Probiotic and Techno-Functional Traits of Lactobacillus pentosus DS2 Isolated from Naturally Fermented Plant Beverage

The research on the isolation of novel lactic acid bacteria (LAB) strains from different fermented plant beverages is receiving immense attention for their signi icant health bene its towards human health. The present study aimed to isolate and categorize various functional attributes of Lactobacillus pentosus DS2 isolated from fermented black carrot beverage. The isolated L.pentosus DS2 strain exhibited resistance to acid and higher salt concentrations. The isolated strain was identi ied by using 16S rRNA gene sequences. L.pentosus DS2 showed high survivability of about 6.75 to 7.02 log CFU/ml from pH (2-8) and at a different salt concentration (1-10%) log CFU/ml ranged from 7.92 to 6.41 log CFU/ml. According to the obtained results, auto-aggregation aswell as cell surface hydrophobicity was about 16.2± 0.35 and 90± 0.21 % respectively, while co-aggregation value was 72.5± 2.12 and 82± 1.41%with Escherichia coli and Staphylococcus aureus respectively. The enzymatic screeningwas performed and estimated as 0.54 ± 0.01, 103 ± 1.41, and 80.5 ± 2.89 U/mL of amylase, protease, and phytase. Cholesterol removal by L. pentosus DS2 was 47.15± 0.41%. The adherence levels by L. pentosus DS2 to different cell lines such as Caco-2 andHT-29 ranged from 17.65± 0.25 to 19.79± 0.31% respectively. Antibiotic susceptibility pattern obtained showed a different degree of antibiotics sensitivity, such as resistance to ampicillin. Thus, the isolated L.pentosus DS2 has all the desired properties to be used as a potential probiotics strain.


INTRODUCTION
Fermented plant beverages (FPB) are a kind of potential functional food that is very common in Asian countries, particularly in China, Japan, and India (Kabak and Dobson, 2011). Plants which include vegetables, fruits, cereals, legumes are materials used to produce fermented plant beverages. FPB is dissimilar from other fermented products such as wine, vinegar, yogurt. The difference is there is no alcohol in fermented plant beverages, or very less amount of alcohol is found in them. LAB are the most prominent group of probiotics which have a bene iciary effect on their host by enhancing intestinal balance, and these are usually integrated with plant beverages. LAB is naturally associated with various dairy and non-dairy fermented food formulations. With the increasing trend of veganism, cholesterol contents, lactose intolerance demand of plant-based probiotic bev-erages has increased particularly fruits and vegetable origin beverages as they contain bioactive nutritive metabolites such as vitamins, minerals, ibers, and non-nutritive constituents which include phenolic substances, lavonoids, bioactive peptides which possess potential health effects along with metabolic and functional versatility of LAB.
LAB contains a group of enzymes and also some metabolic traits which are involved in the biotransformation of natural compounds present in plants by incorporating various metabolic and functional pathways. LAB fermentation affects the nutritional quality of plant-based material by the degradation of toxic or antinutritional factors. LAB isolated from autochthonous microbiota show potential as a starter to ferment plant-based material and ensures the preservation of various attributes: natural color, irmness, antioxidant potential, and other healthpromoting metabolites. This preservation effect may be the result of the modi ication in organic acid pro iles such as the production of lactic acid, acetic acid, and the metabolic breakdown of amino acids.
FPB harbor a large population of LAB. Besides increasing the shelf life, these isolated LAB strains have various bene icial properties on fermented products which include improved functional, sensory, and organoleptic properties. Isolation of LAB strains from fermented fruits and vegetables such as cucumber, radish, sauerkraut, table olives, carrot, etc. hold a great promise as probiotics and contribute fermented vegetables with enormous health bene its like healthy gut microbiota, enhanced immune system, and to increase in functional and nutritional properties via formation of novel bioactive substances or by increasing bioavailability of prevailing one. They have shown great tolerance towards gastrointestinal stresses like subjection to gastric juices, bile salts, antibacterial activity towards Bacillus cereus, Listeria monocytogenes, S.aureus and E.coli, Salmonella enterica). Hence, LAB probiotic strains are obtained from fermented foods as capable probiotics for human utilization. Therefore, the current study aimed to isolate and identify indigenous LAB from fermented black carrot beverage and to determine techno-functional traits.

