Bacterial strains and growth conditions
Healthy human vagina and gastrointestinal tract are important sources of gut-stabilizing beneficial bacteria. Maternal microbiota is the foundation of gut development of the neonate. Thirteen Enterococcus strains used in the study were isolated from the healthy human vagina and fresh meconium of the neonates and identified by 16S rRNA sequencing and submitted to NCBI GenBank under accession numbers KX830968–KX830982. The study was approved by the Ethics Committee of Lokmanya Tilak General Municipal Corporation Hospital, Mumbai, India. The non-virulent strains were previously characterized for in vitro probiotic aspects [1]. The study involved strains indigenous to human source as human origin strains are preferred for probiotic use according to the criterion laid down by Food and Drug Administration (FDA). The strains were grown in MRS broth at 37 °C aerobically for 18 h, preserved in 40% glycerol stocks at − 20 °C and sub-cultured periodically. All the strains were sub-cultured in MRS broth before use for each assay. The commercially well-known probiotic strain Lactobacillus casei Shirota (L. casei YIT 9029) was used as a reference strain for comparison for all the assays.
Starch, Tween 80, and cellulose hydrolysis
The amylolytic and cellulolytic activity was checked by spot inoculating the strain on MRS agar supplemented with 1% starch and 1% carboxy methyl cellulose following incubation at 30 °C, for 4–5 days. Lugol’s solution was added and spread all over the plate and the halo created due hydrolysis of starch and cellulose was measured [15]. The lipolytic activity was checked by streaking culture on MRS medium supplemented with 1% Tween 80 following incubation at 30 °C for 4–5 days. Precipitation with a halo around the colonies indicated lipolytic activity [16].
Gelatinase, urease, and oxidase activity
The culture strain was stab inoculated into gelatin medium (peptone 5 g l−1, beef extract 3 g l−1, gelatin 120 g l−1) and incubated at 37 °C for 5 days. The tubes were then kept at 10 °C for 15–20 min and observed for gelatin liquefaction by tilting the tube [17]. The urease activity was checked by inoculating strains in Christenson’s urea agar plates with phenol red as the indicator. A pink zone around the colony indicated urease activity [18]. For oxidase activity, a well-isolated colony of the test culture was spread on the oxidase disc. A blue color change within 15–30 s was reported as a positive reaction for the presence of oxidase enzyme [19, 20].
Hippurate and esculin hydrolysis
A hippurate impregnated disc was added to BHI broth with the test culture and incubated at 37 °C for 24 h. Two milliliters of the supernatant obtained by centrifugation was mixed with 2 ml of ferric chloride reagent. Precipitation persisting for more or equal to 10 min indicated the presence of hippurate enzyme [21]. For esculin hydrolysis, 18-h-old test culture was swabbed and esculin discs were embedded on bile containing agar, incubated at 37 °C for 72 h. Black precipitation around the disc indicated esculinase activity [22].
Deconjugation of bile salts
For this test, MRS agar supplemented with 0.5% taurodeoxycholic acid and 0.37% calcium chloride was poured into petri plates and placed in anaerobic jars for at least 72 h before use. Wells were cut and inoculated with 10 μl 18-h-old culture. The plates were then incubated anaerobically at 37 °C for 72 h. Highly active strains precipitated bile in 48 h. Precipitation halo around the colonies was measured for bile deconjugation activity. Lactobacillus casei Shirota and E. coli ATCC 8739 were used as positive control and negative control, respectively [23].
Production of biogenic amines
An improved method was applied to check the decarboxylase activity of the strains. The decarboxylase activity was checked by inoculating various amino acids in the Moeller’s decarboxylase medium (Sigma Chemicals, USA). The amino acid discs of ornithine, lysine, tyrosine, proline, histidine, arginine, and serine were used for detecting biogenic amine activity. The disc was inoculated into the broth and overlaid with paraffin oil and incubated at 10 °C, 20 °C, 25 °C, and 37 °C for 10 days, respectively. The color change from yellow to purple was scored as positive for the amine production [24].
Protease, amylase, and lipase activity
The protease activity was evaluated by using casein as the substrate as described by Nguyen et al. One unit of protease activity was determined by the amount of enzyme required to release TCA-soluble fragment giving blue color equivalent to 1 μg of tyrosine [25].
The alpha-amylase activity of enterococci was determined by DNSA (dinitro salicylic acid) method. The supernatant of cells grown in BHI medium was obtained by centrifugation (8000 rpm, 15 min, 4 °C). The crude supernatant was then used for determining the amylase activity. One unit of an enzyme is defined as the amount of enzyme that allows the hydrolysis of 10 mg of starch for 15 min under given conditions [26].
For lipase assay, test culture was grown in MRS broth supplemented with 1% tributyrin for 48 h at 37 °C. The culture was centrifuged (8000 rpm, 10 min, 4 °C), resuspended in 0.05 M of PBS (pH 6.5), and sonicated to obtain cell-free supernatant. The substrate used for analysis consisted of 5% polyvinyl alcohol mixed with 20 ml of olive oil and 80 ml of double distilled water. The mixture was stirred overnight and sterilized prior to use. Then, 2.5 ml of the substrate with 2 ml of buffer and 0.5 ml of supernatant was incubated at 37 °C for 20 min. The reaction was terminated by adding 10 ml acetone. Distilled water was used as a control in the assay. A unit of lipase enzyme can be defined as the amount of 0.05 M NaOH required to neutralize the released after hydrolysis of fatty acids per 1 ml of enzyme per minute [27].
