Coronary heart diseases, are among the most prevalent cardiovascular diseases (CVDs), encompassing various pathological conditions that impact the heart and blood vessels. In 2023, the World Health Organization (WHO) reported that CVDs were responsible for around 17.9 million deaths globally, accounting for approximately 32% of all deaths worldwide (Tsao et al., 2023). Modifiable risk factors for MI include hypercholesterolemia, hypertension, obesity, diabetes, smoking, environmental factors, and modern lifestyle changes. These factors are closely linked to alterations in gut microbiota and its metabolites (Cai & Hui 2022; Ghorbani et al., 2023). Previous research has shown a strong association between CVD pathogenesis and imbalances in intestinal microbiota and inflammatory responses. Probiotics, which positively influence the microbial and metabolic composition of gut microbiota, are considered a potential therapeutic strategy for CVD (Wu & Chiou 2021). The range of cardiovascular-related diseases that can be alleviated through probiotic supplementation has broadened to include conditions such as hypercholesterolemia, atherosclerosis, myocardial infarction, heart failure, type 2 diabetes mellitus, and obesity (Zhao et al., 2021). Probiotics have been shown to protect against CVD by lowering cholesterol levels, reducing oxidative stress, balancing functional and structural changes in gut microbiota, and enhancing immune responses (Oniszczuk et al., 2021).

Oxidative stress plays a crucial role in the progression of CVDs. An increase in reactive oxygen species (ROS) production has been observed in various cardiac conditions (Dubois-Deruy et al., 2020). Reducing oxidative stress is one of the protective effects attributed to probiotics. Probiotics exert antioxidant effects by scavenging ROS, enhancing superoxide dismutase activity, and reducing or preventing lipid peroxidation and ascorbic acid autooxidation (Amaretti et al., 2013; Sadeghzadeh et al., 2017). Specific strains of Lactobacillus and Bifidobacterium spp. exhibit antioxidant properties, helping to mitigate oxidative damage (Avila-Escalante et al., 2020). 

Bile salt deconjugation is a key factor in selecting probiotics due to its impact on cholesterol metabolism (Staley et al., 2017; Foley et al., 2019). Further, probiotics can reduce blood cholesterol by absorbing it, binding it to their cell membranes, or metabolizing it, thereby reducing its absorption in the gut and potentially lowering coronary artery disease risk (Tomaro-Duchesneau et al., 2014). The role of intestinal flora in atherosclerosis has gained attention as a potential target for preventing and treating CVDs. Pancreatic lipase, crucial for digesting 50 to 70% of dietary lipids, breaks down triglycerides for absorption. Finding effective pancreatic lipase inhibitors, particularly natural ones with fewer side effects, is increasingly important. Various lactic acid bacteria (LAB) strains, especially lactobacilli and bifidobacteria, have been shown to inhibit pancreatic lipase in several studies (Park et al., 2014).

Hypertension is a major risk factor for cardiovascular events, driven by excess angiotensin II, a vasoconstrictor produced by angiotensin-converting enzyme (ACE) (Aluko, 2019). Inhibiting ACE is a strategy for preventing and treating conditions like diabetes, heart failure, and other CVDs. Probiotics have shown significant antihypertensive effects, reducing CVD risk by improving lipid levels, bile acid deconjugation, body mass index, nutrient absorption, and lowering plasma glucose levels, all contributing to better blood pressure regulation (Upadrasta & Sudha 2016). Many LAB strains have demonstrated potential hypoglycemic effects in vitro, including the inhibition of α-amylase, α-glucosidase (Wang & Li 2022).

This investigation explores the cardioprotective properties of LAB strains. Strains were screened based on bile deconjugation, antioxidant, lipase inhibition, cholesterol assimilation, ACE-inhibition, proteolytic activities, and antidiabetic potential to identify promising candidates for cardio protection. The strains with strong biofunctional activities were further evaluated for their probiotic potential.

Lactic strains. The strains listed in Table 1 were obtained from the Culture Collection of Dairy Microbiology Department at SMC College of Dairy Science, Kamdhenu University, Anand, Gujarat, India. These strains were verified for purity and propagated in sterilized reconstituted skim milk (11% total solids) at 37ºC/24h before being stored at 7±1ºC. To maintain their activity throughout the study, the strains were regularly transferred to either sterile selective media or sterilized reconstituted skim milk on a weekly basis, depending on the study's requirements.

Table 1: Details of Lactic Acid Bacteria (LAB) strains used in this study.

Assessment of Antioxidant Activity. Antioxidant activity was measured using the ABTS [(2, 2’-Azino-bis (3-ethylbenzothiazoline 6-Sulphonic acid (Sigma-Aldrich, Bangalore, India)] method of Re et al. (1999) with few modifications. The total radical scavenging capacity was assessed by the ability of a compound to scavenge the ABTS radical within 10 minutes. Selected cultures were inoculated in reconstituted skim milk at 2% and incubated at 37ºC for 24 hours. A 200 μL aliquot of culture supernatant, obtained after centrifuging at 14,000 rpm for 30 minutes, was mixed with 2300 μL ABTS and brought to a total volume of 2500 μL. The decrease in absorbance at 734 nm was recorded using PC based double beam spectrophotometer, 2206 (Systronics, Ahmedabad, India) over 10 minutes at 30-second intervals, and percent inhibition was calculated using the following formula.