Isolation and maintenance of strains
Different batches of spontaneously fermented BC juice samples were withdrawn for the isolation of LAB strains. De Man, Rogasa, and Sharpe (MRS) agar (Titan Biotech, India) comprising 6% NaCl was utilized to obtain LAB in the present study (Kingston et al., 2010). To differentiate LAB from inhabitant bacteria diversity, 1% of CaCO3 was incorporated into MRS agar. Fermented BC juice samples were drawn at different time intervals during the fermentation process and were suitably diluted, followed by spreading over the MRS agar plates, which comprised 6% NaCl and 1% CaCO3. MRS plates were kept under incubation at 37 • C for 2 days. Colonies were selected based on their morphology such as small greyish or white colonies, either lat or raised, whether smooth or rough surrounded with clear zone were selected and puri ied by repeated subculture on MRS agar plates. LAB strain was maintained in MRS broth comprising 20% (v/v) glycerol at -20 o C till further use.

Identi ication of lab
Rapid colony PCR and high throughput PCR methods were done for the taxonomic proof of identity (Sharma et al., 2015). Other bacterial sequences were obtained from NCBI mega BlAST (http://blast. ncbi.nlm.nih.gov/Blast.cgi) to analyse the 16S rRNA gene sequence obtained from the isolate for their pairwise identities. DNA baser (http://www.dna baser.com/) software was used to determine the nucleotide constituents of each ampli ied sequence, and the 16S rRNA entry was submitted to GenBank.

Techno-functional and probiotic attributes pH tolerance
The pH resistance of isolated strain was done according to (Pundir et al., 2013) with little variation. Brie ly, cells were cultivated in MRS broth at 37 o C and kept for overnight incubation, and further sub-cultured in fresh MRS media adjusted to pH 1.0-8.0 with hydrochloric acid (5.0 M) and sodium hydroxide (5.0 M), following which 1% (v/v) of preculture 16 hr old LAB was inoculated and cultivated at 37 o C for 4h under shaking conditions at 200 RPM. The survival population of the LAB was determined by the following equation,

Salt tolerance
The NaCl tolerance of LAB isolates was done according to (Jampaphaeng et al., 2017) with some variation. The MRS broth was adjusted at different concentrations of NaCl ranging from 1-10% (w/v), and then 1% (v/v) of 16 hr old pre-culture was inoculated into MRS broth and grown at 37 o C for 24 h. The survival population of the LAB was determined by the following equation:

Cell surface hydrophobicity
The adhesion of LAB cells to hydrocarbon such as hexadecane was estimated. Cells were recovered by centrifugation (5000 rpm for 15 min at 4 o C), washed repeatedly with PBS (pH 7.0), and resuspended in PBS to get roughly 10 8 CFU/mL. Afterwards, 2 mL of LAB culture was placed in close contact with 0.6 mL of xylene. LAB suspension was thoroughly mixed and kept for incubation at 37 o C for 10 mins. OD (A 0 ) of the aqueous phase was analysed at 600nm.
The hydrophobicity (%age) was analysed as,

Cell adhesion
The different cell lines, such as HT-29 and Caco-2, were obtained from NCCS, Pune, India. Both the cell lines were regularly cultured in Dulbecco's modi ied Eagle's minimal essential medium (DMEM) added with 10-20 % (v/v) sterilized foetal bovine serum. The cell lines were also augmented by 1% (v/v) Gentamycin sulfate solution in DMEM. The cell line was kept at 37 o C in the presence of 10 % CO 2 . The cell lines were fed with DMEM medium regularly until the cells reached 80 % con luency.
Adhesion of LAB strain to HT-29 and Caco-2 cells was considered using a modi ied protocol ((née Lehto) and Salminen, 1998). Monolayer cell lines of HT-29 and Caco-2 cells were washed with PBS (pH 7.0) and inoculated with LAB and incubated at 10 % CO 2 at 37 o C.
To count the viable adhered LAB, the monolayer cells were removed by trypsinization for 15 min at 37 o C. The viable cells were counted on MRS agar at 37 o C.

