comparative evaluation of the probiotic and antioxidant potential of indigenous lactic acid Bacteria #

the probiotic and


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Professor and Head

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Assistant Professor, Department of Dairy Technology, CDST Trivandrum *Corresponding author: aparna@kvasu.ac.in,Ph.7558067959end product but also produce a wide range of metabolites that beneficially affect the nutritional, sensorial, and technological properties of fermented food products.The functionality of LAB is primarily attributed to their metabolites, the major one being lactic acid.Others include acetic acid, ethanol, aroma compounds, exopolysaccharides, enzymes, bacteriocins, etc. (Leroy and De Vuyst, 2004).Fermentation by LAB also results in the release of biologically active peptides which are known to have functions like immunomodulatory, angiotensin converting enzyme inhibitory and antioxidant activities (Abubakr et al., 2012).Some LAB are also able to produce antioxidases which results in their antioxidant activity (Zhang et al., 2017).
Fermented milk with health-promoting probiotic properties is one of the oldest functional foods.
Some species in LAB are known to possess probiotic potential which is considered to be more beneficial from a health point of view.Lactobacillus, Bifidobacterium, Leuconostoc, Streptococcus, Enterococcus, Pediococcus, and yeasts like Saccharomyces possess probiotic attributes (Fijan, 2014).According to FAO/ WHO, probiotics are live microorganisms that when administered in adequate amount confer a health benefit on the host.The health benefit is generally acquired by improving or restoring the gut flora.The LAB have been used as probiotics to manage intestinal disorders such as lactose intolerance, acute gastroenteritis, constipation, and inflammatory bowel diseases.Immunomodulating, serum cholesterol lowering, anticarcinogenic, antihypertensive, antidiabetic effects of LAB has also been reported.In addition to these, probiotics also find use in the stabilization of gut flora, recolonisation of bowel following antibiotic treatment, treatment of food allergies, as vaccine adjuvants and improved weight gain (Goldin, 1998).
The probiotic attributes used for the selection of microorganisms are safety, viability/ activity in delivery vehicles, acid tolerance, bile tolerance, resistance to pepsin and pancreatin, ability to adhere to gut epithelial tissue, gastrointestinal tract colonization potential, capacity to stimulate a host immune response, antimicrobial resistance and antimicrobial activity (Pundir et al., 2013;Balamurugan et al., 2014).According to Sharma et al. (2021), acid tolerance, bile salt tolerance, bile salt hydrolase activity, cell surface hydrophobicity, antibiotic susceptibility, antimicrobial activity, haemolytic activity, and production of biogenic amines may be assessed for selecting putative probiotic candidates.Lactobacilli were propagated in de Man Rogosa Sharpe (MRS) broth and Lactococi in M17 broth.One set was maintained as glycerol stock at -20°C by mixing equal volumes (50 µl) each of overnight grown culture and sterilized 50 per cent glycerol.Another set of cultures was propagated and preserved in sterilised reconstituted skimmed milk tubes and stored in the refrigerator.The purity of the cultures was always ascertained before use by Gram's staining and catalase test.The culture of pathogenic bacteria (Escherichia coli) was maintained in nutrient broth.

Acid tolerance
The acid tolerance of the cultures was determined as per Pundir et al. (2013).The cultures were inoculated in sterile MRS broth tubes with pH adjusted to 2.0 and 3.0.After incubation at 37°C optical density was measured at an hour interval for three hours.The pH adjusted broth inoculated with culture was taken as control.

Bile tolerance
The bile tolerance of the cultures was determined as per Pundir et al. (2013).The cultures were inoculated in sterile MRS broth tubes in which the percentage of bile was adjusted to 0.3 and 0.6.the tubes were then incubated at 37°C.After incubation at 37°C optical density was measured at an hour interval for three hours.The bile adjusted broth inoculated with culture was taken as control.

Bacterial cell surface hydrophobicity
The adhesion potential of cultures in terms of cell surface hydrophobicity was determined by Bacterial Adhesion to Hydrocarbons assay using the procedure followed by Collado et al. (2008) with some modifications.The cultures were incubated in MRS broth at 37°C for 16 h.After refrigerated centrifugation at 4°C for 10 min at a speed of 12000 rpm, cells in the stationary phase were collected as pellets.The pellets were washed three times using phosphate buffered saline and then resuspended in the same buffer to achieve an optical density (OD) of 0.25±0.05at 600 nm.An equal volume of xylene was added to 5 mL of this suspension and mixed thoroughly by vortexing for five minutes followed by an immediate measurement of OD at 600nm.The vortexed samples were then held at 37ºC for 1h for phase separation.The aqueous phase of the cell culture was pipetted out and the OD at 600 nm was once more measured.The cell surface hydrophobicity (CSH) in percentage was calculated using the formula.

Autoaggregation
The autoaggregation potential of the cultures was determined as per Kos et al. (2003).The freshly activated culture was added to MRS broth at the rate of one per cent inoculation and incubated at 37°C for 18h.The cells were harvested by refrigerated centrifugation at 5000 g for 15 min.The cell pellets obtained were washed twice with phosphate buffered saline (PBS) and resuspended in the same buffer to attain a final optical density of 0.60±0.02at 600 nm.Four millilitres of this cell suspension was mixed thoroughly by vortexing and then 0.1 millilitre of the undisturbed upper suspension was transferred to another tube with 3.9 mL of PBS.The absorbance (A1) of this suspension was measured at 600 nm.The sample was left undisturbed at 37°C, and the OD of samples (A2) was taken exactly after one hour and six hours.The autoaggregation in percentage was expressed as follows: Where A1: initial optical density, A2: optical density after incubation.