Where, A control = Absorbance of phosphate buffer solution at 734 nm

A Test = Absorbance of bacterial suspension at 734 nm

Assessment of Lipase Inhibitory Activity. Lipase inhibition was evaluated using the method of Gil-Rodriguez and Beresford (2020). Strains were inoculated in reconstituted skim milk at 2% and incubated at 37ºC for 24 hours. A 500 µL fermented milk sample was mixed with 2 mL of Tris-HCl buffer (pH 8.25-8.75), followed by the addition of 50 µL 4-nitrophenyl octanoate and 50 µL lipase. The mixture was incubated at 37ºC for 30 minutes, then 1 mL of clarifying reagent (Sigma-Aldrich, Germany) was added and incubated for another 3 minutes. Absorbance was measured at 412 nm against various controls using PC based double beam spectrophotometer 2206 (Systronics, Ahmedabad, India). Orlistat was used as a positive control, and samples were tested in triplicate. The lipase inhibitory activity was calculated as a percentage relative to the control.

Assessment of Cholesterol Assimilation. Cholesterol assimilation of LAB strains was assessed using a modified method from Ashar and Prajapati (1998). Strains were inoculated at 2% in a selective broth with bile salts (0.2% sodium taurocholate, 0.3% sodium thioglycolate) and 50 µg/mL cholesterol (Himedia Laboratories Pvt. Ltd, Mumbai, India). After anaerobic incubation at 37ºC for 24 hours, the tubes were centrifuged at 10,000 rpm for 10 minutes at 4ºC (Eppendorf centrifuge, US). 0.5 mL of the supernatant was mixed with 3 mL of 95% ethanol and 2 mL of 50% KOH, then heated at 60ºC for 10 minutes. After cooling, 5 mL of hexane(n-hexane 99% AR, LobaChemie Pvt. Ltd, Maharashtra, India) was added, mixed, and then 3 mL of distilled water was added. Phase separation was allowed at room temperature for 15 minutes, and 2.5 mL of the hexane layer was transferred to clean test tubes. The hexane was evaporated overnight at 60ºC. Four mL of O-phthalaldehydereagent (Himedia Laboratories Pvt. Ltd, Mumbai, India) was added to the dried extracts and allowed to stand for 10 minutes at room temperature. Then, 2 mL of concentrated sulfuric acid was added slowly, and the mixture was thoroughly mixed and left for another 10 minutes. Absorbance was measured at 550 nm using a PC-based double beam spectrophotometer (Systronics, Ahmedabad, India). A standard curve of absorbance versus cholesterol concentrations was generated. The percentage of cholesterol assimilation was calculated using the following formula.

Cholesterol assimilated (μg/mL) = [Cholesterol (μg/mL)]0 h – [Cholesterol(μg/mL)]24 h

Assessment of Angiotensin-I-Converting Enzyme (ACE) Inhibitory Activity. ACE-inhibitory activity (%) was measured using the method of Hati et al. (2015). This involves hydrolyzing N-Hippuryl-His-Leu (HHL) into Hippuric Acid (HA) and His-Leu (HL) with ACE. The process includes mixing HHL solution (Sigma, USA) with deionized water and sample, adding ACE enzyme (Sigma, USA), and incubating at 37ºC. After the reaction, it’s terminated with HCl, and HA is extracted using ethyl acetate(Himedia Laboratories Pvt. Ltd, Mumbai, India). The extract is evaporated, dissolved in water, filtered, and its absorbance at 228 nm is measured using spectrophotometer 2202 (Systronics, Ahmedabad, India). ACEi % is calculated by comparing HA produced with and without inhibitors under identical conditions.

HA control: The absorbance of concentration of hippuric acid produced by the ACE in buffer without lactic cultures

HA sample: The absorbance of the concentration of hippuric acid produced by the ACE in the presence of lactic cultures

Assessment of Proteolytic Activity. The degree of proteolysis during milk fermentation was assessed by measuring free NH3 groups using the o-phthaldialdehyde (OPA) method (Hati et al., 2015). 3 mL sample of fermented milk was mixed with 5 mL of 0.75% trichloroacetic acid (TCA) (Himedia Laboratories Pvt. Ltd, Mumbai, India), vortexed (Thermo Fisher Scientific, India), and filtered through WhatmanTM no. 42 paper (Sigma-Aldrich, supplied by Merck Life Science Private Limited, Mumbai, India). The filtrate (200 µL) was combined with 3 mL of OPA reagent, incubated at 20ºC for 2 min, and absorbance was measured at 340 nm using a Systronics spectrophotometer (Systronics, Ahmedabad, India). A standard curve was prepared with leucine.

Assessment of Antidiabetic Activity. Antidiabetic activity was assessed through the inhibition of α-amylase and α-glucosidase enzymes.

