Materials

Chemicals and reagents used in this study were procured from Nice Chemicals Pvt. Ltd. and Sigma-Aldrich Corporation (Sigma, USA). Sterilized reconstituted skim milk with 12% total solids was used for the propagation of lactic cultures and further stored at 7 ± 2°C. Throughout the study, its purity and activity was examined before sub-culturing.

Cultures

Microbial cultures were previously isolated from traditional sweet curd at the Department of Dairy Microbiology, West Bengal University of Animal and Fishery Sciences. The isolates were characterized based on their phenotypic traits, morphological features, physiological properties, and sugar fermentation profiles. From these isolates, 10 Lactobacillus spp. were selected and used in this study. The isolate codes along with the corresponding culture names are given in Table 1.

Table 1.

Traditional sweet curd isolate codes with the corresponding culture names

Activation and propagation of cultures

The selected Lactobacillus spp. were activated in the MRS (De Man–Rogosa–Sharpe) broth at 42°C for 12 h and maintained at 7 ± 2°C. Their microscopy was observed by negative staining. The active Lactobacillus cultures were prepared in the sterile skim milk tubes (42°C/12 h) and stored at 7 ± 2°C for further propagation. Their morphology was microscopically observed with simple staining in skim milk. Thereafter, sub-culturing was done for preparing fermented milk product (fermentate). A 400 ml portion of skim milk was fermented for 24 h at 42°C with the specific Lactobacillus spp. at the rate of 2% (v/v) of inoculum and was divided equally in four parts for analysis of antioxidative and ACE-inhibitory activities of the fermentate on 0th, 3rd, 5th and 7th day of storage at 7 ± 2°C.

Supernatant preparation

For evaluating antioxidative activity as % ABTS inhibition and ACE-inhibitory activity, the fermented samples were centrifuged for 10 min at 10,000 rpm and for assessing the antioxidative activity as % DPPH inhibition, 5 g of fermentate was extracted in 50 ml methanol at 37°C. All the supernatants were then filtered through Whatman No. 42 filter paper.

Determination of antioxidative activity

ABTS [2,2-Azinobis (3-ethylene benzothiazoline) 6-sulphonic acid)] assay

Total radical scavenging capacity was based on the ability of a compound to scavenge the stable ABTS radical in 10 min (Re et al., Reference Re, Pellegrini, Proteggente, Pannala, Yang and Rice-Evans1999) with some modifications. Firstly, the ABTS stock solution was prepared by the reaction between 2.45 mM potassium persulfate and 7 mM ABTS solution in 1:1 ratio and stored in the dark at room temperature for 12–16 h. Then ABTS working solution was made by diluting 1 ml of ABTS stock solution with methanol till it gave an absorbance of 0.70 ± 0.02 at 734 nm. The stock solution of ABTS was stable up to 2 days for analytical purpose. ABTS working solution (3.9 ml) and 0.1 ml of appropriately diluted sample supernatant was mixed and kept at room temperature for 5 min and absorbance at 734 nm was recorded using spectrophotometer. Methanol was used as blank.

The results were expressed as

\begin{align*}\% \text{ABTS free radical inhibition activity} & = (\text{Blank}_{abs} \times \text{Sample}_{abs}) \\ & \times 100/\text{Blank}_{abs},\end{align*}

where Blank_abs is the absorbance of blank sample and Sample_abs is the absorbance of test sample at 734 nm.

DPPH (2,2-Diphenyl-1-picryl hydrazyl) assay

The fermentates were analysed by the method given by Mbunde et al. (Reference Mbunde, Mdegela, Laswai and Mabiki2018) for DPPH radical scavenging ability. Firstly, 0.2 mM DPPH stock solution was prepared by mixing 7.88 mg DPPH powder with 100 ml methanol in an amber coloured reagent bottle and kept in a dark place before 2–3 h of analysis. Then 4 ml of 0.2 mM DPPH solution was added in 1 ml of methanolic extract of sample followed by 2 ml of methanol, mixed properly and was incubated in dark for 90 min at 37°C after covering the test-tubes with aluminium foil. The absorbance of the solution was measured at 517 nm in UV vis-spectrophotometer. Simultaneously, a control (Ac) consisted of 4 ml DPPH solution, and 3 ml methanol was prepared. Methanol was used as blank.