Cholesterol assimilation
The cholesterol assimilation by the isolated Lactobacillus strain was studied as per the method given by (Walker and Gilliland, 1993). The percentage of cholesterol assimilation in supernatant broth as compared to the uninoculated broth was recorded according to the following equation,

Auto-aggregation assay
Aggregation capabilities of isolated LAB strain were estimated by the protocol given by (Collado et al., 2008) with few modi ications. The 16 h old bacterial pr-culture was harvested by centrifugation at 5000×g ,(10 min,4 • C )and washed twice with PBS (pH 7.1). The washed LAB cells were suspended again in PBS. Absorbance was determined at a wavelength of about 600 nm (A 600 ) at two different time intervals (0 or 2h). Aggregation (%) was expressed as, where A t represents absorbance after 2h and A 0 absorbance at 0h.

Co-aggregation assay
Co-aggregation ability of isolated LAB strain with pathogenic bacteria was done according to (Ekmekci et al., 2009) with some modi ication. 2ml of cells from both LAB and pathogenic strains were mixed and kept under incubation at 37 o C. The absorbance of a suspension was determined at a wavelength of 600nm (A 600nm ) at different time intervals (0 or 2h) and co-aggregation was calculated as, where A pat and A LAB designates A 600 nm of the separate LAB suspensions in control tubes and A mix represents the absorbance of the mixed bacterial mix at 2h.

Screening for degradative enzymes
The enzymatic screening of the LAB was performed by spot/well inoculation on agar containing a particular substrate. LAB for amylase production was spotted on modi ied MRS media containing 0.5% peptone, 0.7% yeast extract, 0.2% NaCl, 0.75% starch and 1.5% agar was used. A zone of clearance was observed around the bacterial colonies after incubating plates for 24-48hours followed by looding with iodine solutions. For protease, LAB was spotted on the medium comprising of skim milk (1%) and agar (1.5%). Phytate hydrolysis by LAB was observed by using modi ied Sperbers media containing 10% CaCl 2 and 10% KH 2 PO 4 . After cultivation at 37°C for 48h, the zone of clearance (mm) adjacent to the colony was observed and recorded using a digital micrometer.

Antibiotic sensitivity
Antibiotic susceptibility of isolated LAB was estimated by the protocol given by (Wang et al., 2019) with little variations by using the antibiotic disc diffusion method on Mueller Hinton agar plates. Antibiotic discs (Himedia) containing ampicillin (2mcg), amoxyclav (30mcg), ampicillin/sulbactam (10mcg), gentamycin (10mcg), ertapenem (10mcg), o loxacin (2mcg), piperacillin (75mcg) were used to assess the susceptibility. Overnight bacterial culture of L. pentosusBC2 was spread over the Mueller Hinton agar by three-way swabs, and different antibiotic discs were placed. The plates were kept for incu-bation at 37 • C for 12 to 18 hours and observed for zone formation. This study was conducted in two replications. Results were documented as an average from duplicates, Results for Inhibitory zones were classi ied as resistant R (≤ 14 mm), intermediate susceptible I S (15-19 mm), and susceptible S (≥ 20 mm) towards antibiotic discs. Clinical and Laboratory Standard Institute (Abbey and Deak, 2019) was referred for the interpretation of antibiotic sensitivity.