α-Amylase inhibition assay. α-Amylase inhibition was assessed using a spectrophotometric method with 3,5-dinitrosalicylic acid (Chaudhary & Mudgal 2020). In this assay, 300 μL of sample extract, 70 μL of 50% methanol, 50 μL of enzyme solution, and 1 mL of starch solution were mixed and incubated at 37°C for 5 minutes. After incubation, 2 mL of 3,5-dinitrosalicylic acid (Sigma-Aldrich, Mumbai, India) reagent was added, and the mixture was heated in a boiling water bath for 5 minutes, then cooled to room temperature. Absorbance was measured at 540 nm. Blank and control tubes were prepared by excluding the enzyme and sample, respectively

α-Glucosidase inhibition assay. The assay, based on Chaudhary and Mudgal (2020), involved mixing sample extracts (500 μg/mL) with 1000 μL of 0.1 M phosphate buffer (pH 6.9) containing α-glucosidase (1 U/mL) (Sigma-Aldrich, Mumbai, India) and incubating at 25°C for 10 minutes. After pre-incubation, 500 μL of 5 mM para-nitrophenyl-α-D-glucopyranoside (Sigma-Aldrich, Mumbai, India) in buffer was added, and the mixture was incubated at 25°C for 5 minutes. Absorbance at 405 nm was measured before and after incubation using a UV spectrophotometer and compared to a control with buffer instead of extract. α-glucosidase inhibitory activity (%) was calculated as follows.

Assessment of the probiotic potential of selected strains. Probiotic potential of the strains Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), and Limosilactobacillus fermentum (BM24) was assessed according to ICMR-DBT guidelines (ICMR-DBT, 2011). Evaluation included resistance to gastric acidity and bile acid, antimicrobial activity against pathogenic bacteria, and pathogen adhesion reduction. 

Tolerance of the strains to low pH was assessed using method from Kathiriya et al. (2015). Selective broths were adjusted to pH 1.0, 2.0, 3.0 using 1 N HCl, with pH 6.5 as the control. After mixing, the broth was distributed in 10 mL aliquots. Cultures were activated by inoculating in selective broth (2%) and incubated for 12 hours, then centrifuged at 10,000 rpm for 10 minutes at 4°C. The pellets were washed twice with phosphate buffer saline (PBS) and resuspended in PBS. These cultures (2%) were added to tubes with selective broth at pH 1.0, 2.0, 3.0, and 6.5. After incubation at 37°C, 1 mL samples were taken at 0, 1, 2 and 3 hours, diluted in PBS, and plated on selective agar. Plates were incubated at 37°C for 48-72 hours, and viable cell counts were recorded as log CFU/mL.

Bile tolerance of the strains was assessed using method from Kathiriya et al. (2015) Cultures were activated by inoculating in selective broth (2%) and incubated for 24 hours. After centrifugation at 10,000 rpm for 10 minutes at 4°C, the pellets were washed with PBS and resuspended in PBS. Suspended cultures (2%) were added to 10 mL selective broth containing 0.3%, 0.5%, and 1% bile salt, with a control containing no bile salt. Tubes were incubated at 37°C. Samples (1 mL) were taken at 0, 1, 2, and 4 hours, diluted in PBS, and plated on selective agar. Plates were incubated at 37°C for 48-72 hours, and viable cell counts were recorded as log CFU/mL.

In the methods for adhesion tests (Tuo et al., 2013), each strain was centrifuged at 10,000 rpm for 10 minutes at 4°C, washed with PBS (pH 7.2), and resuspended in PBS buffer. The suspension was incubated at 37°C for 5 hours, and absorbance at 600 nm was measured for both the upper and total suspension. Auto-aggregation percentage was then calculated.

Auto aggregation %=[1 – A (Upper suspension)/A (Total  suspension)]×100

Where,

AUpper suspension=OD at 600nm of upper suspension after 5 hr

A Total suspension=OD at 600 nm of Total bacteria suspension after 5 hr

To determine co-aggregation ability (Tuo et al., 2013), 10 mL of culture was centrifuged at 5,000 rpm for 10 minutes. Equal volumes (2 mL) of each strain and pathogenic bacterial strains were mixed and incubated at 37°C for 5 hours. Optical density of the mixed cultures was measured at 600 nm. The co-aggregation percentage was calculated using the following formula

Co-aggregation %=[(A test bacteria + A strain) – 2 (A mixed strain)/(A test bacteria+ Astrain) ] × 100

Where,

A test bacteria -OD600nm Pathogenic Bacteria

A strain-OD600nm strain

A mixed strain -OD 600 nm of strain+ Pathogenic Bacteria

Antimicrobial activity of the strains was assessed using the agar well diffusion assay following Kathiriya et al. (2015). Cell-free supernatant (CFS) from each strain was tested. Indicator bacteria, grown in nutrient broth at 37°C for 12 hours, were mixed with 100 mL of 1% nutrient agar and poured into Petri dishes. Wells (6 mm diameter) were created in the solidified agar using a sterile borer. 100 µL of CFS was added to each well. Plates were incubated at 37°C for 24 hours and antimicrobial activity was measured by the diameter of the growth inhibition zone around the wells.

