isolates.

Strains	HN4	HN5
Cell morphology	Cocci	Rod
Gram	+	+
Catalase	-	-
Citrate utilization	+	-
Oxidase	-	-
Coagulation of milk	+	+
Nitrate reduction	-	-
Arginine hydrolysis	+	+
Sugar fermentation
Lactose	+	+
Xylose	-	-
Trehalose	-	-
Glucose	+	+
Arabinose	+	+
Mannitol	-	-

+ Positive; - Negative.

3.2. Molecular identification of isolates

The amplification of the 16S rRNA gene and electrophoresis results revealed that the 1.5 kb product size was amplified. The contig of HN4 and HN5 was sequenced and analyzed for similarity using the Basic Local Alignment Search Tool (BLAST). Among the two isolates, one isolate displayed the highest sequence similarity (99.8%) to E. faecium, whereas the other one to L. delbrueckii (99.2%). The consensus sequences were then deposited to NCBI with the accession numbers OM281267 and OM397091, respectively.

3.3. Tolerance to acidic and bile conditions

The results of acid and bile experiments are represented in Figure 1, revealing that both the strains varied with respect to survival at different acidic and bile salt conditions. The L. delbrueckii HN5 initially exhibited a log count between 9.4 and 9.7 log CFU/ml, and a significant 2-fold decrease in viability was noted after 3 h of incubation. In contrast, E. faecium HN4 displayed viability ranging from 9.2 to 9.4 log CFU/ml after 0 h, and it declined to 7.5–8.1 CFU/ml after 3 h of exposure to acidic conditions. Likewise, both the strains displayed a decreasing trend of viability during 2, 6, and 8 h of exposure at different bile salt concentrations. The isolates exhibited initial cell counts in the range of ~8.5 log CFU/ml (0 h), which diminished to 6.5–7.6 log CFU/ml after 8 h of incubation. The HN4 and HN5 strains exhibited a mean survivability rate of 76.4% and 88.3%, respectively. Further, the HN5 strain displayed a significant reduction in cell counts (p < 0.05) compared to the HN4 strain, thereby indicating that the latter had the highest survivability in the simulated gastric environment.

Figure 1.

In vitro resistance to acid and bile stress of E. faecium HN4 and L. delbrueckii HN5. Error bars indicate the standard deviation from three independent experiments.

3.4. Cell surface hydrophobicity and auto-aggregation assay

The findings, as shown in Table 2, indicated that L. delbrueckii HN5 displayed a higher affinity (50.0–61.0%) toward n-hexadecane and xylene, while E. faecium HN4 exhibited low hydrophobicity, i.e., 5.0% and 18.0%, to the abovementioned hydrocarbons. The auto-aggregation ability of the isolates increased with time and was found to be in the range of 37.0–48.0%.

Table 2.

Cell surface hydrophobicity and enzymatic activity.

Strains	Auto-aggregation (%)*		Hydrophobicity (%)*		β-galactosidase (U/mL)*
	1 h	2 h	Xylene	n-hexadecane
L. delbrueckii HN5	36.80 ± 0.05a	47.60 ± 0.01b	60.93 ± 0.97a	50 ± 0.10b	19.60 ± 0.007n
E. faecium HN4	40 ± 0.25a	40.8 ± 0.25b	18.13 ± 0.45a	5.3 ± 0.08b	18.8 ± 0.03n

*Values are the mean of triplicate experiments ±standard deviation. Values with different superscripts (a, b) indicate statistical significance (p < 0.05) as determined by Tukey’s test. Superscript “n” represents no significant difference.

3.5. β-galactosidase assay and antimicrobial activity

The results of the enzymatic assay depicted that the E. faecium HN4 produced 18.8 U/ml, while L. delbrueckii HN5 produced 19.6 U/ml (Table 2). Antagonistic assay results exhibited growth inhibition of pathogens such as E. coli NCDC249, K. pneumoniae MTCC 530, P. aeruginosa MTCC1035, B. subtilis MTCC 1335, B. cereus MTCC 1305, S. typhimurium MTCC 1253, and M. lentus MKS (Figure 2). The data affirm that the HN5 isolate demonstrated excellent antibacterial activity compared to the HN4 isolate.

Figure 2.

Antagonistic activity of cell-free extract against pathogens. Error bars indicate the standard deviation from three independent experiments. A bar with the same superscript (a or b) indicates a significant difference (p < 0.05) as determined by the Tukey test.

3.6. Safety evaluation and functional properties assessment

The isolates did not render lysis of blood cells, and therefore, no clear zone was manifested, indicating the non-hemolytic nature of the strains. Similarly, the gelatinase activity of the isolates was found to be negative. Furthermore, the cultures were not able to degrade mucin in the medium as a sole carbon source, and therefore, no halo around the colonies was observed as compared to the positive control.