Isolation of autochthonous LAB
LAB isolated from different fermented plant products can be utilized for food preservation, food enrichment, and probiotic supplements along with food for human consumption. Isolated colonies were initially Gram-stained and con irmed to produce catalase. Taxonomic identi ication con irmed that ASC isolated in the present study was Lactobacillus pentosus DS2. In the present study, LAB strain isolated from fermented black carrot juice was identi ied as L.pentosus DS2 with accession number MT197323 by using 16S rDNA sequencing. A phylogenetic tree was constructed to ind out the genetic relationship between the L.pentosus DS2 with other strains (Figure 1). The obtained results were consistent with the previous studies suggesting LAB are members of fermented plant products. The other LAB species associated with fermented plant products are Lactobacillus Plantarum, Lactobacillus casei, and Lactobacillus brevis. In a similar study, Pediococcus acidilactici Ch-2 was isolated from fermented apricot called chuli has shown potential probiotic characteristics with many functional properties and novel compounds. In another study, strains of Lb. pentosus,Lb. Plantarum and Lb. paracasei subsp. Paracasei isolated from naturally fermented olives has shown desired invitro probiotic attributes such as resistance towards low pH, high levels of bile salts, Variable ef iciency were observed for adherence towards cell lines like Caco-2 cells which were same regarding strain's susceptibility towards different antibiotics (Argyri et al., 2013).

Techno-functional and probiotic attributes pH and salt tolerance
The selection of probiotic LAB based on pH tolerance is a signi icant criterion because before reaching the intestinal tract where pH lies between 6 to 7.5, LAB should be able to withstand acidic gastric conditions (pH nearly 1-2). In the present study, at pH 1 survival rate of viable counts as zero, and from pH 2-8 survival rate of viable counts increased gradually from 6.75 to 7.02 log CFU/ml and reaching max at pH 7 (Figure 2 A). Other studies have also con irmed that there will be a marked reduction in viable bacterial cells on prolonged exposure to gastric acidic conditions. Incubation time could be another reason for the relatively less rate of survival under acidic conditions (low pH) which was extended by 8 times than that of normal GIT passage in vivo. These results were following previous studies that showed LAB isolated from fermented olives possess the ability to survive at low pH (Zago et al., 2013).
NaCl tolerance of lactic acid bacteria is a vital criterion to ind out the effect of increasing salt concentration on LAB. NaCl can act as an inhibitory substance that may have deleterious effects on the growth of some bacteria. LAB's are unprotected to osmotic stress when higher concentrations of salts are required to be added to a product. The present experimental results showed L.pentosus DS2 was able to grow in the incidence of different NaCl concentrations ranging from 1-10%. Bacterial growth reduction was observed with increasing concentration of NaCl. The survival rate of viable LAB count decreased from 7.92 to 6.41 log CFU/ml (Figure 2 B). These indings were similar to a previous study that showed the growth of LAB isolates from fermented olives gradually showed a decreasing trend with the increase in NaCl concentration, attaining the lowest count when NaCl was 10% Zago et al. (2013)

Cell adherence property (CSH)
Adherence is an important property which enables the LAB to bind with intestinal epithelial cell lining and thus help in inducing cell barrier. LAB shows auto-aggregation property through which the advantageous impact of colonization takes place inside the gastrointestinal tract. Therefore indicating aggregation of lactobacilli a desirable characteristic, especially in the human gut. The CSH is another important anticipated property of probiotic strains which designates signi icant adherence behavior of suitable probiotic candidates. In the present study, L.pentosus DS2 showed a signi icant level of hydrophobicity (90%), and the autoaggregation level was about 16 ± 0.35%. Another probiotic property that is of signi icant importance is co-aggregation in which LAB can interact closely with other pathogens and prevents their colonization. The current study demonstrated signi icant coaggregation property with E.coli (72.5 ± 2.12) and S. aureus (82 ± 1.41) ( Table 1). The present results were similar to the indings previously observed by other studies. According to (Montoro et al., 2016),      both auto and co-aggregation of LAB were strainspeci ic which may involves adhesion, aggregation and mucus binding properties.
One of the prerequisite measures for the selection of promising probiotics is their potential to bind to epithelial cells and mucosal lining for their colonization in the intestine. LAB adherence to the gastrointestinal surface increases their residing time which has a direct impact on the host wellbeing by modulating their intestinal immune system and is also important for the removal of potential pathogens. Various reports have been published with human epithelial cell lines such as HT29 and Caco-2 to determine the adherence potential of probiotic starters (Grover et al., 2011). In the current study, the adherence level of L.pentosus DS2 to Caco-2 and HT-29 cell lines ranged from 17.65 ± 0.25 to 19.79 ± 0.31 % respectively which agrees with the adherence extent reported previously to see the potential of LAB strain in reinstating the gut microbiome composition (Sharma and Kanwar, 2017). LAB displaying variation in adherence potential towards the intestinal cell lines was demonstrated by many workers thus, representing that adherence potential is extremely strain-speci ic ( Figure 3)