Statistical Analysis. Data obtained were analyzed by completely randomized design (CRD) as per the methods described by Steel and Torrie (1980). The significance of the influence of each parameter on the specific characteristic was tested at 5.0% level of significance. 

CVDs are a leading cause of death and disability putting economic burden. While traditional risk factors like hypertension, diabetes, hyperlipidemia, obesity, smoking, and environmental influences are well-known, recent studies have highlighted the significant role of gut microbiota in the development and progression of CVDs. Research suggests that probiotics can mitigate health complications associated with or leading to CVDs. This study investigated the potential of LAB strains to manage CVD risk factors, including hypertension, hyperlipidemia, and oxidative stress. The probable probiotic mechanisms of action can be bile deconjugation, cholesterol assimilation, antioxidant capacity, ACE inhibition, antidiabetic potential and generation of bioactive metabolites through proteolytic activity in addition to being contributing to the gut microbiota health.

Bile deconjugation ability of LAB strains. The ability of the LAB strains to deconjugate bile was determined by measuring the production of free cholic acid from sodium taurocholate. The values were derived using a standard curve. The bile deconjugation ability of the lactic acid strains differed significantly (p<0.05). As shown in Fig. 1 (a), strain V3 exhibited the highest bile deconjugation ability (350.13 μg/mL), followed by MD2 (323.72 μg/mL), F11 (312.95 μg/mL), BM24 (309.62 μg/mL), BM15 (303.97 μg/mL), I4 (296.80 μg/mL), and F7 (291.15 μg/mL) after 24 hours at 37ºC. These results clearly indicate strain-specific variations in bile deconjugation ability.

(a)

(b)

Fig. 1(a) Bile deconjug

ation and (b) Cholesterol assimilation ability of LAB strains. Each observation is a mean±SD of (n=3). Different small case letters in the graph indicate statistically significant (p<0.05) differences. Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), Limosilactobacillus fermentum (BM15), Limosilactobacillus fermentum (BM24), Lactipantibacillus plantarum (F7) and Lactipantibacillus plantarum (F11).

Cholesterol assimilation by LAB strains. Cholesterol assimilation by the strains, as detailed in Fig.1(b), revealed significant differences (p<0.05). Strains V3 (78.49%), MD2 (77.65%), BM24 (79.93%), F7 (79.56%), and F11 (78.84%) exhibited significantly better cholesterol assimilation, with their assimilation abilities being statistically comparable. The lowest cholesterol assimilation was observed in strain BM15 (73.01%), which was also statistically significant.

Bile salt hydrolysis is essential for regulating cholesterol and maintaining cholesterol homeostasis. Liong and Shah (2005) explain that bile salt hydrolase (BSH) enzymes catalyze the hydrolysis of conjugated bile salts into free bile acids and amino acids. At the intestinal lumen's physiological pH, some free bile salts precipitate, reducing serum cholesterol levels by increasing the need for new bile salts synthesized from cholesterol. This deconjugation process, facilitated by BSH enzymes from gut bacteria like Bifidobacterium and Lactobacillus species, reduces bile salt reabsorption in the terminal ileum and increases fecal excretion. BSH activity is noted in Lactobacillus, Bifidobacterium, and Clostridium species (11). Avci (2014) reported that L. delbrueckii subsp. bulgaricus (HL6) and S. thermophilus (HS12) deconjugated sodium taurocholate at 1.0-1.4 mg/ml and 0.9-1.3 mg/ml, respectively (Avci, 2014). Kathiriya et al. (2018) found that L. rhamnosus NS6 had the highest bile deconjugation ability, producing 364 μg/mL of cholic acid from sodium taurocholate, followed by S. thermophilus MD8 with 230 μg/mL. Additionally, Hernandez-Gomez et al. (2021) observed that L. fermentum K73, isolated from traditional fermented sour cream, showed deconjugation activity of 24%.

Kaplan et al. (1998) state that elevated blood cholesterol levels contribute to atherosclerosis, increasing the risk of MI and stroke. Tomaro-Duchesneau et al. (2014) suggest that probiotic bacteria in the gut can assimilate cholesterol, reducing its absorption by enterocytes and promoting its excretion, potentially lowering the risk of CVDs. The primary in vitro mechanisms proposed for how probiotics impact cholesterol levels include cholesterol adherence to cell surfaces and absorption into cellular membranes. Tomaro-Duchesneau et al. (2014) identified L. reuteri NCIMB 11951, L. reuteri NCIMB 701089, and L. acidophilus ATCC 314 as the top cholesterol-absorbing strains, with L. acidophilus ATCC 314 showing the highest assimilation at 41.20±1.92 μg/mL (Tomaro-Duchesneau et al., 2014). In an in vitro study by Ding et al. (2017) L. plantarum (Lp1) exhibited the highest cholesterol assimilation at 75.9%, while Lactococcus lactis subsp. lactis (L1) had the lowest at 61.2% (Ding et al., 2017). Bhat and Bajaj found that among the LAB, L. casei M6 showed the highest assimilation at 82.15%, followed by L. casei M5 at 76.51%, L. paracasei M3 at 67.4%, and L. paracasei M37 at 67.2% (Bhat & Bajaj 2020). Wang et al. (2021) reported significant variability among L. plantarum strains in their cholesterol-lowering abilities. In MRS broth, L. plantarum AR113 and AR171 showed the highest reductions at 27.89% and 19.90%, respectively, while strains AR501 and AR300 exhibited minimal reductions of 0.34% and 0.91%. All strains in our study demonstrated cholesterol reduction of more than 70%, highlighting their potential as probiotics for CVD prevention.