The antibiotic profile of the isolates demonstrated that L. delbrueckii HN5 was resistant to ampicillin, ciprofloxacin, linezolid, nafcillin, penicillin, rifampicin, and trimethoprim, whereas E. faecium HN4 was found resistant to only two antibiotics, cefixime and ceftazidime (Table 3).

Table 3.

Antibiotic resistance profile.

S. No.		Isolates
	Antibiotic Disc	HN4	HN5
1.	Ampicillin (10 µg)	S	R
2.	Azithromycin (15 µg)	S	S
3.	Cefixime (5 µg)	R	S
4.	Ceftazidime (10 µg)	R	S
5.	Ciprofloxacin (5 µg)	S	R
6.	Clindamycin (10 µg)	S	S
7.	Doxycycline hydrochloride (10 µg)	S	S
8.	Erythromycin (10 µg)	S	S
9.	Linezolid (30 µg)	S	R
10.	Methicillin (10 µg)	S	S
11.	Nafcillin (1 µg)	S	R
12.	Penicillin (2Unit)	S	R
13.	Rifampicin (10 µg)	S	R
14.	Tetracycline (10 µg)	S	S
15.	Trimethoprim (25 µg)	S	R
16.	Vancomycin (10 µg)	S	S
17.	Gentamicin (10 µg))	S	S

R- Resistant; S- Susceptible.

The results of the other traits assessed are listed in Table 4. The isolates produced a BSH enzyme that deconjugates glycodeoxycholic acid to liberate glycine, resulting in free bile salt precipitation around the colonies. Regarding antioxidant activity, the CFS of E. faecium and L. delbrueckii had the highest activity (60.4–69.1%) compared to intact cells (52.8–67.3%). As far as acid production ability is concerned, L. delbrueckii is able to produce more lactic acid (1.4%) than E. faecium (0.9%).

Table 4.

Functional properties of human milk isolates.

Strains	Bile salt deconjugation		Free radical scavenging ability*
		Lactic acid (%)*	Cell-free supernatant	Intact cells
E. faecium HN4	++	0.92 ± 0.25a	60.4 ± 0.01a	52.8 ± 0.08b
L. delbrueckii HN5	+	1.35 ± 0.36a	69.18 ± 0.02a	67.31 ± 0.10b

++ (Strong precipitation); + (Weak precipitation).

*Values are mean of triplicate experiments ± standard deviation. Values with different superscripts (a, b) represent statistical significance (p < 0.05) as determined by Tukey’s test.

4. Discussion

HBM is regarded as a vital source of novel probiotic strains that are intended to assist the developing infant’s immune development and gut microbiota [30]. The complex microbial community includes commensal (live harmoniously with the host without exerting benefits) and LAB (promoting health) colonize the infant’s gut, inhibit pathogen adhesion and aid in maintaining metabolic homeostasis, infantile colic, necrotizing enterocolitis, and immunological tolerance [31]. Furthermore, strains isolated from the animal host are most desired because of their proximity to gastrointestinal tract conditions. They can express beneficial traits better than strains from non-animal sources. Hence, this study aims to explore probiotic organisms from Indian mothers' milk.

In the present investigation, two lactic cultures were isolated from HBM, their identity was confirmed using a 16S rRNA metagenomic technique and designated as E. faecium HN4 and L. delbrueckii HN5. Sunera et al. [32] also used the 16S rRNA technology for the identification of the bacterial culture at the species level. Several investigators have documented the abundance of Enterococci and Lactobacilli such as L. delbrueckii WICC-B-02, L. delbrueckii MA-09, L. delbrueckii MKU 10, L. delbrueckii GERU 3, E. faecium 2C, E. faecium CECT8849 in human milk using the genotyping identification technique [33–35]. Our results are in line with those of Jimenez et al. [36] and Kumar et al. [37], who also reported the presence of E. faecalis and L. delbrueckii in human colostrum and breast milk samples. Recently, Bagci et al. [38] reported a higher prevalence of Enterococcus in human colostrum in Turkey. In our previous study, we also successfully isolated some novel strains of S. thermophilus from human milk and identified them utilizing biochemical and metagenomics techniques [39].