β-galactosidase activity
The cell pellet was obtained by centrifuging 18-h-old culture at 6000 rpm, 4 °C for 10 min. The pellet was washed twice, centrifuged again, and resuspended in PBS (0.01 M, pH 7). The cells were sonicated or homogenized using mortar and pestle at 4 °C. The resulting supernatant was then filtered to obtain a cell-free supernatant. A single ONPG disc was added to 1 ml of the supernatant and incubated for 30 min at 37 °C. The reaction was stopped by adding 2 ml Na2CO3 (0.6 M). β-galactosidase cleaves ONPG to release ortho-nitrophenol. Its content was measured by noting the absorbance at 420 nm for 0, 30, 60, and 90 min interval time [28].
Conjugated linoleic acid production
An improved rapid method was employed for CLA producing enterococci. Briefly, test cultures were inoculated in MRS broth supplemented with free linoleic acid (0.5 mg ml−1) and castor oil (1%) and sunflower oil (1%) followed by incubation at 37 °C for 48 h. One percent Tween 80 was added to each tube for production of lipase and free availability of linoleic acid from the substrates. The culture broth (1 ml) was centrifuged at 20,800 g for 1 min and the pellet was discarded. The supernatant was mixed with 2 ml of isopropanol, vortexed and rested for 3 min, following acid extraction by adding 1.5 ml hexane. The CLA content was determined spectrophotometrically at 233 nm by dispensing 230 μl of fat-soluble hexane layer in microtiter plate [29].
Lactic acid production
Briefly, 25 ml of test broth culture was titrated with 0.1 N NaOH using 1 ml phenolphthalein as the indicator until the color change to pink. The lactic acid content was calculated according to AOAC standards (1 ml NaOH = 90.08 mg of lactic acid) [15, 30].
Determination of reduction activity by ferric reducing antioxidant power (FRAP)
The culture was centrifuged at 8000 rpm for 10 min at 4 °C. The cell-free supernatant was obtained and filtered through 0.22 μ Merck-Millipore filter and used for antioxidant assays. Briefly, 0.5 ml of cell-free supernatant was mixed with 0.5 ml of 0.2 M sodium phosphate buffer (pH 6.6) and 0.5 ml of 1% potassium ferricyanide (w v−1). The above mixture was incubated at 50 °C for 20 min. The solution was then cooled rapidly and 0.5 ml of 10% TCA (w v−1) was added. The mixture was then centrifuged at 3000 rpm for 5 min. One milliliter of the upper layer of the mixture was mixed with 1 ml of 0.1% FeCl3 (w v−1). The absorbance was noted after 10 min at 700 nm. Distilled water was used as blank for the experiment. Trolox was used as a standard for reducing power activity. A higher absorbance indicated higher reducing power. Trolox (0.5 mg ml−1) was used as the standard [31].
DPPH free radical scavenging assay
The DPPH (2,2-diphenyl-1-picrylhydrazyl) assay is based on the scavenging of DPPH radicals leading to a decrease in the absorbance at 517 nm. The cell-free supernatant was 1:10 diluted with distilled water (D/W) before use. Around 40 μl of cell-free supernatant was mixed with 140 μl of methanol and 40 μl DPPH (0.15 mM). The absorbance was noted at 517 nm. Trolox (0.5 mg ml−1) was used as a standard in the assay [32].
The percentage inhibition was calculated by the following formula:
$$ \mathrm{DPPH}\ \mathrm{scavenging}\ \mathrm{activity}=\frac{\left(\mathrm{Absorbance}\right)t=0-\left(\mathrm{Absorbance}\right)\ t=15\ }{\left(\mathrm{Absorbance}\right)\ t=0}\times 100, $$
where t0 = absorbance at 0 min and t15 = absorbance after 15 min.
ABTS free radical scavenging assay
The ABTS radical scavenging activity was measured 734 nm using Hitachi Spectrophotometer. The reagent was prepared by mixing 7 mM ABTS in water with 2.45 mM potassium persulfate to produce the radical. This mixture was allowed to stand for 16 h in the dark before use. The reagent was freshly prepared and used within 3 days. The ABTS stock solution was diluted with methanol and set to an absorbance of 0.7 ± 0.020 at 734 nm. The supernatants obtained after centrifugation were diluted (1:10) with methanol to obtain inhibition in the range of 20–95%. Trolox (0.5 mg ml−1) was used as standard for the assay. Forty microliters of test supernatant was mixed with 260 μl of ABTS solution and the absorbance was noted after 6 min at 734 nm. The lowest absorbance indicates the highest antioxidant activity. The percentage inhibition was calculated by the following formula:
$$ \mathrm{Percent}\ \mathrm{inhibition}=1-\frac{As}{Ac}\times 100; $$
where Ac = absorbance of control and As = absorbance of sample [33, 34].
Statistical analysis
All the assays were performed in triplicate. The Statistical Package for Social Sciences (SPSS) software for Windows version (16.0) was used to compare the strains using analysis of variance (ANOVA). Post hoc tests like Duncan were approached to show the significance at p < 0.05.