Antioxidant potential of LAB strains. Antioxidant activity was assessed using the ABTS assay method, with results expressed as a percentage of free radical scavenging activity. The findings, presented in Fig. 2(a), indicate that strain BM24 exhibited significantly (p<0.05) higher antioxidant activity at 73.57% compared to all other strains. On the other hand, strain F7 showed the lowest antioxidant activity at 53.14%, which was statistically similar to that of BM15 at 59.24%. The antioxidant activities of strains V3 (54.48%), F11 (56.62%), BM15 (59.24%), I4 (62.05%), and MD2 (62.76%) were found to be comparable to each other. These results clearly demonstrate that antioxidant activity is highly strain dependent.

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Fig. 2. (a) Antioxidant activity and (b) Lipase Inhibitory activity of LAB strains. Each observation is a mean±SD of (n=3). Different small case letters in the graph indicate statistically significant (p<0.05) differences. Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), Limosilactobacillus fermentum (BM15), Limosilactobacillus fermentum (BM24), Lactipantibacillus plantarum (F7) and Lactipantibacillus plantarum (F11).

Oxidative stress, marked by elevated reactive oxygen species (ROS) and reactive nitrogen free radicals, occurs due to adverse conditions like ischemia and hypoxia, leading to apoptosis and tissue damage, which are risk factors for MI35. Normally, low ROS levels are balanced by detoxification processes that support cellular signaling and function. However, in pathological states such as atherosclerosis or hypertension, excessive ROS production overwhelms the body's antioxidant defenses, causing cell death. Numerous in vivo and in vitro studies have shown that Lactobacilli and Bifidobacteria possess strong antioxidant capacity, offering protection against oxidative stress (Rani et al., 2016.) These bacteria are valuable natural antioxidants, producing enzymes and metabolites that combat free radicals. Amaretti et al. (2013) identified Levilactobacillus brevis, L. acidophilus, and B. lactis as strains with the highest antioxidant activity. Among the 9 LAB strains isolated from traditional Chinese dairy foods, L. paracasei had better scavenging activity of free radicals (Wang & Li 2022).

Lipase inhibitory activity of LAB strains. Significant differences (p<0.05) were observed in the lipase inhibitory activity of the LAB strains, as shown in Fig. 2(b). Among the strains, BM24 demonstrated the highest lipase inhibition at 86.21%, significantly higher than the other strains. This was followed by V3 (75.57%), MD2 (73.72%), I4 (73.03%), F7 (55.67%), BM15 (49.51%), and F11 (44.73%) after 24 hours. Additionally, lipase inhibition by strains V3, MD2, and I4 was statistically similar to each other.

Lipase is essential for the digestion, transport, and processing of dietary lipids. In humans, pancreatic lipase is the key enzyme that breaks down dietary fats in the digestive system. Lipase inhibitors reduce fat absorption in the gastrointestinal tract, leading to fat excretion rather than absorption for energy, potentially resulting in weight loss. These inhibitors are often used to treat obesity (Maqsood et al., 2017). This test was conducted because obese individuals are at a higher risk of developing diabetes and heart-related issues. Mudgil investigated the inhibitory effects of LAB strains from raw camel milk on pancreatic lipase, comparing 11 reference strains and 97 LAB isolates. Among 52 Streptococcus isolates, inhibition ranged from 3.0% to 99.0%, with 11 isolates showing strong effects comparable to or exceeding orlistat (83.0%). Of the 45 Lactobacillus isolates, 13 showed negative inhibition, while 32 exhibited inhibition between 4.0% and 81.0%. Reference cultures had inhibition levels from 3.0% to 37.0%, with L. acidophilus DSMZ 9126 showing the highest and L. gasseri 20243 the lowest (Mudgil et al., 2016). Gil-Rodríguez and Beresford also found varying lipase inhibitory activities among lactic strains in fermented milk, with L. plantarum 70 (37.22%) showing the highest inhibition21. In our study, except BM 15 and F11, others exhibited over 50% lipase inhibition, indicating their potential against CVDs.

ACE-Inhibitory activity of LAB strains. ACE-inhibition ability of the strains is summarized in Fig. 3(a). After 24 hours of incubation, ACE inhibition by the strains ranged from 57.90% to 87.58%. Strain F7 demonstrated the highest ACE-inhibitory activity at 87.58%, followed by MD2 (84.19%), I4 (82.85%), F11 (79.27%), V3 (76.77%), BM15 (74.41%), and BM24 (57.90%). The strains F7, MD2, I4, and F11 showed similar levels of activity, while BM24 exhibited the lowest ACE inhibition, which was significantly different (p<0.05) from the other strains.