Enzyme production
Enzyme production by the probiotic strains is important for their selection, and it also provides information about the enzymatic ability of a strain to their speci ic performance and their effect on the development of organoleptic characteristics of the inal product. In the current case, L.pentosus DS2 was screened qualitatively and quantitatively for commonly known degradative enzymes, i.e. amylase, proteases, and phytase. The production of all above mentioned dietary enzymes was examined by inoculating the L.pentosus DS2 with speci ic media contains the respective substrate. In qualitative screening, the strain was observed for the zone of starch hydrolysis (8.43 ± 0.18mm) when looded with iodine solution for extracellular production amylase. Zone of clearance (14.65 ± 0.21mm) for protease production around the colonies on skim milk agar and extracellular phytase production (11.9 ± 0.68mm) on modi ied Sperbers media. In continuation, quantitative estimation of all the dietary enzymes has been examined, and it was established that 0.54±0.01,103±1.41and 80.5±2.89 U/mL of extracellular production of amylase, protease, and phytase of the L. pentosus DS2 in MRS medium (Table 2). These indings were similar as obtained by previous studies (Pailin et al., 2001).

Cholesterol assimilation
Cholesterol assimilation by probiotic bacteria in the gut through different mechanisms such as assimilation, enzymatic degradation, or by any other mechanisms allows the reduced absorption of cholesterol by enterocytes and more excretion or removal of the cholesterol from the host. Thus, aids in lowering the serum cholesterol level, and thereby helps in the management of hypercholesterolemia related CVDs.In the present study, a signi icant rate of cholesterol removal by L.pentosus DS2 (47.15 ± 0.41%) was obtained and agreed with different reports (Benítez-Cabello et al., 2019).

Antibiotic sensitivity
Probiotic bacteria possess a large number of biotechnological values and have been explicitly selected for the prevention of antibiotic resistance spread. The isolated LAB strain was screened against antibiotics with broad-spectrum and produced inhibiting effects on both protein synthesis as well as on cell wall synthesis ( and Leuconostoc possess strong inherent resistance towards the antibiotics used in the present study (Nawaz et al., 2011). This result agreed with (Halami et al., 2000), who reported that the LAB shows more resistance to the principal types of antibiotics.

CONCLUSIONS
The present study investigated Lactobacillus pen-tosusDS2 isolated from naturally fermented black carrot for their potentiality as a potent probiotic. Findings divulged that the L.pentosus DS2 has a great ability to withstand low pH, higher salt concentration, and has shown higher survival rates. Although, the isolated strain presented numerous cell surface properties, in the case of, hydrophobicity, auto-and co-aggregation. Thus, the cell surfacebinding properties of isolated L.pentosus DS2 will help in attachment and colonizing the gastrointestinal tract, which is an important aspect of antimicrobial effects and other disease treatments. Furthermore, this strain has shown the great ability of cholesterol removal. Also, the isolated strain was able to produce a signi icant amount of enzymes. A signi icant level of adherence was shown on caco2 and HT-29 cell lines. Antibiotic susceptibility shown by isolated strain was satisfactory. LAB strain exhibited complete resistance against ampicillin. LAB isolated from fermented black carrot under in vitro conditions demonstrated promising probiotic characteristics with functional merits and thereby expressing their abilities to be used as probiotics in food and feed formulations. Further in vivo studies are required to be done to determine their health beneits. Safety properties need to be done before their use as probiotics in food industries.