An ideal probiotic species must be capable of surviving during transit from the upper digestive tract to the large intestine without damage. However, E. faecium HN4 has a long history of safe usage and is associated with food fermentation, which still requires careful evaluation and validation of its probiotic properties. The prevalent condition in the gastrointestinal tract is usually low pH and high bile salts that collectively cause an unfavorable environment for a bacterial species to survive [40]. Kumar et al. [37] isolated L. delbrueckii GERU 3 and MKU10 from HBM and reported a 44.3–65.3% survival rate in bile conditions, whereas viability was found in the range of 9.1–36% up to 3 h in acidic solution. Serrano-Nino et al. [41] assessed the survivability of L. delbrueckii from human milk and confirmed that the strain could survive at pH 2.0 and 3.0 after 4 h of treatment. However, the strain failed to survive at 0.2% and 0.4% bile salt concentrations beyond 4 h of incubation. Likewise, six strains of E. faecium from HBM were tested in gastric conditions and were not able to survive at pH 1.0 but showed viability at pH 2.0 and pH 3.0 [42]. Our isolates responded well at low pH (1.5) conditions for up to 3 h, as well as at high bile salt concentration (1.5% w/v) for up to 8 h, demonstrating that they are abundant during passage through the gastrointestinal tract. Generally, LAB isolated from the gastrointestinal environment may endure harsh conditions prevalent during gut transit and have varying survival rates. The plausible rationale behind this observation could be the precipitation of hydrolyzed bile salt by the bsh enzyme, thus improving the survivability of the lactic cultures in the toxic environment of bile acid. Moreover, tolerance to the gastric environment is strictly strain- and species-specific, and multiple factors also affect it, such as H⁺ -ATPase proton pump activity, homeostatic mechanisms, biosynthesis of isoprenoids, electron-chain transport, arginine deiminase, and glutamate decarboxylase [43]. The isolates were then assessed for their in vitro adhesion ability using n-hexadecane and xylene solvent. The findings suggest that E. faecium HN4 possessed low hydrophobicity, while L. delbrueckii HN5 showed high adhesion potential toward xylene and n-hexadecane. The results are in agreement with Ayyash et al. [44], who reported hydrophobicity values of E. faecium from camel milk in the range of 2.9% to 46.9% for xylene and 2.7% to 58.7% for octane. Contradictory results were obtained by Kumar et al. [37] in which L. delbrueckii MKU10 and GERU3 from Indian mother’s milk possessed high hydrophobicity values ranging from 91% to 93.0%. Although cell surface hydrophobicity is a crucial factor in determining the probiotic potential, there may not be a correlation between hydrophobicity and adhesion. For instance, Gharibi et al. 2018 [45] postulated that L. fermentum 88 and Lactobacillus plantarum 9 exhibited medium hydrophobicity toward n-hexadecane but showed strong adhesion to HT29 cell lines.

To out-compete the pathogen adhesion to the gastrointestinal system, the isolates were tested for their inhibitory action against pathogenic strains. The results are in accordance with [46], who proved the high antibacterial activity of L. delbrueckii ASO100 and E. faecium ASO292 against S. aureus, S. typhi, and E. coli. Previous studies on E. faecium and E. faecalis strains derived from human milk, colostrum, saliva, and fecal sources showed considerable antagonistic activity against various pathogens [47–49]. However, Serrano-Nino et al. [41] observed the antimicrobial effect of L. delbrueckii of human origin against E. coli only. The difference in antagonistic activity may be justified by the fact of the production of a variety of different inhibitory metabolites such as organic acids, acetoin, bacteriocins or bactericidal proteins, hydrogen peroxide, and aldehydes, diacetyl (e.g., β-hydroxy-propionaldehyde), which in turn depends on the genetic constitution of the strains [50].

β-galactosidase activity may be beneficial to the infants as the bacterial cells owning the enzyme would release it during gastric transit. The bacterial cells get permeabilized during the gastric passage, which facilitates the release of beneficial enzymes that can aid in digestion and produce the desired effect. Therefore, β-galactosidase of both the isolates was studied, and the results revealed a considerable amount of enzyme production. Ibrahim [51] isolated L. delbrueckii from camel milk with enzymatic activity in the 0.9–1.9 U/ml range. Several species of lactobacilli, such as Limosilactobacillus reuteri ATCC 23,271, Lactobacillus acidophilus ATCC 4346, L. delbrueckii subsp. bulgaricus ATCC 11,842, and Lactobacillus helveticus 15009 were procured from the American Type Culture Collection (ATCC), Rockville, USA, for β-galactosidase activity and found to produce 0.6, 0.4, 0.3, and 0.01 U/ml of the enzyme, respectively [52]. It might be due to the difference in the lactose concentration in the medium and the expression of the corresponding lac gene in the presence of different sugar compounds.

Since the nature of enterococci as a probiotic is still debatable, they must be examined for safety evaluation. Hence, the qualitative assessment of the isolates concerning their ability to hydrolyze gelatin and red blood cells was carried out, as these features are essential virulence factors in pathogenic microorganisms. The results are in line with those of Javed et al. [53], who claimed the non-hemolytic nature of some autochthonous species, i.e., Limosilactobacillus, Enterococcus, and Weisella, isolated from human milk. Similarly, Hussain et al. [54] isolated Enterococcus faecium PL7, L. delbrueckii PL11, and PL13 strains from dairy products and found that no strains showed hemolytic (Gamma hemolysis) and mucin degradation activity. In another study, three strains of L. delbrueckii were isolated from mozzarella cheese that lacked the mucin degradation ability [55]. Likewise, Anjum et al. [56] tested three strains of E. faecalis from breast milk without hemolytic and gelatinase ability.