Co-aggregation
Co-aggregation of cultures was assessed by the method followed by Anandharaj et al. (2015), with slight modifications.Cells were harvested by centrifuging 10 mL culture at 5,000 rpm for 10 minutes.Two millilitres each of LAB strain and pathogenic strain (Escherichia coli) were mixed and incubated at 37ºC for five hours.The optical density of the resultant mixture was taken at 600 nm with either LAB strain or pathogenic strain as a control.The coaggregation percentage was estimated using the formula given below.

Bile salt hydrolase (BSH) activity
A direct plate assay for the detection of BSH activity was carried out according to Lee et al. (2011).The active cultures were streaked on pre-solidified MRS agar containing 0.5% (w/v) bile and 0.37 g/L of CaCl 2 .The plates were then incubated anaerobically in the anaerobic jar at 37ºC for 48 h.BSH activity of the cultures was indicated by the formation of distinctive precipitate around the colonies.

Antioxidant potential
The antioxidant potential of the 765 ____________________________________________________________________________Anija et al.
cultures in skimmed milk was determined as per the procedure followed by Ogunyemi et al. (2021) with slight modifications.The cultures were inoculated in sterilised skim milk and incubated overnight.One gram of fermented skim milk was dissolved in 10 mL ethanol and kept in a shaker incubator for two hours.After incubation, it was centrifuged at 5000 rpm for 20 min and the supernatant was filtered using Whatman no. 1 filter paper.Then 0.5 mL of filtrate was added with 3.5 mL ethanol and 1 mL DPPH reagent (2.4 mg in 100 mL ethanol).Simultaneously, a blank was prepared using 4 mL ethanol and 1 mL DPPH reagent.The absorbances of the solutions were measured at 517 nm after incubation at 37ºC for 30 min.

Result and discussion
The probiotic potentials of cultures were analysed in terms of acid tolerance, bile tolerance, cell surface hydrophobicity, autoaggregation, coaggregation and bile salt hydrolase activity.For effective transit through the stomach and small intestine, potential probiotic strains need to be able to endure acidic conditions and bile secretions (Anandharaj et al., 2015).To be used as a probiotic, bacteria should withstand a low pH of around 3.0 for two hours (Gotcheva et al., 2002).The acid tolerance of LAB cultures determined by evaluating their growth in acidic pH is shown in Fig. 1.Among the 5 cultures, Lacticaseibacillus rhamnosus showed the highest acid tolerance followed by Lactobacillus acidophilus.According to Biswas et al. (2019), Lacticaseibacillus rhamnosus was found to tolerate prolonged acidic conditions which support current observations.Bile tolerance of the cultures is depicted in Fig. 2. The level of bile salt in the intestine is around 0.3 per cent and can reach extremely up to 2 per cent during the start of digestion.Therefore, bile resistance for probiotic potential is usually assessed in 0.1-0.5 per cent bile (Gotcheva et al., 2002).Lactiplantibacillus plantarum exhibited considerable bile tolerance in 0.6 per cent bile.As per Hamon et al. (2011) this species was having some strains with reasonable growth in broth containing bile which strengthens our findings.Lacticaseibacillus rhamnosus also showed good bile tolerance and Lactobacillus acidophilus showed slight tolerance at 0.3 per cent level but not at 0.6 per cent.Lactobacillus bulgaricus and Streptococcus thermophilus were found to be sensitive to bile even at 0.3 per cent.
As probiotics are meant to inhabit the intestine of the host, aggregation ability and cell surface hydrophobicity are advantageous.Bacterial cell surface hydrophobicity was established by adherence to apolar solvent xylene.The bacterial cell surface hydrophobicity of cultures in chloroform and xylene are shown in Fig. 3. Lacticaseibacillus rhamnosus (83.24 in xylene and 65.78 in chloroform) was found to have the highest cell surface hydrophobicity succeeded by Lactobacillus acidophilus in xylene as well as chloroform.Strains of Lacticaseibacillus rhamnosus showed high hydrophobicity in a study by Harty et al. (1993) which is similar to the present observation.Also, Lactobacillus acidophilus was found to have good hydrophobicity (Reid et al., 1992).Higher hydrophobicity of bacterial cell surface can be attributed to the presence of (glycol-) proteinaceous material on the cell surface (Collado et al., 2008).Autoaggregation seems to be important for the adhesion of probiotic organisms to the intestinal epithelial cells while coaggregation hinders colonisation by pathogenic microorganisms by creating a barrier (Sabir et al., 2010).The autoaggregation potential of cultures are presented in Fig. 4. Maximum percentage of autoaggregation (78.405% in 6h and 35.34% in 1h) was demonstrated by Lacticaseibacillus rhamnosus followed by Lactobacillus acidophilus.The high autoaggregation of Lacticaseibacillus rhamnosus strains can be attributed to their surface proteins (Polak-Berecka et al., 2014).The coaggregation of cultures with E. coli was assessed and the results were shown in Fig. 5. Lactobacillus acidophilus and also Lacticaseibacillus rhamnosus and Lactiplantibacillus plantarum were found to have good coaggregation potentials of 26.67, 25.89 and 25.65 per cent respectively.Similarly, Lactobacillus acidophilus showed 766 Probiotic and antioxidant potential of lactic acid bacteria ________________________________________ J. Vet.Anim. Sci. 2023. 54 (3) : 763-771   Ekmekci et al. (2009).Bile salt hydrolase (BSH) activity is responsible for deconjugating bile salts in the intestine and helps in colonisation (Anandharaj et al., 2015).consumers are getting receptive to 'Probiotic movement' and the study emphasis the need for culture screening for its probiotic attributes even though it belongs to LAB.

Fig. 1 .
Fig. 1.Optical density at 600 nm of lactic acid bacteria at different pH

Fig. 2 .
Fig. 2. Optical density at 600 nm of lactic acid bacteria at different concentrations of bile