The results were expressed as

\begin{equation*}\% {\text{ DPPH scavenging activity}} = \left( {{\text{Ac}}-{\text{A}}} \right) \times 100/\left( {{\text{Ac}}} \right)\end{equation*}

where Ac is the absorbance of control and A is the absorbance of test sample.

Determination of ACE-inhibitory activity

The assay for the angiotensin-converting enzyme (ACE)-inhibitory activity based on the liberation of hippuric acid from hippuric-L-histidyl-L-leucine (HHL) catalysed by ACE and measured by the method of Cushman and Cheung (Reference Cushman and Cheung1971) with slight modifications done by Solanki and Hati (Reference Solanki and Hati2018).

A 110 μl portion of the supernatant of the fermentates was taken, 50 μl of 5 mM HHL solution and 100 μl of 0.1 M sodium borate buffer (pH 8.3) were added. Thereafter, 20 μl of ACE was added and incubated at 37°C for 30 min. The reaction was stopped by the addition of 1 ml of 1 N HCl. The resulting hippuric acid formed in the enzymatic process was extracted with 1 ml of ethyl acetate by centrifugation at 3000 rpm for 10 min and evaporated by heating at 100°C for 10 min. The dried residue containing hippuric acid was dissolved in 2 ml of distilled water, and the absorbance was measured spectrophotometrically at 250 nm against blank. The blank was prepared by adding HHL, sodium borate buffer sample and distilled water (ACE volume was replaced by an equal volume of distilled water). The control was prepared by adding HHL, ACE, sodium borate buffer and distilled water (volume of sample was replaced by distilled water).

The extent of inhibition is calculated as follows:

\begin{align*}\% {\text{ ACE inhibition}} =& \left[ \left( {{{\text{A}}_{{\text{control}}}}-{{\text{A}}_{{\text{sample}}}}} \right)\right.\\ & \left./\left( {{{\text{A}}_{{\text{control}}}}-{{\text{A}}_{{\vphantom{p}\text{blank}}}}} \right) \right] \times 100\end{align*}

Antioxidative activity

The antioxidative activity of fermented skim milk were estimated using two assay systems in vitro: % ABTS inhibition and %DPPH inhibition.

% ABTS inhibition

The antioxidative activity of Lactobacillus-fermented skim milk increased significantly (p < 0.05) over the 7-day storage period (Table 2). Among the cultures tested, the fermentate derived from the BM₆ culture exhibited the highest antioxidative potential, showing 17.02 ± 0.36% ABTS radical cation inhibition on the 7th day of storage. On the initial (0th) day, however, Lactobacillus delbrueckii subsp. indicus (JSL₂ P₂) demonstrated the greatest activity (14.57 ± 0.67% ABTS inhibition). The observed inter-strain variability in antioxidative capacity (p < 0.05) can be attributed to strain-specific proteolytic systems and the differential release of antioxidative peptides during fermentation.

Table 2.

Changes in antioxidative activity (% ABTS inhibition) of Lactobacillus fermentates during storage at 7 ± 2°C

Notes: 1. Values are the mean of three replicates along with standard error (Average ± SE).

2. a, b, c different superscripts in a row denotes significant (p < 0.05) differences between storage days of each isolates.

3. A, B, C, D different superscripts in a column denotes significant (p < 0.05) differences between isolates at different storage day.

During storage at 7 ± 2°C, the antioxidative activity of JSL₂ P₂ fermentate increased from 14.57 ± 0.67% to 16.08 ± 0.48%, suggesting continued peptide modification and stabilization of bioactive compounds over time. Similar enhancement in antioxidative activity during refrigerated storage has been documented in fermented dairy matrices such as misti dahi, where TEAC values increased significantly (p < 0.05) from 72.99 ± 0.33 to 206.55 ± 4.04 µM Trolox/100 g dry weight, likely due to metabolic activity of lactic cultures and post-acidification effects (Chatterjee et al., Reference Chatterjee, Ranjan, Sen and Mandal2022).

The current findings are consistent with previous observations by Yoon et al. (Reference Yoon, Sung, Jhoo and Kim2019), who reported that yogurt fermented at a lower temperature (27°C for 27 h) showed ABTS inhibition ranging between 31.08 ± 3.69% and 79.23 ± 4.32%, depending on sample concentration. The enhancement of antioxidative potential is generally attributed to the liberation of low-molecular-weight peptides and free amino acids during proteolysis of milk caseins, which act as hydrogen donors or metal chelators (Korhonen & Pihlanto, Reference Korhonen and Pihlanto2006; Sharma et al., Reference Sharma, Garg and De2020).