Antibiotic susceptibility is desired in the probiotic isolates as the incidence of human milk isolated enterococci containing the antibiotic-resistance gene is higher. Therefore, these genes could transfer among the microbial community of the lumen of the host. Our findings confirm that E. faecium HN4 is sensitive to most antibiotics, including vancomycin, which is desirable. In addition, L. delbrueckii displayed susceptibility to azithromycin, cefixime, ceftazidime, clindamycin, erythromycin, methicillin, vancomycin, doxycycline hydrochloride, and gentamicin. The results are in accordance with the earlier report of Tougay et al. [57], who demonstrated that E. faecalis spp. from HBM origin were vancomycin-sensitive. Contradictory results were observed by Mahmoud et al. [46], who found that L. delbrueckii ASO100 from human milk was found to be resistant to vancomycin and neomycin. The resistance property of LAB against antibiotics may be intrinsic in nature; therefore, the chances of horizontal transfer would be minimal.

The antioxidant activity is of paramount importance as the LAB strains play a role in alleviating the progression of many chronic conditions due to elevated levels of reactive oxygen species (ROS). The results are consistent with Rastogi et al. 2020 [58], who found that the CFS of Lactobacillus mucosae SRV5 and SRV10 strains had higher antioxidant activity than intact cells. Asan Ozusauglam and Gunyakti [35] recently isolated L. delbrueckii MA-9 from human milk with 43.4% antioxidant capacity. Likewise, E. faecium 27321 originated from cheese and showed an antioxidant ability of 32.9% [59]. Various studies have demonstrated that DNA hydroxylation, protein denaturation, and lipid peroxidation cause oxidative stress to harm cells. However, the formation of exopolysaccharides, bioactive peptides, and antioxidant enzymes helps to overcome the stress conditions [60].

The BSH activity of probiotic cultures has the ability to reduce levels of serum cholesterol in hypercholesterolemic patients. Our results corroborated with the observation of Anjum et al. [56], who reported the BSH activity of different LAB strains originated from mother’s milk. Similarly, Kusada et al. [61] confirmed the presence of the BSH enzyme in L. gasseri JCM 1131. The production of lactic acid is another desirable trait of probiotic bacteria that delivers texture and flavor to fermented food products and also suppresses the growth of undesirable bacterial populations. Our findings manifested the acidification abilities of L. delbrueckii HN5 and E. faecium HN4, suggesting that they could be utilized in the food industry. Similarly, E. faecalis strains isolated from Spanish cheese produced lactic acid, ranging between 0.4% and 0.6% after 24 h of fermentation. In concurrence, Cardenas et al. [34] also stated that the E. faecium CECT 8849 originated from human milk produced 0.8% lactic acid after 24 h. This aspect could be employed in fermented foods to enhance the flavor of cheese and its organoleptic properties.

5. Conclusions

This study concluded that two bacterial species, viz., E. faecium HN4 and L. delbrueckii HN5 from the breast milk of Indian mothers, possess probiotic features such as tolerance to simulated gastric conditions, inhibitory action against pathogens, BSH activity, and adhesion capacity. The functional attributes of the isolates, including antioxidant, β-galactosidase activity, and lactic acid production, mark them as multifaceted agents in promoting overall health. Lactic cultures have the potential to promote gut health and prevent infections. Additionally, the strains manifested safety parameters like susceptibility to various antibiotics, including gentamicin and vancomycin, non-hemolysin producers, absence of gelatinase activity, and non-mucolytic behavior, suggesting their nonpathogenic nature and thus have the potential to be used as new probiotics. However, to determine their suitability for human consumption, this study recommended further in vivo testing.

Funding Statement

The first author received financial support from the Indian Council of Medical Research (ICMR), New Delhi, India [Grant No.: 3/1/3/JRF-2017/HRD-LS/50430/89].

Article highlights

• In this study, bacterial species were isolated from human milk, and their identity was established using metagenomic techniques. It was inferred that the isolates belonged to E. faecium and L. delbrueckii.

• A comprehensive in vitro analysis of probiotic attributes manifested that both cultures could survive under simulated gastrointestinal conditions.

• The data on functional properties further demonstrated that the isolates lack the pathogenic traits, have colonization capacity, and could prevent cardiovascular disease and pathogen infection.

• The findings suggested that both isolates have probiotic potential and may be considered for further testing for their potential use in industry.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Ethical declaration

This study was approved by the Research Committee, DCRUST, Murthal (Reference No. DCRUST/regn./6.3/2019/200). Written informed consent was obtained from all participants.