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Fig. 3. (a) ACE-inhibitory and (b) Proteolytic activity of LAB strains. Each observation is a mean±SD of (n=3), Different small case letters in the graph indicate statistically significant (p<0.05) differences. DH=Degree of Hydrolysis. Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), Limosilactobacillus fermentum (BM15), Limosilactobacillus fermentum (BM24), Lactipantibacillus plantarum (F7) and Lactipantibacillus plantarum (F11).

Hypertension contributes to cardiovascular disorders like arteriosclerosis, heart failure, coronary heart disease, MI, and stroke. The enzyme ACE produces angiotensin II, a vasoconstrictor that raises blood pressure. ACE inhibitors lower blood pressure by blocking the conversion of angiotensin I to angiotensin II and may also prevent the breakdown of bradykinin, which induces vasodilation. These inhibitors are used to manage and treat conditions such as diabetes, heart failure, MI, nephropathy, and other CVDs (Li et al., 2017). Hati et al. (2015) reported the ACE-inhibitory activity of Lactobacillus rhamnosus (NS4) and Lactobacillus bulgaricus (009) strains to be 79.66% and 67.09%, respectively. According to Zhao et al. (2021) Lactobacillus helveticus, used in dairy fermentation, has long-term hypotensive effects in hypertensive patients. It produces two tripeptides, Val-Pro-Pro and Ile-Pro-Pro, that inhibit angiotensin-I converting enzyme and lower blood pressure without adverse effects. Furthermore, L. helveticus KII 13 isolated from fermented milk produced hypotensive peptides. Parmar et al. (2020) investigated five Lactobacillus strains L. rhamnosus (NK2) (KR080695), L. casei (NK9) (KR732325), L. fermentum (M5) (KU366365), L. paracasei (M16) (KU366368), and L. fermentum TDS030603 (MTCC 25067) (LF) from fermented goat milk for their ACE-inhibitory activities. After 24 hours, NK9 significantly outperformed the other cultures in terms of ACE-inhibitory activity (66.99%, p<0.05) (Parmar et al., 2020).

Proteolytic activity of LAB strains. Proteolytic activity of the strains ranged from 5.65% to 8.75% during 24 hours of incubation [Fig. 3(b)]. There was no significant difference observed in the proteolysis among the strains. BM15 demonstrated the highest proteolytic activity at 8.75%, followed by I4 (7.94%), F11 (7.59%), V3 (7.53%), F7 (6.14%), BM24 (6.06%), and MD2 (5.65%) after 24 hours at 37ºC. 

LAB can degrade milk proteins through their proteolytic system, producing peptides with health benefits, including ACE-inhibitory bioactive peptides (Hati et al., 2015). Ahmed and Bousmaha-Marroki (2014) reported the highest proteolytic activities in Lactobacillus plantarum strains LbMS16 (15.50%) and LbMS21 (19.00%), as well as in Lactobacillus rhamnosus strain LbMF25 (25.00%). Hati et al. (2015) found that the proteolytic activity of Lactobacillus rhamnosus (NS4 and NS6), Lactobacillus helveticus MTCC 5463 (V3), Lactobacillus delbrueckii (009), Enterococcus faecalis (ND3 and ND11), and Lactobacillus rhamnosus (SH8) increased in skim milk when cultured at a rate of 1% at 37ºC for 24 hours. Among these, NS4 released the highest number of amino acids after 24 hours of fermentation at 37ºC, with 009 and ND3 also showing efficient amino acid release. In contrast, V3, NS6, SH8, ND11, and I4 exhibited comparatively lower proteolytic activity. Their study concluded that NS4, 009, and ND3 had the most robust proteolytic systems, with the greatest capacity for producing proteolytic enzymes in skim milk.

Antidiabetic potential of LAB strains. Diabetes is recognized as a modifiable risk factor for CVDs. Inhibiting α-amylase activity can help mitigate postprandial blood glucose spikes. Additionally, α-glucosidase, an enzyme found in the intestine, hydrolyzes various sugars into glucose; thus, inhibiting α-glucosidase can help lower blood glucose levels (Wang & Li 2022).

α-amylase inhibition by LAB strains. α-amylase inhibitory activity of the LAB strains is shown in Fig. 4(a). Among the strains BM24 (94.28%) and MD2 (92.16%) showed significantly high inhibition followed by V3 (80.16%), I4 (83.05%), F7 (60.82%), BM15 (47.21%) and F11 (38.42%). Least inhibition was seen for strain F11. The α-amylase inhibitory activity of strains V3 and I4 were at par with each other.

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Fig. 4. (a) Alpha amylase inhibition and (b) Alpha glucosidase inhibition by LAB strains. Each observation is a mean±SD of (n=3), Different small case letters in the graph indicate statistically significant (p<0.05) differences. Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), Limosilactobacillus fermentum (BM15), Limosilactobacillus fermentum (BM24), Lactipantibacillus plantarum (F7) and Lactipantibacillus plantarum (F11).