Additionally, lactic acid fermentation is known to increase the production of exopolysaccharides and bioactive metabolites such as γ-aminobutyric acid (GABA) and conjugated linoleic acid (CLA), which contribute synergistically to the overall antioxidant activity (Li et al., Reference Li, Zhao, Zhang, Zhang, Huang and Li2021; Zeng et al., Reference Zeng, Xu, Zhang, Chen and Zhao2022). The progressive increase observed during storage may also result from the continuous action of residual enzymes or peptide–peptide interactions enhancing radical scavenging capacity (Raveschot et al., Reference Raveschot, Fiore, Faverzani, Cudennec, Coutte, Flahaut, Fremont, Drider and Dhulster2020).

Overall, these results reaffirm that Lactobacillus-mediated fermentation of milk is a promising strategy to enhance the antioxidant potential of dairy products through the generation of bioactive peptides and metabolites, with variations depending on strain specificity, fermentation conditions and storage period.

% DPPH inhibition

The average antioxidative activity of Lactobacillus-fermented skim milk, measured as percent DPPH radical inhibition, increased significantly (p < 0.05) over the 7-day storage period, ranging from 8.25 ± 0.18% to 19.02 ± 0.49% (Table 3). Among the cultures tested, Lactobacillus delbrueckii subsp. indicus (JSL₂P₂) exhibited the highest antioxidative potential, increasing from 14.43 ± 0.72% on day 0 to 19.02 ± 0.49% by day 7. The observed enhancement in DPPH radical scavenging activity during storage suggests continuous generation or stabilization of bioactive compounds, such as low-molecular-weight peptides, amino acids and other fermentation-derived metabolites.

Table 3.

Changes in antioxidative activity (% DPPH inhibition) of Lactobacillus fermentates during storage at 7 ± 2°C

Notes: 1.Values are the mean of three replicates along with standard error (Average ± SE).

2. a, b, c, d different superscripts in a row denotes significant (p < 0.05) differences between storage days of each isolates.

3. A, B, C, D, E, F, G different superscripts in a column denotes significant (p < 0.05) differences between isolates at different storage day.

These findings are consistent with those of Verma (Reference Verma2020), who observed a significant (p < 0.05) increase in in-vitro antioxidative activity in various Lactobacillus strains fermented in skim milk after 24 h of incubation at 37°C, demonstrating the potential of LAB to produce antioxidative metabolites capable of mitigating oxidative stress. Similarly, Roy (Reference Roy2023) reported a rise in DPPH inhibition values (from 10.21 ± 0.03% to 15.90 ± 0.08%) in whey-fortified lassi stored at 7 ± 2°C for 7 days, suggesting that cold storage may promote post-fermentative biochemical reactions leading to higher antioxidant activity.

The present results also align with Biadała and Adzahan (Reference Biadała and Adzahan2021), who found a significant improvement in antioxidant potential (DPPH assay) in goat milk fermented with Lactobacillus plantarum, L. acidophilus and L. rhamnosus during 14 days of refrigeration at 4 ± 1°C. The increase in antioxidant capacity during refrigerated storage has been attributed to the ongoing proteolytic activity of LAB, resulting in the gradual release of antioxidant peptides from casein and whey proteins (Korhonen & Pihlanto, Reference Korhonen and Pihlanto2006; Raveschot et al., Reference Raveschot, Fiore, Faverzani, Cudennec, Coutte, Flahaut, Fremont, Drider and Dhulster2020).

Mechanistically, LAB fermentation enhances antioxidant activity through multiple pathways. The hydrolysis of milk proteins liberates bioactive peptides rich in hydrophobic and aromatic amino acids (e.g., tyrosine, tryptophan and phenylalanine) that donate hydrogen atoms or electrons to neutralize free radicals (Pihlanto, Reference Pihlanto2006; Sharma et al., Reference Sharma, Garg and De2020). In addition, fermentation may increase the levels of bioactive metabolites such as exopolysaccharides, CLA and GABA, which exert synergistic antioxidant effects (Li et al., Reference Li, Zhao, Zhang, Zhang, Huang and Li2021; Zeng et al., Reference Zeng, Xu, Zhang, Chen and Zhao2022). The observed increase during storage could also be influenced by the redox stability of these peptides and their ability to interact with residual enzymes, maintaining or enhancing radical scavenging activity over time.