α-glucosidase inhibition by LAB strains. α-glucosidase inhibition by the LAB strains is shown in Fig. 4(b). Among the strains I4 (57.73%) exhibited the highest (p<0.05) inhibition followed by MD2 (45.51%), BM24 (36.20%), V3 (26.75%) and others.  Least inhibition was seen for strains BM15 (9.29%), F7 (10.56%) and F11 (15.60%) which were at par with each other.

Wang and Li (2022) reported that among all the LAB strains they studied, Lactobacillus plantarum, Lactobacillus delbrueckii, Lactobacillus paracasei and Lactobacillus casei exhibited significantly higher inhibition activity (p<0.05), with Lactobacillus plantarum showing the highest inhibition rate at 83.36% for α-amylase. Additionally, the CFS of Lactobacillus plantarum demonstrated the highest α-glucosidase inhibitory activity at 85.16%. Ayyash et al. (2018) reported that the probiotic cultures L. reuteri KX881777, L. plantarum KX881772, and L. plantarum KX881779 exhibited α-amylase inhibitions of over 34% in milk medium. Kwun et al. (2020) found that L. sakei MBEL1397, isolated from kimchi, exhibited an α-glucosidase inhibitory activity of 3.91±0.25%.

Probiotic potential of the selected strains. The strains V3, MD2, I4 and BM24 were capable of surviving at pH 2.0 after 3h of incubation and they had shown viability of 4.24, 4.11, 4.57 and 4.24 log CFU/mL respectively (Fig. 5). Similarly, the bile tolerance of the strains at 1% bile concentration after incubation period of 4h were 3.70, 3.78, 3.74 and 4.47 log CFU/mL respectively (Fig. 6).

Fig. 5. pH tolerance of the strains. (a) Lactobacillus helveticus MTCC 5463 (V3), (b) Streptococcus thermophilus MTCC 5460 (MD2), (c) Lacticaseibacillus rhamnosus MTCC 5462 (I4), (d) Limosilactobacillus fermentum (BM24). Each observation is a mean±SD of (n=4).

Fig. 6. Bile tolerance of the strains. (a) Lactobacillus helveticus MTCC 5463 (V3), (b) Streptococcus thermophilus MTCC 5 (MD2), (c) Lacticaseibacillus rhamnosus MTCC 5462 (I4), (d) Limosilactobacillus fermentum (BM24). Each observation is a mean±SD of (n=4).

The results (Table 2) of the antimicrobial activity of the strains revealed that 3 strains had significant (p<0.05) antimicrobial activity against all 5 pathogens except for MD2, which has shown antimicrobial activity only against S. enterica paratyphi MTCC 735 and B. cereus MTCC 1272. V3 had shown highest inhibition against Salmonella enterica ser. paratyphi MTCC 735 (10.00±3.29), and Bacillus cereus MTCC 1272 (10.00±1.00). I4 showed highest inhibition against Bacillus cereus MTCC 1272 (10.33±1.52).

Table 2: Antimicrobial activity of LAB strains against pathogens (Inhibition zone in mm).

Each observation is a mean±SD of (n=4). Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), Limosilactobacillus fermentum (BM24). Staphylococcus aureus subsp. aureus MTCC 737, Escherichia coli MTCC 1687, Salmonella enterica ser. paratyphi MTCC 735, Bacillus cereus MTCC 1272, Shigella flexneri MTCC 1457

In order to exert its beneficial effect on the host, a probiotic strain must be able to survive the gut passage of humans to reach to the action site in viable state and should be in sufficient population (ICMR-DBT, 2011). Acid tolerance of 255 isolates of LAB isolated from Greek traditional fermented products revealed that 133 isolates out of 255, exhibited final counts of ≥103 CFU/mL at low pH for 3h. Probiotic potential of Lactobacillus rhamnosus NK2, Lactobacillus casei NK9, Lactobacillus rhamnosus NK10, Lactobacillus pentosus M20 and Lactobacillus plantarum M22 has been reported by Kathiriya et al. (2015). These isolates were able to tolerate upto pH 2.0 for 3h. Pino et al. (2019), in their investigation on detection of vaginal lactobacilli as probiotic candidates reported survival rates of ≥80% for lactobacilli isolate at both pH 3.0 and pH 2.0 conditions.

Bile salt tolerance is generally considered a prerequisite for the colonization and metabolic activity of bacteria in the host’s intestine (CMR-DBT, 2011). The average concentration of bile salts in the small intestine ranges from 0.2% to 0.3%, but it can rise to as high as 2% (w/v) depending on the individual and the type and amount of food consumed (Menconi et al., 2014). Nagyzbekkyzy et al. (2016) examined the bile tolerance of Lactobacillus strains isolated from Kazakh dairy products after 24 hours of exposure to 1.0% bile acid and found that the cultures responded differently in the presence of bile acids. Several of the most tolerant LAB isolates showed growth percentages ranging from 40% to 71%, while the remaining isolates demonstrated intermediate tolerance, with cell growth ranging from 20% to 40% (Nagyzbekkyzy et al., 2016). In a study by Pino et al. (2019), investigating vaginal lactobacilli as potential probiotics, it was found that a bile salt concentration of 0.5% (w/v) had no effect on most strains, except for Lactobacillus crispatus P10 and Lactobacillus plantarum C11 and V7 strains. At a bile salt concentration of 1.0% (w/v), 86% and 79% of the strains exhibited bile tolerance after 2 and 4 hours, respectively.

Bacterial aggregation, whether involving the same strain (auto-aggregation) or different species and strains (co-aggregation), plays a crucial role in the human gut, where probiotics are intended to function. A probiotic strain's ability to adhere to the oral cavity, gastrointestinal system, and urogenital tract is largely determined by its auto-aggregation capacity, while its co-aggregation ability helps form a barrier that prevents pathogen colonization (Divya et al., 2012).

Fig. 7. (a) Autoaggregation and (b) Co-aggregation of the strains. Each observation is a mean±SD of (n=4). Lactobacillus helveticus MTCC 5463 (V3), (b) Streptococcus thermophilus MTCC 5460 (MD2), (c) Lacticaseibacillus rhamnosus MTCC 5462 (I4), (d) Limosilactobacillus fermentum (BM24). Staphylococcus aureus subsp. aureus MTCC 737, Escherichia coli MTCC 1687, Salmonella enterica ser. paratyphi MTCC 735, Bacillus cereus MTCC 1272, Shigella flexneri MTCC 1457.

Li et al. (2015) studied the aggregation and adhesion abilities of 18 LAB strains isolated from traditional fermented foods and found significant (p<0.05) differences in co-aggregation among the strains. All tested LAB exhibited some level of co-aggregation with Salmonella sp., ranging from 5.15% to 29.54%, highlighting strain-specific characteristics. Lactobacillus fermentum 9 showed the highest co-aggregation ability (29.54%) with Salmonella sp., followed by Lactobacillus fermentum AB4 (19.45%). Enterococcus faecalis 5 displayed the lowest co-aggregation ability. Among the 18 strains, 66.67% showed co-aggregation above 15%, while only 11.11% had co-aggregation below 10%.

LAB are well known for producing a variety of antimicrobial compounds that have significant antagonistic effects against various microorganisms, including pathogenic and spoilage organisms. These antimicrobial compounds include organic acids such as lactic acid and acetic acid, along with substances like hydrogen peroxide, acetoin, diacetyl, reuterin, helveticin, carbon dioxide, and bacteriocins. Menconi et al. (2014) identified and characterized LAB in a commercial probiotic culture, discovering that the strains LAB 18 and LAB 48 demonstrated in vitro antibacterial activity against Salmonella enterica serovar Enteritidis, Escherichia coli O157, and Campylobacter jejuni (Divya et al., 2012). Shokryazdan et al. (2014) explored the probiotic potential of Lactobacillus strains with antimicrobial activity against several human pathogens, finding that nine Lactobacillus strains could be considered potential probiotics. Many of these strains exhibited stronger antagonistic effects against the test pathogens compared to the reference strain Lactobacillus casei Shirota. Notably, L. casei BF1, L. casei BF2, and L. casei BF3 showed significantly higher inhibitory effects on Helicobacter pylori and Staphylococcus aureus than L. casei Shirota. Karimi et al. (2017) evaluated the antimicrobial effects of probiotic bacterial strains isolated from various natural sources against two pathotypes of pathogenic E. coli. They observed that Lactobacillus plantarum, Lactobacillus gasseri, Enterococcus faecium, Bacillus subtilis, and Weissella paramesenteroides strains exhibited considerable antimicrobial effects against the Escherichia coli O157strain but had no inhibitory effect against Enterohemorrhagic Escherichia coli.

Among the seven LAB strains evaluated for cardioprotective potential, Lactobacillus helveticus MTCC 5463 (V3), Streptococcus thermophilus MTCC 5460 (MD2), Lacticaseibacillus rhamnosus MTCC 5462 (I4), and Limosilactobacillus fermentum (BM24) stood out for their superior performance across a range of bio-functional properties. These strains showed the most effective bile deconjugation, potent antioxidant and lipase inhibition, significant cholesterol assimilation, strong ACE inhibition, notable proteolytic activity, and antidiabetic potential. As a result, they have been identified as promising candidates for use as a multi-strain probiotic. The observed variation in biological activities among LAB strains highlights the importance of selecting specific strains based on their unique properties to address targeted health issues. Leveraging the unique capabilities of individual LAB strains will be key to optimizing therapeutic efficacy and expanding their use in health-promoting and disease-preventing products.

Further in vivo studies using animal models, along with human clinical trials, are essential to confirm the cardioprotective effects of these strains. Research should prioritize understanding their mechanisms of action, optimizing effective dosages, and evaluating long-term safety to ensure regulatory compliance and build consumer confidence.