Overall, the results clearly demonstrate that fermentation with specific Lactobacillus strains substantially improves the antioxidative potential of skim milk, making such fermented products valuable functional foods with potential health-promoting properties related to oxidative stress mitigation. Future studies employing peptide profiling and metabolomic analyses could help identify the key compounds responsible for the enhanced antioxidant activity observed in this study.

ACE-inhibitory activity (% ACE inhibition)

The ACE-inhibitory activity of all Lactobacillus-fermented skim milk samples increased significantly (p < 0.05) during the 7-day storage period (Table 4). Among the tested cultures, the fermentate produced with Lactobacillus delbrueckii subsp. indicus (JSL₂ P₂) exhibited the highest ACE-inhibitory capacity, starting at 47.86 ± 1.95% on day 0 and rising to 53.78 ± 0.83% by day 7. This progressive increase suggests continued enzymatic proteolysis during storage, resulting in the release and accumulation of bioactive peptides with antihypertensive potential.

Table 4.

Changes in ACE-inhibitory activity (% ACE inhibition) of Lactobacillus fermentates during storage at 7 ± 2°C

Notes: 1. Values are the mean of three replicates along with standard error (Average ± SE).

2.a, b different superscripts in a row denotes significant (p < 0.05) differences between storage days of each isolates.

3.A, B, C, D, E, F, G different superscripts in a column denotes significant (p < 0.05) differences between isolates at different storage day.

The increase in ACE-inhibitory activity during refrigerated storage aligns with the findings of Hati et al. (Reference Hati, Sreeja, Solanki and Prajapati2015), who demonstrated that fermentation of skim milk by different Lactobacillus spp. at 37°C for 24 h significantly enhanced ACE inhibition, attributed to strain-specific proteolytic systems and their ability to hydrolyze casein into low-molecular-weight peptides. Similarly, Abdel-Hamid et al. (Reference Abdel-Hamid, Romeih, Gamba, Nagai, Suzuki and Koyanagi2019) reported a gradual rise in ACE-inhibition during cold storage of probiotic fermented milk, confirming that post-fermentative biochemical reactions can further enhance bioactive peptide formation.

The current study’s results are also consistent with Yang et al. (Reference Yang, Zhang, Zhang, Wang and Liu2023), who observed an increasing trend in ACE-inhibitory activity in goat milk yogurt fermented with probiotic cultures and stored for 28 days at refrigeration temperature. Their work emphasized that the antihypertensive properties of fermented dairy products are primarily due to peptides generated during fermentation rather than the presence of live probiotic cells alone.

Mechanistically, the enhancement of ACE-inhibitory activity during storage can be attributed to several factors. Firstly, Lactobacillus strains possess diverse proteolytic systems – including cell-envelope proteinases and intracellular peptidases – that hydrolyse milk caseins into small peptides capable of inhibiting ACE (Pihlanto, Reference Pihlanto2020; Raveschot et al., Reference Raveschot, Fiore, Faverzani, Cudennec, Coutte, Flahaut, Fremont, Drider and Dhulster2020). These peptides often contain hydrophobic amino acid residues (e.g., proline, phenylalanine and tryptophan) at the C-terminal region, which interact with the active site of ACE, reducing its activity (Gupta et al., Reference Gupta, Sharma and De2021). Secondly, residual enzymatic activity during storage may continue to modify peptide profiles, thereby increasing the concentration of bioactive fragments with higher affinity for ACE-binding sites (Chen et al., Reference Chen, Zhao, Chen, Yaqiong and Jianxin2022).

Furthermore, the fermentation-induced acidification of milk can improve peptide solubility and stability, preventing their degradation and enhancing bioavailability (Sharma et al., Reference Sharma, Garg and De2020). The gradual increase in bioactivity during storage thus reflects the dynamic interplay of microbial enzymes, storage temperature, and peptide–protein interactions.

Overall, these findings confirm that specific Lactobacillus strains – particularly Lactobacillus delbrueckii subsp. indicus (JSL₂P₂) – can generate and sustain potent ACE-inhibitory activity in fermented skim milk, positioning such products as promising functional foods for hypertension management. Future studies incorporating peptide sequencing and in vivo validation are recommended to identify and characterize the specific bioactive peptides responsible for this activity.